emr – lipid: enhanced matrix removal for fatty samples · emr – lipid: enhanced matrix removal...
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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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