QuEChERS - 101 - The Basics and Beyond
QuEChERS - 101 - The Basics and Beyond
QuEChERS - 101 - The Basics and Beyond
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Sample Preparation
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Sample Preparation
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Sample Preparation
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QuEChERS
(Pronounced “catchers”)
• Quick, Easy, Cheap, Effective, Rugged, and Safe
• Portmanteau: blend of 2 or more words
• www.quechers.com
• Introduced in 2003: M. Anastassiades, S.J. Lehotay, D.
Stajnbaher, and F.J. Schenck, J. AOAC Int 86 (2003) 412
• Validated in 2005, with subsequent modification in 2007
• AOAC 2007.01 and European Method EN 15662
• Streamlined approach that makes it easier and less
expensive to examine pesticide residues in food
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QuEChERS
• Alternative to existing methods: LLE, SPE, GPC
• QuEChERS is still a very young; being adopted worldwide
• Detector availability: MS and MS/MS (selective and sensitive)
• Automated solution for QuEChERS on the market today:
ChemSpeed, Gerstel, and Anatune
• QuEChERS process substantially decreases cost per sample
Luke method, traditional SPE, or QuEChERS QuEChERS Benefits!
GPC
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QuEChERS: Prepping the Sample for Processing
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QuEChERS: Prepping the Sample for Processing
• Representative sampling
• Homogenizing the sample
Uniform size
Uniform distribution
High surface area
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QuEChERS Procedure
10
Agilent Improvements to the Extraction of Samples
Step 1: Extraction salts with comminuted fruit/vegetable
• Consistency in shaking, everyone shakes differently
• Variability in QuEChERS applications, recovery and RSDs
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QuEChERS Extraction: With and Without Ceramic
Homogenizers
CH no CH CH no CH
Ceramic Homogenizers
• Inert ceramic material
• No loss in pesticide recovery
• Consistent recovery with RSD
Comparison of QuEChERS Method AOAC and EN With and Without
Ceramic Homogenizers
120
100
80
Recoveries
60
AOAC Method with Ceramic Homogenizers
EN Method with Ceramic Homogenizers
40 AOAC Method without Ceramic Homogenizers
EN Method without ceramic Homogenizers
20
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240
220
200
Save Time with Ceramic Homogenizers:
180
20 second shaking is all it takes
160
140
120
Percent Recovery
100
80
60
40
20
0
0 10 20 30 40 50 60 70
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Temperature Graphs from AOAC and EN
Extraction Kits
60 60
50 50
40
Temperature ºC
40
Temperature ºC
EN Add Buffer Salts to
30 AOAC Add Buffered Salts to 30 Sample
Sample
20 AOAC Sample Added to 20
Buffered Salt EN Sample Added to
10 10 Bufferd Salt
0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
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 is added to the extraction salt in the 50 mL PP tube versus when the
extraction salt from an anhydrous pouch is added to the produce sample
Basic AOAC Method
Weigh 15 g comminuted sample ( 0.1g) in 50 mL centrifuge tube
Add 100 µL of IS (TPP) solution, and QC spike solution if necessary, vortex 1min
Analyze by GC/MS
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Basic EN Method
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Comparison of the 3 Extraction Methods
How to Choose?
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Historical Perspective
• Unbuffered method first published in 2003
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Ionic Strength: Salt Buffering
• AOAC buffered method
• Relatively strong acetate buffering conditions (pH 4.8)
• EN buffered method
• Weaker citrate buffering conditions (pH 5-5.5)
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Three Methods: How To Choose
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AOAC Buffered Method
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EN Buffered Method
- M. Anastassiades
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Matrix Effects:
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Analytical Chemist have a common saying:
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QuEChERS: Rule of Thumb
Use matrix match calibration standards
Extent of matrix effects can be measured
• Compare calibration standards of same concentration is solvent to those in
matrix extracts (~ 16%)
• The matrix can negatively or positively effect individual compounds
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Extraction Choice
• AOAC versus EN versus Original method
• identify the compounds of interest
• pH stability
• Cleanest extract, for all matrices without unacceptably affecting recoveries even
for structurally planar pesticides
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QuEChERS Modifications:
• Low water content products
• fish, meats, grains, rice, spices, oils
• add water to initial homogenized sample
• facilitates the partitioning and compounds of interest
into the ACN layer
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Instruments Used in the Analysis
• LC/UV/FLD
• Based on compounds being analyzed
• LC/QQQ
• Selectivity and sensitivity
• GC/MS
• Selectivity and sensitivity
• Back flush strongly suggested
• Large volume injections
• Solvent exchange: Not the first choice, but an option
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QuEChERS: Current Food Research Areas
• Food Contamination
• Agrochemical
• chemicals used in agriculture practices and animal
husbandry
• pesticides, plant growth regulators, veterinary
drugs, bovine somatotropin (rBST)
• Environmental
• Chemicals are in the environment where food is grown,
harvested, transported, stored, packaged, processed,
and consumed
•PAH, PCB, dioxins, PBDE
•Mycotoxins, toxins
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QuEChERS Applications: Beyond Produce
Application Literature
Veterinary Drugs (antibiotics) in animal tissues 5990-5085EN, 5990-5086EN, 5990-5395EN
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QuEChERS: Analysis Examples
LC/MS/MS, GC/MS, LC/FLD
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A
10 11 B
4
6
3
7
2
5 9
1 8
LC/MS/MS Chromatograms of A) liver blank extract, and B) 5 ng/g fortified liver extract (LOQ).
Peaks identification: 1. Pipemidic acid, 2. Ofloxacin, 3. Ciprofloxacin, 4. Danofloxacin, 5. Lomefloxacin,
6. Enrofloxacin, 7. Sarafloxacin, 8. Cinoxacin, 9., Oxolinoc acid, 10. Nalidixic acid, 11. Flumequine,
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Recovery
10
20
30
50
60
70
80
90
40
100
0
Sulfadizine
Sulfathiazole
Sulfamerazine
Sulfamethizole
Sulfamethazine
Sulfamethoxypyridazine
Recovery and Reproducibility
Sulfachloropyridazine
Sulfamethoxazole
Sulfadimethoxin
Recoveries for the 9 Sulfonamides in Bovine Liver
Excellent Signal to Noise Ratios at Trace Levels
1. Napthalene
2. Acenaphthylene
3. Acenaphthene
4. Fluorene
5. Phenanthrene
Abundance
6. Anthracene
2400 3 7. Fluoranthene
8. Pyrene
2200
9. Benz[a]anthracene
10. Chrysene
2000
11. Benzo[b]fluoranthene
1 2
1800 12. Benzo[k]fluoranthene
4 13. Benz[a]pyrene
1600 5 14. Indeno[1,2,3-c,d]pyrene S/N = 11.5
15. Dibenz[a,h]anthracene
1400 6 16. Benzo[g,h,i]perylene 14
1200
1000 7 8
800 10
16
14 15
600 9 12 13
11
400
200
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
Time
GC/MS SIM Chromatogram of 10 ppb PAHs spiked in fish matrix blank extract
GC/MSD: 7890/5975B with purged ultimate union
Column: DB-5ms UI 20 m 0.18 mm 0.18 µm
Restrictor : Siltek 0.7m x 0.15mm ID (Col 2)
MMInlet: 0.5µL, 320°C, splitless , purge flow 50mL/min at 0.8min
gas saver 30mL/min at 2min
Carrier: Helium, 1.7mL/min cnst flow col 1
PCM 1=3.8 psi cnst pressure, col 2=3.8ml/min flow @ 50°C
Oven: 50°C (0.4 min) to 195°C (25°C/min) hold 1.5 min,
8°C/min to 265°C 20°C/min to 315°C (1.25 min)
Postrun backflush 7 min @320°C
MSD: Transfer line 340°C Source 340°C Quad 150°C
Recovery and Repeatability of PCBs
in Fortified Red Snapper Fish
with Agilent J&W DB-5ms UI column
25 ng/mL fortified QC 250 ng/mL fortified QC 500 ng/mL fortified QC
Analytes %Recovery RSD (n=6) %Recovery RSD (n=6) %Recovery RSD (n=6)
Naphthalene 80.35 3.29 96.77 4.23 98.64 1.88
Acenaphthylene 95.28 2.30 103.36 2.80 101.02 2.27
Acenaphthalene 92.28 2.51 101.18 2.87 100.69 2.34
Fluorene 95.98 2.99 105.94 2.82 105.00 1.28
Phenanthrene 100.51 3.46 104.93 2.71 103.25 1.70
Anthracene 107.38 3.51 105.95 3.45 105.38 1.74
Fluoranthene 113.27 3.87 105.76 3.33 103.64 1.81
Pyrene 113.55 3.51 103.99 3.24 102.29 1.94
Benz[a]anthracene 129.79 3.41 101.45 3.91 100.61 3.24
Chrysene 116.75 4.01 98.55 4.17 95.95 5.61
Benzo[b]fluoranthene 131.20 3.70 98.77 4.08 98.08 3.24
Benzo[k]fluoranthene 139.45 2.52 99.13 3.98 95.31 4.54
Benzo[a]pyrene 125.30 3.68 95.33 3.89 96.82 1.80
Indeno[1,2,3-cd]pyrene 119.51 3.47 94.57 3.23 93.71 2.55
Dibenz[a,h]anthracene 126.35 3.54 98.55 3.50 98.85 2.24
Benzo[g,h,i]perylene 114.91 4.93 97.30 3.37 95.63 1.83
Dual Column GC-µECD Primary Column Analysis
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GC-µECD Second Column: Confirmation Analysis
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PAHs in Soil Employing QuEChERS Extraction and
HPLC-FLD Analysis
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PAHs in Fish Employing QuEChERS
Extraction and HPLC-DAD Analysis: Recovery
and RSDs
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Agilent QuEChERS Kits
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Conclusion: Agilent QuEChERS Advancements
Excellent results
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Thank You…..Questions!
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