Implementation of Modified QuEChERS Techniques for Modern ... · AOAC 2007.01 5982-5755CH 20 Select...

Implementation of

Modified QuEChERS

Techniques for Modern

Applications

Derick Lucas, Ph.D.

Agilent Technologies

1

Today’s Agenda

QuEChERS – A Brief Overview

Preservatives Analysis in Pet Food

Pesticide Analysis in Juice Concentrates

Pesticide Analysis in Chicken

Pesticide Analysis in Herbal Teas

Conclusions and Summary

2

Today’s Agenda







3

What is QuEChERS (pronounced “Catchers”)

Quick, Easy, Cheap, Effective, Rugged and Safe

• Developed jointly by USDA and Food Regulatory Agencies as a

sample preparation method for multi-residue analyses

• Simplified extraction and cleanup approaches that reduce use of

expensive and/or dangerous solvents

• Primarily for preparing fruits and vegetables for pesticide analysis

• Rapidly being extended to other food types and compound classes

What are the Benefits of QuEChERS?

• QuEChERS Approach: Extract +250 compounds at one time

• Final extract amenable to GC/MS or LC/MS

• Reduced solvent, reduced labor, increased lab

productivity

QuEChERS Approach Advantages

30 minutes to extract multiple samples at once

Minimal solvent usage per sample: 10-15 mL

Chlorinated Solvents: None

If you can weigh, shake and have a centrifuge, you can perform

QuEChERS

Why Choose QuEChERS?

• Lives up to its name

• Flexibility and adaptability

• Works with solids and liquids

• Perfect complement to tandem MS instruments

• Called out in many regulatory methods and norms

6

Sample Homogenization – Pre-Preparation

QuEChERS – Easy as 1-2-3

Today’s Agenda







9

QuEChERS Application Examples: Preservatives in

Pet Food

Looked at BHA, BHT and ethoxyquin in pet food

Used QuEChERS sample prep approach to preparing pet foods

for LC-MS/MS analysis

Developed LC-MS/MS method

Results:

Incurred sample analysis proved suitability of the technique

Difficult to find pet food samples that would be free of

preservatives

Method optimization was required

10

We will get back to the method optimization in a moment

Extraction Procedure: General QuEChERS Method

• Add 2 g pet food, either dry or wet, to a 50 mL centrifuge tube

• Add 2 ceramic homogenizers (to help facilitate extraction)

• Add 8 mL water, vortex, 1 min at 2500 rpm

• Add 10 mL ACN, vortex, 1 min at 2500 rpm

• Add QuEChERS extraction salts

• Shake vigorously for 1 min, or vertical shake at 1000 rpm

• Centrifuge at 5000 rpm, 5 min

11

Various Pet Foods After QuEChERS Extraction

(Step 1)

12

d-SPE (Step 2)

• Transfer 6 mL to 15 mL d-SPE tube

• Vortex 1 min at 2500 rpm

• Centrifuge at 5000 rpm for 5 min

• Transfer 1 mL to tube for evaporation

• Reconstitute in 1 mL ACN:water (1:4)

• Filter sample with 0.45 μm PP

• Analyze by LC-MS/MS

13

LC-MS/MS Conditions

• LC conditions:

– Poroshell 120 SB-C18, 2.7 μm, 2.1 x 50 mm

• Mobile phase A: Water (0.1% FA)

• Mobile Phase B: ACN (0.1% FA)

• Time 0 25% B

0.1 25% B

4.0 90% B

4.5 90% B

4.6 90% B

• MS Settings:

14

Compound MRM Channels

(m/z)

Dwell Vol Fragmentor

(V)

CE

(V)

Ethoxyquin

TS1 EMV 300

218 > 174

218 > 160

6

6

30

30

27

31

BHA

TS3 EMV 1000

181.1 > 166

181.1 > 138

100

20

89

89

11

19

BHT

TS4 EMV 0

221.2 > 165

221.2 > 41.1

6

6

84

84

8

27

Propyl Paraben IS*

TS2 EMV 500

181 > 139

181 > 120.9

20

20

57

57

3

15

3 x10

0

0.5

1

1.5

2

2.5

3 1 1 2 2 3 3 4 4

4 x10

0

2

4

6

8 1 1 2 2 3 3 4 4

2 x10

0

1

2

3

4

5

6

1 1 2 2 3 3 4 4

2 x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75 1 1 2 2 3 3 4 4

Counts vs. Acquisition Time (min)

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6

400 ppb Post-Spiked Sample

15

Ethoxyquin

IS

BHA

BHT

400 ppb Pre-Spiked Sample

16

3 x10

0

0.2

0.4

0.6

0.8

1 1 1 2 2 3 3 4 4

4 x10

0

0.5

1

1.5

2

2.5

3

3.5

4 1 1 2 2 3 3 4 4

2 x10

0.5

1

1.5

2

2.5

3 1 1 2 2 3 3 4 4

2 x10

0.25

0.5

0.75

1

1.25

1.5

1.75

2 1 1 2 2 3 3 4 4


0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6

Ethoxyquin

IS

BHA

BHT

Incurred Samples

17

3 x10

0

2

4

6

1 1 2 2 3 3 4 4

4 x10

0

0.5

1

1.5

2

2.5 1 1 2 2 3 3 4 4

3 x10

0

2

4

6

8 1 1 2 2 3 3 4 4

4 x10

0

1

2

3

4

5 1 1 2 2 3 3 4 4

3 x10

0

1

2

3

4

5 1 1 2 2 3 3 4 4


0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6

Raw Pet Food: Organic

Product E: high grade, dry

Product M: High grade, wet

Product P: Lower grade, dry

Product PP: Lower grade, wet

QuEChERS Method Development Approaches

• Evaluate extraction salt mixtures

• Evaluate dSPE cleanup mixtures

18

We will use the pet food example to highlight method development

and optimization approaches

Optimization of QuEChERS Procedure: Pet Food

Extraction

• Three variations of the QuEChERS extraction salts were

investigated

– Non-buffered: 4 g MgSO4, 1 g NaCl

– AOAC: 6 g MgSO4, 1.5 g NaAc

– EN: 4 g MgSO4, 1 g NaCl, 1 g NaCitrate, 0.5 g disodium citrate

sesquihydrate

19

Use one dSPE mixture and

keep this part the same for

the extraction salt

optimization

TIP!

QuEChERS dSPE Method Optimization Summary

dSPE Mix 1

EN Extraction Salts

5982-5650CH

Original

5982-5550CH

AOAC 2007.01

5982-5755CH

20

Select Representative Sample

Homogenize

Weigh

Mix with salts, solvent

Centrifuge

Transfer aliquots of supernatant to each of the dSPE tubes

Mix

Centrifuge

Transfer supernatant to vials

Analyze

Identify best extraction salts

Use one dSPE type with three salt types to identify the best combination for

the application

Method Development

Process Method Development

Products – Fruits &

Vegetables

Optimization of QuEChERS Procedure: Pet Food

Cleanup

• Four variations of the d-SPE material were investigated

– 25 mg PSA, 150 mg MgSO4

– 25 mg C18, 150 mg MgSO4

– 25 mg PSA, 25 mg C18, 150 mg MgSO4

– 25 mg PSA, 2.5 mg GCB, 150 mg MgSO4

21

Use the extraction salt mix

identified in the first

experiment and keep this

part the same

TIP!

QuEChERS dSPE Method Optimization Summary

EN Extraction Salts

5982-5650CH

dSPE Mix 1

5982-5021CH

dSPE Mix 2

5982-4921CH

dSPE Mix 3

5982-5121CH

dSPE Mix 4

5982-5221CH

22

Select Representative Sample

Homogenize

Weigh

Mix with salts, solvent

Centrifuge

Transfer aliquots of supernatant to each of the dSPE tubes

Mix

Centrifuge

Transfer supernatant to vials

Analyze

Identify best dSPE sorbent

Use one extraction tube and 4 dSPE tubes to identify the best combination for

the application

Method Development

Process Method Development

Products

Pet Food QuEChERS Optimization Results

Extraction:

Use the EN extraction salts: 4 g MgSO4, 1 g NaCl, 1 g

NaCitrate, 0.5 g disodium citrate sesquihydrate (PN 5982-

5650CH)

Cleanup:

d-SPE: 25 mg PSA, 2.5 mg GCB, 150 mg MgSO4 (PN 5982-

5221CH)

23

In a minimal number of steps and experiments, the ideal match of

extraction and dSPE processes can be determined

Results for Spike Samples

Compound

400 ppb

Recovery RSD

(%) (%)

600 ppb

Recovery RSD

(%) (%)

800 ppb

Recovery RSD

(%) (%)

Ethoxyquin 64 4 57 6.5 55 5.4

BHA 101 1.5 100 3.6 100 1.0

BHT 103 7.6 110 6.5 130 6.9

24

Incurred Samples

25

3 x10

0

2

4

6

1 1 2 2 3 3 4 4

4 x10

0

0.5

1

1.5

2

2.5 1 1 2 2 3 3 4 4

3 x10

0

2

4

6

8 1 1 2 2 3 3 4 4

4 x10

0

1

2

3

4

5 1 1 2 2 3 3 4 4

3 x10

0

1

2

3

4

5 1 1 2 2 3 3 4 4


0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6

Raw Pet Food: Organic

Product E: high grade, dry

Product M: High grade, wet

Product P: Lower grade, dry

Product PP: Lower grade, wet

Today’s Agenda







26

QuEChERS Method Optimization – Multi-residue


A GC/MS/MS multiple reaction monitoring method was developed on

the Agilent 7890A/7000 GC Triple Quadrupole Mass Spectrometer

system(GC/QQQ) for 84 pesticide residues in lemon and orange juice

concentrates provided by a world wide beverage company.

Sample Preparation process follows the QuEChERS EN extraction

with modifications to account for pH and sample volume.

Results: Lemon juice concentrate was acidic (pH = 2) and treatment

with NaOH gave better results for several pesticides. Analyte

protectants (AP) were required to obtain good peak shapes by GC-

MS. The method recoveries for the majority of the compounds were

between 70-120% with RSD below 10%.

Optimized QuEChERS Sample Preparation for Juice Concentrates

28

Transfer 1 mL of upper ACN layer to Dispersive SPE 2 mL tube (EN fruits & Vegetables; p/n: 5982-5021)

Weigh 4 g Juice Concentrate in EN 50 mL centrifuge tube

Add QC spike solution as necessary, shake on mechanical shake for 10 min

Centrifuge @ 4000rpm for 2 min

Vortex 10 s then centrifuge @ 4000 rpm for 2 min

Add 6 mL of H2O (and 2 mL 5N NaOH for lemon juice conc.)

Cap and load extraction tubes on SPEX ShaQer for 60 seconds (or by hand)

Add 10 µL of AP, 20 µL of STD mix (ACN for recovery samples), 30 µL of

ISTD mix, and 250 µL of supernatant from dSPE tubes to autosampler vial.

Vortex vial and place in sample tray for GC-MS/MS analysis

Add 10 mL ACN and vortex briefly

Add 2 ceramic homogenizers and QuEChERS EN salt packet

*Analyte protectant (AP) mixture: 10 mg/mL D-sorbitol and 20 mg/mL L-gulonolactone in ACN

**ISD mix: 500 ng/mL in ACN + 1% AcOH

Improving Peak Shape with pH

29

Piperonyl Butoxide

0 mL

5 N NaOH

0.6 mL

5 N NaOH

1 mL

5 N NaOH

2 mL

5 N NaOH

Atrazine

QuEChERS Optimization – AP (Analyte Protectant)

7/26/2013 30

Diazinon

Hexachlorobenzene

Ethion

with AP without AP

APs are a must in multi-residue pesticide analysis

Precision & Accuracy for Lemon Juice Conc.

(CAL range: 2 – 200 ppb, n=6 for %REC and %RSD)

31

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Lemon Juice Concentrate

Precision & Accuracy for Orange Juice Conc.

(CAL range: 2 – 200 ppb, n=6 for %REC and %RSD)

0

10

20

30

40

50

60

70

80

90

100

110

120

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Today’s Agenda







33

QuEChERS Method – Pesticides in Chicken using

GC-Tandem MS

A GC/MS/MS multiple reaction monitoring method was

developed on the Agilent 7890/7000 GC Triple Quadrupole

Mass Spectrometer system(GC/QQQ) for 54 pesticide residues

in chicken.

Sample Preparation process follows the QuEChERS AOAC

extraction. Two dispersive cleanup options were evaluated.

Results: Chicken samples were analyzed to verify the

calibration coefficient, recovery and reproducibility. The

detection limit of most compounds is down to 5 µg/kg (5 ppb).

The method recoveries for all of the compounds were between

70-120% with RSD below 15%.

Sample Preparation

Transfer 8 mL of upper ACN layer to Bond Elut Dispersive-SPE* 15 mL tube

Weigh 7.5 g chicken muscle sample (±0.1g) in 50 mL centrifuge tube and add 7.5 mL water

Add Internal Standard (TPP) solution, and QC spike solution if necessary, vortex 1 min

Centrifuge @ 4000rpm and 4 ℃for 5 min

Vortex 1min, centrifuge @ 4000 rpm and 4 ℃ for 5 min

Add 15mL of ACN containing 1% AcOH

Cap and shake vigorously for 30 seconds

Transfer 4 mL extract to tube and dry under nitrogen

Reconstitute with 1 mL of ACN and analysis with GC-QQQ

Add Bond ElutEN QuEChERS Extraction salt packet

* Two different Dispersive-SPE kits were used.

Bond Elut QuEChERS Dispersive Kit for other food methods, 5982-4956CH

Bond Elut QuEChERs Dispersive Kit for all food types, 5982-0029CH

Clean up with

Bond Elut

QuEChERS

Dispersive Kit for

all food types

Clean up with

Bond Elut

QuEChERS

Dispersive Kit

for other food

methods

Picture of the Solutions after Sample

Preparation

C18, MgSO4

PSA, C18, GCB, MgSO4

TIC of spike 10 ug/kg pesticides in chicken muscle with Bond Elut QuEChERS AOAC kits.

Clean up with Bond Elut QuEChERS Dispersive Kit for other food methods.

TIC of chicken muscle sample blank with Bond Elut QuEChERS AOAC kits.

Clean up with Bond Elut QuEChERS Dispersive Kit for other food methods.

TIC of spike 10 ug/kg pesticides in chicken muscle with Bond Elut QuEChERS AOAC kits.

Clean up with Bond Elut QuEChERS Dispersive Kit for all food types.

TIC of chicken muscle sample blank with Bond Elut QuEChERS AOAC kits.

Clean up with Bond Elut QuEChERS Dispersive Kit for all food types.

Results – Recoveries, Continued

39

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%

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Recoveries for the pesticides of 50 µg/kg spiked in chicken muscle sample with

QuEChERS and clean up with Bond Elut Dispersive Kit for other food methods.

Results – Recoveries

40

Recoveries for the pesticides of 50 µg/kg spiked in chicken muscle sample with

QuEChERS and clean up with Bond Elut Dispersive Kit for all food types.

0.0%

20.0%

40.0%

60.0%

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100.0%

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Today’s Agenda







41

QuEChERS Method – Multi-residue analysis of

Pesticides in Herbal Tea

An LC/MS/MS multiple reaction monitoring method was developed on the Agilent 1290/6410 LC Triple Quadrupole Mass Spectrometer system(LC/QQQ) for 83 pesticide residues in herbal green and black teas.

Sample Preparation process follows the QuEChERS original 10 g extraction. Several dispersive cleanup sorbent options were evaluated.

Results: Tea samples were analyzed to verify the calibration coefficient, recovery and reproducibility. Planar pesticide recoveries are affected by carbon sorbent mass and type. The method recoveries for the majority of the compounds were between 70-120% with RSD below 15%.

Sample Preparation

Transfer 5 mL of upper ACN layer to Dispersive-SPE, 15 mL tube

Weigh 1.00 g dried tea (±0.01g) in 50 mL centrifuge tube and add 10 mL water

Add QC spike solution as necessary, vortex 1 min

Centrifuge @ 4000rpm for 5 min

Vortex 1 min, centrifuge @ 4000 rpm for 5 min

Add 10 mL of ACN containing I.S. (50 ppb dimethoate-d6)


Transfer 2 mL extract to tube and dry under nitrogen

Reconstitute with 1 mL of 70/30 Water/ ACN, syringe filter and analysis by LC-QQQ


Add Bond Elut Original QuEChERS Extraction salt packet

Chromatogram

Chromatogram of 100 ppb spike in black tea.

Green and Black Tea dSPE Cleanup

Black Tea Green Tea

A Extract A Extract B B C C

A = GCB, B = Alternative Carbon Sorbent, C = Chlorophyll Specific Sorbent

46

0

20

40

60

80

100

0-70 70-120 >120 ND

% P

es

tic

ide

s

0-70 70-120 >120 ND

Black Tea 10 ppb Spike

0

20

40

60

80

100

0-70 70-120 >120 ND

% P

es

tic

ide

s

0-70 70-120 >120 ND

Green Tea 10 ppb Spike

Black Tea 100 ppb Spike

Green Tea 100 ppb Spike

% Recovery

%

Reco

very

■ GCB, ■ Alternative carbon sorbent, ■ No carbon, ■ Commercially available, chlorophyll

specific, polymeric sorbent, ■ High GCB, ■ High alternative carbon

Comparison of Recoveries for Planar Pesticides

47

0.0

20.0

40.0

60.0

80.0

100.0

%R

ec

ove

ry

Black Tea Green Tea

■ GCB, ■ Alternative carbon sorbent, ■ Commercially available, chlorophyll specific, polymeric

sorbent, ■ High GCB, ■ High alternative carbon

Today’s Agenda







48

QuEChERS Conclusions and Summary

• QuEChERS is a flexible, simple, yet powerful tool for

preparing a wide variety of food products for analysis by GC

and LC-MS.

• A straightforward approach to method development and

optimization is easy with pre-packaged kits and bulk

sorbents.

• QuEChERS reproducibility and ruggedness can be improved

with products designed for optimal performance.

• Agilent offers the QuEChERS expertise along with the range

of products needed for successful QuEChERS sample

preparation.

49

Questions

50

Acknowledgements

51

Agilent Technologies SPP Team

• Angelica Riemann

• Trisa Robarge

• Mike Chang

• Andy Zhai

• Irina Dioumaeva

Special thanks to Jon Wong

Thank you for your time and attention

52

Addition of Anhydrous Extraction Salts

• The extraction addition step is very crucial to recovery of pesticides

• The addition of the anhydrous extraction salt to the homogenized food sample that has

already been mixed with the extraction solvent ACN produces the greatest recoveries and

smallest relative standard deviation

• The sealed foil package easily facilitates this requirement versus the extraction salts being

in a 50 mL PP tube, where the extraction salt integrity is questionable

• Extraction salts are easily poured directly into the matrix extract, minimizing exothermic

effects or possible errors in adding the matrix to the salts

Exothermic Temperature Tracking Apparatus

Sample and

Buffered

Extraction

Salts

A 50 mL Polypropylene (PP) tube cap was modified to

accommodate a thermocouple

The thermocouple (Omega: HSTC-TT-K-24S-36-SMPW-CC) is

attached to a digital thermometer (FLUKE 52 K/J Thermometer)

recording temperature as a function of time

The produce sample and extract are added to the 50 mL PP

tube according to experiment 1 or 2

The 50 mL PP is vigorously shaken either by hand or by a

mechanical vertical grinder Geno/Grinder®

The temperature is recorded over a period of time: 60 seconds

Averages were taken over several experiments and the results

are shown in graphs 1 and 2

The Geno/Grinder is a registered

trademark of SPEX Sample Prep®

Temperature Graphs from AOAC and EN Extraction

Kits

0

10

20

30

40

50

60

0 10 20 30 40 50 60

Te

mp

era

tu

re

ºC

Time (seconds)

AOAC Add Buffered Salts to Sample

AOAC Sample Added to Buffered Salt

0

10

20

30

40

50

60

0 10 20 30 40 50 60

Te

mp

era

ture

ºC

Time (seconds)

EN Add Buffer Salts to Sample

EN Sample Added to Bufferd Salt

These graphs represent the thermal data acquired from the AOAC and EN

extraction kits. A substantial increase in temperature is observed (increase of

approx 25 ºC for the AOAC method and approx 10 ºC for the EN method) when

the produce sample was added to the extraction salt in the 50 mL PP tube

versus when the extraction salt from an anhydrous pouch was added to the

produce sample.

Pesticides used to Evaluate the Effect of

Temperature on Recovery

The following four pesticides were used to evaluate the thermal effects that

are prevalent when the produce is added to the extraction salts

Fenthion Nicotine* Isofenphos Isocarbophos

The 4 pesticides were chosen based on their labile chemical structures,

nicotine being stable control

Analysis was performed on a Agilent 6410 Triple Quad LC/MS using a

Phenyl- Hexyl column 2.1 x 50 mm, 3.5 µm

Relative Recoveries for the Labile Pesticides

Represents the negative effect adding the produce matrix to the salt has

on recovery during the QuEChERS extraction step, AOAC method

0

10

20

30

40

50

60

70

80

Rec

over

y %

Pre-Spiked Extraction Salts Added

to Matrix AOAC Method

Pre-Spiked Matrix Added to

Extraction Salts AOAC Method

Improvements to the Extraction of Samples

Step 1: Extraction salts with homogenized food sample

• Consistency in shaking: Everyone shakes differently

• Variability in QuEChERS applications, recovery and RSDs

• Bond Elut Ceramic Homogenizers

• Reduces shaking time from 1 minute to <20 seconds!

• Consistent extraction of the sample with the salts

• Breaks up salt agglomerates

• Facilitates homogenization with angle cut

• Increase recovery of pesticides from sample

• 3 different sizes: 50 mL extraction tube and for dispersive tubes (15 and 2 mL)

QuEChERS Extraction: With and Without Ceramic

Homogenizers

CH no CH CH no CH

0

20

40

60

80

100

120

Reco

verie

s

Comparison of QuEChERS Method AOAC and EN With and Without Ceramic Homogenizers

AOAC Method with CeramicHomogenizers

EN Method with CeramicHomogenizers

AOAC Method without CeramicHomogenizers

EN Method without ceramicHomogenizers

Ceramic Homogenizers

• Inert ceramic material

• No loss in pesticide recovery

• Consistent recoveries

•Less individual variation

Pesticides used in study: Acephate, Carbaryl, Carbendazim, Cyprodinil, Imidacloprid, Imazalil, Methamidophos,

Penconazole, Propoxur, Pymetrozine, Thiabendazole, Thiophanate-methyl, Ethoprophos, Kresoxim-methyl ; Apple matrix

0

20

40

60

80

100

120

140

160

180

200

220

240

0 10 20 30 40 50 60 70

Pe

rce

nt

Re

co

ve

ry

Time in Seconds of Shaking Extract with Ceramic Homogenizers

Organophosphates Carbamates Benzimidazoles Anilinopyrimidines

Neonicotinoids Triazoles Strobilurins

Average Recovery with 20 Second Shaking

Extraction and Ceramic Homogenizers

Pre-Packaged Kits or Bulk and In-House

Preparation?

Pre-Packaged Kits

Quality is “free” – built into kits

Reduced labor costs

Increased productivity

UV and moisture protection

maintain quality of product

Consistent results

Less flexibility for method

development

In-House Preparation

Increased labor costs

Hygroscopic materials left open

may reduce effectiveness

Inconsistency in measurement –

variability in results

Reduced productivity

Good for method development,

low sample volumes

62

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