Foodomics Applications with High Resolution MS - Waters Corporation Food Research
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Transcript of Foodomics Applications with High Resolution MS - Waters Corporation Food Research
copy2015 Waters Corporation 1
Foodomics Applications Improving consumer well being health and
knowledge using the latest -omic techniques
copy2015 Waters Corporation 2
Overview
Introduction to foodomics and nutrimetabolomics
ndash Influence of diet on health (eg Dietary flavonoids)
Metabolomic approaches for molecular characterisation of foods
ndash Characterisation of nutritionally valuable natural products (Passiflora example)
ndash Investigation of flavonoid markers of dietary intake
Rapid Evaporative Ionisation Mass Spectrometry (REIMS)
ndash Direct analysis for rapid profiling
Summary
Acknowledgements
copy2015 Waters Corporation 3
Foodomics
ndash Refers to the metabolite profiling of
foods prior to consumption
(biomarkers of consumption)
Nutritional metabolomics has
emerged with two major goals
ndash (1) to determine the effects of
dietary compounds on host
metabolism after consumption
(biomarkers of effect)
ndash (2) identify metabolic disease that is
influenced by nutrients amp to develop
targeted diet-based treatments
(disease risk biomarkers)
Foodomics and nutrimetabolomics
Heart disease
Alheizmers
Cancer
Asthma
Diabetes
copy2015 Waters Corporation 5
Links between diet and healthhellip
copy2015 Waters Corporation 6
Functional foods
Flavonoids are one of the largest widespread class of plant secondary
metabolites with diverse biological amp pharmacological properties
Highly bioactive and play a wide variety of different roles in health of
pants animals and human health
Many flavonoid containing plants are utilized as functional foods amp
phytomedicines
Functional foods represent one of the fastest growing markets with
interest in the systematic characterization of flavonoids in plant crops
Flavonoids
Reduce blood
pressure
Antioxidants
Anti-inflammator
y action
Improve endothelial
function
Reduce platelet activity
Enzymatic Modulation
copy2015 Waters Corporation 7
Characterisation of functional foods
Functional foods amp phytomedicines
ndash Natural product profiling - flavonoids
ndash Analytical challenges
Technology overview
ndash Introduction to CCS
ndash HRAMS with ion mobility
ndash Reducing sample complexity (spectral cleanup)
ndash Increased selectivity with ion mobility
Profiling of Passiflora species (marker flavonoids) using ion
mobility with UNIFI processing
copy2015 Waters Corporation 9
Natural product profiling Analytical challengeshellip
Sample complexity
Crude (non-selective) sample preparation
Generic chromatography conditions (no chromatography)
ndash gt30k features
Spectral interpretation
Isomeric compounds
ndash different pharmacological affects
Structural elucidation of ldquounknownsrdquo
Identification of authentic product
ndash Identification of active components
ndash Quantitation of active components
copy2015 Waters Corporation 10
IMS-MS for natural product profiling
IMS-MS can help overcome the challenge of matrix complexity
IMS is a rapid separation approach orthogonal to UPLC that provides
increased peak capacity
Separation can help spectral decongestion for complex samples
improving selectivity amp specificity
Can separate isomeric species and provide structural confirmatory
information
Spectral clarity improves confidence in identification and aids in
structural elucidation of unknowns
copy2015 Waters Corporation 11
Introduction to ion mobility amp Synapt G2-Si
technology
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 2
Overview
Introduction to foodomics and nutrimetabolomics
ndash Influence of diet on health (eg Dietary flavonoids)
Metabolomic approaches for molecular characterisation of foods
ndash Characterisation of nutritionally valuable natural products (Passiflora example)
ndash Investigation of flavonoid markers of dietary intake
Rapid Evaporative Ionisation Mass Spectrometry (REIMS)
ndash Direct analysis for rapid profiling
Summary
Acknowledgements
copy2015 Waters Corporation 3
Foodomics
ndash Refers to the metabolite profiling of
foods prior to consumption
(biomarkers of consumption)
Nutritional metabolomics has
emerged with two major goals
ndash (1) to determine the effects of
dietary compounds on host
metabolism after consumption
(biomarkers of effect)
ndash (2) identify metabolic disease that is
influenced by nutrients amp to develop
targeted diet-based treatments
(disease risk biomarkers)
Foodomics and nutrimetabolomics
Heart disease
Alheizmers
Cancer
Asthma
Diabetes
copy2015 Waters Corporation 5
Links between diet and healthhellip
copy2015 Waters Corporation 6
Functional foods
Flavonoids are one of the largest widespread class of plant secondary
metabolites with diverse biological amp pharmacological properties
Highly bioactive and play a wide variety of different roles in health of
pants animals and human health
Many flavonoid containing plants are utilized as functional foods amp
phytomedicines
Functional foods represent one of the fastest growing markets with
interest in the systematic characterization of flavonoids in plant crops
Flavonoids
Reduce blood
pressure
Antioxidants
Anti-inflammator
y action
Improve endothelial
function
Reduce platelet activity
Enzymatic Modulation
copy2015 Waters Corporation 7
Characterisation of functional foods
Functional foods amp phytomedicines
ndash Natural product profiling - flavonoids
ndash Analytical challenges
Technology overview
ndash Introduction to CCS
ndash HRAMS with ion mobility
ndash Reducing sample complexity (spectral cleanup)
ndash Increased selectivity with ion mobility
Profiling of Passiflora species (marker flavonoids) using ion
mobility with UNIFI processing
copy2015 Waters Corporation 9
Natural product profiling Analytical challengeshellip
Sample complexity
Crude (non-selective) sample preparation
Generic chromatography conditions (no chromatography)
ndash gt30k features
Spectral interpretation
Isomeric compounds
ndash different pharmacological affects
Structural elucidation of ldquounknownsrdquo
Identification of authentic product
ndash Identification of active components
ndash Quantitation of active components
copy2015 Waters Corporation 10
IMS-MS for natural product profiling
IMS-MS can help overcome the challenge of matrix complexity
IMS is a rapid separation approach orthogonal to UPLC that provides
increased peak capacity
Separation can help spectral decongestion for complex samples
improving selectivity amp specificity
Can separate isomeric species and provide structural confirmatory
information
Spectral clarity improves confidence in identification and aids in
structural elucidation of unknowns
copy2015 Waters Corporation 11
Introduction to ion mobility amp Synapt G2-Si
technology
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 3
Foodomics
ndash Refers to the metabolite profiling of
foods prior to consumption
(biomarkers of consumption)
Nutritional metabolomics has
emerged with two major goals
ndash (1) to determine the effects of
dietary compounds on host
metabolism after consumption
(biomarkers of effect)
ndash (2) identify metabolic disease that is
influenced by nutrients amp to develop
targeted diet-based treatments
(disease risk biomarkers)
Foodomics and nutrimetabolomics
Heart disease
Alheizmers
Cancer
Asthma
Diabetes
copy2015 Waters Corporation 5
Links between diet and healthhellip
copy2015 Waters Corporation 6
Functional foods
Flavonoids are one of the largest widespread class of plant secondary
metabolites with diverse biological amp pharmacological properties
Highly bioactive and play a wide variety of different roles in health of
pants animals and human health
Many flavonoid containing plants are utilized as functional foods amp
phytomedicines
Functional foods represent one of the fastest growing markets with
interest in the systematic characterization of flavonoids in plant crops
Flavonoids
Reduce blood
pressure
Antioxidants
Anti-inflammator
y action
Improve endothelial
function
Reduce platelet activity
Enzymatic Modulation
copy2015 Waters Corporation 7
Characterisation of functional foods
Functional foods amp phytomedicines
ndash Natural product profiling - flavonoids
ndash Analytical challenges
Technology overview
ndash Introduction to CCS
ndash HRAMS with ion mobility
ndash Reducing sample complexity (spectral cleanup)
ndash Increased selectivity with ion mobility
Profiling of Passiflora species (marker flavonoids) using ion
mobility with UNIFI processing
copy2015 Waters Corporation 9
Natural product profiling Analytical challengeshellip
Sample complexity
Crude (non-selective) sample preparation
Generic chromatography conditions (no chromatography)
ndash gt30k features
Spectral interpretation
Isomeric compounds
ndash different pharmacological affects
Structural elucidation of ldquounknownsrdquo
Identification of authentic product
ndash Identification of active components
ndash Quantitation of active components
copy2015 Waters Corporation 10
IMS-MS for natural product profiling
IMS-MS can help overcome the challenge of matrix complexity
IMS is a rapid separation approach orthogonal to UPLC that provides
increased peak capacity
Separation can help spectral decongestion for complex samples
improving selectivity amp specificity
Can separate isomeric species and provide structural confirmatory
information
Spectral clarity improves confidence in identification and aids in
structural elucidation of unknowns
copy2015 Waters Corporation 11
Introduction to ion mobility amp Synapt G2-Si
technology
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 5
Links between diet and healthhellip
copy2015 Waters Corporation 6
Functional foods
Flavonoids are one of the largest widespread class of plant secondary
metabolites with diverse biological amp pharmacological properties
Highly bioactive and play a wide variety of different roles in health of
pants animals and human health
Many flavonoid containing plants are utilized as functional foods amp
phytomedicines
Functional foods represent one of the fastest growing markets with
interest in the systematic characterization of flavonoids in plant crops
Flavonoids
Reduce blood
pressure
Antioxidants
Anti-inflammator
y action
Improve endothelial
function
Reduce platelet activity
Enzymatic Modulation
copy2015 Waters Corporation 7
Characterisation of functional foods
Functional foods amp phytomedicines
ndash Natural product profiling - flavonoids
ndash Analytical challenges
Technology overview
ndash Introduction to CCS
ndash HRAMS with ion mobility
ndash Reducing sample complexity (spectral cleanup)
ndash Increased selectivity with ion mobility
Profiling of Passiflora species (marker flavonoids) using ion
mobility with UNIFI processing
copy2015 Waters Corporation 9
Natural product profiling Analytical challengeshellip
Sample complexity
Crude (non-selective) sample preparation
Generic chromatography conditions (no chromatography)
ndash gt30k features
Spectral interpretation
Isomeric compounds
ndash different pharmacological affects
Structural elucidation of ldquounknownsrdquo
Identification of authentic product
ndash Identification of active components
ndash Quantitation of active components
copy2015 Waters Corporation 10
IMS-MS for natural product profiling
IMS-MS can help overcome the challenge of matrix complexity
IMS is a rapid separation approach orthogonal to UPLC that provides
increased peak capacity
Separation can help spectral decongestion for complex samples
improving selectivity amp specificity
Can separate isomeric species and provide structural confirmatory
information
Spectral clarity improves confidence in identification and aids in
structural elucidation of unknowns
copy2015 Waters Corporation 11
Introduction to ion mobility amp Synapt G2-Si
technology
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 6
Functional foods
Flavonoids are one of the largest widespread class of plant secondary
metabolites with diverse biological amp pharmacological properties
Highly bioactive and play a wide variety of different roles in health of
pants animals and human health
Many flavonoid containing plants are utilized as functional foods amp
phytomedicines
Functional foods represent one of the fastest growing markets with
interest in the systematic characterization of flavonoids in plant crops
Flavonoids
Reduce blood
pressure
Antioxidants
Anti-inflammator
y action
Improve endothelial
function
Reduce platelet activity
Enzymatic Modulation
copy2015 Waters Corporation 7
Characterisation of functional foods
Functional foods amp phytomedicines
ndash Natural product profiling - flavonoids
ndash Analytical challenges
Technology overview
ndash Introduction to CCS
ndash HRAMS with ion mobility
ndash Reducing sample complexity (spectral cleanup)
ndash Increased selectivity with ion mobility
Profiling of Passiflora species (marker flavonoids) using ion
mobility with UNIFI processing
copy2015 Waters Corporation 9
Natural product profiling Analytical challengeshellip
Sample complexity
Crude (non-selective) sample preparation
Generic chromatography conditions (no chromatography)
ndash gt30k features
Spectral interpretation
Isomeric compounds
ndash different pharmacological affects
Structural elucidation of ldquounknownsrdquo
Identification of authentic product
ndash Identification of active components
ndash Quantitation of active components
copy2015 Waters Corporation 10
IMS-MS for natural product profiling
IMS-MS can help overcome the challenge of matrix complexity
IMS is a rapid separation approach orthogonal to UPLC that provides
increased peak capacity
Separation can help spectral decongestion for complex samples
improving selectivity amp specificity
Can separate isomeric species and provide structural confirmatory
information
Spectral clarity improves confidence in identification and aids in
structural elucidation of unknowns
copy2015 Waters Corporation 11
Introduction to ion mobility amp Synapt G2-Si
technology
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 7
Characterisation of functional foods
Functional foods amp phytomedicines
ndash Natural product profiling - flavonoids
ndash Analytical challenges
Technology overview
ndash Introduction to CCS
ndash HRAMS with ion mobility
ndash Reducing sample complexity (spectral cleanup)
ndash Increased selectivity with ion mobility
Profiling of Passiflora species (marker flavonoids) using ion
mobility with UNIFI processing
copy2015 Waters Corporation 9
Natural product profiling Analytical challengeshellip
Sample complexity
Crude (non-selective) sample preparation
Generic chromatography conditions (no chromatography)
ndash gt30k features
Spectral interpretation
Isomeric compounds
ndash different pharmacological affects
Structural elucidation of ldquounknownsrdquo
Identification of authentic product
ndash Identification of active components
ndash Quantitation of active components
copy2015 Waters Corporation 10
IMS-MS for natural product profiling
IMS-MS can help overcome the challenge of matrix complexity
IMS is a rapid separation approach orthogonal to UPLC that provides
increased peak capacity
Separation can help spectral decongestion for complex samples
improving selectivity amp specificity
Can separate isomeric species and provide structural confirmatory
information
Spectral clarity improves confidence in identification and aids in
structural elucidation of unknowns
copy2015 Waters Corporation 11
Introduction to ion mobility amp Synapt G2-Si
technology
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 9
Natural product profiling Analytical challengeshellip
Sample complexity
Crude (non-selective) sample preparation
Generic chromatography conditions (no chromatography)
ndash gt30k features
Spectral interpretation
Isomeric compounds
ndash different pharmacological affects
Structural elucidation of ldquounknownsrdquo
Identification of authentic product
ndash Identification of active components
ndash Quantitation of active components
copy2015 Waters Corporation 10
IMS-MS for natural product profiling
IMS-MS can help overcome the challenge of matrix complexity
IMS is a rapid separation approach orthogonal to UPLC that provides
increased peak capacity
Separation can help spectral decongestion for complex samples
improving selectivity amp specificity
Can separate isomeric species and provide structural confirmatory
information
Spectral clarity improves confidence in identification and aids in
structural elucidation of unknowns
copy2015 Waters Corporation 11
Introduction to ion mobility amp Synapt G2-Si
technology
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 10
IMS-MS for natural product profiling
IMS-MS can help overcome the challenge of matrix complexity
IMS is a rapid separation approach orthogonal to UPLC that provides
increased peak capacity
Separation can help spectral decongestion for complex samples
improving selectivity amp specificity
Can separate isomeric species and provide structural confirmatory
information
Spectral clarity improves confidence in identification and aids in
structural elucidation of unknowns
copy2015 Waters Corporation 11
Introduction to ion mobility amp Synapt G2-Si
technology
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 11
Introduction to ion mobility amp Synapt G2-Si
technology
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 12
Ion mobility separation
Separation of ionic species as they drift through a gas under the influence of an electric field
Rate of drift is dependent on ionrsquos mobility in the gas
Leads to separation based on 3D molecular conformation size amp charge
Isobaric ions
IMS
-9 -8 -7 -6
TOF MS LC
-5 -4 -3 -2 -1 0 1 2 3 4 n
10n seconds
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 13
SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF
1 Increased sensitivity
2 Ion mobility 3 Accurate mass
measurement
15mm
5mm
~11mm
CID amp ETD
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 14
HDMSE Unlimited Product Ion Acquisition
Co-eluting precursor ions
Mobility separation
Drift time aligned precursors and products
Drift time
mz
Drift time
mz
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 16
MSE ndash (no mobility separation)
Low Energy
High Energy
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 17
HDMSE ndash MSE with a mobility separation
Low Energy
High Energy
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 18
Collision Cross Section (CCS)
Measured drift time can be converted into CCS
CCS is an important distinguishing characteristic of an ion which is related to
ndash chemical structure
ndash 3-dimensional conformation
ndash Charge
CCS is a physicochemical property of an ion
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 19
Combining analytical discrimination
Analytical measurement
rt
mz
dt
Property of molecule
KD
mass
CCS
UPLC
Mass spectrometer
Ion mobility
cell
Peak capacity
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 20
Profiling analysis of Passifloraceae spp
Also known as passion flower or passion vine
~ 500 species of flowering plants
Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery
ndash Herbal remedies amp supplements
Sedative properties of some species used to alleviate nervous anxiety and insomnia
Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 21
Product profiling - analytical strategy
Component detection
Determine chemical structures
Assign identifications for knownunknowns look for unknowns
Resolve analytes chromatographically OR
direct analysis
Component identification
isobaric analytes isomers different charge states resolve co-eluting analytes
UPLC separation for sensitivity and resolution
Infusion ASAP
Synapt G2-Si ESI [pos amp neg]
HDMSE unbiased data acquisition
UNIFI 18 Exact mass precursor amp fragments isotope
pattern rtime drift time (CCS)
Chemical elucidation toolset Elemental Composition
Mass Fragment for structure assignment amp confirmation Chemspider library searches
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 22
Answering the challenge of natural product
characterisation
Sample complexity
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 23
UPLC separation Passiflora edularis
gt10 000 components
detected
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 24
Ion mobility separation Increased peak capacity
Conventional UPLC separation
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 25
Answering the challenge of natural product
characterisation
Spectral interpretation
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 26
MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins
Conventional HRMS Vitexin
Low energy
High energy
Vitexin
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 27
HDMSE single component precursor and ion mobility product ion spectra
Vitexin (mz 431) retention time 8395 mins drift time 427 ms
Resolved from chromatographically coeluting components using ion mobility
Ion mobility spectral clean-up Vitexin
Vitexin
Low energy
High energy
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 28
Answering the challenge of natural product
characterisation
Isomers
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 29
Active marker flavonoid structures
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
OH
O
O
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
OH
OH
OH
Isoorientin Orientin
Isovitexin Vitexin
6C glycocides 8C glycocides
6
8
6
8
Luteolin-8-C-glucoside C21H20O11
Luteolin-6-C-glucoside C21H20O11
Apigenin-6-C-glucoside C21H20O10
Apigenin-8-C-glucoside C21H20O10
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 30
Orientinisoorientin isomer separation amp CCS values
Isoorientin
19768Ǻ2
Orientin
18765Ǻ2
Identical twins with shared retention time amp accurate mass BUT different DRIFT times
C21H20O11
448377
∆ =10 Ǻ2
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 31
Answering the challenge of natural product
characterisation
Authenticity amp active compounds
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 32
Identification of active compounds
CAERULEA EDULIS
INCARNATA ALATA
Which species has the most potent sedative effects
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 33
Comparative analysis tools Pedulis and Palata
Region of interest isoorientin amp orientin
Region of interest isovitexin amp vitexin
Mirror plots
Species comparison Passiflora edulis (reference) Passiflora alata (control)
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 34
Passiflora edulis +++
Passiflora alata +
Vitexin
Isovitexin
Chromatographic view shows intensity differences for active compounds between species
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 35
PE unique region of
interest mz 577
Comparative analysis tools Pedulis and Palata
Drift time
mz
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 36
Passiflora Edulis
Passiflora Alata
Different profiles for the markers at mz 577 Candidates for further investigation
Comparative analysis tools Pedulis and Palata
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 37
Summary
Natural product profiling is challenged by complex samples with many isomeric
compounds
Combined with UPLC and HRMS ion mobility offers advantages for the
characterisation of these samples
ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak
capacity
ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers
ndash Drift times are measured for all precursor ions
ndash Simultaneous fragmentation data is obtained for all components
ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate
single component fragment ion spectra for all precursors
UNIFI software provides all the required informatics for interpretation of these
comprehensive datasets
ndash UNIFI automatically generates collision cross sections (CCS) for all components
ndash HDMS data viewer and comparison tools allows drift time mapping
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 38
Markers of dietary intake
Monitoring a flavanol-rich diet
Dr Vanessa Garcia Larsen
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 39
Project background
Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease
Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous
More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures
Aims ndash Can an accurate method be developed to estimate flavonoid markers
of dietary intake
o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)
o Can any biomarkers metabolites of flavonoid intake in vivo be identified
o Is it possible to develop a method of analysis that can be used routinely in studies in humans
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 40
Experimental Approach
Dietary assessment of flavonoid intake
ndash Food Frequency Questionnaire (FFQ)
ndash Covered 12 months of dietary intake
ndash High and low dietary intake
Sampling
ndash Serum
Analytical set-up
ndash ACQUITY I-Class
ndash SYNAPT G2-Si HDMS
ndash Progenesis QI
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 41
Dietary flavonoid subclasses
ndash Flavanones (eriodictyol hesperidin
and naringenin)
ndash Anthocyanins (cyanidin delphinidin
malvidin pelargononidin petunidin
peonidin)
ndash Flavan-3-ols (catechins epicatechin)
ndash Flavonols (quercetin kaemferol
myricetin isohamnetin)
ndash Flavones (luteolin apigenin)
ndash Flavonoid polymers
(proanthocyanidins theaflavins
thearubigins)
Flavonoids amp their polymers
constitute a large class of food
constituents amp alter metabolic
process amp have a positive impact
on health
Specific groups of foods are rich
sources of 1 or more subclasses
Proposed compound classes Polyphenolics
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 42
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 43
UPLC Chromatogram ESI negative trace
Flavanoids Non-polar metabolites
Gallic acid C7H6O5
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 44
Unsupervised PCA analysis Low vs high grape consumption
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 45
S-Plot Low vs high grape consumption
Potential biomarkers for the high samples
Potential biomarkers for the low samples
Visualisation of covariance amp correlation between the
metabolites and the class designation
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 46
Progenesis QI Marker identification ndash database searching phase
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 47
Initial results amp future work
43000 compounds of interest = comprehensive data set to
determine the most appropriate markers for the study
ndash Data interpretation Progenesis and Ezinfo
Further evaluation planned to test the model with a larger study
group
Use the data to construct a routine assay using MS
ndash Three biomarkers have already been identified and will be used as an
objective tool to estimate dietary intake of flavonoids
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 48
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 49
Sample
REIMS instrument configuration
iKnife and bipolar forceps Hand-held
sampling device(s)
Diathermy generator
Informatics system
Source
REIMS is an emerging technique that allows rapid characterisation of
biological tissues
Xevo G2-XS Qtof and
Synapt G2Si
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 50
How does it work
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 51
REIMS workflow Direct analysis with chemometric profiling
Xevo G2-XS Qtof or Synapt G2Si
Model Generation
Multivariate statistical analysis
Different tissues
Different species
Different production
Biomarker ID amp verification
Rapid screening of unknowns by similarity to database
entries
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 52
Direct analysis for flexible amp rapid screening
Sub-sampling Determination
(5s)
Data analysis (2s)
Report
Sample to report in seconds
Point-of-control analysis
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 53
Genotype influences phenotypic characteristics
Ca 22k genes
20 different amino acids combined to give gt100k
expressed proteins
gtgt post-translational modifications
Metabolites are close to the phenotype
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 54
Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 55
Polyunsaturated fatty acid (PUFA) content
Ca x16
Arachidonic acid Ω6
C20H32O2
Eicosapentaenoic acid Ω3
C20H30O2
Ca x7
Good ratio of Ω36 acids
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 56
Multi-species muscle tissue ndash MVA models
2 biological replicates of each species 10 technical replicates per sample
PCA model
Non-ruminants
Ruminants
Buffalo Bovine
Porcine
Ovine
Gallus gallus
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 57
PROTOTYPE real-time recogniser software
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 58
Food Fraud amp authenticity applications
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 59
REIMS spectra
Kaempferol
Apigenin
Oleanolic acid ursolic acid
Herbs amp spices
PDO status Belgian butter
Botanical origin of honey
Essential Ω6 fatty acid
Ω9
Galangin Myricetin
Geographical origin of coffee
Caffeine Tanzania
Peru
Kenyan
Colombian
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 60
Butter fatty acid profile 220-290 mz Tof MS neg ion
Myristic acid C140
C14H27O2
[M-H]- Δ-06
Palmitoleic acid
C161 (9Z) C16H29O2
[M-H]- Δ 07
Palmitic acid C160
C16H31O2
[M-H]- Δ 06
Linoleic acid C182 (9Z12Z)
C18H31O2
[M-H]- Δ 28
Oleic acid C181 (9Z)
C18H33O2
[M-H]- Δ 08
Stearic acid C180
C18H35O2
[M-H]- Δ 06
Pentadecanoic acid C150
C15H29O2
[M-H]- Δ 02
Heptadecanoic acid C17H33O2
[M-H]- Δ 05
Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and
oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1
α-Linoleic acid C183 (9Z12Z 15Z)
C18H29O2
[M-H]- Δ 13
Δ mDa error
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 61
Rosmarinus officinalis Tof MS neg ion
Kaempferol C15H10O6
-016ppm
Apigenin C15H10O5
-22ppm
Oleanolic acid ursolic acid
C30H48O3
-184ppm
Quinic acid C7H16O6
123ppm
57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7
-394ppm
Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids
2-hydroxybenzoic acid (salicyclic acid) C7H6O3
146ppm
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 62
Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 63
Summary
There is a link between food and health
ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease
ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]
Metabolomic approach using HRMS screening
ndash Allows a holistic view of food composition amp contaminants
ndash Ion mobility separation is orthogonal to chromatography
o Enhanced peak capacity from ion mobility reduces sample complexity
REIMS emerging technique for direct analysis
ndash Non-targeted analysis with real-time chemometric profiling
ndash Point-of-control analysis
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells
copy2015 Waters Corporation 64
Acknowledgements
Prof Clare Mills
Mike McCullagh
Antonietta Wallace
Lee Gethings
Martin Wells