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Joan Stevens, Ph.D. Sample Preparation Applications Scientist, Chemistries and Supplies Division, Agilent Technologies, Inc.
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Joan Stevens, Ph.D. Sample Preparation Applications Scientist, Chemistries and Supplies Division, Agilent Technologies, Inc.
Outline
Background information: sample preparation
LLE, and SPE
QuEChERS Procedure
Extraction/partitioning Dispersive SPE Simplicity of the sample preparation process Analysis LC/MS/MS, GC/MS, and GC/MS/MS
QuEChERS modifications
Low water content products Very polar compounds
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Sample Preparation
LLE: liquid liquid extraction
Advantages
Inorganic salts easily removed Short Method development time
Disadvantages
Labor intensive Large volumes of organics
Low Cost
Difficult to automate
Emulsion formation
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Sample Preparation
SPE: Solid phase extraction
Advantages
Very selective Effective with variety of matrix
Disadvantages
Greater complexity/difficult to master Lengthy method development
Concentration effect
High recoveries High reproducibility
Costly
Many choices
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Sample Preparation
Combinations of sample preparation techniques
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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 GPC Estimated Time to process 6 samples (min) 120 QuEChERS QuEChERS Benefits! 30 4x faster
90 mL
30 mL
10mL
none
9 x less solvent
safer, greener, less costly No additional supplies needed
None
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QuEChERS Procedure
Add homogenized sample to tube. Add 2 ceramic homogenizers Add ACN, vortex Add Extraction packet Shake, Shake, Shake! Centrifuge
Transfer to dispersive-SPE
Vortex, Centrifuge Analyze
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SampliQ Ceramic Homogenizers Reduces shaking time from 1 minute to <20 seconds!
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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
40
20
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Percent Recovery
100 80 60 40
20
0 0 10 20 30 40 50 60 70
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50
Temperature C
50
Temperature C
40 30 20 10
0
0 10 20 30 40 50 60
10
20
30 Time (seconds)
40
50
60
Time (seconds)
Represent the thermal data acquired from the AOAC and EN extraction kits. A substantial increase in temperature is observed (increase of approx 25 C for the AOAC method and approx 10 C 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
Spike 100 L of IS solution vortex 1 min Add 10 mL of ACN, and shake 1 min Add Agilent QuEChERS extraction salt bag Cap and shake vigorously 1min Centrifuge @ 4000 rpm 5 min
Transfer upper ACN layer to Agilent QuEChERS dispersive-SPE tube, 1 mL/2 mL tube or 8mL /15 mL tube
Vortex 1min then centrifuge
Transfer 200 L extract to autosampler vial, dilute with 800 L appropriate solution Sample are ready for LC/MS/MS analysis
Figure QuEChERS AOAC sample preparation procedures flow chart Flow Chart of 1. the Agilent QuEChERS AOAC Extraction Procedure
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Basic EN Method
Weigh 10 g comminuted sample ( 0.05g) in 50 mL centrifuge tube
Spike 100 L of IS spike solution, vortex 1 min Add 10 mL of ACN, and shake 1 min Add Agilent EN extraction packet, and shake vigorously by hand for 1 min
Vortex 1min, centrifuge @ 13000 rpm for 2 min for 2 mL tubes or @ 4000 rpm for 5 min for 15 mL tubes
Transfer 200 L extract to autosampler vial, add 10 L of 1% FA in ACN, dilute with 800 L water Samples are ready for LC/MS/MS analysis Chart of the Agilent QuEChERS EN procedure Flow Flow Chart of the Agilent QuEChERS ENextraction Extraction Procedure
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Historical Perspective
Unbuffered method first published in 2003 2 interlaboratory validated versions AOAC official method 2007.01 EN official method 15662 www.quechers.com
All 3 methods give excellent results: average 98% recoveries with 10% RSDs
Unbuffered method have a negative effect on few pH-dependent pesticides
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Pymetrozine: AOAC 82% (7% RSD) EN ~ 30% (51% RSD) Unbuffered (100% RSD) Buffering at pH 5 during extraction gave optimum balance and acceptable recoveries > 70%
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EN Buffered Method
Acetate buffer system resulted in visibly worse cleanup results compared to the original QuEChERS method - M. Anastassiades
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Matrix Effects:
S. Lehotay: J. Chromatography A 1217 (2010), 2548-2560 Slight differences in color and color intensity: AOAC versus EN 1) Determine by gravimetric measurements 2) Chromatography 3) Determination of matrix effects on quantitation Extract: unbuffered = 0.23% citrate buffered = 0.17% acetate buffered = 0.13%
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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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Literature
5990-5085EN, 5990-5086EN, 5990-5395EN
Acrylamides in Fried Food and Oil Pesticides in Baby Food Pesticides in Green Tea PCBs in Fish and Fish Oil Supplements Pesticides in Essential Oils Hormones in Shrimp
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10 4
6
11 B
3 7 2 1 5 8 9
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
100 110 10 20 30 40 50 60 70 80 90
Pipemidic acid
Ofloxacin
Ciprofloxacin
Danofloxacin
Lomefloxacin
Enrofloxacin
Sarafloxacin
Cinoxacin
Oxolinic acid
1
1800 1600 1400 1200 1000 800 600 400 200 4.00
2 4 5 6
S/N = 11.5
14
8 10 9 16
11
12
13
14 15
6.00
8.00
10.00
12.00 Time
14.00
16.00
18.00
20.00
GC/MS SIM Chromatogram of 10 ppb PAHs spiked in fish matrix blank extract
GC/MSD: Column: Restrictor : MMInlet: Carrier: Oven: MSD: 7890/5975B with purged ultimate union DB-5ms UI 20 m 0.18 mm 0.18 m Siltek 0.7m x 0.15mm ID (Col 2) 0.5L, 320C, splitless , purge flow 50mL/min at 0.8min gas saver 30mL/min at 2min Helium, 1.7mL/min cnst flow col 1 PCM 1=3.8 psi cnst pressure, col 2=3.8ml/min flow @ 50C 50C (0.4 min) to 195C (25C/min) hold 1.5 min, 8C/min to 265C 20C/min to 315C (1.25 min) Postrun backflush 7 min @320C Transfer line 340C Source 340C Quad 150C
Recovery and Repeatability of PCBs in Fortified Red Snapper Fish with Agilent J&W DB-5ms UI column
25 ng/mL fortified QC Analytes %Recovery RSD (n=6) Naphthalene 80.35 3.29 Acenaphthylene 95.28 2.30 Acenaphthalene 92.28 2.51 Fluorene 95.98 2.99 Phenanthrene 100.51 3.46 Anthracene 107.38 3.51 Fluoranthene 113.27 3.87 Pyrene 113.55 3.51 Benz[a]anthracene 129.79 3.41 Chrysene 116.75 4.01 Benzo[b]fluoranthene 131.20 3.70 Benzo[k]fluoranthene 139.45 2.52 Benzo[a]pyrene 125.30 3.68 Indeno[1,2,3-cd]pyrene 119.51 3.47 Dibenz[a,h]anthracene 126.35 3.54 Benzo[g,h,i]perylene 114.91 4.93 250 ng/mL fortified QC %Recovery RSD (n=6) 96.77 4.23 103.36 2.80 101.18 2.87 105.94 2.82 104.93 2.71 105.95 3.45 105.76 3.33 103.99 3.24 101.45 3.91 98.55 4.17 98.77 4.08 99.13 3.98 95.33 3.89 94.57 3.23 98.55 3.50 97.30 3.37 500 ng/mL fortified QC %Recovery RSD (n=6) 98.64 1.88 101.02 2.27 100.69 2.34 105.00 1.28 103.25 1.70 105.38 1.74 103.64 1.81 102.29 1.94 100.61 3.24 95.95 5.61 98.08 3.24 95.31 4.54 96.82 1.80 93.71 2.55 98.85 2.24 95.63 1.83
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PAHs in Fish Employing QuEChERS Extraction and HPLC-DAD Analysis: Recovery and RSDs
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Easy Product Selection: QuEChERS Kits Identified by method and matrix Lit number: 5990-3562EN QuEChERS Poster: Easy as 1-2-3 Lit number: 5990-5324EN QuEChERS Food Application Notebooks: Lit number: 5990-4977EN Technical support: Over 33 application notes on QuEChERS: All available on our website: www.agilent.com/sampliQ
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o Excellent results
o Green and very cost effective technique
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Thank you!
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