EMR – Lipid: Enhanced Matrix Removal for Fatty Samples · EMR – Lipid: Enhanced Matrix Removal...

EMR – Lipid: Enhanced Matrix Removal for Fatty Samples

Joan Stevens, Ph.D.Senior Applications ScientistAgilent Technologies

Today’s Agenda

Introduction

EMR-Lipid - Enhanced Matrix Removal• Background, workflows, and results

Applications• Pesticides in avocado• Veterinary drugs in beef liver• PAHs in salmon

Summary and Conclusions

2

The challenges and goals of most sample analyses:

Accurate, consistent data

Keep the instruments running

Process as many samples for as little cost as possible

Remove interferences

3

Remove interferences…

• To acquire desired sensitivity/selectivity• To reduce contamination/carryover issues• Use of sensitive and expensive instruments: Protect your

investment!!!

Pesticides in Avocado with SPPesticides in Avocado without SP

4

Lipids

5

Matrices and Approximate Total Lipid Content<2% 4-12% >12%

Beef Liver (4%)

Canola Oil (100%)

Nut Butters (16%)Spinach (0%)Strawberry (0%)

Onion (0%)

Canned Pet Food (~10%)

Avocado (21%)

Chocolate (8%)

Soy Milk (4%)

Pork Liver (4%)

Soy Oil (100%)

Avocado Oil (100%)

Coconut Oil (100%)

Paprika (1%)

Hops (2%)

Cumin (2%)

Salmon (27%)Corn (4%)

Rice (2%)

Wheat (3%)

Catfish (12%)

Sea Bass (3%)

Carp (12%)

Trout (8%)

Tilapia (2%)

Pork (8%)

Plasma or whole blood(10 – 20 %)

Cow’s Milk (5%)

Low Med High

6

Current Procedures for Lipid Removal

Method How Lipids are removed Weakness

Dilute and Shoot No lipid removal, only dilution No lipid removal

Protein Precipitation PPT followed by centrifugation Insufficient lipid removal

PPT followed by filtration with or without sorbent

Insufficient lipid removal; low analyterecovery

QuEChERS PSA/C18 sorbent (dSPE) Not selective; insufficient lipid removal; analyte loss

Zr-containing sorbent Low total lipid capacity; analyte recovery

Freeze sample Time needed; loss of analyte

SPE/SLE Load and elute Time needed; solvent usage; extensive method development

SEC/GPC Chromatographic separation Uses copious amounts of solvent and time; capital expense

7

EMR-Lipid Enhanced Matrix Removal

EMR: As easy to use as QuEChERS; as clean as SPE

8

EMR fits into current sample preparation workflows

EMR-Lipid (p/n 5982-1010)

EMR-Polish (p/n 5982-0101)Extraction Tube

EMR Product offering

9

EMR Sorbent - What is it?

When “activated” by water… - The materials selective

hydrophobic interactions increase.

- Suspension of nano particles (high surface area).

- Rapidly interacts with straight chain, “lipid-like” functional groups.

Centrifugation preferably used to separate precipitate from solution (not filtration).

EMR-Lipid Mechanism – Size exclusion and hydrophobic interaction.

10

1.0 g EMR in 15 mL tube

… and what does it do?

EMR sorbent removes LipidsWhat are Lipids?

A class of naturally occurring hydrocarbon containing compounds commonly known as fats and oils

Free Fatty Acids

Triglycerides

Cholesterol Phospholipids

11

What Does EMR NOT Interact With?

EMR does NOT remove analytes of interestExceptions?

Compounds containing long aliphatic functional groups (e.g. prostaglandins)

Fluoroquinolones Tetracyclines

Vitamin DPAHsOrganochlorine Pesticides

Imidazole pesticides

12

EMR Protocols for Applications

13

EMR Fits into Existing Workflows

QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe)• Easy-to-use sample preparation for food testing, solid

samples (e.g. vegetables, fruits, meat, seafood, etc.)EMR Applications: - Pesticide Residues in Avocado,

Modified Liquid Extraction (Protein Precipitation)• Proteins are removed by a “crash” step prior to injection

or cleanup (e.g. milk, meat, seafood, etc.)EMR Applications: - PAHs in Salmon,

- Veterinary Drugs in Bovine Liver

14

Pros• Fast and inexpensive• Takes minimal experience• Doesn’t require special equipment• Accommodates multiple matrices• Accommodates large analyte groupsCons• Large amount of coextractives

Improving dSPE in QuEChERS1. Extraction 2. Dispersive SPE

Pros• Same as extractionCons• Minimal cleanup provided• Can remove analytes• Lipids are challenging to remove

selectively

15

Traditional QuEChERS versus QuEChERS EMR-Lipid

Transfer to A/S Vial Transfer to A/S Vial

Standard QuEChERS EMR QuEChERSExtraction/Partition (6 steps) Extraction/Partition (6 steps)

↓ ↓Mix and Centrifuge Mix and Centrifuge

↓ ↓Transfer Sample to dSPE

(sorbents)Transfer Sample to dSPE

(sorbents & H20)↓ ↓

Mix and Centrifuge Mix and Centrifuge↓ ↓

Evaporate and reconstitute or dilute Polish Tube↓ ↓

Filter out precipitate Evaporate and reconstitute or dilute

16

Improving Liquid Extraction Workflows

2. Add food sample.1. Add crash solvent.(ACN)

SaltProteinLipidAnalyte

3. Mix to precipitate proteins.

4a. EMR dSPE and Polish. 4b. SPE and/or GPC.

Transfer for analysis

17

Centrifuge

Traditional versus EMR-Lipid Liquid Extraction

Transfer to A/S Vial Transfer to A/S Vial

Liquid Extraction with SPE/GPC EMR Liquid ExtractionAdd Sample and Acetonitrile Add Sample and Acetonitrile

↓ ↓Mix and Centrifuge Mix and Centrifuge

↓ ↓

Sample Pretreatment Transfer Sample and H2O to EMR dSPE

↓ ↓SPE and/or GPC Mix and Centrifuge

↓ ↓Evaporate and reconstitute or dilute Polish Tube

↓ ↓Filter out precipitate Evaporate and reconstitute or dilute

18

EMR – Lipid – dSPE Cleanup

A B C D

E

A. QuEChERS or Liquid ExtractB. Add H2O to EMR tube (“activation”)C. Transfer extractD. Vortex and centrifugeE. Supernatant (1:1; extract: H2O)

19

EMR – Polish – ACN/H2O Phase Separation

A B C D

E

A. Add supernatant to EMR – Polish tubeB. Vortex immediatelyC. Phase separation after centrifugeD. Transfer upper layer for analysisE. Final samples split for GC and LC analysis

20

Tips for Success: EMR-Lipid Protocols

Extraction Solvent• *Acetonitrile – preferred for broad extraction and water miscibility.

• Acetone – also amenable to EMR workflow.

• Do not use…- Water immiscible solvents (e.g. hexane, ethyl acetate, DCM) – no

interaction without initial miscibility.

- Alcohols – can dissolve salts, EMR, and give poor partitioning.

10/20/2015

Agilent Confidential

21

Tips for Success: EMR-Lipid Protocols

dSPE “Activation”• Addition of water for dSPE

- Increases hydrophobic interaction between EMR-Lipid and matrix components.

• “Activation Strength”- 1 to 1; extract/water (recommended) – Excellent matrix removal

and analyte recovery.

- 1 to >1; extract/water – marginal increase in matrix removal, lower hydrophobic analyte recovery.

- 1 to <1; extract/water – decrease matrix removal, improve hydrophobic analyte recovery.

10/20/2015

Agilent Confidential

22

Tips for Success: EMR-Lipid Protocols

Polishing Step (Partition)• Contains 2.0 g NaCl/MgSO4 (1:4; same as QuEChERS)• Applied AFTER EMR-Lipid dSPE for phase separation

Purpose• Remove unwanted water from “activation”• Removes dissolved solids• Improves recoveries for non-polar compounds

Additional Information• Use the Polish salts after EMR-Lipid• Mix immediately – avoid clumping• Customer may chose bulk salts

Separate Product but HIGHLY RECOMMENDED for optimal results

10/20/2015

Agilent Confidential

23

Tips for Success with EMR-Lipid

Recommend Centrifugation NOT Filtration • Filter membranes put EMR Samples at Risk for Analyte

Retention• Analytes with properties that risk retention on hydrophobic membranes• Less matrix leaves analytes “susceptible” for retention• If filtration must be used, choose a hydrophilic membrane such as

regenerated cellulose.

Recommend Immediate Mixing• Avoid Clumping; Immediately vortex extract/water in the EMR dSPE

tube and EMR Polish tube.

10/20/2015

Agilent Confidential

24

GC-MS Fullscan Avocado

Avocado – Untreated

25

GC-MS Fullscan Avocado

Avocado – UntreatedAvocado – EMR Treated

26

GC-MS Fullscan Pet Food

Pet Food – Untreated

27

GC-MS Fullscan Pet Food

Pet Food – UntreatedPet Food – EMR Treated

28

GC-MS Full Scan– Avocado Oil

Avocado Oil Crude ExtractEMR – Lipid Extract

29

GC-MS Spinach Fullscan

30

Spinach matrix blank w/ EMR cleanup

Spinach matrix blank w/o cleanup

Spinach matrix blank w/ Q-Pigment dSPE cleanup

Spinach matrix blank w/ Q-Universal dSPE cleanup

5.00 10.00 15.00 20.00 25.00 30.00 35.000

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

Time-->

Abundance

GC-MS Red Pepper Fullscan

31

5.00 10.00 15.00 20.00 25.00 30.00 35.000

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

Red Pepper matrix blank w/ EMR cleanup

Red Pepper matrix blank w/o cleanup

Red Pepper matrix blank w/ Q-General dSPE cleanup

Red Pepper matrix blank w/ Q-Universal dSPE cleanup

Suggested Workflow for Multi-Residue Analysis

• Sample Preparation: EMR-Lipid with QuEChERS/Modified Liquid Extraction workflow

• System: Agilent LC or GC MS/MS (6400, 6500, 5977, 7000, 7010, 7200 series)

• Columns: - LC: Agilent Poroshell 120 EC-C18, 2.7µm- LC: ZORBAX RRHD Eclipse Plus C18, 1.8 µm- GC: Agilent J&W DB-5ms Ultra Inert

• Agilent MassHunter Software

• Experimental setup: Matrix-matched Calibration

October 20, 2015

Confidentiality Label

32

Matrix Matched Calibration

What is matrix matched calibration?• A sample blank is added to final calibration standards to correct any ion

enhancement/suppression due to co-extracted matrix.

- Example: 950 µL blank extract + 25 µL working standard + 25 µL internal standard = 1000 µL matrix matched calibration standard

Matrix Matched Calibration and EMR-Lipid• We HIGHLY RECOMMEND the use of matrix matched calibration

standards for complex food samples.

- Small amounts of matrix can cause poor results.

10/20/2015

Agilent Confidential

33

Benefits to Instrumental Flowpath

34

Comparison of Analytes Response Consistency over Multiple Avocado Sample Injections

TPP

Commonly used IS

RT: 18.3 min

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

TPP

Peak

Are

a

Injections of Avocado Samples on GC-QQQ

TPP Peak Area over 100 Injections

EMR cleanup

Z-Sep+ cleanup

C18 dSPE cleanup

Linear (EMR cleanup)

Linear (Z-Sep+ cleanup)

Linear (C18 dSPE cleanup)

Response drops ~ 50% after 100 avocado sample injections

Response increases ~ 35% after 100 avocado sample injections

Response drops ~ 4% after 100 avocado sample injections

35

Analytes Responses Reproducibility on GC/MS/MS over 100 Injections of Avocado Samples

36

MS Source Critical Tuning Parameters

0

200

400

600

800

1000

1200

1400

1600

0

10

20

30

40

50

60

70

80

90

100

110

120

130

0 500 1000 1500 2000 2500

EM V

olat

age

Rela

tive

Abun

danc

e fo

r Tun

ing

Ion

(%)

Injections of Avocado Samples with EMR cleanup on GC/MS

MS (GC/MS) Performance Monitoring over Multiple Avocado Sample Injections

Rel Abundance for Ion 219, Minimum of 70 needed

Rel Abundance for Ion 502, minimum of 5 needed

EM voltage, higher than 1400 indicated needs to replace

37

Pesticides in Avocado

38

72 Pesticides Analysis in Avocado by LC and GC-QQQ

10/20/2015

Agilent Confidential

39

Accurately weigh 15 g comminuted Avocado sample in 50 mL centrifuge tube

Centrifuge @ 5000 rpm for 5 min

Transfer 5 mL of upper ACN extract and 5 mL of water to EMR dSPE 15 mL tubes

Add 15 mL of 1% Acetic Acid in Acetonitrile, and AOAC QuEChERS extraction kit

Cap and shake vigorously on mechanical shaker for 2 min

Vortex and Centrifuge

QuEChERS-EMR Protocol for Multi-residue Analysis of Pesticides in Avocado

Combine 200 µL of ACN extract and 800 µL water, vortex, and centrifuge if needed

Transfer 5 mL supernatant to EMR polish tube

Vortex, centrifuge, and transfer the upper ACN layer to another vial

Samples are ready for GC-QQQ analysis Samples are ready for LC-QQQ analysis

40

CleanupAmount of co-

extractives(mg, n= 3)

Amount of co-extractives removed by

cleanup (mg, n = 3)

% of matrix co-extractives removed by

cleanup

No further cleanup 14.7 --

C18/PSA Cleanup 9.5 5.2 35.4

EMR-Lipid Cleanup 4.2 10.5 71.4

Zirconia sorbent Cleanup 7.0 7.7 52.4

% Matrix Co-extractives Removed by Cleanup = 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝑜𝑜 𝐶𝐶𝐴𝐴−𝑒𝑒𝑒𝑒𝐴𝐴𝑒𝑒𝑒𝑒𝑒𝑒𝐴𝐴𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑅𝑅𝑒𝑒𝐴𝐴𝐴𝐴𝑒𝑒𝑒𝑒𝑅𝑅 𝑒𝑒𝑜𝑜𝐴𝐴𝑒𝑒𝑒𝑒 𝐶𝐶𝐶𝐶𝑒𝑒𝑒𝑒𝐴𝐴𝐴𝐴𝐶𝐶𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝑜𝑜 𝐶𝐶𝐴𝐴−𝑒𝑒𝑒𝑒𝐴𝐴𝑒𝑒𝑒𝑒𝑒𝑒𝐴𝐴𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑒𝑒𝐴𝐴𝑤𝐴𝐴𝐴𝐴𝐴𝐴 𝐶𝐶𝐶𝐶𝑒𝑒𝑒𝑒𝐴𝐴𝐴𝐴𝐶𝐶

× 100%

The use of EMR material cleanup removes extra 20-30% of Avocado co-extractives in comparison to traditional QuEChERS and/or competitor’s cleanup

Comparison of Avocado Co-extractives by Weight

41

Comparison of GC/MS Full-scan Chromatogram for Matrix Background

The use of EMR material cleanup provides significantly cleanup chromatographic sample background.

42

Comparison of GC/MS/MS MRM Chromatogram for Matrix Background

43

6x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

Counts vs. Acquisition Time (min)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

6x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Matrix blank w/ C18/PSA cleanup

Matrix blank w/ zirconia sorbent cleanup

Matrix blank w/ EMR cleanup

Chromatographic Benefits of Matrix Removal Provided by EMR Cleanup

ExampleEPN in Avocado on LC-QQQ

+ MRM (151.0 -> 79.1) AV MBPI-a-CD C2 spike 50ppb-1.D

Acquisition Time (m

12.7 12.8 12.9 13 13.1 13.2 13.3 13.4 13.5 13.6

Coun

ts 4x10

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

13.163 min.

+ MRM (151.0 -> 79.1) AV Z-Sep+ C5 spike 50ppb-1.D

Acquisition Time (m

12.7 12.8 12.9 13 13.1 13.2 13.3 13.4 13.5 13.6

Coun

ts 5x10

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

13.163 min.

+ MRM (183.1 -> 168.1) AV MBPI-a-CD C2 spike 50ppb-1.D

Acquisition Time (m

20.4 20.5 20.6 20.7 20.8 20.9 21 21.1 21.2

Coun

ts 4x10

-0.25

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

3.25

3.5

3.75

4

4.25

4.5

4.75

520.788 min.

+ MRM (183.1 -> 168.1) AV Fatty dSPE spike 50ppb-1R.D

Acquisition Time (m

20.4 20.5 20.6 20.7 20.8 20.9 21 21.1 21.2

Coun

ts 4x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

20.833 min.

+ MRM (183.1 -> 168.1) AV Z-Sep+ C5 spike 50ppb-1.D

Acquisition Time (m

20.4 20.5 20.6 20.7 20.8 20.9 21 21.1 21.2

Coun

ts 4x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

20.819 min.

Captan in Avocado on GC-QQQ

Permethrin in Avocado on GC-QQQ

EMR-Lipidcleanup

Zirconia sorbent cleanup

C18/PSAcleanup

BenefitReduced matrix suppression Improved S/N ratio Less interferences for

accurate integration

44

Selected Problematic Pesticides for Recovery Comparison

45

■ Zirconia cleanup

0

20

40

60

80

100

120

Methamidophos Thiophanatemethyl

Bentazone 2,4-D Acid Dichlorprop Penconazole TPP EPN Captafol

Anal

ytes

Rec

over

y %

(n=6

) in

50pp

b pe

stic

ides

fort

ified

Avo

cado

sam

ples

Selected Pesticides Recovery Comparison from Multi-residue Pesticides Analysis in Avocado by LC/QQQ or GC/QQQ

EMR cleanup C18/PSA cleanup Zirconia sorbent cleanup

Statistical Recovery Results Comparison

Page 46

Method Accuracy and Precision

47

Veterinary Drugs in Bovine Liver

48

Representative Vet Drugs for Evaluation

Representative Vet Drug

Drug ClassRepresentative Vet

DrugDrug Class

Representative Vet Drug

Drug Class

Amoxicillin Pednisone Corticosteroid Tylosin MacrolideDifloxacin Oxyphenylbutazone NSAID Oxytetracline

Florfenicol 2-Thiouracil Thyreostat DoxycyclineChloramphenicol Metronidazole-OH Nitroimidazole ChlortetracyclineSulfamethizole Fenbendazole Acepromazine

Sulfamethoxypridazine

LavamisoleChlorpromazine

Lincomycin Lincosamide Morantel KetoprofenCiprofloxacin Bithionol Cefazolin CephalosporinNorfloxacin Clorsulon Melengesterol other

Danofloxacin Niclosamide Ractopamine β-AgonistFlukicide

β-Lactam

Phenicol

Sulfonamide

Fluoroquinolone

Tetracycline

TranquilizerAnthelmintic

49

EMR-Lipid Protocol for Multi-residue Analysis of Vet Drugs in Bovine Liver

Accurately weigh 2 g comminuted Bovine Liver sample in 50 mL centrifuge tube

Centrifuge @ 5000rpm for 5 min

Transfer 5 mL of upper ACN extract and 5 mL of buffer* to EMR dSPE 15 mL tubes

Add 10 mL of 5% Formic Acid in ACN

Cap and shake vigorously on mechanical shaker for 2 min

Vortex and Centrifuge

Combine 200 µL of upper ACN final extract and 800 µL buffer*, vortex

Transfer 5 mL supernatant to EMR polish tube

Vortex and centrifuge, and for matrix blank samples transfer upper ACN layer to another tube

Samples are ready for LC-QQQ analysis

50

* 5 mM Ammonium Acetate, pH 5.0

CleanupAmount of co-

extractives(mg, n= 3)

% of matrix co-extractives removed by

cleanup

No further cleanup 12.1 --

EMR dSPE 5.3 56.2

Zirconia dSPE 6.0 50.4

C18 dSPE 7.8 35.5

% Matrix Co-extractives Removed by Cleanup = 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝑜𝑜 𝐶𝐶𝐴𝐴−𝑒𝑒𝑒𝑒𝐴𝐴𝑒𝑒𝑒𝑒𝑒𝑒𝐴𝐴𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑅𝑅𝑒𝑒𝐴𝐴𝐴𝐴𝑒𝑒𝑒𝑒𝑅𝑅 𝑒𝑒𝑜𝑜𝐴𝐴𝑒𝑒𝑒𝑒 𝐶𝐶𝐶𝐶𝑒𝑒𝑒𝑒𝐴𝐴𝐴𝐴𝐶𝐶𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝑜𝑜 𝐶𝐶𝐴𝐴−𝑒𝑒𝑒𝑒𝐴𝐴𝑒𝑒𝑒𝑒𝑒𝑒𝐴𝐴𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑒𝑒𝐴𝐴𝑤𝐴𝐴𝐴𝐴𝐴𝐴 𝐶𝐶𝐶𝐶𝑒𝑒𝑒𝑒𝐴𝐴𝐴𝐴𝐶𝐶

× 100%

The use of EMR material cleanup removes extra 20% of liver co-extractives in comparison to tradition QuEChERS cleanup.

Bovine Liver Matrix Co-Extractives Comparison by Weight

51

GC-MS Fullscan Beef Liver

Beef Liver – Untreated

52

GC-MS Fullscan Beef Liver

Beef Liver – UntreatedBeef Liver – EMR Treated

53

Selected Problematic Vet Drugs for Recovery Comparison

54

0

20

40

60

80

100

120

2-Thiouracil Lincomycin Norfloxacin Ciprofloxacin Cefazolin Chlortetracycline Doxycycline

Aver

age

Reco

very

% (n

=6) i

n 50

ppb

Vet D

rugs

spik

ed B

ovin

e Li

ver S

ampl

es

Selected Vet Drugs Recovery Comparison from Multi-residue Vet Drugs in Bovine Liver by LC-QQQ

EMR-Lipid QuEChERS w/ C18 dSPE QuEChERS w/ Zirconia sorbent

Statistical Recovery Comparison for Vet Drugs in Bovine Liver

55

0

5

10

15

20

25

30

EMR-Lipid QuEChERS w/ C18 QuEChERS w/ zirconia

Num

ber

of V

et D

rugs

test

ed in

Bov

ine

Live

r Statistical Recovery Results for Multi-residue Vet Drugs in Bovine Liver by LC-QQQ

REC <70%

REC in 70-120%

REC > 120

Method Accuracy and Precision

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

110.0

120.0

130.0

140.0

Ove

rall

Anal

ytes

Acc

urac

y of

Low

, Mid

and

Hig

h Q

Cs (n

= 2

4)Analysis of Multi-residue Vet Drugs in Bovine Liver -- Overall Average Accuracy

Error bar = 95% CI

*Results include combined multi-vet drug and tetracycline protocols

56

PAHs in Salmon

57

Target AnalytesGC-MS (SIM)

Compound RT Target IonNaphthalene 3.89 128.0Acenaphthalene 5.37 152.0Fluorene 6.05 166.0Phenanthrene 7.25 178.0Anthracene 7.34 178.0Pyrene 10.31 202.0Benz[a]anthracene 13.83 228.0Chrysene 13.93 228.0Benzo[b]fluoranthene 16.99 252.0Benzo[k]fluoranthene 17.08 252.0Benzo[a]pyrene 17.85 252.0Perylene 18.09 252.0indo[1,2,3-cd]pyrene 20.72 276.0Dibenz[a,h]anthracene 20.87 278.0Benzo[g,h,i]pyrene 21.29 276.0

Naphthalene-d8 3.87 136.0Acenaphthalene-d10 5.52 162.0Phenanthrene-d10 7.22 188.0Chrysene-d12 13.86 240.0Perylene-d12 18.03 264.0

Color coded according to internal standard being used

58

Accurately weigh 5 g comminuted salmon sample in 50 mL centrifuge tube

Centrifuge @ 5000 rpm for 5 min

Transfer 8 mL of ACN extract to EMR dSPE 15 mL tubes

Add 10 mL of ACN

Cap and shake vigorously on mechanical shaker for 2 min

Vortex and Centrifuge

EMR Modified Protocol for Analysis of PAHs in Salmon

Transfer 8 mL supernatant to EMR polish tube

Vortex and centrifuge, transfer upper ACN layer to another vial

Samples are ready for GC-MSD analysis

59

EMR – New Procedure Results

0

20

40

60

80

100

120

140 Accuracy PAH in Salmon

0

20

40

60

80

100

120 Recovery PAH in Salmon

00.5

11.5

22.5

33.5

44.5

5 %RSD PAH in Salmon

S1 25 ppb spike EMR S2 100 ppb spike EMR S3 500 ppb spike EMR

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■ 25 ng/mL ■ 100 ng/mL ■ 500 ng/mL

Summary and Conclusion• EMR-Lipid provides the most complete lipid removal of any sorbent on

the market.

• Achieve SPE cleanliness with dSPE simplicity.• EMR is a one size fits all sorbent for a variety of sample types and analytes.• EMR fits easily into existing workflows including QuEChERS and modified

liquid extraction.

• Key applications were validated with EMR and demonstrate better recovery, better precision, and decreased matrix impact to the instrument and results.

• Less re-runs, Less time manual data integration, Less time/cost unexpected instrument maintenance (matrix)

• More efficient and productive laboratory = reducing cost/sample!

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