©2012 Waters Corporation 1
Sample CleanSample Clean--up up ApproachesApproachesfor for Food AnalysisFood Analysis
[email protected]: +358-9-5659 6288Fax: +358-9-5659 6282
Waters FinlandKutomotie 1600380 Helsinki
©2012 Waters Corporation 2
Food Analysis ChallengeFood Analysis ChallengeSample Pretreatment & PreparationSample Pretreatment & Preparation
Sample pretreatment and preparation requires a systematic approach to answer the question:
Sample Extract?
to
How do we get from….
©2012 Waters Corporation 3
OutlineOutline
� Food analysis methods�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
©2012 Waters Corporation 4
Food Analysis MethodsFood Analysis MethodsSystematic ApproachSystematic Approach
� Food analysis could be classified into 3 critical steps
SamplePretreatment
SamplePreparation
Sample Analysisby Instrument
� Some may consider both steps asa single step of sample preparation
� Depending on the analytes and sample matrix, sample pretreatment may not be necessary.
©2012 Waters Corporation 5
OutlineOutline� Food analysis methods�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
©2012 Waters Corporation 6
Food Analysis MethodsFood Analysis MethodsWhy Sample PretreatmentWhy Sample Pretreatment??
� Obtain representative, consistent sample– homogenization by cutting, chopping, blending
� To remove moisture – Drying sample by heat or drying reagents
Blender Polytron®Homogenizer
©2012 Waters Corporation 7
Potential Problems of MatrixPotential Problems of MatrixCommon Types of Matrix InterferencesCommon 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
©2012 Waters Corporation 8
Potential Problems of MatrixPotential Problems of MatrixSample consistencySample 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
©2012 Waters Corporation 9
Food Analysis MethodsFood Analysis MethodsWhy Sample PretreatmentWhy Sample Pretreatment??
To adjust the sample conditions for the NEXT STEP of the sample preparation
� Sample conditions can be adjusted using:
– Dilution
– Extraction
– Solvent exchange into instrument compatible solvents
– pH adjustment
©2012 Waters Corporation 10
Why Sample Pretreatment? Why Sample Pretreatment? To Adjust Sample pHTo Adjust Sample pH
� pH adjustment can provide:– Optimized ionization of the analytes for SPE sample preparation
o Neutral species for reversed-phaseo Ionized species for ion-exchange
– Stabilization of pH labile compounds
– Reduced matrix interferenceso Protein precipitation (PPT) by adding acids or organic solvent
– Elimination of protein binding by adding acids or bases o TFA, formic acid, phosphoric acid or ammonia
©2012 Waters Corporation 11
OutlineOutline� 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
©2012 Waters Corporation 12
Sample PreparationSample PreparationNonNon--Chromatographic TechniquesChromatographic 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(LLE)
• Best non-chromatographiccleanup
• Enrichment
• Cumbersome• Expensive• Lots of solvent usage
©2012 Waters Corporation 13
Sample Preparation by SPE Sample Preparation by SPE A Chromatographic CleanupA Chromatographic Cleanup
Techniques Advantages Disadvantages
Solid Phase Extraction(SPE)
• Best cleanup• Enrichment• Fast• Easy to automate• Many sorbents availablefor optimum cleanup
• May need multiple steps• Not well understood
©2012 Waters Corporation 14
Why Use SPE for Food Analysis ?Why Use SPE for Food Analysis ?� Sample Cleanup– Significant cleanup with specific SPE sorbent
o Japanese multi-pesticide residue method– 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 extractionSPE requires much less solvent per sample
(often 10X less solvent used for SPE)
©2012 Waters Corporation 15
General Pretreatment Procedures General Pretreatment Procedures for SPE Sample Preparationfor SPE Sample Preparation
SampleType ?
Liquid
Solid
Filter/CentrifugeSample*
Solid Extraction
Liquid
SPE
SampleHomogenized
pHadjustment
* sample with suspended solids, e.g. orange juice
Liquid
Filter/CentrifugeSample
Solids
Liquid
SPE
pHadjustment
Liquid
Extraction
Clearfluid
Extraction
©2012 Waters Corporation 16
General Pretreatment Procedures General Pretreatment Procedures for SPE Sample Preparationfor SPE Sample Preparation
SampleType ?
Liquid
Solid
Filter/CentrifugeSample*
Solid Extraction
Liquid
SPE
SampleHomogenized
pHadjustment
Liquid
Filter/CentrifugeSample
Solid
Liquid
SPE
pHadjustment
Liquid
Extraction
Clearfluid
Extraction
Often performed in one step
©2012 Waters Corporation 17
OutlineOutline� 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
©2012 Waters Corporation 18
Factors that Influence SPE Factors that Influence SPE Performance Performance
� Interactions between Solvent, Analyte, Sorbent and Matrixdetermine the SPE method performance.
� Interactions of all factors need to be considered together when developing SPE method.
� Optimized SPE methods maximize specific interactions while minimizing unwanted interactions.
Analyte
Sorbent Matrix
Solvent
©2012 Waters Corporation 19
Important Properties of Analytes for Important Properties of Analytes for SPE PerformanceSPE Performance
Key properties of analytes to be considered
– Polar functional groups consisting of O, N, S or P– The numbers and positions of the polar groups– Any ionizable functional groups
o cationic and/or anionic– 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 analyte interactionswith solvent, matrix and the SPE sorbent
©2012 Waters Corporation 20
Solvent
SolventSolvent
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
©2012 Waters Corporation 21
MatrixMatrixKey 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-up and analyte extraction
©2012 Waters Corporation 22
Sorbent
SPE SorbentsSPE SorbentsKey considerations for SPE Sorbents
� Sorbents can be chosen to interact with analyte or sample matrix
� Sorbent interactions may be enhanced or weakened by solvent manipulation
� In general, there are 4 sorbent classes:– Reversed-phase– Normal-phase– Ion-exchange– Mix-mode
©2012 Waters Corporation 23
� 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 sorbent based on:– Level of cleanup required– Sensitivity required– Analyte interaction– Other specific requirements of method
SPE Sorbent SelectionSPE Sorbent Selection
©2012 Waters Corporation 24
� Normal-Phase Sorbents– Silica, Alumina, Florisil®, Aminopropyl silica, PSA, Diol silica, � Reversed-Phase Sorbents– Oasis® HLB – 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 AnalysisSPE Sorbents for Food Analysis
©2012 Waters Corporation 25
� Normal-Phase Sorbents– Silica, Alumina, Florisil®, Aminopropyl silica, PSA, Diol silica,� Reversed-Phase Sorbents– Oasis® HLB – 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 AnalysisSPE Sorbents for Food Analysis
©2012 Waters Corporation 26
� Normal-Phase Sorbents– Silica, Alumina, Florisil®, Aminopropyl silica, PSA, Diol silica, � Reversed-Phase Sorbents– Oasis® HLB – C18, C8 (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 AnalysisSPE Sorbents for Food Analysis
©2012 Waters Corporation 27
OutlineOutline� 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
©2012 Waters Corporation 28
Common Sample Matrix InterferencesCommon Sample Matrix Interferences
� Fats, Oils, Lipids and Hydrocarbons– These interferences are non-polar– They have high solubility in non-polar solventso hexane, ethers
– Common found in:o meatso nutso dairy productso manufactured goods,
• chips, cookies and chocolate
©2012 Waters Corporation 29
SPE Considerations to Remove:SPE Considerations to Remove:Fats, Oils, Lipids and HydrocarbonsFats, 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 but optimization is usually difficult
©2012 Waters Corporation 30
Sample Pretreatment to Remove: Sample Pretreatment to Remove: Fats, Oils, Lipids and HydrocarbonsFats, Oils, Lipids and Hydrocarbons
SampleHomogenized with organic solvent. Add buffer if necessary
ExtractionBy other solvent
Discardsolvent
Analyte insample
Yes
No Discardsolid fraction
SampleType ?
Liquid
Solids
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Solids
Centrifugesample SPE
©2012 Waters Corporation 31
Sample Pretreatment to Remove: Sample Pretreatment to Remove: Fats, Oils, Lipids and HydrocarbonsFats, Oils, Lipids and Hydrocarbons
SampleHomogenized with organic solvent. Add buffer, if necessary
Extract analytes in solid usingother solvent
Discardsolvent
Analyte inLiquid phase
No
Yes Discardsolid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPELiquid
©2012 Waters Corporation 32
Remove Fats, Oils, Lipids Remove Fats, Oils, Lipids Example: HexaneExample: Hexane
Homogenize Sample with
Solvent
Re-extract sampleusing other solvent
DiscardSolvent
Analyte inLiquid phase
No
Yes Discard solid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPELiquid
Hexane: Non-polarAnalyte: Polar
Fats and Oils(Hexane)
©2012 Waters Corporation 33
Remove Fats, Oils, Lipids Remove Fats, Oils, Lipids Example: HexaneExample: Hexane
Homogenize Sample with
Solvent
Re-extract sampleusing other solvent
DiscardSolvent
Analyte inLiquid phase
No
Yes Discard solid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPELiquid
Hexane: Non-polarAnalyte: Polar
Fats and Oils(Hexane)
PolarAnalytes
©2012 Waters Corporation 34
Remove Fats, Oils, Lipids Remove Fats, Oils, Lipids Example: HexaneExample: Hexane
Homogenize Sample with
Solvent
Re-extract sampleusing other solvent
DiscardSolvent
Analyte inLiquid phase
No
Yes Discard solid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPELiquid
Hexane: Non-polarAnalyte: Non-Polar
Fats and Oils(Hexane)
©2012 Waters Corporation 35
Remove Fats, Oils, Lipids Remove Fats, Oils, Lipids Example: HexaneExample: Hexane
Homogenize Sample with
Solvent
Re-extract sampleusing other solvent
DiscardSolvent
Analyte inLiquid phase
No
Yes Discard solid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPELiquid
Hexane: Non-polarAnalyte: Non-Polar
Fats and OilsNon-Polar Analytes
©2012 Waters Corporation 36
Remove Fats, Oils, Lipids Remove Fats, Oils, Lipids Example: HexaneExample: Hexane
Homogenize Sample with
Solvent
Re-extract sampleusing other solvent
DiscardSolvent
Analyte inLiquid phase
No
Yes Discard solid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPELiquid
Hexane: Non-polarAnalyte: Non-Polar
Fats and OilsNon-Polar Analytes
©2012 Waters Corporation 37
Common Sample Matrix InterferencesCommon 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 cause precipitation)
©2012 Waters Corporation 38
Sample Pretreatment to Remove: Sample Pretreatment to Remove: ProteinsProteins
SampleType ?
Liquid
Solids
SPE
Centrifugesample
Proteinbinding ?
No
Yes
Add Acetonitrileor Acids
Centrifugesample
Dilute Sample with solvent
SampleHomogenized with organic solvent. Add buffer if necessary
Centrifugesample
SPE
SPE
Protein Precipitation
©2012 Waters Corporation 39
Sample Pretreatment to Remove: Sample Pretreatment to Remove: ProteinsProteins
SampleType ?
Liquid
Solids
SPE
Centrifugesample
Proteinbinding ?
No
Yes
Dilute Sample with solvent
SampleHomogenized with organic solvent. Add buffer if necessary
Centrifugesample
SPE
Add Acetonitrileor Acids
Centrifugesample SPE
Protein Precipitation
©2012 Waters Corporation 40
Sample Pretreatment to Remove: Sample Pretreatment to Remove: ProteinsProteins
SampleType ?
Liquid
Solids
SPE
Centrifugesample
Proteinbinding ?
No
Yes
Dilute Sample with solvent
SampleHomogenized with organic solvent. Add buffer if necessary
Centrifugesample
SPE
Add Acetonitrileor Acids
Centrifugesample SPE
Protein Removal Step
©2012 Waters Corporation 41
SPE Considerations to Remove:SPE Considerations to Remove:ProteinsProteins
� Choose sorbents that maximize analyte retention
� Large protein molecules usually pass-through reversed-phase sorbent without retaining– Cannot access pores in sorbent
� Eliminate proteins-analyte interactions prior to the SPE step
©2012 Waters Corporation 42
OutlineOutline
� 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
©2012 Waters Corporation 43
How Much Sample Cleanup is Needed?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 usedo Selective detection, e.g. MS or MS/MS
• Simple cleanup of major interference may be sufficiento Less selective detection, e.g. LC-UV or GC-FID
• More cleanup may be required
©2012 Waters Corporation 44
General SPE Cleanup StrategyGeneral SPE Cleanup Strategy
Evaluate analytestructures and
properties, matrixand cleanup requirement
Pass-through SPE(Matrix retained by
sorbent)
Optimizedcleanup
Max. Sensitivity
Matrix Removal
ScreeningMethod?
Yes Low Dispersive- SPE(d-SPE)
Pass-through SPE by cartridge
Retention-cleanup-elution SPE (Analytes initially
retained by sorbent, lastly eluted by strong solvent)
Better cleanup than d-SPE in general
Some enrichment via solvent evaporation
No Moderate
©2012 Waters Corporation 45
Dispersive-SPE(d-SPE) Pass-through SPE Retention-cleanup-
elution
Cleanup Quick, simple, easy More effective than d-SPE in general
Most effective and very selective
Analyte Non-retained Non-retained Initially retained and then eluted
Matrix Mostly retained Mostly retained Non-retained or removed by washing
SorbentSelection
Maximize: Matrix retention
Minimize: Analyte retention
Maximize: Matrix retention
Minimize: Analyte retention
Maximize: Analyte retention
Minimize: Matrix retention
Enrichment No, in general Limited by solvent evaporation
Yes
Analysis Multiresidueanalysis
Multiresidueanalysis
Compounds with similar structures and/or properties
Comparison of the SPE StrategiesComparison of the SPE Strategies
©2012 Waters Corporation 46
OutlineOutline� Food analysis methods�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
©2012 Waters Corporation 49
Milk CompositionMilk Composition
� Typical Cow’s Milk– Approximately 14 % solids
o 4 % fato 4 % proteino 5 % sugar (lactose)o 85 % water
©2012 Waters Corporation 50
General SPE Cleanup StrategyGeneral SPE Cleanup Strategy
Evaluate analytestructures and
properties, matrixand cleanup requirement
Pass-through SPE(Matrix retained by
sorbent)
Optimizedcleanup
Max. Sensitivity
Matrix Removal
ScreeningMethod?
Yes Low Dispersive- SPE(d-SPE)
Pass-through SPE by cartridge
Retention-cleanup-elution SPE (Analytes initially
retained by sorbent, lastly eluted by strong solvent)
Better cleanup than d-SPE in general
Some enrichment via solvent evaporation
No Moderate
©2012 Waters Corporation 51
Veterinary Residues in MilkVeterinary Residues in MilkMultiresidue LC/MS AnalysisMultiresidue 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 SPE cleanupo excellent protein precipitationo poor recovery of tetracyclines, beta-adrenergics, polar sulfonamides
o good recovery of most other compounds
*) The CRC Handbook of Biochemistry gives the composition of McIlvaine buffer as a mixture of 0.1 molar citric acid and 0.2 Mdisodium phosphate. Depending on the pH you want, you mix varying amounts of the two components together. For example, 98 ml of citric acid + 2 ml of Na2HPO4 gives pH 2.2, 42 ml of citric acid + 58 ml Na2HPO4 gives pH 5.6, and 2.75 ml citric acid and 97.25 ml Na2HPO4 gives pH 8.0 (all pH determinations given at 21 oC).
©2012 Waters Corporation 52
Typical Recoveries From MilkTypical Recoveries From MilkComparison of Precipitation/Extraction TechniquesComparison of Precipitation/Extraction Techniques
Drug Class 3:1 ACN Aq Buffer 1:1 ACN*β-adrenergic <10 ~100 >80Tetracycline <25 >70 >25Fluoroquinolone >50 >50 >50Macrolide >60 <35 >60Beta-Lactam >70 <30 >70Steroid >70 <10 >70
*Conclusion:-Procedure chosen for this study -Extraction/precipitation of milk with an equal volume of acetonitrile provides recovery of the widest range of compoundsHowever - insufficient protein precipitation
See also: Stolker et. al., Anal. Bioanal. Chem. 391, 2309 (2008)
©2012 Waters Corporation 53
Analytical Method For This StudyAnalytical Method For This StudyMilk Milk –– 2 ml Sample2 ml Sample
Initial Extraction/PrecipitationPipet 2 ml sample into centrifuge tube
Add 2 ml acetonitrileCentrifuge @ 8000 x gTake 2 ml supernatant
Protein PrecipitationAdd 3 ml acetonitrile(0,2 % formic acid)
Centrifuge @ 8000 x gTake 1 ml supernatant
SPE CleanupSep-Pak C18 (1 cc, 100 mg) Evaporate and reconstitute
provides good recovery of most compoundsminimal extraction of fatmuch protein in extract
secondary protein precipitation step removes most residual protein without significant loss of polar analytes
©2012 Waters Corporation 54
SPE Cleanup SPE Cleanup SepSep--Pak C18 (passPak C18 (pass--thru mode)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/Reconstitute 0,2 ml 25:75 acetonitrile/buffer
(25 mM ammonium formate buffer @ pH 4,5)
add 0.25 ml 200 mM ammonium formate in 50:50 ACN/methanol*
* buffers sample to protect acid labile analytes
©2012 Waters Corporation 55
Effect of Buffering Prior to Effect of Buffering Prior to EvaporationEvaporation
Analyte Recovery Without Buffer Recovery With BufferSulfamerizine 70-80 70-80Lincomycin < 25 80-100Erythromycin < 25 60-80Penicillin < 40 75-85
©2012 Waters Corporation 57
Some Examples:Some Examples:MultiMulti--Residue ScreeningResidue Screening
�Method Objectives:– Need to screen a wide variety of pesticides
– Need moderate Sensitivityo Sensitivity is instrument driven
– Need moderate to low sample cleanupo Optimize system performanceo Maximize the number of commodities tested
©2012 Waters Corporation 58
SPE FlowchartSPE Flowchart
Pass-through SPE(Matrix retained by
sorbent)
Optimizedcleanup
Max. Sensitivity
Matrix Removal
ScreeningMethod?
Yes Low Dispersive- SPE(d-SPE)
Pass-through SPE by cartridge
Retention-cleanup-elution SPE (Analytes initially
retained by sorbent, lastly eluted by strong solvent)
Better cleanup than d-SPE in general
Some enrichment via solvent evaporation
No Moderate
Evaluate analytestructures and
properties, matrixand cleanup requirement
OBJECTIVES:Screen a wide variety of pesticides
Need moderate sensitivityRequire limited sample cleanup
©2012 Waters Corporation 59
SPE FlowchartSPE Flowchart
Pass-through SPE(Matrix retained by
sorbent)
Optimizedcleanup
Max. Sensitivity
Matrix Removal
ScreeningMethod?
Yes Low Dispersive- SPE(d-SPE)
Pass-through SPE by cartridge
Retention-cleanup-elution SPE (Analytes initially
retained by sorbent, lastly eluted by strong solvent)
Better cleanup than d-SPE in general
Some enrichment via solvent evaporation
No Moderate
Evaluate analytestructures and
properties, matrixand cleanup requirement
OBJECTIVES:Screen a wide variety of pesticides
Need moderate sensitivityRequire limited sample cleanup
©2012 Waters Corporation 60
Dispersive Sample PreparationDispersive Sample PreparationMethod OutlineMethod 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
©2012 Waters Corporation 61
DisQuE™ KitDisQuE™ KitDispersive Sample PreparationDispersive 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 sulfate�50 mg of PSA (Primary-Secondary Amine SPE sorbent)
Tube 1
Tube 2
©2012 Waters Corporation 62
DisQuE Product LineDisQuE Product Line
Clean Up Tubes (Tube 2 - 2 ml Option)AOAC Configuration CEN Configuration
150 mg Magnesium Sulphate50 mg PSA
150 mg Magnesium Sulphate50 mg PSA50 mg C18
150 mg Magnesium Sulphate
25 mg of PSA
150 mg Magnesium Sulphate
25 mg of PSA25 mg C18
Extraction Tubes (Tube 1)AOAC Configuration CEN Configuration1,5 g Sodium Acetate
6 g Magnesium Sulphate4 g Magnesium Sulphate1 g Sodium Chloride1 g Trisodium Citrate0,5 g Disodium Citrate
Clean Up Tubes (Tube 2 - 15 ml Option)
900 mg Magnesium Sulphate150 mg of PSA
900 mg Magnesium Sulphate150 mg of PSA150 mg C18
©2012 Waters Corporation 63
Homogenize SampleSample Extraction
15 g sample15 ml 1 % Acetic Acid in ACN
Liquid FractionationShake for 1 minute
Centrifuge > 500 x g
CollectionRecover acetonitrile for clean-up
using tube 2
DisQuE ExtractionTube 1
Analytes remain
in Supernatant
Tube 1
Tube 1
DisQuE Procedure DisQuE Procedure –– Tube 1Tube 1AOAC MethodAOAC Method
©2012 Waters Corporation 64
Transfer1 ml Extract
Tube 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 FractionationShake for 1 minute
Centrifuge >1500 x g
CollectionRemove acetonitrile for clean-up
DisQuE ExtractionTube 1
Tube 1
Tube 2
Analytesremain
in Supernatant
Transfer Supernatant
to Tube 2
Tube 2
DisQuE Procedure DisQuE Procedure -- Tube 2Tube 2AOAC MethodAOAC Method
©2012 Waters Corporation 65
Cleanup Tube 2Cleanup Tube 2� Provides additional cleanup
� Sorbent choices – PSA removeso 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)
©2012 Waters Corporation 66
DisQuEDisQuEGraphitized Carbon BlackGraphitized Carbon Black
� Uses for QuEChERS: – Removes Chlorophyll and Pigments
– Also removes ANALYTES – CAREFUL!!
PSA Only
PSA +2.5 mgGCB
PSA +12.5 mgGCB
NoCleanup
PSA +25 mgGCB
PSA +50 mgGCB
PSA +7.5 mgGCB
©2012 Waters Corporation 67
DisQuEDisQuEGraphitized Carbon Black Graphitized Carbon Black –– BE CAREFUL!!BE CAREFUL!!
Pesticide Recovery in Grape
0
20
40
60
80
100
120
140
160
Atrazin
eAzo
xyst
robin
Carba
ryl
Cypro
dinil
Dichlo
rvos
Imaz
alil
Imida
clopr
idLi
nuro
n
Met
hamido
phos
Met
homyl
Pymet
rozin
eTeb
ucon
azole
Thiaben
dazo
leToly
fluanid
Pesticides
% R
eco
very
PSA PSA+C18 PSA+GCB
©2012 Waters Corporation 69
Some Examples:Some Examples:Targeted Residue AnalysisTargeted Residue Analysis
Determining melamine and cyanuric acid in infant formula
�Method Objectives:– Specific for melamine and cyanuric acid
– Need maximum sample cleanupo Optimize system performanceo Eliminate false positives and negatives
– Need sensitivity and sample enrichment
©2012 Waters Corporation 70
Pass-through SPE(Matrix retained by
sorbent)
Optimizedcleanup
Max. Sensitivity
Matrix Removal
ScreeningMethod?
Yes Low Dispersive- SPE(d-SPE)
Pass-through SPE by cartridge
Retention-cleanup-elution SPE (Analytes initially
retained by sorbent, lastly eluted by strong solvent)
Better cleanup than d-SPE in general
Some enrichment via solvent evaporation
No Moderate
Evaluate analytestructures and
properties, matrixand cleanup requirement
OBJECTIVES:Selective for melamine/cyanuric Acid
Need moderate sensitivityNeed limited sample cleanup
Targeted Residue Analysis Targeted Residue Analysis SPE Flowchart:SPE Flowchart: Method SelectionMethod Selection
©2012 Waters Corporation 71
Targeted Residue AnalysisTargeted Residue AnalysisOasis 2x4: Starting Protocol for MelamineOasis 2x4: Starting Protocol for Melamine
MelamineWeak Base
Use a Strong Cation Exchanger
Oasis MCX
©2012 Waters Corporation 72
Targeted Residue AnalysisTargeted Residue AnalysisOasis 2x4: Starting Protocol for Cyanuric AcidOasis 2x4: Starting Protocol for Cyanuric Acid
Cyanuric AcidWeak Acid
Use a StrongAnion Exchanger
Oasis MAX
©2012 Waters Corporation 73
Sample PretreatmentSample PretreatmentChallenges and Analyte ConsiderationsChallenges 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
©2012 Waters Corporation 74
Sample PretreatmentSample PretreatmentSample Extraction: Melamine/Cyanuric AcidSample Extraction: Melamine/Cyanuric Acid
Sample Extraction
5 g liquid infant formula or 1 g dry infant 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
©2012 Waters Corporation 75
Sample Pretreatment FlowchartSample Pretreatment FlowchartExtraction Details: Melamine/Cyanuric AcidExtraction Details: Melamine/Cyanuric Acid
SampleHomogenized with organic solvent. Add buffer, if necessary
Extract analytes in solid usingother solvent
Discardsolvent
Analyte inLiquid phase
no
yes Discardsolid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPELiquid
Sample PreparationWater: Dissolve the infant formulaAcetonitrile: Precipitate proteins
©2012 Waters Corporation 76
Sample Pretreatment FlowchartSample Pretreatment FlowchartExtraction Details: Melamine/Cyanuric AcidExtraction Details: Melamine/Cyanuric Acid
SampleHomogenized with organic solvent. Add buffer, if necessary
Extract analytes in solid usingother solvent
Discardsolvent
Analyte inLiquid phase
No
Yes Discardsolid fraction
SampleType ?
Liquid
Solid
Dilute withnon-polar solvent SPE
Centrifugesample
Liquid
SPE
Centrifugesample SPELiquid
Sample PreparationWater: Dissolve the infant formulaAcetonitrile: Precipitate proteins
©2012 Waters Corporation 77
Sample PretreatmentSample PretreatmentOptimized Sample ExtractionOptimized Sample Extraction
Sample Extraction
5 g liquid infant formula or 1 g dry infant 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 extractionAcetonitrile for protein precipitationSolvent is compatible with SPE
Solid powder dissolved in water
Removed solids and precipitated proteins
To enhance precision
©2012 Waters Corporation 78
Targeted Residue Analysis Targeted Residue Analysis SPE Flowchart:SPE Flowchart: Method SelectionMethod Selection
Pass-through SPE(Matrix retained by
sorbent)
Optimizedcleanup
Max. Sensitivity
Matrix Removal
ScreeningMethod?
Yes Low Dispersive- SPE(d-SPE)
Pass-through SPE by cartridge
Retention-cleanup-elution SPE (Analytes initially
retained by sorbent, lastly eluted by strong solvent)
Better cleanup than d-SPE in general
Some enrichment via solvent evaporation
No Moderate
OBJECTIVES:Targeted melamine and cyanuric acidNeed maximum sample cleanupNeed sensitivity and sample enrichment
Evaluate analytestructures and
properties, matrixand cleanup requirement
©2012 Waters Corporation 79
Condition5 ml 0,1M NaOH in ACN5 ml 0,1M HCl in ACN
5 ml ACNEquilibrate
5 ml 4 % FA in waterLoad
2 ml sample supernatant Diluted with 3 ml 4 % FA in water
Wash5 ml ACN
5 ml 0,2 % DEA in ACN
Elute4 ml 2,0 % DEA in ACN
Targeted Residue AnalysisTargeted Residue AnalysisPutting It All TogetherPutting It All Together
Melamine Cleanup by Oasis® MCX
Pre-clean SPE cartridge from any environmental 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 to put it in its un-ionized form.Acetonitrile is used for subsequent LC by HILIC mode
Condition
Melamine Analysis
©2012 Waters Corporation 80
Condition5 ml 0,1M HCl in ACN
5 ml 0,1M NaOH in ACN5 ml ACN
Equilibrate5 ml 5 % NH4OH in water
LoadDilute 2 ml sample supernatant
with 3 ml 5 % NH4OH in waterWash
5 ml ACNElute
2 ml 4,0 % FA in ACN
Cyanuric Acid Cleanup by Oasis® MAX
Pre-clean SPE cartridge from any environmental contaminants 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
Targeted Residue AnalysisTargeted Residue AnalysisPutting It All TogetherPutting It All Together
Cyanuric Acid Analysis
©2012 Waters Corporation 81
Targeted Residue AnalysisTargeted Residue AnalysisSummary of the Melamine MethodSummary of the Melamine Method
� The method goals were obtained by using:– Simple sample pretreatmento Removed protein and fat interferences
– Specific SPE cleanup for melamine and cyanuric acido Removed carbohydrates interferenceso Easy modification 2x4 methodology
• Acetonitrile was chosen for HILICo Required two SPE sorbents for analyte specificity
©2012 Waters Corporation 82
SPE Method Development StrategySPE Method Development StrategySummarySummary
� Sample pretreatment and preparation requires a systematic approach– Many factors govern the final method
– Common interferences can be removed by a selective use of solvent and SPE sorbents
– Charts and flow paths help gain an understanding of the reasoning behind the method choices