Sample Preparation in Food Safety Applications

Anshu Kumari SinghProduct SpecialistAgilent Technologies

Agenda1. Introduction to SampliQ

2. Basics of sample preparation

3. Selecting the right SPE cartridge for your application

4. Application examples

• Sulfadrugs in milk

• Tetracyclines in milk and chicken

5. QuEChERS

Introducing SampliQ!

Complete SPE solution from Agilent with our trusted technical expertise and quality supporting the line• NEW! Polymer Technologies – Featuring high

retention, outstanding recovery and excellent reproducibility across a wide range of solvents and solutions.

• Silica-Based – Available in reversed (non-polar), normal (polar) and ion exchange phases with trifunctional bonding to provide more stability.

• Other Phases – Florisil PR, aluminas and carbon phases address specialty applications.

• Specialty mixed-mode sorbent beds that provide dual retention mechanisms unmatched by single sorbents

• Bulk and QuEChERS – Plentiful selection of bulk sorbents to pack your own cartridges, as well as a full line of QuEChERS sorbents

If You Use One of These Try Agilent SampliQ Products

Cross Reference of Comparable Phases by Manufacture r

Try this…

Agilent Phenomenex Waters Varian SupelcoSampliQ Strata Oasis Bond-Elut Plexa Supelclean/Discovery

OPT Strata-X HLB PlexaDVB

ENV PS-DVB SDB-L ENV ENVI-Chrom PSCX Strata-XC MCX Plexa PCXSAX MAX

Try this…

Agilent Phenomenex Waters Varian SupelcoSampliQ Strata Sep-Pak Bond-Elut Supelclean/DiscoveryC18EC C18-E tC18 C18 ENVI-18, DSC-C18, LC-18

C18 C18-U C18 C18OHC8 C8 C8 C8 DSC-8, Envi-8, LC-8C2 tC2 C2

Phenyl Phenyl (PH) PH DSC-Ph, LC-PhEvidex Screen-C Certify-IC8/SCX DSC-MCAXSilica Si-1 Silica SI DSC-Si, LC-SI

Florisil PR FL-PR Florisil FL LC and ENVI FlorisilAmino NH2 Amino Propyl NH2 DSC-NH2, LC-NH2

Diol 2OH DSC-Diol, LC-DiolCyano CN Cyano Propyl CN-U DSC-CN, LC-CN

Alumina A, B, N Alumina A, B, N Al - A, B, N LC-Alumina A, B, NSi-SAX SAX AccellPlus QMA SAX DSC-SAX, LC-SAXSi-SCX SCX AccellPlus CM SCX DSC-SCX, LC-SCXCarbon Carbon ENVI-Carb

If your customer is using one of these...

If your customer is using one of these...

Polymers

Silica-Based and Other Sorbents

SampliQ Product Portfolio – Sorbents onlySilica

Reversed Phase (Non-Polar) Normal Phase (Polar) Mixed Mode Ion Exchange

C18 C18EC C8 C2Si-

Phenyl Silica Diol Cyano AminoSi-

C8/SCX EVDX Si-SAX Si-SCX1ml 100mg, 100/pk 5982-1111 5982-1311 5982-1011 5982-1411 5982-1511 5982-2211 5982-1711 5982-1811 5982-1911 5982-2011 5982-2111

3ml 200mg, 50/pk 5982-1132 5982-1332 5982-1032 5982-1432 5982-1532 5982-2232 5982-1832 5982-1932 5982-2332 5982-2032 5982-2132

3ml 500mg, 50/pk 5982-1135 5982-1335 5982-1035 5982-1435 5982-1535 5982-2235 5982-1635 5982-1835 5982-1935 5982-2035 5982-2135

6ml 500mg, 30/pk 5982-1165 5982-1365 5982-1065 5982-1465 5982-2265 5982-1765 5982-1865 5982-1965 5982-2364* 5982-2065 5982-2165

6ml 1000mg, 30/pk 5982-1160 5982-1360 5982-1460 5982-2260 5982-1760 5982-1860 5982-1960 5982-2060 5982-2060

96 Well Plate 50mgBulk 25g Bottle 5982-1182 5982-1382 5982-1082 5982-1582 5982-2282 5982-1882 5982-2082

* 6ml/400mg

Additional Sorbents QuEChERS BulkAlumina

AcidAlumina

BasicAlumina Neutral Florisil Carbon

Magnesium Sulfate PSA Carbon C18

1ml 100mg, 100/pk 5982-4411 25 gram bottle 5982-8382 5982-4482 5982-1182

3ml 200mg, 50/pk 5982-4332 5982-4432* 100 gram bottle 5982-8082

3ml 500mg, 50/pk 5982-4035 5982-4135 5982-4235 5982-4335

6ml 500mg, 30/pk 5982-4365 5982-4465

6ml 1000mg, 30/pk 5982-4060 5982-4160 5982-4260 5982-4360

96 Well Plate 50mgBulk 25g Bottle 5982-4082 5982-4182 5982-4282 5982-4382 5982-4482

* 3ml/250mg

PolymersOPT DVB SCX SAX PS-DVB

1ml 30mg, 100/pk 5982-3013 5982-3113 5982-3213 5982-3313

3ml 60mg, 50/pk 5982-3036 5982-3136 5982-3236 5982-3336

6ml 150 mg, 30/pk 5982-3067 5982-3167 5982-3267 5982-3367

6ml 500 mg, 30/pk 5982-3465

6ml 1000 mg, 30/pk 5982-3460 Replacement of Accubond Products96 Well Plate 10mg 5982-3096 New Product

Why is Sample Preparation Required?

Current Sample = Unsuitable for further analysis!!! …

Why?

Too dilute - analyte(s) not concentrated enough for quantitative detection

Too dirty - contains other sample matrix components that interfere with the analysis

Too dangerous- Contaminants can be ‘column killers’

Collected Sample Analyze

X

Where does sample preparation fit is the workflow o f food analysis?

Sample Collection Sample Prep Separation Detection Data Analysis

Analytical Sample Workflow

Sample preparation is what allows us to go from sample to quality dataWith sample preparation we can clean-up the sample and concentrate the target compounds

Real World & Real Samples

0 2 4 6Time (min)

Pesticides in Avocado without SPE

0 2 4 6Time (min)

Pesticides in Avocado with SPE

Checklist for Selecting a Sample Preparation Method

� Know the Identity and chemical properties of the analytes and potential chemical forms

� Know the concentration range(s) of the analytes and the detection limit requirements

� The chemical and physical composition of the sample matrix

� The availability of apparatus and equipment

� The sample size that is available or required

� The potential for contamination during some part of the sample preparation process

What is SPE? • Can be thought of as digital chromatography –compounds either bind or flow through

•Has a wide choice of sorbents with selectivities similar to sorbents used in HPLC, but it is not HPLC

•Many samples can be analyzed in parallel. Manifolds of 10 and 20 ports are readily available.

•Methods are simple to perform, but the process can be time-consuming, however SPE can be readily automated

•SPE methods give high selectivity, recovery and reproducibility

In addition SPE is widely used for:•Desalting (reversed-phase principles)•Solvent Exchange (for better chromatography)•Sample Preservation and Storage (analytes stabilize d)

How is SPE performed in the laboratory?

Prepared sample in aqueous solution is loaded onto the cartridge

Vacuum is applied, but flow rate is critical. Too fast a flow will not give the compounds of interest time to interact with the sorbent. Irreproducible flow results in poor RSD’s

Matrix contaminants are washed off

Target compounds are collected in a small volume of solvent

Sample preparation is vital to successful trace ana lysis of compounds in food.

Choose an appropriate sample

Homogenize and spikeWith internal standard

Clarify by centrifugation

Solvent exchange to aqueous solution

rotovapSPESolvent exchange toAqueous solution

Mass spec analysis Quantitative Analysis

1-8 hours depending on number of samples

1-2 hours1-2 hours

2-6 hours 0.2-0.5 hour per sample

6-8 hours

1-2 hours

Total Experimental Time: 2-3 days

Optimize chromatographyAnd MS/MS

SPE Modes—”Digital Chromatography”

Matrix Adsorption (Interference Removal)

Analyte(s) unretained (K D ~ 0)

Matrix retained (K D >> 1)

No preconcentration advantage

Eluates may not be as clean

Sample loading often gravity fed

Used less often than analyte adsorption

Analyte Adsorption (Bind-Elute)

Analyte(s) retained (K D >> 1)

Matrix unretained (K D ~ 0)and/or strongly retained (K D >> 1)

Preconcentration factor

Cleaner extracts

Load at 1-3 drops/sec (recovery ∝1/flow)

Capacity issues may be more important

SPE Modes—”Digital Chromatography”

Matrix Adsorption (Interference Removal)

Analyte Adsorption (Bind-Elute)

= Analyte of interest

= Matrix/Interferences

Analyte elutes at step 3 Analyte flows through

Condition Load Wash Elute

Prepare cartridge to accept sample

Load sample and rinse reservoir(s)

Wash with solvent that won’t elute analyte

Elute analyte in smallest volume possible

1. MeOH or ACN

2. Weak solvent(water, buffer)

Weakly retained matrix compds

elute

Analyte and othermatrix compds

retained

Elute analyteleaving highly

retained compds

1 2

Fundamental Steps for the ‘Bind-Elute’ SPE Experim ent

Optimizing Steps for the ‘Bind-Elute’ SPE Experime nt (non-polar example)

1. Conditioning: Solvent is passed through the SPE material to wet the bonded functional groups => ensures consistent interaction . (Use methanol)

2. Equilibration: Sorbent/ phase is treated with a solution that is s imilar (in polarity, pH, etc.) to the sample matrix => maximiz es retention. (Use water or the same aqueous solution that the sample is prepared i n).

3. Sample Load: Introduction of the sample = analytes of interest a re bound/ extracted onto the phase/ sorbent. Must be an aque ous solvent (no organic)

4. Washing: Use the ‘strongest’ aqueous solution that will NOT elute the target compounds. Increasing the % organic, increasing or decreasing the pH, changing the ionic strength are all tips for increa sing clean-up. Dry the cartridge to remove all water.

5. Elution: Use the smallest volume of organic solvent that wil l elute ALL of the target analyte. Use the ‘weakest’ organic solvent that will remove ALL of the target analyte. As a general rule the ‘strength’ of the solvent is directly related to the target compound. Polar target compounds elu te best in polar solvents so in order of polarity try: methanol>acetonitrile>et hylacetate>acetone>THF. Modify the pH, increase the ionic strength.

6. Solvent exchange: If the subsequent analyses are HPLC, the organic el ution solvent should be evaporated and the sample reconst ituted in starting mobile phase. If the next analysis is GC, then methanol i s the reconstitution solvent. In all cases the reconstitution must be to the same volume.

Phase Selection

Is your sample water soluble?

Yes, Water Soluble

If ionic will be using an ion exchange sorbent

If non-ionic will be using a reversed phase type sorbent for neutral compounds, or normal phase type sorbents for water soluble polar compounds.

The other sorbents are used for matrix-adsorption clean-up methods.

Phase Selection

Is your sample soluble in organic?

Yes, Soluble in organic solvent

Are the target molecules soluble in polar, moderately polar or non polar organic solvents?

If nonpolar, a reversed phase type sorbent will work best.

For methanol soluble compounds, frequently the reversed phase type sorbents will be effective, but greater selectivity can be obtained with a normal phase type sorbent

As with water soluble compounds, the other sorbents are used for matrix-adsorption clean-up methods.

What is Mixed-Mode?

There are 2 types of mixed mode cartridges .

1. Cartridges which contain two types of packing material such as carbon and C8 or C6 and SCX

2. The new polymer resins which have the ability to bind compounds over a wide range of polarities and pKa’s. These polymer resins effectively bind both ionic and neutral, polar and neutral compounds. Examples of the mixed-mode polymers are SampliQ OPT, Oasis HLB, SampliQ SCX and SAX, Oasis MCX and MAX.

Wash

Eluent or

Wash 2 EluentThis figure shows a general example of how a mixed mode extraction could be used.

Using Polymer SAX for Selective Fractionation of Neutral and Acidic Compounds

91 9471

97 98 9876 81

45

92 89 89

020406080

100120

secobarbital nortriptyline vitamin B3 Ketoprofen Naproxen Ibuprofen

% r

ecov

ery

SCX Brand X

• For acidic and neutral compounds, two different pol ymer SAX cartridges show similar recoveries.

• The neutral compounds (secobarbital and nortriptyli ne) are isolated in a methanol eluent. The other compounds (all acids) a re isolated in an acidic methanol eluent.

SAX

Polymer Performance is Robust

• Highly reproducible recoveries wet or dry

– Cartridges dried under vacuum for 10 minutes before the equilibration step • RSD’s of the recoveries for each of the compounds (n=5) very low • Compounds range from very polar, basic compounds to hydrophobic, neutral

0

20

40

60

80

100

120

140

1acetaminophen

propranolol

brompheniramine

mianserin

doxepin

fluoxetine

Dihydroxynaphthalene

DRY DRY DRYDRY DRY DRY

DRYWET WET WETWET WET WET

WET

acetaminophen

propranolol

brompheniramine

mianserin

doxepin

fluoxetine

Dihydroxynaphthalene

DRY DRY DRYDRY DRY DRY

DRYWET WET WETWET WET WET

WET

Other Considerations Important in Choosing SPE Devices?*A device refers to cartridge, disk, or 96-well plate. Decision is based on:

1. Batch-to-batch and cartridge-to-cartridge reprodu cibility2. Quality: low extractables, high recovery3. Degree of sample cleanliness needed for their analysis4. Selectivity of SPE phase (ie. range of chemistries)5. Sample capacity of SPE device (ie packing wt. & volume)6. Cost per device7. Automation capability8. Availability of SPE method for their analyte/matrix9. Availability of literature methods10. Applications support/methods development assistance11. Brand name12. Availability of free samples

* LC/GC Magazine Survey

Set-up Extraction

Sample Pre-treatment

None Wash 1 2mL 0.5M HCl

Cartridge Polymer SCX Wash 22mL 5% methanol

in water

Spike 0.5 ug/mL Wash 32mL 20% methanol

in water

Condition 2mL 2% formic acid in methanol

Wash 4 2x 2mL methanol

Equilibrate 2mL 2% formic acid in water

Dry 3 min. under vacuum

Load 5mL whole milk Elute2.5mL 5% ammonia

in methanol

Dry 30 sec. under vacuum FinalEvaporate and

reconstitute in mobile phase

10 Sulfadrugs in Milk Extracted Using Polymer SCX

Sulfadrugs in milk

spike

control

blank

LC/MS/MS Chromatogram of 10 Sulfadrugs

All 10 sulfa drugs at ~0.03mg/mL in solution

A: 0.1% formic acid/H20 (pH = 2.74)B: 0.1% formic acid/ACN

Gradientt %B0 103.5 107 60

Tetracyclines in Milk

Condition ：：：：

5ml methanol

Rinse：：：：

5ml TFA (pH=2.13-2.16)

load：：：：

5 ml sample@ >10min

wash：：：：

3ml TFA solution (pH=4.0)

elute：：：：

6ml 10mmol oxalic acid in methanol

blow-dry with N2, dissolve residue in 1 ml

Sample preparation:Dissolved 1g of milk in 10ml 0.1 mol/L Na2EDTA-Mcllyaine buffer solution (pH=4.0±0.05). Spiked 0.8ug/g standard in the solution, then mixed for 1 min and ultrasonic extracted in ice water bath for 10 min, then centrifuged at a rotate speed of 4000 r/min for 10 min ( below 15℃), filtrated with fast filter paper.

Dry cartridge under vacuum 3 minutes before elute

Clean-up of milk is excellent with the exception of a trace interference for oxytetracycline

OTC

impurity

sample

blank

std MNO TC

DMCTC

CTC DCMC

OTC

Condition：：：：5ml methanol

Rinse：：：：5ml TFA (pH=2.16)

load：：：：10ml sample@ >20min

wash：：：：3ml TFA solution (Ph=4.5)

elute：：：：8ml 10mmol oxalic acid in methanol

blow-dry with N2, dissolve residue in 1 ml

Sample preparation:Dissolved 5g of chicken(homogenized) in 50ml 0.1 mol/L Na2EDTA-Mcllyaine buffer solution (pH=4.0±0.05). Spiked standard (1ug/g) in the solution, then mixed for 1min and ultrasonic extracted in ice water bath for 10min, then centrifuged at a rotate speed of 4000r/min for 10 min ( below 15℃), filtrated with fastfilter paper.

Dry cartridge under vacuum 3 minutes before elute

Tetracyclines in Chicken Tissue

m in2 4 6 8 10 12 1 4

m A U

- 8

- 6

- 4

- 2

0

2

D A D1 A , S ig=3 50,4 Ref =of f (8 -22 \8- 22 2 006- 08- 22 19- 10- 06\3 .D)

m in2 4 6 8 10 12 1 4

m A U

- 8

- 6

- 4

- 2

0

2

4

6

D A D1 A , S ig=3 50,4 Ref =of f (8 -22 \8- 22 2 006- 08- 22 19- 10- 06\1 E G -08 01.D )

Chromatograms of TCs in chicken

Spiked 0.1ug/g standards

Blank

MNOOTC TC DMCTC CTC

MC DC

Impurity 1

Impurity 1 Impurity 2

Impurity 2

CompoundMilk

0.8ug/g

Chicken

0.2ug/g

GBT

0.2ug/g

Milk (independent publication)

Minocycline(MNO) 73.1 76.9 99.5

Oxytetracycline (OTC) 88.0* 62.9 99.5 67.7

Tetracycline(TC) 83.3 75.8 93.5 68.5

Demeclocycline(DMCTC) 93.6 90.2 89.2

Chlorotetracycline (CTC) 90.2 99.0 91.0 67.3

Methacycline(MC) 81.6 82.7 87.0

Doxycycline(DC) 92.0 97.1 93.5

% Recovery of Tetracyclines in Food Using SampliQ O PT in Comparison to an Independent Report Using a Different Non-Polar Poly mer Cartridge

Conclusions

1) Trace level detection of analytes in food samples is going to require sample clean-up.

2) SPE method development requires knowledge of your target analytes and chemical characteristics of your matrix. With that information it is possible to select an appropriate cartridge

3) Most cartridges have a simple ‘starting point’ method. From there it is simple to adjust, pH, solvent and ionic strength to optimize recovery and precision.

� extraction

� clean-up

� quantitation

� confirmation

Introduction to QuEChERSMulticlass, Multiresidue Analytical Approach

QuEChERS method

Quick

Easy

Cheap

Effective

Rugged

Safe GC-MS

LC-MS/MSSlide adapted from Kate Mastovska, USDA-ARS

Introduction to QuEChERS

1) Shake sample withsolvent and salts

TomatoTomatoSpinachSpinach

GrapeGrapeStrawberryStrawberry

2) Centrifuge for 1 min

3) Mix aportionwith asorbent

TomatoTomato

SpinachSpinach GrapeGrape

StrawberryStrawberry

4) Centrifuge for 1 min5) Analyze Pesticides

A Low Cost, Highly Effective Sample Preparation Technique for Pesticide Residue Analysis

QuEChERS Extract of a Mixed Vegetable Sample: DRS Report (Sample was not spiked)

Reportin 90 seconds

Courtsey of E. Blanke, S Bracht, P. Furst, C-K Meng

Introducing SampliQ QuEChERS Kits (4/09)

Provides food testing labs with a simplified approach to preparing samples for pesticide analysis

No guesswork, no measuring: Pre-packed Extraction Kits and Dispersive SPE Kits are assembled to suit specific food types and screening protocols

Extraction salts are pre-weighed and provided in anhydrous packets, so you can conveniently add them at the correct time and improve results by reducing analyte degradation

Sorbents and salts for the dispersive SPE step are supplied in 2 mL or 15 mL centrifuge tubes, for 1 mL, 6 mL or 8 mL aliquot volumes, as specified by the various QuEChERS methodologies in use today

Agilent’s tight quality control processes ensure that all QuEChERS salts, sorbents and supplies are high quality, and free of contaminants, so your results are accurate and reproducible

Agilent ConfidentialPage 41

Selecting the Right Kit is Easy

Choose your Extraction Kit based on the QuEChERS method you’re using, and the pesticides you’re screening for

– All salts and sorbents are pre-measured according to the specific method you are using.

Choose the Dispersive SPE Kit based on the type of food you’re analyzing and the method you are following

– A look-up reference of foods and kits can be found at www.agilent.com/chem/quechers

Agilent ConfidentialPage 42

QuEChERS Dispersive SPE Kits

Agilent ConfidentialPage 43

Qty per pack tube size sample size Sorbent 1 Sorbent 2 S orbent 3 Sorbent 4

100 2ml 1ml 25mg PSA 150mg MgSO4 ----- -----

100 2ml 1ml 50mg PSA 150mg MgSO4 ----- -----

50 15ml 6ml 150mg PSA 900 mg MgSO4 ----- -----

50 15ml 8ml 400mg PSA 1200 mg MgSO4 ----- -----

100 2ml 1ml 25mg PSA 25mg C18EC 150mg MgSO4 -----

100 2ml 1ml 50mg PSA 50mg C18EC 150mg MgSO4 -----

50 15ml 6ml 150mg PSA 150mg C18EC 900 mg MgSO4 -----

50 15ml 8ml 400mg PSA 400mg C18EC 1200 mg MgSO4 -----

100 2ml 1ml 25 mg PSA 2.5mg carbon 150mg MgSO4 -----

100 2ml 1ml 50mg PSA 50mg carbon 150mg MgSO4 -----

50 15ml 6ml 150mg PSA 15mg carbon 900 mg MgSO4 -----

50 15ml 8ml 400mg PSA 400mg carbon 1200mg MgSO4 -----

100 2ml 1ml 25 mg PSA 7.5mg carbon 150mg MgSO4 -----

50 15ml 6ml 150mg PSA 45mg carbon 900 mg MgSO4 -----

100 2ml 1ml 50mg PSA 50mg carbon 150mg MgSO4 50mg C18

50 15ml 6ml 400mg PSA 400mg carbon 1200 mg MgSO4 400mg C18

Fruits and Vegetables with Pigments and Fats, AOAC Method 2007.01, 2ml

Highly Pigmented Fruits and Vegetables, European EN Method 15662, 2ml Tube

Fruits and Vegetables with Pigments, European EN Method 15662, 15ml Tube

Fruits and Vegetables with Pigments, AOAC Method 2007.01, 15ml

Most Fruits and Vegetables: Removes polar organic acids, some sugars and lipids

Fatty Fruits and Vegetables, European EN Method 15662, 2ml Tube

Fruits and Vegetables with Pigments, European EN Method 15662, 2ml Tube

Fruits and Vegetables with Pigments and Fats, AOAC Method 2007.01, 15ml

Highly Pigmented Fruits and Vegetables, European EN Method 15662, 15ml Tube

General Fruits and Vegetables, European EN Method 15662, 2ml Tube

General Fruits and Vegetables, AOAC Method 2007.01, 2ml Tube

Fatty Fruits and Vegetables, European EN Method 15662, 15ml Tube

Fatty Fruits and Vegetables, AOAC Method 2007.01, 2ml Tube

For Use WithDescription

Fruits and Vegetables w/ pigments and fats; Removes polar organic acids, some sugars and lipids plus Carotinoides and Chlorophyll, not for

use with planar pesticides

Fruits and Vegetables w/ High levels of pigments;Removes polar organic acids, some

sugars and lipids plus high levels of Carotinoides and Chlorophyll, not for use with planar pesticides

Fruits and Vegetables w/ pigments; Removes polar organic acids, some sugars and lipids plus

Carotinoides and Chlorophyll, not for use with planar pesticides

Fruits and Vegetables with >1% Fat, Removes polar organic acids, some sugars, lipids and

sterols

General Fruits and Vegetables, European EN Method 15662, 15ml Tube

Fruits and Vegetables with Pigments, AOAC Method 2007.01, 2ml

General Fruits and Vegetables, AOAC Method 2007.01, 15ml Tube

Fatty Fruits and Vegetables, AOAC Method 2007.01, 15ml Tube

QuEChERS Advantages

�� A batch of 6-12 extracts can be prepared in 30-40 m in by a single analyst with ≈$1-3 of disposable materials per sample and generate <12 mL solvent waste and only a single re-usable item (FEP tube) for cleaning.

� Consistently high recoveries (mostly 90-110% with R SDs < 5%) of a wide range of GC- and LC-amenable pesticide s are achieved from many matrices.

� A low cost, highly effective sample preparation tec hnique which can be used on most food matricies for a wide range of pesticide classes

Questions??

Acknowledgements

•Karyn Usher and Carol Gonzalez, West Chester Universi ty, W. Chester, PA for the studies on sulfadrugs in mil k.

•Andy Zhai and Yun Zou, Agilent, China for the studi es on tetracyclines in chicken and milk