1. No category

Revisiting the Advantages
of the QuEChERS Approach
to Sample Preparation
Steven J. Lehotay
Outline of the Presentation
• Introduction to QuEChERS
• QuEChERS for pesticides in foods
• Other QuEChERS applications and options
• Conclusions
What is QuEChERS?
www.quechers.com
http://en.wikipedia.org/wiki/Quechers
The Quechers method is a streamlined
approach that makes it easier and less
expensive for analytical chemists to examine
pesticide residues in food. The name is a
portmanteau word formed from "Quick, Easy,
Cheap, Effective, Rugged, and Safe."
Multiclass, Multiresidue
Analytical Approach:
 extraction
 clean-up
 quantification
 identification
Quick
Easy
QuEChERS method Cheap
Effective
Rugged
Safe
GC-MS(/MS)
LC-MS/MS
Slide Devised by Katerina Mastovska
Sample Size and Comminution
• Numerous studies have shown that the sample
processing step is crucial (IUPAC, FAO/IAEA, CSL, FDA,
USDA, EU studies, check samples).
Conclusions from the Studies:
• Use proven choppers of appropriate size
• Chop frozen samples (dry ice or liquid N2)
• 10-20 g subsample gives representative portion
Minimizing Sample Size
Minimizes Effort and Expense
Minimizing Sample Size
Minimizes Effort and Expense
Freeze Sample (-20°C),
Add dry ice, Homogenize
to produce a flowable
powder (-25 to -30°C)
Improved homogeneity
Slide adapted from Richard Fussell, FERA (UK)
QuEChERS Approach
Tomato
Grape
Spinach Strawberry
1) Shake sample with
solvent and salts
2) Centrifuge for 1 min
Spinach
Tomato
Grape
Strawberry
4) Centrifuge for 1 min
5) Analyze Pesticides
3) Mix a
portion
with a
sorbent
The Birth of QuEChERS
QuEChERS was born of two fathers at the USDAARS-ERRC, after 40 years of labor in the residue
community, 8 years of labor of the fathers apart,
and 2 years of their labor together.
Birth announcement:
4th European Pesticide Residue Workshop
Rome, Italy May 28-31, 2002
A rose by any other name would
smell as sweet?
QuEChERS without Rugged is:
Fast
Easy
Cheap
Effective
Safe
(FECES)
Curtain plate after injection of 25 samples with
extracts from raisins without cleanup
Picture from Lutz Alder, BfR, Germany
Traditional
QuEChERS
Blending
Shaking
Filtration
Centrifugation
Large solvent volumes
Small solvent volumes
Multiple partitioning steps
Single partitioning
Transfers of entire extract
Take aliquots (use I.S.)
Lot of glassware
Single vessel
Evaporation/Concentration
Large volume injection
Classical SPE
Dispersive SPE
QuEChERS Features and Impact
• A single extract can be prepared in 10 min or a batch of 20 in an
hr by a single analyst with ≈$1-3 of disposable materials per
sample and generate <12 mL nonchlorinated solvent waste.
• Consistently high recoveries (mostly 90-110% with RSDs < 10%)
of a wide range of GC- and LC-amenable pesticides are achieved
from many food matrices.
• Countless labs have implemented QuEChERS successfully for up
to 500 pesticides in food and increased efficiency (faster, less
labor, lower cost, less waste, saves space, less labware, higher
throughput).
• QuEChERS concepts have spread to other applications.
Recoveries in the QuEChERS method
229 pesticides analyzed by GC/MS and LC/MS-MS
90%
Percentage of Pesticides
80%
70%
60%
50%
40%
10 ng/g Lettuce
10 ng/g Orange
25 ng/g Orange
50 ng/g Lettuce
100 ng/g Lettuce
100 ng/g Orange
30%
20%
10%
0%
<20
20-49
50-69
70-79
80-89
%Recovery
90-110
111-120
>120
Repeatabilities in the QuEChERS method
229 pesticides analyzed by GC/MS and LC/MS-MS
80%
Percentage of Pesticides
70%
10 ng/g Lettuce
10 ng/g Orange
25 ng/g Orange
50 ng/g Lettuce
100 ng/g Lettuce
100 ng/g Orange
60%
50%
40%
30%
20%
10%
0%
>0-5
>5-10
>10-15
>15-20
%RSD
>20-30
>30
A QuEChERS Advantage
QuEChERS
“Waste”
Mini-Luke
Waste
Same Number of Extractions and 15 g Sample Size
(Nonhalogenated)
(Halogenated)
QuEChERS at General Mills
vs. Traditional Approach
Reduced cost per sample
About 40-50% reduction in sample prep and operational costs
($72 savings per sample analyzed)
Reduced and less hazardous waste
1:100 reduction; no chlorinated solvents
Increased sample throughput
About 3-fold increase
Increased scope of pesticides
- Successfully validated >180 pesticides
Slide Devised by
Katerina Mastovska
Number of results reported in EU PT samples by method
1200
1000
800
2006:
2007:
2008:
2009:
2010:
2011:
2012:
129 labs x 16 pesticides = 2064 total
137 x 19 = 2603
132 x 18 = 2376
151 x 21 = 3171
153 x 18 = 2754
154 x 19 = 2926
167 x 18 = 3006
QuEChERS
600
EtOAc
400
200
Mini-Luke
Specht
0
2006
MeOH
MeCl2
2007
2008
2009
2010
2011
2012
Year
Slide adapted from Paula Medina Pastor
QuEChERS in the Literature
225
200
Citations to 1st Paper
175
QuEChERS & d-SPE Pubs
669 Total
659 Total
*
Number
150
125
100
According to
ISI Web of KnowledgeSM
75
50
25
0
2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Year
*search conducted on Jan. 17, 2013
QuEChERS/d-SPE Applications
Application
Publications
Pesticides
≈ 425
Veterinary drugs
≈ 50
Environmental (e.g. PAHs)
≈ 35
Mycotoxins
≈ 15
Natural Products (e.g. alkaloids)
≈ 15
Reviews
≈ 15
Other
≈ 15
(e.g. BPA, acrylamide, PFOS/PFOA, nerve agents, phthalates,
dyes/ink, sildenafil, melamine, seafood toxins, Cu)
Matrices
Fruits/Vegetables, Grains, Processed Foods (e.g. baby food), Honey, Nuts,
Animal Tissues (meat, liver, kidney), Fish/Seafood, Milk, (Vegetable) Oils,
Soil/Sediment, Dried Fruits, Feeds, Eggs, Tea, Juices/Beverages, Plants,
Wine/Beer, Mushrooms, Blood/Serum, Water, Cactus, Compost, Roots,
Tobacco, Dietary Supplements, Chinese Medicines
Vendors of QuEChERS Products
b e k o lu t
QuEChERS Update
Steve and Angelo “Interviewed” by Ron Majors
QuEChERS a Sample Preparation
Technique that is “Catching On”:
An Up-to-Date Interview with the
Inventors, LC GC North America,
July, 2010
The QuEChERS Revolution,
LC GC Europe, Sept., 2010
Available from ChromatographyOnline.com
What’s New with QuEChERS?
•
•
•
•
•
•
•
•
•
More vendors and formats (e.g. DPX)
GCB or ChloroFiltr for chlorophyll reduction
Type of MgSO4: can use 97% purity
Shakers – e.g. Spex and Glas-Col
Automation with robotic autosampler
“Unified” method to undergo AOAC update
d-SPE has a life of its own
More applications (e.g. environmental, blood)
Veterinary drug residue methods
Limitations of QuEChERS?
•
•
•
•
•
•
•
•
Too many modified versions
Cereals require a separate protocol
Still problems with captan, folpet, captafol
Spices, tea, and oils give problems
Works best with modern MS systems
Need PTV or solvent exchange for low LOD
Matrix effects in complicated matrices
Even simpler sample prep possible
QuEChERS Methods
2003
Anastassiades et al.
2005
Lehotay et al.
2007
Anastassiades et al.
Original
AOAC 2007.01
CEN 15662
10-15 g sub sample
10-15 g sub sample
10-15 g sub sample



10-15 mL
1% HOAc in MeCN
10-15 mL MeCN
 shake
 shake
 centrifuge
shake & centrifuge
0.4 g/mL anh.MgSO4
0.1 g/mL NaCl
0.1g/mL Na3Cit2H2O
0.05 g/mL Na2Cit1.5H2O
0.4 g/mL anh.MgSO4
0.1 g/mL NaOAc
 shake

 shake
 shake
0.4 g/mL anh.MgSO4
0.1 g/mL NaCl
150 mg/mL anh.MgSO4
25 mg/mL PSA
10-15 mL MeCN
Option:
+ 50 mg
C18 &
7.5 mg
GCB
 centrifuge
150 mg/mL anh.MgSO4
50 mg/mL PSA

 shake
Option:
+ 50 mg
C18 &
2.5-7.5
mg GCB
shake & centrifuge
 centrifuge
150 mg/mL anh.MgSO4
25 mg/mL PSA

shake & centrifuge
Option: Scale-Up & Conc. in Toluene
Slide Devised by Urairat Koesukwiwat
Comparison of QuEChERS Methods
Dimethoate
120%
Recovery
100%
80%
60%
Original QuEChERS
40%
CEN (Citrate buffer)
20%
0%
Apple/Berry
Peas
Limes
AOAC (Acetate buffer)
No significant difference in recoveries for the
majority of pesticides.
Comparison of QuEChERS Methods
Tolylfluanid
F
120%
Cl
S
100%
Cl
O
N
Recovery
S
N
80%
O
60%
Original QuEChERS
40%
CEN (Citrate buffer)
20%
0%
Apple/Berry
Peas
Limes
AOAC (Acetate buffer)
Base-sensitive pesticides remain problematic
in matrices with a higher pH.
Comparison of QuEChERS Methods
Pymetrozine
120%
N
Recovery
100%
80%
60%
N
NH
N
O
N
Original QuEChERS
40%
20%
0%
CEN (Citrate buffer)
AOAC (Acetate buffer)
Apple/Berry Peas
Limes
For basic pesticides in low-pH matrices,
the AOAC method with acetate buffer gives higher
and more consistent recoveries.
Unified QuEChERS Method
per 1 g sample, 1 mL of MeCN w/ 1% HOAc
for fruits and vegetables (10-15 g)
add internal standard
per g sample, add 0.4 g anh. MgSO4
+ 0.1 g anh. NaOAc
shake or blend
centrifuge
per mL of the upper layer:
150 mg MgSO4 + 50 mg PSA
+ 50 mg C18 + 7.5 mg GCB
mix and centrifuge
QuEChERS for Grains, Nuts, Doughs
2.5 - 5 g sample + 10 mL water*
+ 10 mL MeCN + internal standards
shake for 1 h
add 4 g MgSO4 + 1 g NaCl
shake vigorously for 1 min
centrifuge for 1 min
*15 mL water for 5 g of rice
1 mL of the upper layer
+ 150 mg PSA + 50 mg C18 + 150 mg MgSO4
mix for 30 s
centrifuge for 1 min
Slide adapted from Kate Mastovska
Buffered vs. Non-Buffered Approach
9.00E+04
GC-TOF MS
Endrin
8.00E+04
7.00E+04
Dieldrin
6.00E+04
5.00E+04
4.00E+04
3.00E+04
Slide by Kate
Mastovska
2.00E+04
1.00E+04
0.00E+00
1020
1040
1060
1080
1100
Time (s)
1120
1140
1160
Is a Freeze-Out Step Worthwhile?
Effect of Freezing Out Lipids from QuEChERS Flaxseed
Extracts
Co-Extractives by Weight
2.5%
Before d-SPE
After d-SPE
2.0%
1.5%
1.0%
0.5%
0.0%
22
4
-10
-20
"Freeze-Out" Temperature (deg C)
-85 (-45)
Pesticide Recoveries vs. Fat Content
120%
Pesticide Recovery
100%
80%
60%
chlordane
40%
20%
DDE
HCB
lettuce/orange
avocado
0%
1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06
Pesticide Solubility in Water (mg/L)
Pesticide Recoveries vs. Fat Content
120%
Recovery
100%
penconazole
chlorpyrifos-methyl
lindane
heptachlor epoxide
permethrin
chlordane
DDE
hexachlorobenzene
80%
60%
40%
20%
orange
lettuce
milk
0%
0%
eggs
5%
10%
Fat content
avocado
15%
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Recovery
6 Data Sets from 3 Types of Flaxseeds
QuEChERS of Milled Flaxseeds
100%
Why not correct for recoveries?
80%
60%
40%
20%
0%
n = 26
“Just-Enough” Selectivity in Extraction
Volatility
dichlorvos
methiocarb sulfone
deltamethrin
pKa
Polarity
pymetrozine
Objective: consistent recoveries at low LOQ
Factors in Extraction
Sample Type
Extraction Solvent(s)
Sample:Solvent Ratio
Type(s) of Salt Added
pH
Temperature
Comminution
Water Content
Extraction Method
Amt. of Salt(s) Added
Time of Extraction
Pressure
All Adjustable Parameters were Isolated and
Measured using Quantifiable Factors
(Recoveries, NMR, Gravimetry, GC/MS, Color)
Extraction/Cleanup for
Acrylamide in Foods
FEP tube
discard ...... removal of fat
hexane layer
MeCN layer
matrix
take 1 mL aliquot for
dispersive SPE clean-up
water layer
excessive salts
Slide by
Katerina Mastovska
Effect of NaCl Addition
NaCl modifies extraction selectivity
(amount of polar co-extractives):
LC-MS/MS analysis (m/z 72  55) of potato chips extract
no cleanup
dispersive PSA cleanup
Intensity (cps)
Intensity (cps)
6.0e5
Acrylamide
7.0e4
Interference
6.0e4
5.0e5
Amount of NaCl:
0g
0.5 g
1g
Acrylamide
1.5 g
2g
4.0e5
3.0e5
2.0e5
1.0e5
0.0
5.0e4
4.0e4
Interference
3.0e4
2.0e4
1.0e4
0.0
4.6
5.0
5.4
5.8
6.2
6.6
7.0
7.4
7.8
4.6
5.0
Time (min)
5.4
5.8
6.2
6.6
Time (min)
Slide by Katerina Mastovska
7.0
7.4
7.8
Optimized Method for Acrylamide Analysis
1 g sample in a 50 mL FEP tube
add d3-acrylamide at 500 ng/g
e
ra
t
x
c
n
o
ti
add 5 mL hexane, vortex
add 10 mL water + 10 mL MeCN
+ 4 g MgSO4 + 0.5 g NaCl
shake vigorously for 1 min
centrifuge for 5 min at 3450 rcf
discard the hexane layer
e
cl
a
p
u
n-
1 mL of the upper layer
+ 50 mg PSA + 150 mg MgSO4
mix for 30 s
centrifuge for 1 min at 3450 rcf
Slide adapted from Katerina Mastovska
Cleanup
Solid-Phase Extraction (SPE) is a common
efficient and effective approach to cleanup
of extracts in residue analysis.
Slide Devised by
Katerina Mastovska
Dispersive-SPE
• Why use an SPE apparatus for “chemical filtration?”
• Dispersive-SPE involves the mixing of the sorbent
with the extract in a mini-centrifuge tube to
retain matrix interferants, but not analytes.
Pictures by Michelangelo Anastassiades
Advantages of Dispersive-SPE
• No SPE Apparatus, No SPE Cartridges, No
Vacuum, No pretreatment, No Channeling,
No Drying Out, No Collection Tube, No Flow
Control, No Elution Solvent, No Dilution of
Extract, No Solvent Evaporation, No
Knowledge or Skill Needed
• Less Sorbent, Less Time, and Less Cost
• Fast, Easy Procedure (no need for automation)
Disadvantages: less cleanup than SPE;
half of extract is lost;
“chemical filtration” applications only
Comparison of Sorbents in Dispersive-SPE
Mixture of Apple, Orange, Grape, Strawberry,
Spinach, Carrot, Lettuce, and Tomato
Dispersive-SPE clean-up:
Mixed
Extract
None C18 Nexus NH2 SAX PSA AL-N GCB
Evaporated Mixed Extracts
Before and After Dispersive-SPE
No
Cleanup
PSA
PSA
+
GCB
2.2 mg
residue
12.5 mg
residue
4.2 mg residue
The Problem with GCB
%Recoveries of pesticides when GCB used for cleanup
Pesticide
o-Phenylphenol
Chlorothalonil
Vinclozolin
Cyprodinil
Thiabendazole
Coumaphos
Orange
92
30
94
48
44
41
Tomato
93
4
92
28
27
7
Strawberry
84
24
88
28
17
18
GCB Strongly Retains Planar Analytes
Spiked spinach QuEChERS extracts (at 625 ng/mL)
w/o GCB clean-up (5 μL PTV injections)
Not OK after
100 injections
Spiked spinach QuEChERS extracts (at 625 ng/mL)
w/o GCB clean-up (5 μL PTV injections)
OK after
100 injections
Comparison of 16B (ChloroFiltr) and GCB
HCB in Lettuces (QuEChERS w/MeCN)
16B - chlorophyll removal
GCB - chlorophyll removal
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
16B - recovery
GCB - recovery
5-10 mg GCB is
like 50 mg 16B
0
10
20
30
40
50
Amount of sorbent (mg per mL extract)
Comparison of GCB-Type Sorbents
Chlorophyll %Removed Measured by Absorbance @ 680 nm
Recovery of Hexachlorobenzene (HCB) at 150 ng/g in Kale
Sorbent (7.5 mg/mL)
ENVI-Carb
Hypercarb
CarbonX
CarboPrep-90
CarboPrep-200
GCB “A”
GCB “B”
GCB “C”
ChloroFiltr*
%Removed
89 ± 5
96 ± 9
61 ± 1
92 ± 2
94 ± 2
93 ± 7
95 ± 6
93 ± 5
90 ± 4
* 50 mg/mL extract in d-SPE
HCB %Recovery, n=4
71 ± 6
73 ± 12
89 ± 12
63 ± 0
68 ± 10
65 ± 17
42 ± 3
47 ± 3
78 ± 4
Use of MeCN/EtOAc with GCB
Hexachlorobenzene
100%
EtOAc
MeCN
Recovery
80%
60%
40%
20%
0%
2.5
5.0
7.5
10
25
50
Amount of GCB sorbent (mg per mL extract)
Our MMM for Veterinary Drugs
2 g tissue in a 50 mL tube, add IS mix
add 10 mL of 4/1 (v/v) MeCN/water
vortex briefly, shake for 5 min
centrifuge for 5 min >3500 rcf
supernatant + 500 mg C18 + 10 mL hexane sat’d
w/MeCN; mix for 30 s, centrifuge for
5 min > 3500 rcf; aspirate hexane to waste
evaporate 5 mLs extract and
dilute with water to a final volume of 1 mL
add atrazine
filter extract with the syringeless PVDF filter vial
UHPLC-MS/MS analysis
Effectiveness of Clean-Up
in Removing Co-Extractives
Comparison of Sorbents
60%
% Co-Extractives Removed
Cleanup of Beef Muscle Tissue
50%
40%
30%
20%
10%
0%
Z-Sep
Z-Sep + hexane
Hybrid + hexane
Average Recoveries and Reproducibilities
80%
70%
For VD Group 2 – end-capped C18
was found to be more effective for
quantitative results
60%
40%
70 – 120%
30%
25%
RSD
50%
20%
10%
87 analytes
0%
0%
25%
50%
75%
100%
Recovery
125%
150%
175%
Method Logistics
1 chemist was able to process 60 pre-homogenized
samples in an 8-hr day for an overnight sequence
(longest step was 1 hr to evaporate MeCN)
No glassware to be cleaned afterwards
Cost of materials ≈ $3/sample (using bulk C18)
Waste = 10 mL hexane and 5 mL MeCN
(and two 50 mL and one 15 mL PP tubes)
Conclusions
• This presentation has barely scratched the
surface to discuss what is possible with the
“QuEChERS” template.
• QuEChERS doesn’t work for everything, but
it is easy, fast, and cheap to try, and little is
lost if it fails.
• Even so, the QuEChERS template is so
flexible that conditions can be found to work
eventually.
Acknowledgments
• Michelangelo Anastassiades
• Katerina Mastovska
• Lucía Geis-Asteggiante
• Alan Lightfield
• Urairat Koesukwiwat
• Brian Kinsella
• Yelena Sapozhnikova
+
+
Tanks Berry Mulch!
!
Recoveries of Tylosin (100-400 ng/g)
100%
90%
80%
Reproducibility = 21% RSD
70%
60%
50%
Marilyn
Christine
n = 150
20%
Alan
30%
Lucia
40%
Steve
Recovery
n = 30 each day
(10 reps x 3 levels)
Avg. Repeatability = 12% RSD
Day 1
Day 2
Day 3
Day 4
Day 5
Average
10%
0%
Average Recoveries of the 62 Drugs
Matrix-matched stds
w/o internal stds
n = 150 per drug
(10 reps x 3 levels x 5 days)
20 different kidneys
No. of Drug Analytes
60
50
40
30
20
10
0
70-100
50-69
%Recovery
<50
Precision in the Analysis of the 62 Drugs
No. of Drug Analytes
60
Reproducibility
50
Repeatability
n = 150 per drug
(10 reps x 3 levels x 5 days)
20 different kidneys
40
30
20
10
0
≤15
16-20
%RSD
21-25
>25
Using matrix-matched stds w/o normalization to internal stds
ME Profiles Before and After Clean-Up
Z-Sep + hexane shows
enhancement
Suppression is not reduced
the front of the run
Effect of Clean-Up on Recoveries
Effect of Clean-Up on Repeatabilities
Dilution Effects on ME Profiles
 Extracts obtained after C18 + hexane clean-up were diluted by
a dilution factor (DF) of 2 and 4.
Numbers of false
negatives at lowest
spiking level
increased vs. DF
Comparison of Extraction Solvents
EtOAc Acetone MeCN
High Recoveries
Avoid Lipids
Avoid Proteins
Avoid Sugars
Water Removal Ease
GC Compatible
RP-LC/SPE Compatible
GPC Compatible
Ease of Evaporation
Low Cost
Low Toxicity
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Comparison of Extraction Solvents
Co-Extracted
Matrix (mg/g)
3.5
2.8
2.1
1.4
0.7
0.0
Ac
t
E
QuEChERS Method
of Mixed Produce
CN
e
M
ac
:
CN
e
M
e
n
o
et
ac
e
n
o
et
Extraction Solvent
w/o PSA SPE Cleanup
w/PSA SPE Cleanup
Problematic Pesticide/
Solvent Combinations
Pesticide(s)
Solvent(s)
Dicofol
acetone, MeCN
Chlorothalonil
acetone, MeCN
Pesticides with a thioether group
(fenthion, phorate, disulfoton etc.)
EtAc, acetone
Pyrethroids
(deltamethrin, cyhalothrin-lambda)
acetone, MeCN
N-trihalomethylthio pesticides
(captan, folpet, dichlofluanid)
potentially MeCN
Slide by Katerina Mastovska
Effect of pH and Extraction Solvents
%Recoveries in pH-adjusted apple juice
Thiabendazole Imazalil
EtOAc MeCN EtOAc MeCN
pH 2.5 57
92
58
101
pH 3
54
90
51
92
pH 4
85
90
73
94
pH 5
96
84
84
86
pH 6
104
90
94
90
pH 7
104
94
94
89
Addition of 0.1% acetic acid to extracts improves stability of
folpet, dichlofluanid, captan, captafol and other pesticides
or
ot
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en
to alo
do ly
su lflu nil
lfa an
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su id
lo ph lf
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a
yr osa te
ifo lo
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m
et
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co fo l
um lpe
t
pr
oc aph
ym os
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pt ne
ac
h
di lor
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cy rifo
pr
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od
at inil
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et e
m ho
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sch ph
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Recovery
Comparison of MeCN and EtOAc
Acetate-Buffered QuEChERS (GC-MS)
140%
120%
MeCN
EtOAc
100%
80%
60%
40%
20%
0%
Extraction of Produce, n = 36
The Extraction/Partitioning Step
• MeCN forms a strong partition with water by
adding salt(s) (also sugars).
• No dilution of the extract with nonpolar cosolvents as needed with acetone.
• Amount and type of salts controls MeCN:water
partitioning to affect polarity range.
• MeCN:water extracts polars, MeCN after
partitioning can extract nonpolars.
• MeCN is immiscible with nonpolar solvents to give
the possibility to remove lipid co-extractives.
MgSO4 Removes Water from MeCN
Better than Other Dessicants
Dessicant
NaCl
Na2SO4
Mol. Sieves
MgSO4
*NaCl
Amt. Added
2 g per 27 mL
0.8 g per 4 mL
0.8 g per 4 mL
0.8 g per 4 mL
Initial %H2O
27
6.0
6.0
6.0
Final %H2O
6.0*
3.9
3.2
0.6
was used to form 2 layers in a MeCN:H2O solution;
6% H20 remained in upper layer
Solubility of MgSO4 in Different Solvents
eC
N/
wa
te
eC
N/
wa
t
55
/45
M
M
/2
98
r
er
eC
N
M
c
Et
A
ne
to
Ac
e
eO
H
M
ne
16
14
12
10
8
6
4
2
0
To
l ue
solubility (mg/mL)
Solubility of MgSO4 in different solvents
Use of Different Types of MgSO4
Matrix
Lime
Broccoli
Type of MgSO4 Temp (°C)
% Matrix
Removed
anh. MgSO4
≥99% purity
45
57
anh. MgSO4
≥97% purity
39
55
anh. MgSO4
≥99% purity
48
62
anh. MgSO4
≥97% purity
39
67
Effect of Extraction Temperature
Broccoli No ice
Broccoli Ice
Lime No ice
Lime Ice
120%
Recovery
100%
80%
60%
40%
20%
0%
Phthalimide
Tetrahydrophthalimide
Chlorothalonil
Captan
Folpet
Use of Ice to Lower Extraction Temp. may Help for
Base-Sensitive Analytes in Broccoli (non-acidic),
but it has No Effect in Lime (acidic)
How Salting Out Effects Recoveries
Results in Different Commodities
MgSO4 NaCl Water
(g)
(g) (mg/mL)
0
1
124
0
2
83
0
3
61
2
0
195
4
0
138
6
0
128
1
2
76
3
2
63
5
2
62
Methamid.
(%recov.)
41
43
53
79
98
94
51
79
88
Acephate Omethoate
(%recov.) (%recov.)
44
44
43
45
53
64
97
96
102
103
99
106
57
67
88
92
85
88
Higher Recoveries were Achieved with MgSO4
than NaCl in Combination or Alone...
How Salting Out Effects Selectivity
5000000
4500000
2 g NaCl
4000000
3500000
1 g NaCl
3000000
0.5 g NaCl
2500000
2000000
1500000
1000000
500000
fructose
degradant
(HMF)
Apple extracts
5 g MgSO4 used in all cases
0 g NaCl
0
6.00 8.0010.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.00
… but
Time-->Combination of MgSO4 with NaCl Improved
Selectivity by Avoiding Sugar Co-Extractives
Evaluation of Incurred Samples
• FSIS provided 10 kidneys found in their monitoring
program to contain drug residues.
• We analyzed the samples in blind fashion
(unknown drugs and unknown levels).
• These were each analyzed in duplicate using the
different features of 3 multiclass, multiresidue
methods (MMMs) to compare and assess their
performances on real samples.
Analysis of Incurred Kidney (2 g)
Pirlimycin in Incurred Kidney (no IS)
Concentration (ng/g)
250
5 min shake; w/o acid
5 min shake; w/ acid
30 min shake; w/o acid
30 min shake w/ acid
60 min heat; w/o acid
60 min heat; w/ acid
200
150
100
50
0
1
2
3
4
5
6
Kidney Sample
7
8
9
10
Analysis of Incurred Kidney (2 g)
Sulfamethazine in Incurred Kidney (no IS)
180
5 min shake; w/o acid
5 min shake; w/ acid
30 min shake; w/o acid
30 min shake w/ acid
60 min heat; w/o acid
60 min heat; w/ acid
Concentration (ng/g)
160
140
120
100
80
60
40
20
0
1
2
3
4
5
6
Kidney Sample
7
8
9
10
Analysis of Incurred Kidney (2 g)
Beta/Dexamethasone in Incurred Kidney (no IS)
450
5 min shake; w/o acid
5 min shake; w/ acid
30 min shake; w/o acid
30 min shake w/ acid
60 min heat; w/o acid
60 min heat; w/ acid
Concentration (ng/g)
400
350
300
250
200
150
100
50
Beta/Dexamethasone was missed by FSIS
0
1
2
3
4
5
6
7
Kidney Sample
8
9
10
Analysis of Incurred Kidney (2 g)
Flunixin in Incurred Kidney (no IS)
Concentration (ng/g)
600
5 min shake; w/o acid
5 min shake; w/ acid
30 min shake; w/o acid
30 min shake w/ acid
60 min heat; w/o acid
60 min heat; w/ acid
500
400
300
200
100
0
1
2
3
4
5
6
7
Kidney Sample
8
9
10
Analysis of Incurred Kidney (2 g)
Flunixin vs. Flunixin-d3 IS in Incurred Kidney
700
Concentration (ng/g)
5 min shake; w/o acid
600
5 min shake; w/ acid
500
30 min shake; w/o acid
30 min shake w/ acid
400
60 min heat; w/o acid
60 min heat; w/ acid
300
200
100
0
1
2
3
4
5
6
7
Kidney Sample
8
9
10
Analysis of Incurred Kidney (2 g)
Penicillin G vs. PenG-d7 IS in Incurred Kidney
Concentration (ng/g)
60
5 min shake; w/o acid
30 min shake; w/o acid
60 min heat; w/o acid
50
40
5 min shake; w/ acid
30 min shake w/ acid
60 min heat; w/ acid
No need for long shake nor heat,
so fast and cool is fine!
30
Spikes were ok, but incurred
penicillins can’t tolerate acids
20
10
0
1
2
3
4
5
6
7
Kidney Sample
8
9
10
Comparison of Cleanup
45%
40%
% Removed
35%
30%
25%
20%
15%
10%
5%
0%
Cleanup of Beef Kidney Tissue
<4% of matrix co-extracted initially
“QuEChERS” w/o Partitioning
2.5 g sample + 15 mL MeCN/H2O
(75/25) with 1% formic acid
Vortex 30 min
Centrifuge at 3000 rcf 5 min
Based on Mol et. al.
(2008) Anal. Chem.
80, 9450-9459.
Take a 2 mL aliquot
Partition with 2 mL of hexane
saturated in MeCN
Take a 0.5 mL aliquot
Dilute with 0.5 mL of H20
Filter using 0.45 μm
PVDF filter vials
Inject in LC-MS/MS
Slide adapted from Lucía Geis-Asteggiante
Mycrocystins (MCs) and Nodularin
D-Glu
Mdha
D-Ala
Adda
L-Leu
MC-LR
Nodularin
D-MeAsp
L-Arg
Two variable L-amino acids
Slide adapted from Lucía Geis-Asteggiante
MCs Recoveries and Precision:
4 levels (10, 25, 50, and 100 ng/g), 5 replicates each on
5 different days (n=42)
Slide adapted from Lucía Geis-Asteggiante
QuEChERS for Anthelmintics
Place 10 g tissue / 10 ml milk into tube
Add 10 ml MeCN + 4 g MgSO4 + 1 g NaCl
Add internal standard (Cyprodinil + 2,4D)
Shake for 1 min
Centrifuge for 5 min
Place 1 ml supernatant into 2 ml centrifuge tube
containing 150 mg MgSO4 and 50 mg sorbent (C18)
Mix for 1 min
Centrifuge for 1 min
Place 0.5 ml into auto sampler vial
Add QC spike (TPP)
Inject onto LC-MS/MS (ESI+ and ESI-)
Slide adapted from Brian Kinsella
Recoveries of 39 Anthelmintics
120
110
100
90
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Recovery
80
Slide adapted from Brian Kinsella
Anthelmintics
ESI +
Triphenyl Phosphate (IS)
Cyprodinil (QC spike)
Abamectin
Doramectin
Emamectin
Eprinomectin
Moxidectin
Ivermectin
Selamectin
Dichlorvos
Coumaphos
Coumaphos-Oxon
Haloxon
Morantel
Levamisole
ESI –
Albendazole
Albendazole-Sulfoxide
Albendazole-Sulfone
Albendazole-Amino-Sulfone
Cambendazole
Flubendazole
Amino-Flubendazole
Hydroxy-Flubendazole
Fenbendazole
Fenbendazole-Sulfone
Oxfendazole
Mebendazole
Amino-Mebendazole
Hydroxy-Mebendazole
Oxibendazole
Thiabendazole
5-Hydroxy-Thiabendazole
Triclabendazole
Triclabendazole-Sulfoxide
2,4
Dichlorophenoxyace
tic acid (IS)
Bithionol
Clorsulon
Closantel
Niclosamide
Nitroxynil
Oxyclozanide
Rafoxanide
Triclabendazole
Slide adapted from Brian Kinsella