Nordic Sample Pre-treatment and Preparation for Food Analysis
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Transcript of Nordic Sample Pre-treatment and Preparation for Food Analysis
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2012 Waters Corporation 1
Sample Clean-up approaches for Food
Analysis
Euan Ross
BDM Chemical Analysis
CO Europe and India
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Food Analysis ChallengeSample Pretreatment & Preparation
Sample pretreatment and preparation requires asystematic approachto answer the question:
Sample Extract?
to
How do we get from.
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Outline
Food analysis methodsWhy sample pretreatment?
Sample preparation techniques
Factors influence SPE performance
Procedures for removing sample interferences
SPE cleanup strategies for sample preparation
Dispersive SPE strategy and example
Pass-through SPE strategy and example Retention-cleanup-elution SPE strategy
and example
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Food Analysis MethodsSystematic Approach
Food analysis could be classified into 3 critical steps
Sample
Pretreatment
Sample
Preparation
Sample Analysis
by Instrument
Some may consider both steps as a single step of samplepreparation
Depending on the analytes and sample matrix, samplepretreatment may not be necessary.
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Outline
Food analysis methodsWhy sample pretreatment?
Sample preparation techniques
Factors influence SPE performance
Procedures for removing sample interferences
SPE cleanup strategies for sample preparation
Dispersive SPE strategy and example
Pass-through SPE strategy and example Retention-cleanup-elution SPE strategy and example
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Food Analysis MethodsWhy Sample Pretreatment?
Obtain representative, consistent sample
homogenization by cutting, chopping, blending
To remove moisture
Drying sample by heat or drying reagents
Blender PolytronHomogenizer
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Potential Problems of MatrixCommon Types of Matrix Interferences
Fats, oils, lipids, and proteins
Infant formula, milk, meat
Carbohydrates and polysaccharides
Fruits and cereals
Salts
Snack foods
Surfactants
Naturally occurring phospholipids
Synthetic used in ice cream to improve mouth feel
Pigments
Chlorophyll in leafy vegetables
http://images.google.com/imgres?imgurl=http://www.geekologie.com/2008/05/06/meat-face.jpg&imgrefurl=http://www.geekologie.com/mt/mt-search.cgi?tag=meat&blog_id=1&usg=__orBUtWD9gEKqqk3JByb-24xdkbI=&h=340&w=450&sz=34&hl=en&start=19&um=1&tbnid=xKM1Mr16ziKDyM:&tbnh=96&tbnw=127&prev=/images?q=meat&hl=en&um=1http://images.google.com/imgres?imgurl=http://www.giftlebanon.com/catalog/images/RoyalFruitBasket.jpg&imgrefurl=http://www.giftlebanon.com/catalog/index.php?cPath=36&usg=__pF7SlkCtgfIJKJf9wbUuayr-Bns=&h=480&w=393&sz=41&hl=en&start=5&um=1&tbnid=Ng4GxbGRSELIGM:&tbnh=129&tbnw=106&prev=/images?q=fruit&hl=en&sa=X&um=1http://images.google.com/imgres?imgurl=http://www.nandyala.org/mahanandi/images/vegetables/spinach1.jpg&imgrefurl=http://www.nandyala.org/mahanandi/?p=154&usg=__sJLk9aguxeGwLMzmT-4oT3HoFt4=&h=338&w=450&sz=41&hl=en&start=6&um=1&tbnid=30bCJW6onPvuMM:&tbnh=95&tbnw=127&prev=/images?q=spinach&hl=en&um=1http://images.google.com/imgres?imgurl=http://thewaffleplace.co.uk/content_files/Ice%20Cream.JPG&imgrefurl=http://thewaffleplace.co.uk/?p=10&c=&usg=__jfyFnjKWu3hK_mrxqvajmP6rAd4=&h=949&w=604&sz=52&hl=en&start=2&um=1&tbnid=076y2igcPqJ22M:&tbnh=148&tbnw=94&prev=/images?q=ice+cream&hl=en&um=1 -
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Potential Problems of MatrixSample consistency
Emulsions
Milk - The milk fats are dispersed in water
Butter- is an emulsion of water particles
dispersed in milk fats
Turbidity
pulpy orange juice
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Food Analysis MethodsWhy Sample Pretreatment?
To adjust the sample conditions
for the NEXT STEP of the sample preparation
Sample conditions can adjusted using:
Dilution
Extraction
Solvent exchange into instrument compatible solvents
pH adjustment
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Why Sample Pretreatment?To Adjust Sample pH
pH adjustment can provide: Optimized ionization of the analytes for SPE sample preparation
o neutral species for reversed-phase
o Ionized species for ion-exchange
Stabilization of pH labile compounds
Reduced matrix interferences
o protein precipitation by adding acids
Elimination of protein binding by adding acids or bases
o TFA, formic acid, phosphoric acid, or ammonia
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Outline
Food analysis proceduresWhy sample pretreatment?
Sample preparation techniques
Factors influence SPE performance
Procedures for removing sample interferences
SPE cleanup strategies for sample preparation
Dispersive SPE strategy and example
Pass-through SPE strategy and example Retention-cleanup-elution SPE strategy and example
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Sample PreparationNon-Chromatographic Techniques
Techniques Advantages Disadvantages
Dilution
Simple
Cheap
High throughput
No cleanup
No enrichment
Filtration
Simple
Fast
No enrichment
Potential analyte binding
Centrifugation
Simple cleanup
High throughput
No enrichment
Cumbersome
Liquid-Liquid
Extraction
Best non-chromatographic
cleanup
Enrichment
Cumbersome
Expensive
Lots of solvent usage
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Sample Preparation by SPEA Chromatographic Cleanup
Techniques Advantages Disadvantages
Solid Phase
Extraction(SPE)
Best cleanup
Enrichment
Fast
Easy to automate
Many sorbents available
for optimum cleanup
May need multiple steps
Not well understood
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Why Use SPE for Food Analysis ?
Sample Cleanup Significant cleanup with specific SPE sorbent
Optimum cleanup by using multiple SPE cartridges
o Melamine analysis
Sample Enrichment For sub-ppb detection limits, enrichment factors (100X) may be
needed.
o Sudan dye analysis
Compared to liquid-liquid extraction
SPE requires much less solvent per sample(often 10X less solvent used for SPE)
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General Pretreatment Proceduresfor SPE Sample Preparation
SampleType ?
Liquid
Solid
Filter/Centrifuge
Sample*
SolidExtraction
Liquid
SPE
SampleHomogenized
pHadjustment
* sample with suspended solids, e.g. orange juice
Liquid
Filter/CentrifugeSample
Solids
Liquid
SPE
pHadjustment
Liquid
Extraction
Clearfluid
Extraction
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General Pretreatment Proceduresfor SPE Sample Preparation
SampleType ?
Liquid
Solid
Filter/Centrifuge
Sample*
SolidExtraction
Liquid
SPE
SampleHomogenized
pHadjustment
Liquid
Filter/CentrifugeSample
Solid
Liquid
SPE
pHadjustment
Liquid
Extraction
Clearfluid
Extraction
Often performed in one step
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Outline
Food analysis procedures
Why sample pretreatment?
Sample preparation techniques
Factors influence SPE performance
Procedures for removing sample interferences
SPE cleanup strategies for sample preparation
Dispersive SPE strategy and example
Pass-through SPE strategy and example Retention-cleanup-elution SPE strategy and example
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Factors that Influence SPEPerformance
Interactions between Solvent,Analyte, Sorbentand Matrixdetermine the SPE methodperformance.
Interactions of all factors need tobe considered together whendeveloping SPE method.
Optimized SPE methods
maximize specific interactionswhile minimizing unwantedinteractions.
Analyte
Sorbent Matrix
Solvent
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Important Properties of Analytes forSPE Performance
Key properties of analytesto be considered
Polar functional groups consisting of O, N, S, or P?
The numbers and positions of the polar groups
Any ionizable functional groups? Are they cations or
anions?
Approximate pKa of these functional groups
The solubility in water and the solvents for the
sample preparation method (Kow)
Analyte
The above properties will determine the analyteinteractions
with solvent, matrixand the SPE sorbent
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Solvent
Solvent
Key Functions of the solvent
Extract analytes
Remove matrix interference
Change the polarity and ionic strength of
the sample extract
Change the ionization status of analytes,
sample matrix, or SPE sorbents
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Matrix
Key considerations of the sample matrix
The relative polarity of the matrix
compared to analytes, solvent or sorbent
Potential interferences with the analyte
analysis
Potential binding of analytes with the
matrix components (proteins)
Matrix
Matrix interactions influence the sample clean-upand analyte extraction
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Sorbent
SPE Sorbents
Key considerations for SPE Sorbents
Sorbents can be chosen to interact withanalyte or sample matrix
Sorbent interactions be enhanced or weakenby solvent manipulation
In general, there are 4 sorbent classes:
Reversed-phase
Normal-phase
Ion-exchange
Mix-mode
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Based on the solvents selection
Organic solvents normal-phase
Aqueous solvents and buffers reversed-phase
Based on the strategy of the SPE method
Pass-through cleanup Retain-wash-elute cleanup
If there are more than one sorbent suitable, pick the sorbentbased on:
Level of cleanup required
Sensitivity required
Analyte interaction
Other specific requirements of method
SPE Sorbent Selection
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Normal-Phase Sorbents Silica, Alumina, Florisil, Aminopropyl silica, PSA, Diol silica,
Reversed-Phase Sorbents
Oasis HLB
C18, C8 etc (alkyl bonded silica) Graphitized carbon and activated carbon
Ion Exchange
Accell Plus CM, QMA
Mixed Mode (ion-exchange/reversed-phase) Oasis MAX, Oasis WAX (strong and weak anion-exchange)
Oasis MCX, Oasis WCX (strong and weak cation-exchange)
SPE Sorbents for Food Analysis
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Normal-Phase Sorbents Silica, Alumina, Florisil, Aminopropyl silica, PSA, Diol silica,
Reversed-Phase Sorbents
OasisHLB
C18, C8 (alkyl bonded silica) Graphitized carbon and activated carbon
Ion Exchange
Accell Plus CM, QMA
Mixed Mode (ion-exchange/reversed-phase) Oasis MAX, Oasis WAX (strong and weak anion-exchange)
Oasis MCX, Oasis WCX (strong and weak cation-exchange)
SPE Sorbents for Food Analysis
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Normal-Phase Sorbents Silica, Alumina, Florisil, Aminopropyl silica, PSA, Diol silica,
Reversed-Phase Sorbents
Oasis HLB
C18, C8 etc (alkyl bonded silica) Graphitized carbon and activated carbon
Ion Exchange (silica based)
Accell Plus CM, QMA
Mixed-Mode (ion-exchange/reversed-phase) Oasis MAX, Oasis WAX (strong and weak anion-exchange)
Oasis MCX, Oasis WCX (strong and weak cation-exchange)
SPE Sorbents for Food Analysis
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Outline
Food analysis procedures
Why sample pretreatment?
Sample preparation techniques
Factors influence SPE performance
Procedures for removing sample interferences
SPE cleanup strategies for sample preparation
Dispersive SPE strategy and example
Pass-through SPE strategy and example Retention-cleanup-elution SPE strategy and example
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Common Sample Matrix Interferences
Fats, Oils, Lipids and Hydrocarbons These interferences are non-polar
They have high solubility in non-polar solvents
o hexane, ethers
Common found in:o meats
o nuts
o dairy products
o manufactured goods,
chips, cookies, and chocolate
http://images.google.com/imgres?imgurl=http://home.wickedlocal.com/wp-content/uploads/2009/03/cookie.jpg&imgrefurl=http://home.wickedlocal.com/2009/03/04/girl-scout-cookie-sales-crumble/&usg=__WEGQoMxs8acprcVrYtiJhLOk05g=&h=565&w=755&sz=135&hl=en&start=136&um=1&tbnid=UiGi9Br-qc5C5M:&tbnh=106&tbnw=142&prev=/images?q=cookie&ndsp=20&hl=en&sa=N&start=120&um=1http://images.google.com/imgres?imgurl=http://img.dailymail.co.uk/i/pix/2008/05_03/MaraSnicksDM_468x308.jpg&imgrefurl=http://www.dailymail.co.uk/news/article-565849/Farewell-Snickers-Marathon-bars-make-comeback.html&usg=__XiH76QhRUKHK-j_bFuDQcUVGdYE=&h=308&w=468&sz=36&hl=en&start=34&um=1&tbnid=ZhkIc8cjBMWsjM:&tbnh=84&tbnw=128&prev=/images?q=chocoloate+bar&ndsp=20&hl=en&sa=N&start=20&um=1 -
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SPE Considerations to Remove:Fats, Oils, Lipids and Hydrocarbons
Normal-phase sorbents are usually selected to remove fats
and lipids
Silica, Alumina, Florisil
The sample will need to be diluted with solvent compatible
with normal-phase sorbents
Hexane, ethyl acetate, dichloromethane
Reversed-phased sorbents are sometimes used butoptimization is usually difficult
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Remove Fats, Oils, LipidsExample: Hexane
Homogenize Samplewith
Solvent
Re-extract sample
using other solvent
DiscardSolvent
Analyte inLiquid phase
no
yes Discardsolid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent
SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPE
Liquid
Hexane: Non-polar
Analyte: Polar
Fats and Oils(Hexane)
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Remove Fats, Oils, LipidsExample: Hexane
Homogenize Samplewith
Solvent
Re-extract sample
using other solvent
DiscardSolvent
Analyte inLiquid phase
no
yes Discardsolid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent
SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPE
Liquid
Hexane: Non-polar
Analyte: Polar
Fats and Oils(Hexane)
PolarAnalytes
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Remove Fats, Oils, LipidsExample: Hexane
Homogenize Samplewith
Solvent
Re-extract sample
using other solvent
DiscardSolvent
Analyte inLiquid phase
no
yes Discardsolid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent
SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPE
Liquid
Hexane: Non-polar
Analyte: Non-Polar
Fats and Oils(Hexane)
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Remove Fats, Oils, LipidsExample: Hexane
Homogenize Samplewith
Solvent
Re-extract sample
using other solvent
DiscardSolvent
Analyte inLiquid phase
no
yes Discardsolid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent
SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPE
Liquid
Hexane: Non-polar
Analyte: Non-Polar
Fats and Oils
Non-Polar Analytes
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Remove Fats, Oils, LipidsExample: Hexane
Homogenize Samplewith
Solvent
Re-extract sample
using other solvent
DiscardSolvent
Analyte inLiquid phase
no
yes Discardsolid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPE
Liquid
Hexane: Non-polar
Analyte: Non-Polar
Fats and Oils
Non-Polar Analytes
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Common Sample Matrix Interferences
Proteins They are typically present in meats, tissues, dairy products
They have high molecular weights
They are sensitive to pH and organic solvents (addition of
acidified acetonitrile to breakup protein binding and causeprecipitation)
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Sample Pretreatment to Remove:Proteins
SampleType ?
Liquid
Solids
SPE
Centrifugesample
Proteinbinding ?
No
Yes
Add Acetonitrileor Acids
Centrifugesample
Dilute Samplewith solvent
SampleHomogenized withorganic solvent.
Add buffer ifnecessary
Centrifugesample
SPE
SPE
Protein Precipitation
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Sample Pretreatment to Remove:Proteins
SampleType ?
Liquid
Solids
SPE
Centrifugesample
Proteinbinding ?
No
Yes
Dilute Samplewith solvent
SampleHomogenized withorganic solvent.
Add buffer ifnecessary
Centrifugesample
SPE
Add Acetonitrileor Acids
Centrifugesample
SPE
Protein Precipitation
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Sample Pretreatment to Remove:Proteins
SampleType ?
Liquid
Solids
SPE
Centrifugesample
Proteinbinding ?
No
Yes
Dilute Samplewith solvent
SampleHomogenized withorganic solvent.
Add buffer ifnecessary
Centrifugesample
SPE
Add Acetonitrileor Acids
Centrifugesample
SPE
Protein Removal Step
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SPE Considerations to Remove:Proteins
Choose sorbents that maximize analyte retention
Large protein molecules usually pass-through sorbent
reversed-phase without retaining
Cannot access pores in sorbent
Eliminate proteins-analyte interactions prior to the SPE step
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Outline
Food analysis proceduresWhy sample pretreatment?
Sample preparation techniques
Factors influence SPE performance
Procedures for removing sample interferences
SPE cleanup strategies for sample preparation
Dispersive SPE strategy and example
Pass-through SPE strategy and example Retention-cleanup-elution SPE strategy and example
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How Much Sample Cleanup is Needed?
The Level of Cleanup is determined by
The required selectivity of analytes
The Limit of Detection (LOD) for the method
The method is dictated by the analytical technique used
o Selective detection, e.g. MS or MS/MS
Simple cleanup of major interference may be sufficient
o
Less selective detection, e.g. LC-UV or GC-FID More cleanup may be required
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General SPE Cleanup Strategy
Evaluate analytestructures and
properties, matrix,and cleanuprequirement
Pass-through SPE(Matrixretained by
sorbent)
Optimizedcleanup
Max. Sensitivity
MatrixRemoval
ScreeningMethod?
Yes LowDispersive
- SPE(d-SPE)
Pass-throughSPE by
cartridge
Retention-cleanup-elutionSPE (Analytesinitially
retained by sorbent, lastlyeluted by strong solvent)
Better cleanup thand-SPE in general
Some enrichment viasolvent evaporation
No Moderate
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Dispersive-SPE
(d-SPE) Pass-through SPE
Retention-cleanup-
elution
Cleanup Quick, simple, easyMore effective than
d-SPE in general
Most effective and
very selective
Analyte Non-retained Non-retainedInitially retained and
then eluted
Matrix Mostly retained Mostly retainedNon-retained or
removed by washing
Sorbent
Selection
Maximize:
Matrix retention
Minimize:
Analyte retention
Maximize:
Matrix retention
Minimize:
Analyte retention
Maximize:
Analyte retention
Minimize:
Matrix retention
Enrichment No, in generalLimited by solvent
evaporation
Yes
AnalysisMultiresidue
analysis
Multiresidue
analysis
Compounds with
similar structures
and/or properties
Comparison of the SPE Strategies
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Outline
Food analysis methodsWhy sample pretreatment?
Sample preparation techniques
Factors influence SPE performance
Procedures for removing sample interferences
SPE cleanup strategies for sample preparation
Dispersive SPE strategy and example
Pass-through SPE strategy and example Retention-cleanup-elution SPE strategy and example
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Some Examples
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Screening of Veterinary
Drugs In Milk
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Milk Composition
Typical Cows Milk
Approximately 14 % solids
o 4 % fato 4 % protein
o 5 % sugar (lactose)
o 85 % water
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General SPE Cleanup Strategy
Evaluate analytestructures and
properties, matrix,and cleanuprequirement
Pass-through SPE(Matrixretained by
sorbent)
Optimizedcleanup
Max. Sensitivity
MatrixRemoval
ScreeningMethod?
Yes LowDispersive
- SPE(d-SPE)
Pass-throughSPE by
cartridge
Retention-cleanup-elutionSPE (Analytesinitially
retained by sorbent, lastlyeluted by strong solvent)
Better cleanup thand-SPE in general
Some enrichment viasolvent evaporation
No Moderate
i id i ilk
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Veterinary Residues in MilkMultiresidue LC/MS Analysis
Typical Sample Preparation Strategies
Precipitation/extraction with strong buffer (McIlvaine pH 4)followed by SPE
o good for tetracyclines, beta-adrenergics, polar sulfonamides,
fair for fluoroquinolones,o poor recovery of most other compounds
Precipitation/extraction with 3:1 acidic acetonitrile with SPEcleanup
o excellent protein precipitation
o poor recovery of tetracyclines, beta-adrenergics, polarsulfonamides
o good recovery of most other compounds
Typical Recoveries From Milk
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Comparison of Precipitation/ExtractionTechniques
Drug Class 3:1 ACN Aq Buffer 1:1 ACN*
-adrenergic 80
Tetracycline 70 >25
Fluoroquinolone >50 >50 >50
Macrolide >60 60
Beta-Lactam >70 70
Steroid >70 70
*Conclusion:-Procedure chosen for this study-Extraction/precipitation of milk with an equal volume ofacetonitrile provides recovery of the widest range of compounds
However - insufficient protein precipitation
See also: Stolker et. al.,Anal. Bioanal. Chem.391, 2309 (2008)
A l ti l M th d F Thi St d
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Analytical Method For This StudyMilk 2 mL Sample
Initial Extraction/PrecipitationPipet 2 mL sample into centrifuge tube
Add 2 mL acetonitrile
Centrifuge @ 8000 xg
Take 2 mL supernatant
Protein Precipitation
Add 3 mL acetonitrile(0.2% formic acid)
Centrifuge @ 8000 xg
Take 1 mL supernatant
SPE CleanupSep-Pak C18 (1 cc, 100 mg)Evaporate and reconstitute
provides goodrecovery of mostcompounds
minimal extractionof fat
much protein inextract
secondary proteinprecipitation step
removes mostresidual proteinwithout significantloss of polar
analytes
SPE Cl
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SPE CleanupSep-Pak C18 (pass-thru mode)
Condition1 mL 80:20 acetonitrile/water
Pass-Thru/Collect1 mL protein ppt sample
1 cc 100 mg
install collection tubes
Rinse/Collect0.5 mL 80:20 acetonitrile/water
Evaporate/Reconstitute0.2 mL 25:75 acetonitrile/buffer
(25 mM ammonium formate buffer @ pH 4.5)
add 0.25 mL 200 mM ammonium formatein 50:50 ACN/methanol*
* buffers sample to protect acid labile analytes
Effect of Buffering Prior to
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Effect of Buffering Prior toEvaporation
Analyte Recovery Without Buffer Recovery With Buffer
Sulfamerizine 70-80 70-80
Lincomycin
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Multi-Residue Pesticide Screening
S E l
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Some Examples:Multi-Residue Screening
Method Objectives: Need to screen a wide variety of pesticides
Need moderate Sensitivity
o Sensitivity is instrument driven
Need moderate to low sample cleanup
o Optimize system performance
o Maximize the number of commodities tested
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SPE Flowchart
Pass-through SPE(Matrixretained by
sorbent)
Optimized
cleanupMax. Sensitivity
MatrixRemoval
ScreeningMethod?
Yes Low Dispersive- SPE
(d-SPE)
Pass-throughSPE by
cartridge
Retention-cleanup-elutionSPE (Analytesinitially
retained by sorbent, lastlyeluted by strong solvent)
Better cleanup than
d-SPE in generalSome enrichment viasolvent evaporation
No Moderate
Evaluate analytestructures and
properties, matrix,and cleanuprequirement
OBJECTIVES:Screen a wide variety of pesticides
Need moderate sensitivity
Require limited sample cleanup
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SPE Flowchart
Pass-through SPE(Matrixretained by
sorbent)
Optimized
cleanupMax. Sensitivity
MatrixRemoval
ScreeningMethod?
Yes Low Dispersive- SPE
(d-SPE)
Pass-throughSPE by
cartridge
Retention-cleanup-elutionSPE (Analytesinitially
retained by sorbent, lastlyeluted by strong solvent)
Better cleanup than
d-SPE in generalSome enrichment viasolvent evaporation
No Moderate
Evaluate analytestructures and
properties, matrix,and cleanuprequirement
OBJECTIVES:Screen a wide variety of pesticides
Need moderate sensitivity
Require limited sample cleanup
Dispersive Sample Preparation
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Dispersive Sample PreparationMethod Outline
1. Combine sorbent, sample matrix and solvent into a vessel
2. Sample is filtered or centrifuged
Matrix interferences are retained by sorbent
3. Filtrate or supernatant is collected for analysis
Analytes are in the filtrate or supernatant
DisQuE Kit
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DisQuE KitDispersive Sample Preparation
DisQuE Extraction Tube 1:50 mL centrifuge tube containing;
1.5 g anhydrous sodium acetate
6 g of anhydrous magnesium sulfate
DisQuE Clean-Up Tube 2:
2 mL centrifuge tube containing;
150 mg anhydrous magnesium sulfate50 mg of PSA (Primary-Secondary Amine SPE sorbent)
Tube 1
Tube 2
DisQuE Product Line
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DisQuE Product Line
Clean Up Tubes (Tube 2 - 2 mL Option)AOAC Configuration CEN Configuration
150mg Magnesium
Sulphate
50mg PSA
150mg Magnesium
Sulphate
50mg PSA
50mg C18
150mg Magnesium
Sulphate
25mg of PSA
150mg Magnesium
Sulphate
25mg of PSA
25mg C18
Extraction Tubes (Tube 1)
AOAC Configuration CEN Configuration1.5 g Sodium Acetate
6 g Magnesium Sulphate
4 g Magnesium Sulphate
1 g Sodium Chloride
1 g Trisodium Citrate
0.5 g Disodium Citrate
Clean Up Tubes (Tube 2 - 15 mL Option)
900 mg Magnesium Sulphate
150 mg of PSA
900 mg Magnesium Sulphate
150 mg of PSA
150 mg C18
DisQuE Procedure Tube 1
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Homogenize Sample
Sample Extraction15 g sample15 mL 1% Acetic Acid in ACN
Liquid FractionationShake for 1 minute
Centrifuge >1500 x g
CollectionRecover acetonitrile for clean-up
using tube 2
DisQuE ExtractionTube 1
Analytes
remainin
Supernatant
Tube 1
Tube 1
DisQuE Procedure Tube 1AOAC Method
DisQuE Procedure - Tube 2
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Transfer
1 mL ExtractTube 1 to Tube 2
Shake vigously for 1 minuteCentrifuge >1500 x g
TransferCollect to autosampler vial
Dilute if necessary
DisQuE Clean-UpTube 2
Prepare SampleHomogenize15 g sample
15 mL 1% Acetic Acid in ACN
Liquid Fractionation
Shake for 1 minuteCentrifuge >1500 x g
CollectionRemove acetonitrile for clean-up
DisQuE ExtractionTube 1
Tube 1
Tube 2
Analytes
remain
in
Supernatant
Transfer
Supernatant
toTube 2
Tube 2
DisQuE Procedure - Tube 2AOAC Method
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Cleanup Tube 2
Provides additional cleanup
Sorbent choices
PSA removes
o Acidic interferences (ion-exchange mechanism)
o Carbohydrates and sugars (HILIC mechanism)
Graphitized Carbon Black (GCB) Removes
o Chlorophyll and Pigments
C18 Removes
o Non Polar Interferences
Acetonitrile Layer (ANALYTES ARE HERE)
Sorbent (INTERFERENCES)
DisQuE
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DisQuEGraphitized Carbon Black
Uses for QuEChERS:
Removes Chlorophyll and Pigments
Also removes ANALYTES CAREFULL!!
PSAOnly
PSA +2.5 mgGCB
PSA +12.5 mgGCB
NoCleanup
PSA +25 mgGCB
PSA +50 mgGCB
PSA +7.5 mgGCB
DisQuE
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DisQuEGraphitized Carbon Black BE CAREFULL!!
Pesticide Recovery in Grape
0
20
40
60
80
100
120
140
160
Atrazin
e
Azoxystrobin
Carbaryl
Cyprodinil
Dichlorvos
Imazalil
Imidaclop
rid
Linuron
Methamidoph
os
Methomyl
Pymetrozine
Tebuconazole
Thiabendazole
Tolyfluanid
Pesticides
%Re
cover
PSA PSA+C18 PSA+GCB
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Targeted Residue Analysis
Melamine
Some Examples:
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Some Examples:Targeted Residue Analysis
Determining melamine and cyanuric acid in infant formula
Method Objectives: Specific for melamine and cyanuric acid
Need maximum sample cleanupo Optimize system performance
o Eliminate False positives and negative
Need sensitivity and sample enrichment
Targeted Residue Analysis
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Pass-through SPE(Matrixretained by
sorbent)
Optimized
cleanupMax. Sensitivity
MatrixRemoval
ScreeningMethod?
Yes Low Dispersive- SPE
(d-SPE)
Pass-throughSPE by
cartridge
Retention-cleanup-elutionSPE (Analytesinitially
retained by sorbent, lastlyeluted by strong solvent)
Better cleanup than
d-SPE in generalSome enrichment viasolvent evaporation
No Moderate
Evaluate analytestructures and
properties, matrix,and cleanuprequirement
OBJECTIVES:Selective for melamine/cyanuric Acid
Need moderate sensitivity
Need limited sample cleanup
Targeted Residue AnalysisSPE Flowchart:Method Selection
Targeted Residue Analysis
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Targeted Residue AnalysisOasis 2x4: Starting Protocol for Melamine
MelamineWeak Base
Use a StrongCation Exchanger
Oasis MCX
Targeted Residue Analysis
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Targeted Residue AnalysisOasis 2x4: Starting Protocol for Cyanuric Acid
Cyanuric AcidWeak Acid
Use a Strong
Anion Exchanger
Oasis MAX
Sample Pretreatment
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Sample PretreatmentChallenges and Analyte Considerations
Maximum cleanup due to complex matrix
Proteins
Fat
Sugars
Very Polar analytes
Melamine, weak base pKa ~ 9
Cyanuric acid, weak acid pKa ~ 5
Melamine
Cyanuric Acid
Sample Pretreatment
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Sample PretreatmentSample Extraction: Melamine/Cyanuric Acid
Sample Extraction
5g liquid infant formula or 1g dryinfant formula add 4 mL water
Add internal standards
Add 20 mL 50:50 ACN:H2O
Shake for 10 -20 minCentrifuge @ 3400 rpm for 10 min
Sample Pretreatment Flowchart
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Sample Pretreatment FlowchartExtraction Details: Melamine/Cyanuric Acid
SampleHomogenized with
organic solvent. Addbuffer, if necessary
Extract analytes in
solid usingother solvent
Discardsolvent
Analyte inLiquid phase
no
yesDiscard
solid fraction
SampleType ?
Liquid
Solid
Dilute with
non-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPE
Liquid
Sample PreparationWater: Dissolve the infant formulaAcetonitrile: Precipitate proteins
Sample Pretreatment Flowchart
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Sample Pretreatment FlowchartExtraction Details: Melamine/Cyanuric Acid
SampleHomogenized with
organic solvent. Addbuffer, if necessary
Extract analytes in
solid usingother solvent
Discardsolvent
Analyte inLiquid phase
no
yesDiscard
solid fraction
SampleType ?
Liquid
Solid
Dilute with
non-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPE
Liquid
Sample PreparationWater: dissolve the infant formulaAcetonitrile: precipitate proteins
Sample Pretreatment
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Sample PretreatmentOptimized Sample Extraction
Sample Extraction
5g liquid infant formula or 1g dryinfant formula add 4 mL water
Add internal standards
Add 20 mL 50:50 ACN:H2O
Shake for 10 -20 minCentrifuge @ 3400 rpm for 10 min
Polar solvent for analyte extraction
Acetonitrile for protein precipitationSolvent is compatible with SPE
Solid powder dissolved in water
Removed solids andprecipitated proteins
To enhance precision
Targeted Residue Analysis
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SPE Flowchart: Method Selection
Pass-through SPE(Matrixretained by
sorbent)
Optimized
cleanupMax. Sensitivity
MatrixRemoval
ScreeningMethod?
Yes Low Dispersive- SPE
(d-SPE)
Pass-throughSPE by
cartridge
Retention-cleanup-elutionSPE (Analytesinitially
retained by sorbent, lastlyeluted by strong solvent)
Better cleanup than
d-SPE in generalSome enrichment viasolvent evaporation
No Moderate
OBJECTIVES:Targeted melamine and cyanuric acidNeed maximum sample cleanupNeed sensitivity and sampleenrichment
Evaluate analytestructures and
properties, matrix,and cleanuprequirement
Targeted Residue Analysis
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Condition5 mL 0.1M NaOH in ACN5 mL 0.1M HCl in ACN
5 mL ACN
Equilibrate5 mL 4% FA in water
Load2 mL sample supernatant
Diluted with 3 mL 4% FA in water
Wash5 mL ACN5 mL 0.2% DEA in ACN
Elute4 mL 2.0% DEA in ACN
g yPutting It All Together
Melamine Cleanup by OasisMCX
Pre-clean SPE cartridge from anyenvironmental contaminants of melamine
Solvate sorbent with loading solvents
Reduce organic strength of sampleIonize the melamine
Remove acidic and neutral interferencesRemove weak basic interference
Elute melamine with stronger base toput it in its un-ionized form.Acetonitrile is used for subsequent LCby HILIC mode
Condition
Melamine Analysis
Targeted Residue Analysis
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Condition5 mL 0.1M HCl in ACN
5 mL 0.1M NaOH in ACN
5 mL ACN
Equilibrate5 mL 5% NH4OH in water
LoadDilute 2 mL sample supernatant
with 3 mL 5% NH4OH inwater
Wash5 mL ACN
Elute2 mL 4.0% FA in ACN
Cyanuric Acid Cleanup by OasisMAX
Pre-clean SPE cartridge from any environmentalcontaminants of cyanuric acid
Solvate sorbent with loading solvents
Reduce organic strength of sampleIonize the cyanuric acid
Remove basic and neutral interferences
Elute cyanuric acid with strongeracid to put it in its un-ionized form
Acetonitrile is used for HILIC
Condition
g yPutting It All Together
Cyanuric Acid Analysis
Targeted Residue Analysis
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g ySummary of the Melamine Method
The method goals were obtained by using: Simple sample pretreatment
o Removed protein and fat interferences
Specific SPE cleanup for melamine and cyanuric acid
o Removed carbohydrates interferences
o Easy modification 2x4 methodology
Acetonitrile was chosen for HILIC
o Required two SPE sorbents for analyte specificity
SPE Method Development Strategy
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p gySummary
Sample pretreatment and preparation requires a systematic
approach
Many factors govern the final method
Common interferences can be removed by a selective use ofsolvent and SPE sorbents
Charts and flow paths help gain an understanding of the
reasoning behind the method choices
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Thank You?