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Metabolite Extraction and Platforms for Metabolomic Studies
Andrew D. Patterson, PhDAssociate Professor of Molecular ToxicologyPenn State [email protected]
Resources
• Metabolomics Workbench – www.metabolomicsworkbench.org– Large resource of experimental protocols, datasets, and other resources
• XCMS Institute– https://xcmsonline.scripps.edu/institute– Great tutorials on chromatography, platforms, databases
• Metabolomics Society Forums– http://www.metabolomics-forum.com/
• Twitter – #metabolomics
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Objectives
At the conclusion of this lesson, students will be able to:
• Define factors that influence metabolite extraction and describe their impact on metabolomic studies
• Explain the value of orthogonal approaches for improved metabolite identification and quantitation
Which parts of the metabolomic process might influence your data?
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Other Classes of Metabolites
• Where might you have trouble extracting everything from a particular class of metabolites?
• Example: Are all bile acids the same in terms of general solubility in aqueous or organic solvents?
Matrix Effects
• Challenges with urine–
–
• Challenges with blood, serum, or plasma–
–
• Challenges with tissue–
–
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Extraction of Metabolites
Nucleic Acid Extraction
What do we know about our target analyte?
• Negatively charged phosphate backbone (polar)• Need to remove proteins, lipids, etc
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About Solvents
OH
PHENOL
1.07 g/cm3
O
H H
WATER
1.00 g/cm3
Cl
Cl
HCl
CHLOROFORM
1.49 g/cm3
POLARITY
Protein Chemistry
https://en.wikipedia.org/wiki/Proteinogenic_amino_acid
Denature proteins
• Hydrophobic amino acids face phenol:chloroform
• Phe, Tyr, Leu
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Liquid Liquid Extraction
DNA or RNA(aqueous)
Protein
Lipids (phenol:chloroform)
DNA Extraction Protocol
• Disrupt tissue in phenol:chloroform
**chloroform prevents small amounts of water in phenol from dissolving mRNA**adjust pH to favor DNA (basic) or RNA (acidic) isolation
• Centrifuge to separate layers• Dehydrate with alcohol
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What could go wrong?
• Solvents not appropriate or prepared incorrectly
– pH incorrect
– Ratios incorrect
• Contamination of solvents or buffers
• What else can you think of?
Standard Operating Procedures
• Saves time and prevents mistakes
• Consistent results
• Checking in samples (sample lists, location)
• Labeling and storing samples (aliquot)
• Metabolite extraction (targeted or global)
• Acquiring data on various platforms (MS, NMR)
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Even with SOPs…
Note influence of individual preparing pooled samples. Easy to see who prepared what.
DECAF COFFEE
SUMATRACOFFEE
COLUMBIAN COFFEE
Common Solvents for Metabolomics
• Methanol
• Acetonitrile
• Chloroform
• Methyl tert-butyl ether (MTBE)
• Water
NH3C
CH3HO
Cl
Cl
HCl
H3C
H3C
CH3
CH3O
O
H
H
List is not inclusive
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Methanol
• Relatively inexpensive compared to acetonitrile
• Not regulated like ethanol
• Easy to evaporate
• Extracts polar and (some) non-polar molecules – why?
Acetonitrile
• Advantages mostly for chromatography– Reduced absorbance for UV based methods
– Reduced pressure compared to methanol
– Greater elution strength (generally)
– HILIC applications
• Expensive– Isolated as a byproduct not produced directly
– Shortages can influence price and availability
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Chloroform vs MTBE
• Chloroform densitiy – 1.49 g/cm3
• MTBE density – 0.740 g/cm3
• Toxicity of chloroform (check out ATSDR.CDC.GOV)
• Still made need to tailor specific extractions for lipid classes (hexane for TAGs or MTBE for Cer)
– More detail checkout cyberlipid.org or lipidmaps.org
SO WHAT?
Metabolomics
Metabolomics is the systematic analysis of the unique chemical fingerprints left behind by specific cellular processes
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FOOD
PESTICIDES
COSMETICS
POLLUTANTS
DRUGS
XEN
OB
IOTI
C
MET
AB
OLI
SM
END
OG
ENO
US
MET
AB
OLI
SM
METABOLOME
GUT MICROBIOTA
Metabolomics
All “-omics” based scientific disciplines aim at the collectivecharacterization and measurement of their particularconstituent molecules
• A comprehensive approach to study complete pools ofbiological molecules
• Defines the structure, function and dynamics of anorganism
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Metabolomics
Vast chemical diversity among small molecule metaboliteshas made extended coverage of the metabolomechallenging
• Size (50 – 1500 Da)
• Concentration ( pM – mM)
• Physicochemical properties (diverse log P values)
• Stereochemistry (distinct biological activity)
EHP 2014 Vol 122 Number 8http://ehp.niehs.nih.gov/1308015/
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http://journal.frontiersin.org/article/10.3389/fimmu.2016.00044/full
Metabolite Extraction
• Currently no analytical technique exists that is capable ofmeasurement of all classes of cellular metabolites
• Metabolite extraction is a crucial step in any metabolomics study
– Critical to both targeted and global based profiling strategies
• Optimized extraction methodology should fulfill several criteria:
– Extract the largest number of metabolites
– Unbiased and non-selective - physical or chemical propertiesof a molecule
– Non-destructive - no modification of metabolites
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organic phase
aqueous phase
KD
HAorg
HAaq+ H20 H30+ + A-Ka
organic phase
aqueous phase
KD
HAorg
HAaq
Separation of Metabolites
• Mass spectrometry usually requires some form of chromatographic separation
• Most systems use either liquid or gas chromatography
• Fractionation of sample components simplifies the resulting mass spectra while ensuring more accurate compound identification
• Capacity factor (k) is critical to optimizing resolution
• Increased resolution allows longer MS dwell times resulting in better signal/noise ratios
• Inadequate chromatographic separation of metabolites results in:
• signal suppression – ion suppression
• compromised metabolite quantification
• reduced metabolite coverage
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Ceramide Physicochemical Properties
• Ceramides are a family of waxy lipid molecules.– Name derived from the latin word: cera = waxy + amide
• Ceramides are comprised of: – sphingosine: 18 carbon unsaturated amino alcohol – fatty acid moiety – amide bond
• Ceramides are not water soluble:– Very hydrophobic– Confined to cellular membranes– Participate in lipid raft formation– >200 structurally distinct species have been identified in
mammalian cells
Ceramide General Structure
• Ceramide (d18:1/16:0)
• 2-amino-1,3-octadec-4-ene-diol
• Amino alcohol (sphingoid) backbone
• Palmitic acid
• Fatty acyl group
• Ceramide (d18:1/24:1(15Z))
• 2-amino-1,3-octadec-4-ene-diol• Amino alcohol (sphingoid) backbone
• 15-tetracosenoic acid• Fatty acyl group
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Mol. BioSyst., 2015, 11, 698--713 | 699
Structures and Nomenclature
Ceramide Biosynthesis
Front. Endocrinol., 30 July 2014 | https://doi.org/10.3389/fendo.2014.00127
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Ceramide Biochemistry
Ceramides are found in high concentration in the membrane of cells
• Structural component of the lipid bilayer
• Bioactive lipid - implicated in a variety of physiological functions including:
• Apoptosis and cell growth arrest
• differentiation and cell senescence
• cell migration and adhesion
Ceramides are converted rapidly to more complex sphingolipids:
• Sphingomyelin
• Glycosylceramides
• Little accumulation observed
– Except for the skin (50% of total lipids can be ceramides)
Biosynthesis of Ceramides
De novo biosynthesis
• Ceramide synthases couple sphinganine + long chain fatty acid to form
dihydroceramide
• Double bond introduced into position 4 of the sphingoid base
ceramide synthases 5 and 6 generate are specific for palmitic acid
ceramide synthases 1 (brain and skeletal muscle) specific for stearic acid
ceramide synthases 2 specific for very long chain CoA-thioesters (C20-C26)
ceramide synthases 3 unusual ceramides of skin & testes
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Biosynthesis of Ceramides
Catabolism of complex sphingolipids:
• Sphingomyelinases/phospholipase C breakdown sphingomyelin in
animal tissues
• Many factors can stimulate the hydrolysis of sphingomyelin to
produce ceramide:
Cytokines :TNF-a, IFN-g & various interleukins
1,25-dihydroxy-vitamin D3
endotoxin
nerve growth factor
ionizing radiation & heat
LC Method DevelopmentWhere to Start?
• Designing and optimizing an LC method involves choosing appropriate:
1. Separation mechanism: NPC, RPLC, HILIC, size exclusion ion, exchange etc
2. Column chemistry: C2, C4, C8, C18, cyanopropyl, phenyl, biphenyl, amide, SiOH etc
3. Column properties: pore size, particle size & column dimensions
4. Stationary and mobile phase combinations
• Critical to optimizing the chromatographic efficiency, retention, resolution & selectivity of analytes
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Ceramide Scouting Gradients on Waters BEH C18
Steep gradient; large change in organic modifier.
Provides initial information about analyte retention & resolution.
Aids in expediting the development of an optimized LC method
Poor Resolution (R)
Long Retention (Tr) Time
1. C16:02. C18:13. C18:04. C20:05. C24:16. C22:07. C24:0
1 2 3 4 7
5 6
Fractionation of Ceramide Metabolites on Waters CSH C18 Column
Column: 100x2.1mm 1.7u Solvent System:A= MeCN:water (60:40 v/v)B= 2-propanol:MeCN (90:10 v/v)10mM NH4OAc + 0.1% formic acidBallistic gradient0.4ml/min @ 55oC
UPLC-ESI-QTOF-MS
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0 5 1 0 1 5 2 0
2 0
4 0
6 0
8 0
1 0 0
G ra d ie n t C o m p a r is o n
t im e (m in )
% o
rga
nic
b a llis t ic
s ta n d a r d
Ballistic Gradient:
• Ultra-fast gradient
• Rapid change in organic modifier
• Separations performed at high flow rates
• High linear velocity
Fractionation of Ceramide Metabolites on Waters CSH C18 Column
1. C16:02. C18:13. C18:04. C20:05. C24:16. C22:07. C24:0
UPLC-ESI-MS-MS
Column: 100x2.1mm 1.7u Solvent System:A= MeCN:water (60:40 v/v)B= 2-propanol:MeCN (90:10 v/v)10mM NH4OAc + 0.1% formic acidBallistic gradient0.4ml/min @ 55oC
QQQ MS Detector
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Ceramide Extraction
Extraction protocols and LC-MS methodsadapted from Shaner RL et al JLR 2009
• Add 50 mg of liver tissue to 50% aqueous methanol– Why not chloroform directly?
• Homogenize in Bertin Precellys at 6500 rpm with ~10 zirconium beads for 30 seconds
Ceramide Extraction – cont’d
• Add 1 mL of CHCl3:MeOH (2:1, v/v) containing 20 μl of C17:0 internal standard solution (use 1 mM stock solution)– Why internal standard at this point?– Should we use glass or plastic? Does it matter?– What if you swap chloroform for hexane or isopropanol?
• Homogenize again and centrifuge at 18,000xg for 10 min to separate phases
• Transfer organic phase to a new tube (#2) and repeat extraction of left over material – Why repeat?
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Ceramide Extraction – cont’d
• Combine organic phases and dry down in a vacuum centrifuge
• Solubilize residuals in 50 μl of CHCl3:MeOH (2:1, v/v)– Why chloroform here?
• Saponification or acid hydrolysis of residuals to release ceramides
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Ceramide Extraction – cont’d
• Incubate residuals with 0.5 mL of 1M HCl in MeOH @ 50°C for 1 hr (or base for sapn)
• Cool samples and re-extract
• Solubilize with in 30 μl of CHCl3:MeOH (2:1, v/v), sonicate for 5 minutes in sonicatingwater bath– Why sonicate?
Ceramide Extraction – cont’d
• Dilute 10 fold with acetonitrile:isopropanol:water (1:1:1, v/v)
• Centrifuge to remove any particulates and transfer to autosampler tube
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0
11 0 3
21 0 3
31 0 3
41 0 3
E ffe c t o f S o lv e n t S y s te m o n C 1 6 :0 C e ra m id e R e c o v e ry fro m M u r in e L iv e r
C 1 6 :0 C e ra m id e
pe
ak
are
a/m
g l
ive
r t
iss
ue
C H C l3 :M e O H
E tO A c
H e x a n e :E tO A c
H e x a n e :IP A
M tB E
0
11 0 3
21 0 3
31 0 3
41 0 3
E ffe c t o f S o lv e n t S y s te m o n C 2 4 :0 C e ra m id e R e c o v e ry fro m M u r in e L iv e r
C 2 4 :0 C e ra m id e
pe
ak
are
a/m
g l
ive
r t
iss
ue
C H C l3 :M e O H
E tO A c
H e x a n e :E tO A c
H e x a n e :IP A
M tB E
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Ileum Serum
Lipidomics Reveals Ceramides are Decreased inFXR Intestine-null Mice
J Lipid Res. 2011 Aug; 52(8): 1583–1594. doi: 10.1194/jlr.D015586
Ceramide Labeling
0500
10001500200025003000
1 2 3 4
C16 cer
C16 Cer C16 Cer+16 C16 Cer+32
0
5000
10000
15000
20000
25000
1 2 3 4
c16sm
c16sm c16 sm+16 c16 sm+32
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Direct energy source
Anabolic substrates
Regulatory roles
Short Chain Fatty Acids• SCFAs are those carboxylic acids that contain aliphatic tails less than 6 carbon atoms
• In humans, SCFAs are derived in large part from fermentation of carbohydrates and proteins in the colon
• By this process, the host is able to salvage energy from foods that cannot be processed normally in the upper parts of the gastrointestinal tract
• SCFAs serve as direct energy source, anabolic substrates and signaling molecules involve metabolic and immunological regulation.
SCFA Extraction and Detection
• What do we know about SCFAs?
• How do we detect them?
• What problems might influence SFCA measurement?
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3 .0 0 4 .0 0 5 .0 0 6 .0 0 7 .0 0 8 .0 0 9 .0 0 1 0 .0 0 1 1 .0 0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
3 0 0 0 0 0
3 5 0 0 0 0
4 0 0 0 0 0
4 5 0 0 0 0
5 0 0 0 0 0
5 5 0 0 0 0
6 0 0 0 0 0
T im e -->
A b u n d a n c e
T IC: JC1 6 0 1 1 5 _ 0 6 .D \ d a ta .m s
aceticacid
butyricacid
propionicacid
pyridine
aceticacidbutyricacidpropionicacid
4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
T ime-->
Abundanc e
T IC: JC160606_new 13.D \ data.ms
caproic acid-6,6,6-d3(IS)
caproic acid-6,6,6-d3(IS)
carbonicacid
Total ion chromatogram of 100 µg/ml
SCFAs standard mixture with 10
µg/ml caproic acid-6,6,6-d3 as
internal standard (IS) by (top) propyl
esterification method and (bottom)
acidified water method.
Notice change in retention time
between the two methods.
Derivatized
Native
2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 1 0 5 1 1 00
2 0 0 0 0
4 0 0 0 0
6 0 0 0 0
8 0 0 0 0
1 0 0 0 0 0
1 2 0 0 0 0
1 4 0 0 0 0
1 6 0 0 0 0
1 8 0 0 0 0
2 0 0 0 0 0
2 2 0 0 0 0
2 4 0 0 0 0
2 6 0 0 0 0
m / z -->
A b u n d a n c e
A v e ra g e o f 2 .7 2 2 to 2 .7 3 8 m in .: JC 1 6 0 1 1 5 _ 2 9 .D \ d a ta .m s4 4 .0
6 2 .0
7 4 .0
3 1 .0
8 7 .95 4 .9 1 0 1 .9
2.60 2.65 2.70 2.75 2.80 2.85 2.90 2.950
50000
100000
150000
200000
250000
300000
Time-->
Abundance
Ion 44.00 (43.70 to 44.70): JC160115_29.D\ data.ms
2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 1 0 5 1 1 00
2 0 0 0 0
4 0 0 0 0
6 0 0 0 0
8 0 0 0 0
1 0 0 0 0 0
1 2 0 0 0 0
1 4 0 0 0 0
1 6 0 0 0 0
1 8 0 0 0 0
2 0 0 0 0 0
2 2 0 0 0 0
2 4 0 0 0 0
2 6 0 0 0 0
2 8 0 0 0 0
3 0 0 0 0 0
3 2 0 0 0 0
3 4 0 0 0 0
3 6 0 0 0 0
m / z - - >
A b u n d a n c e
A v e r a g e o f 2 . 7 2 8 t o 2 . 7 4 1 m in . : J C 1 6 0 1 1 5 _ 0 7 . D \ d a t a . m s4 3 . 0
6 1 . 0
7 3 . 0
3 1 . 0
8 7 . 05 3 . 0 1 0 0 . 82.5 5 2 .60 2 .65 2 .7 0 2 .75 2 .80 2 .85 2 .90 2 .950
5000 0
1 0000 0
1 5000 0
2 0000 0
2 5000 0
3 0000 0
3 5000 0
4 0000 0
T ime -->
A bu ndanc e
Ion 4 3 .00 (42 .70 to 4 3 .70 ): JC160115_ 07 .D \ da ta .ms
propylacetate(aceticacid)
1-13Cpropylacetate(1-13Caceticacid)
13C
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SCFA by 1H NMR
ppm
0.70 .80 .91 .01 .11.21 .31 .41 .51 .61 .71 .81 .92 .02 .12 .2 ppm
15
20
25
30
35
40
acetaten-butyrate
n-butyrate
n-butyrate
propionatepropionate
Acetate#1
Propionate#1
Propionate#2
Butyrate#1
Butyrate#2
Butyrate#3
1H-13C HSQC NMR
spectroscopy of mice feces.
Reference spetra were
obtained from Human
Metabolome Database
(HMDB). Acetate
(HMDB00042), Propionate
(HMDB00237) and Butyrate
(HMDB00039)
GC-MS Propyl Esterification Method
CompoundsSpiked amount
(µg/ml)
Amount recovered
(µg/ml)%Recovery %RSD %Matrix effect
Acetic Acid
10 6.7±1.0 66.9 14.9 76.3
100 108.3±15.0 108.3 13.9 94.4
250 254.5±16.3 101.8 5.9 92.7
Propionic Acid
10 7.2±1.2 72.3 16.9 74.2
100 100.2±17.6 100.2 17.5 84.2
250 244.5±36.6 97.8 15.0 92.7
Butytic Acid
10 6.7±1.1 67.0 11.2 77.7
100 99.4±20.4 99.4 20.6 88.2
250 249.7±38.1 99.8 15.3 97.4
GC-MS Acidified Water Method
CompoundsSpiked amount
(µg/ml)
Amount recovered
(µg/ml)%Recovery %RSD %Matrix effect
Acetic Acid
10 7.5±1.9 74.8 25.1 116.8
100 108.7±14.9 108.7 13.7 95.7
250 226.1±19.2 90.4 8.5 76.2
Propionic Acid
10 7.5±1.2 75.4 16.0 95.1
100 106.3±10.7 106.3 10.1 80.3
250 297.2±23.1 118.9 7.8 76.2
Butyric Acid
10 5.2±0.4 52.2 6.8 89.6
100 87.6±17.0 87.6 20.6 76.2
250 291.7±24.3 116.7 8.3 79.7
1H NMR Quantification Relative to TSP Method
CompoundsSpiked amount
(µg/ml)
Amount recovered
(µg/ml)%Recovery %RSD %Matrix effect
Acetic Acid
10 8.4±0.4 83.7 4.2 90.6
100 97.7±4.3 97.7 4.4 118.5
250 271.7±16.0 108.7 5.9 111.7
Propionic Acid
10 10.1±0.6 101.4 6.0 107.2
100 116.6±5.5 116.6 4.7 116.1
250 332.1±15.3 132.8 4.6 104.8
Butyric Acid
10 5.4±0.8 54.2 14.7 64.1
100 92.9±4.6 92.9 5.0 118.1
250 263.2±14.7 105.3 5.6 106.8
1H NMR Quantification with Calibration Curve Method
CompoundsSpiked amount
(µg/ml)
Amount recovered
(µg/ml)%Recovery %RSD
Acetic Acid
10 8.6±0.4 85.6 4.2
100 98.7±5.1 98.7 5.2
250 277.9±16.3 111.2 5.9
Propionic Acid
10 8.0±0.48 80.2 6.0
100 91.4±4.6 91.4 5.0
250 262.7±12.1 105.1 4.6
Butyric Acid
10 5.4±0.8 54.1 14.7
100 92.0±4.7 92.0 5.1
250 263.0±14.7 105.2 5.6
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GC-MSPropylEsterificationMethod
Compounds Intraday(%RSD) Interday (%RSD)
10µg/ml 25µg/ml 100µg/ml 250µg/ml 10µg/ml 25µg/ml 100µg/ml 250µg/ml
AceticAcid 4.3 2.7 6.1 3.0 4.5 3.5 10.0 3.4
PropionicAcid 12.2 5.7 7.7 5.6 21.3 6.5 11.2 8.4
ButyricAcid 1.2 3.5 5.8 3.6 7.7 5.1 4.6 6.9
GC-MSAcidifiedWaterMethod
Compounds Intraday(%RSD) Interday (%RSD)
10µg/ml 25µg/ml 100µg/ml 250µg/ml 10µg/ml 25µg/ml 100µg/ml 250µg/ml
AceticAcid 12.6 6.8 5.1 4.5 9.8 5.6 5.7 5.3
PropionicAcid 6.4 7.2 4.9 5.4 8.9 7.9 7.8 7.9
ButyricAcid 2.4 4.2 5.0 5.5 4.7 6.4 6.0 7.7
1HNMRQuantificationRelativetoTSP
Compounds Intraday(%RSD) Interday (%RSD)
10µg/ml 25µg/ml 100µg/ml 250µg/ml 10µg/ml 25µg/ml 100µg/ml 250µg/ml
AceticAcid 1.7 4.6 0.3 1.9 2.6 5.3 0.3 0.6
PropionicAcid 3.8 0.9 2.4 2.8 1.4 2.4 1.4 2.0
ButyricAcid 4.3 4.5 1.4 2.1 4.5 2.6 0.7 4.0
1HNMRQuantificationwithCalibrationCurve
Compounds Intraday(%RSD) Interday (%RSD)
10µg/ml 25µg/ml 100µg/ml 250µg/ml 10µg/ml 25µg/ml 100µg/ml 250µg/ml
AceticAcid 1.1 6.7 1.7 1.8 1.6 4.3 0.3 0.6
PropionicAcid 2.0 0.7 2.3 2.7 0.7 1.7 1.3 1.9
ButyricAcid 2.3 3.2 1.3 2.1 2.4 1.9 0.7 3.9
How do the methods compare?
0
400
800
2000
4000
µg
/gF
ec
es
GC-MS Propyl Esterification Method
GC-MS Acidified Water Method1H NMR Quantification Method
Fecal SCFAs Concentration
NS
NS
NS
Biological concentrations of SCFAs in mice fecal samples measured by GC-MS propyl
esterification method, GC-MS acidified water method and 1H NMR quantification
method. Values are expressed as the mean ± 95% CI. (n=10). One-way ANOVA.
Biological Concentrations measured by different methods were not significantly
different.
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SCFA Measurement in the Real World
0
2
4
50
100
200
1400
2600
3800
µg
/gGF
CONV-R****p<0.0001
****p<0.0001 ****p<0.001
Biological concentrations of
SCFAs in germ free(GF) and
conventionally raised (CONV-R)
fecal samples measured by GC-
MS propyl esterification
method. Values are expressed
as the mean ± sd. (n=5). Two-
tailed t-test.
Conclusions
• Extraction protocols can impact metabolomic data sets considerably
• Solvent system composition and pH exhibit the most dramatic effects on metabolite recovery – The magnitude of these effects depend on metabolite
class
– Some classes of metabolites
• The number of extraction repetitions also plays a role in enhancing metabolite recovery– Tradeoff - longer sample prep time
– Larger sample volumes to process (evaporate)
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Conclusions
• Traditional RPLC methods can provide efficient separation of acyl-carnitine, bile acid and CoA thioester mixtures.
– Advancements in hybrid particle technologies
– Allowing for extremes in mobile phase pH and temperature – manipulate selectivity
– Complex ligand stationary phase interactions
• HILIC methods are superior at separating highly polar metabolites.
– Nucleotides and derivatives
– Small polar metabolites – sugars, organic acids, amino acids, hydrophilic vitamins
Conclusions – cont’d
• There’s no one “perfect” extraction or LC method available capable of efficiently extracting or resolving, respectively, all components or features in the metabolome
• Advanced column chemistries (amide, aminopropyl, biphenyl, graphite, phenyl-hexyl) and alternative chromatographic methodologies (HILIC) can provide enhanced coverage of the metabolome
• Different platforms can provide greater confidence in metabolite measurement
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Acknowledgments
Penn State University
• Chris Chiaro
• Philip Smith
• Jared Correll
• Jingwei Cai
• Jingtao Zhang
National Cancer Institute
• Frank J. Gonzalez
• Kris Krausz
• Changtao Jiang
• Fei Li
NIEHS R01 ES022186
MRC
• Julian Griffin
• Elizabeth Stanley
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