Nordic Sample Pre-treatment and Preparation for Food Analysis

8/9/2019 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







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
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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



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



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



Outline

Food analysis proceduresWhy sample pretreatment?









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



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



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)



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



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



Outline

Food analysis procedures

Why sample pretreatment?









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



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



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



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



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





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 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)




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Outline

Food analysis procedures

Why sample pretreatment?








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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
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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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Solvent

Re-extract sample

using other solvent

DiscardSolvent


no


SampleType ?

Liquid

Solid


SPE

Centrifugesample

Liquid

SPE


Liquid

Hexane: Non-polar

Analyte: Polar


PolarAnalytes


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Solvent

Re-extract sample

using other solvent

DiscardSolvent


no


SampleType ?

Liquid

Solid


SPE

Centrifugesample

Liquid

SPE


Liquid

Hexane: Non-polar

Analyte: Non-Polar



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Solvent

Re-extract sample

using other solvent

DiscardSolvent


no


SampleType ?

Liquid

Solid


SPE

Centrifugesample

Liquid

SPE


Liquid

Hexane: Non-polar

Analyte: Non-Polar

Fats and Oils

Non-Polar Analytes


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Solvent

Re-extract sample

using other solvent

DiscardSolvent


no


SampleType ?

Liquid

Solid

Dilute withnon-polar solvent SPE

Centrifugesample

Liquid

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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SampleType ?

Liquid

Solids

SPE

Centrifugesample

Proteinbinding ?

No

Yes




Centrifugesample

SPE


Centrifugesample

SPE



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SampleType ?

Liquid

Solids

SPE

Centrifugesample

Proteinbinding ?

No

Yes




Centrifugesample

SPE


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?








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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









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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




sorbent)

Optimizedcleanup

Max. Sensitivity

MatrixRemoval

ScreeningMethod?

Yes LowDispersive

- SPE(d-SPE)

Pass-throughSPE by

cartridge



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


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


sorbent)

Optimized

cleanupMax. Sensitivity

MatrixRemoval

ScreeningMethod?

Yes Low Dispersive- SPE

(d-SPE)

Pass-throughSPE by

cartridge



Better cleanup than

d-SPE in generalSome enrichment viasolvent evaporation

No Moderate



OBJECTIVES:Screen a wide variety of pesticides

Need moderate sensitivity

Require limited sample cleanup


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SPE Flowchart


sorbent)

Optimized


MatrixRemoval

ScreeningMethod?


(d-SPE)

Pass-throughSPE by

cartridge



Better cleanup than


No Moderate



OBJECTIVES:Screen a wide variety of pesticides


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



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sorbent)

Optimized


MatrixRemoval

ScreeningMethod?


(d-SPE)

Pass-throughSPE by

cartridge



Better cleanup than


No Moderate



OBJECTIVES:Selective for melamine/cyanuric Acid


Need limited sample cleanup

Targeted Residue AnalysisSPE Flowchart:Method Selection



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Targeted Residue AnalysisOasis 2x4: Starting Protocol for Melamine

MelamineWeak Base

Use a StrongCation Exchanger

Oasis MCX



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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


no

yesDiscard

solid fraction

SampleType ?

Liquid

Solid

Dilute with

non-polar solvent SPE

Centrifugesample

Liquid

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


no

yesDiscard

solid fraction

SampleType ?

Liquid

Solid

Dilute with

non-polar solvent SPE

Centrifugesample

Liquid

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



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SPE Flowchart: Method Selection


sorbent)

Optimized


MatrixRemoval

ScreeningMethod?


(d-SPE)

Pass-throughSPE by

cartridge



Better cleanup than


No Moderate

OBJECTIVES:Targeted melamine and cyanuric acidNeed maximum sample cleanupNeed sensitivity and sampleenrichment





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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



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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



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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?

Nordic Sample Pre-treatment and Preparation for Food Analysis

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Transcript of Nordic Sample Pre-treatment and Preparation for Food Analysis