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Pesticide Analysis Using QuEChERS Extraction: A

Comparison of Manual and Automated
Approaches

Conference Paper October 2012

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 Pesticide Analysis Using QuEChERS
Extraction: A Comparison of Manual
and Automated Approaches

Abstract
QuEChERS is a Quick-Easy-Cheap-
Effective-Rugged-Safe extraction method
that has been developed for the
determination of pesticide residues in
agricultural commodities. QuEChERS itself
has evolved from the original unbuffered
method to a pair of multi-laboratory validated
methods using citrate buffering (EN
15662:2008) or acetate buffering (AOAC
Official Method 2007.01). While originally
developed for plant matrices, the technique
has since been adapted to be used in
several applications and in many additional
matrices. The rise in popularity of the
technique and the increase in sample testing
loads have lead to automation as a possible
solution to increase productivity. The
method, as mentioned, is a simple and fast extraction when compared to traditional semi-volatile
extraction techniques. However, it still requires dedicated manpower, extraction equipment, manual
spiking, manual pipetting, and a wide range of reagents and consumables. The analytical and practical
challenges of automating this technique will be discussed and a comparison of extraction recoveries
achieved both manually and by automation will be evaluated. Data will be presented that justifies
automation as a possible solution to increase sample throughput, reduce costs, and improve the accuracy
and precision of the extraction.
Introduction
Increasing globalization of the food industry has lead to increased concerns about food safety. This has
resulted in an increased number of pesticides residues which must be regulated and monitored by
governing bodies. The QuEChERS method was published in 2003 for the extraction of pesticide residues
1
in agricultural commoditites. Modifications to the method have expanded the scope to include many
additional matrices and target analytes. After validation, the methods were adopted as AOAC Official
2
Method 2007.01, and EN 15662:2008. These methods require several manual steps, such as addition of
extraction solvent, salts/buffer addition, spiking, shaking, mixing, centrifugation, transferring to a
dispersive solid phase extraction (dSPE) step, and finally measuring and transferring a portion of the
3
extract.
With the increasing amounts of samples required to be analyzed for pesticide residues, Teledyne Tekmar
has developed the AutoMate-Q40. This revolutionary system is designed and optimized to automate the
QuEChERS sample extraction workflow. The AutoMate-Q40 is configured to perform two of the most
popular QuEChERS extraction methods. This poster will compare results from both the AutoMate-Q40
and manual QuEChERS extractions using the AOAC Method 2007.01.

www.teledynetekmar.com 1 4736 Socialville Foster Rd

Mason, OH 45040 USA
Experimental-Instrument Conditions
Analyses were performed on a Thermo Trace Ultra GC equipped with an ISQ mass spectrometer.
Instrument conditions can be seen below in Tables 1 and 2. The QuEChERS extractions were performed
by the AutoMate-Q40 and manually by an analyst. Figure 1 shows the flow chart the AutoMate-Q40
followed for the AOAC sample extraction procedure.

GC Conditions Thermo Trace Ultra GC

Column Restek 5MS-Sil 30.0 m x 0.25 mm x 0.25 m

Column Constant Flow 1.0 mL/min

Oven Program 40.0 C,(1.5 min), 25 C/min to 150 C,

(0.0 min), 7.0 C/min to 250 C (0.0 min),
25.0 C/min to 290.0 C (10.0 min)

S/SL Temperature 250 C

S/SL Mode Splitless with Surge Pressure

Split Flow 50 mL/min

Transfer Line Temperature 290 C

Table 1: Thermo Trace Ultra GC conditions

Analyte SIM RT (min)

Trifluralin 306.0, 264.0 10.74
Atrazine 200.0, 215.0 11.81
Chlorothalonil 266.0, 264.0 12.45
Chloropyrophos-methyl 286.0, 125.0 13.52
Cyprodinil 224.0, 225.0 15.64
Procymidone 283.0, 285.0 16.12
2,4-DDD 235.0, 237.0 17.55
Kresoxim-methyl 116.0, 131.0 17.61
Triphenyl Phosphate (TPP) 77.0, 65.0 19.96
Bifenthrin 181.0, 165.0 20.69
Lambda-Cyhalothrin 181.0, 197.0 21.62

Cis-Permethrin 183.0, 163.0 22.31

Trans-Permethrin 183.0, 163.0 22.43

Table 2: Thermo ISQ acquisition parameters used for the analysis of pesticides by GC/MS

www.teledynetekmar.com 2 4736 Socialville Foster Rd

Mason, OH 45040 USA
Sample Preparation
A. Sampling
Six pounds of apples were purchased from a local grocery store in Mason, Ohio. All six pounds were
chopped into small cubes as prescribed in the method. The whole apple was used but the seeds were
discarded. The chopped apple cubes were then placed into a plastic bag and frozen overnight. On the
day of analysis the required amount of sample was removed and then carefully blended.
B. Extraction/Cleanup
For the extraction, a 15 g (+/-0.1 g) homogenized apple sample was placed into a 50 mL centrifuge tube.
The AutoMate-Q40 then moved the centrifuge tubes to a de-capping station where the caps were
removed. Using a dual pump liquid handling system, the instrument was programmed to add extraction
solvent and spiking solutions to the samples. 15 mL of 1%HAc in ACN and 75 Ls of internal standard
(20 g/mL) were added to each sample to yield a 100 ng/g concentration. QC samples were fortified with
50 uL and 250 L of the QC spiking solution (6 g/mL), yielding 20 ng/g and 100 ng/g check samples. 7.5
g of AOAC extraction salts were added by the solids dispenser. The samples were automatically capped
and moved to the shaker where they were shaken vigorously for 1 minute to ensure complete mixing.
Once the mixing was complete the samples were centrifuged for 5 minutes at 4000 rpm.
Upon completion, the centrifuged samples were transferred to the VialVision (patent pending) station.
Once there, a camera determines the positions of the layers in the vial and calculates the available
supernatant that can be transferred to a dSPE cleanup vial. The samples are then transferred from the
VialVison station to a pipetting station where the necessary 15 mL dSPE cleanup tube waits. The
centrifuge tubes are uncapped, and using the air displacement pipetter (ADP), an 8 mL aliquot is
transferred from the extraction tube to the 15 mL dSPE tube containing 400 mg of PSA and 1200 mg of
returned to the tray while the 15 mL
dSPE tube is moved to the shaker and
shaken for 1 minute. Once the mixing is
complete the sample is then moved to
the centrifuge and spun for 5 minutes at
4000 rpm.
Once centrifuging is complete, the 15 mL
dSPE cleanup tube is moved to the
VialVision station to determine the
amount of extract available to be
transferred to the final extract tube. The
15 mL dSPE cleanup tube is moved from
the VialVison station to a shuttle where
the corresponding 15 mL final extract
tube waits. The cleanup and final extract
tubes are uncapped and, using the ADP
5 mL of the final extract is transferred to
the final tube. Then an aliquot from the
final extract tube is manually placed into Figure 1: Flow chart of AutoMate-Q40 AOAC QuEChERS extraction
an autosampler vial, and analyzed by GC/MS.
Figure 1 shows the flow chart for the AOAC QuEChERS extraction procedure for both the AutoMate-Q40
and the manual hand extraction.

www.teledynetekmar.com 3 4736 Socialville Foster Rd

Mason, OH 45040 USA
Results and Discussion
By automating the liquid handling, addition of salts/buffers, sample mixing, centrifugation, pipetting and
liquid level sensing (VialVision), the QuEChERS extraction process is fast, and easy. The AutoMate-Q40
offers time and labor savings, while improving consistency and repeatability when compared to manual
QuEChERS extractions. Two sets of data were analyzed to illustrate the differences between using the
AutoMate-Q40 and manual QuEChERS extraction.
A. Linearity
AOAC Method 2007.01 specifies preparation of a matrix matched calibration to compensate for potential
interferences. Six matrix blanks were extracted with internal standard and surrogates and were spiked
with a prescribed amount of a 2 g/mL calibration stock standard. The calibration ranges for all
compounds were performed at 5-400 ng/g. The calibrations were extracted both by hand and by
automation (Table 3). Each calibration curve was prepared at levels of 5, 10, 20, 50, 100, and 400 ng/g.
The results for both sets of extractions are shown in Table 4.

Amount of 2 g/mL AOAC Amount of Final Final Concentration in 2

Pesticide Stock Standard (L) Extract Used (L) mL sample

5.0 995 5.0 ng/g

10.0 990 10.0 ng/g

20.0 980 20.0 ng/g

50.0 950 50.0 ng/g

100.0 800 100 ng/g

400.0 600 400 ng/g

Table 3: Calibration framework for both hand and automated QuEChERS extractions

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Mason, OH 45040 USA
 AutoMate-Q40 Hand Extraction
Component in Apple Matrix Linearity (R2) Linearity (R2)

Trifluralin 0.9976 0.9930

Atrazine 0.9992 0.9969

Chlorothalonil 0.9990 0.9926

Chloropyrophos-methyl 0.9980 0.9955

Cyprodinil 0.9970 0.9986

Procymidone 0.9987 0.9961

2,4-DDD 0.9981 0.9970

Kresoxim-methyl 0.9991 0.9986

Triphenyl Phosphate (TPP)

Bifenthrin 0.9988 0.9987

Lambda-Cyhalothrin 0.9974 0.9993

Cis-Permethrin 0.9995 0.9994

Trans-Permethrin 0.9998 0.9992

Average 0.9971 0.9962

Table 4: Linearity study for both the AutoMate-Q40 and manual extractions

B. Recovery and Reproducibility

A precision and accuracy study was performed using both manual and automated QuEChERS
extraction techniques. A 6 g/mL stock AOAC QC pesticide standard was used to fortify the
homogenized apple samples. Using the AutoMate-Q40, the system spiked the apples samples with 50
and 250 L of the AOAC QC check standard yielding 20 and 100 ng/g check samples. The same
process was done for the manual extractions using a micro syringe. These QC samples were
quantitated against their corresponding matrix-spike calibration curve. The analyses were performed in
replicates of five (n=5).

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Mason, OH 45040 USA
 120.0
Recovery for 20 ng/g Spike
110.0
100.0
90.0
80.0
70.0
Avg Recovery (ng/g)

60.0
50.0
40.0
30.0
20.0
10.0
0.0
Chloropyr Lambda- Cis- Trans-
Chlorothal Procymid Kresoxim-
Trifluralin Atrazine ophos- Cyprodinil 2,4-DDD Bifenthrin Cyhalothri Permethri Permethri
onil one methyl
methyl n n n
Manual Extraction 95.7 82.8 103.4 78.0 103.0 100.1 101.9 100.0 89.5 89.1 95.0 96.6
AutoMate Q40 107.9 101.4 98.7 106.8 108.1 100.7 104.6 98.0 93.4 103.2 92.5 93.1

Table 5: Recovery for 20 ng/g fortified apple.

Precision for 20 ng/g Spike

10.0

9.0

8.0

7.0

6.0
%RSD (n=5)

5.0

4.0

3.0

2.0

1.0

0.0
Chloropy Kresoxi Lambda- Cis- Trans-
Triflurali Chloroth Cyprodin Tolylflua Procymi Bifenthri
Atrazine rophos- 2,4-DDD m- Cyhaloth Permeth Permeth
n alonil il nid done n
methyl methyl rin rin rin
Manual Extraction 5.0 4.7 8.6 4.0 5.1 6.2 3.4 4.1 7.6 4.2 5.2 7.4 5.3
AutoMate Q40 3.1 6.1 5.5 3.5 3.6 3.8 5.4 3.5 6.2 5.9 1.9 3.5 1.8

Table 6: Precision for 20 ng/g fortified apple. (n=5)

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Mason, OH 45040 USA
 120.0
Recovery for 100 ng/g Spike
110.0
100.0
90.0
80.0
70.0
Avg Recovery (ng/g)

60.0
50.0
40.0
30.0
20.0
10.0
0.0
Chloropy Lambda- Cis- Trans-
Chloroth Cyprodini Tolylflua Procymid Kresoxim Bifenthri
Trifluralin Atrazine rophos- 2,4-DDD Cyhalothr Permethr Permethr
alonil l nid one -methyl n
methyl in in in
Manual Extraction 83.0 86.6 87.1 85.5 86.3 106.9 95.2 91.2 95.5 92.1 99.0 93.4 95.7
AutoMate Q40 93.7 97.4 100.9 96.7 89.9 116.8 91.4 91.0 95.3 90.3 97.0 102.0 99.3

Table 7: Recovery for 100 ng/g fortified apple.

Precision for 100 ng/g Spike

9.0

8.0

7.0

6.0
%RSD (n=5)

5.0

4.0

3.0

2.0

1.0

0.0
Chloropy Kresoxi Lambda- Cis- Trans-
Triflurali Chloroth Cyprodin Tolylflua Procymi Bifenthri
Atrazine rophos- 2,4-DDD m- Cyhaloth Permeth Permeth
n alonil il nid done n
methyl methyl rin rin rin
Manual Extraction 8.2 7.5 6.3 7.1 4.3 3.4 4.1 4.6 2.5 4.7 3.1 3.7 2.2
AutoMate Q40 6.5 1.4 8.2 3.3 2.5 2.2 5.8 2.0 2.1 3.3 2.5 2.9 1.9

Table 8: Precision for 100 ng/g fortified apple. (n=5)

www.teledynetekmar.com 7 4736 Socialville Foster Rd

Mason, OH 45040 USA
 Tables 5, 6, 7 and 8 show the recoveries and precision (%RSD) for both the manual and automated
QuEChERS extractions. It can be seen from the results that when using the AutoMate-Q40 that all of the
pesticides exhibited excellent recovery (average of 100.8% for 20 ng/g spike and 97.1% for 100 ng/g
spike) and precision (average of 4.1%RSD for the low spike and 3.4%RSD for the high spike). In
comparison, the results of the manual QuEChERS exhibit slightly lower recoveries (average of 87.3% for
20 ng/g spike and 92.1% for 100 ng/g spike) and comparable precision (RSD 4-5% on average for both
spikes). Figure 2 shows a chromatographic comparison of a 100 ng/g fortified apple extract extracted by
both the AutoMate-Q40 and manual QuEChERS.

Figure 2: 100 ng/g fortified apple extract, extracted by the AutoMate-Q40 (A) and by manual QuEChERS (B).

Conclusions
By automating the liquid handling, the addition of salt/buffers, sample mixing, pipetting, and liquid level
sensing using the patent pending VialVision, the extraction process is fast, and easy. The AutoMate-Q40
not only offers time and labor savings, but also improves extraction precision and accuracy. As shown in
the data above, all of the pesticides give excellent spike recoveries on average 100.8% for the 20 ng/g
spike and 97.1% for the 100 ng/g spike and excellent precision on average of 3-4%RSD.
References
1. AOAC Official Method 2007.07 Pesticide Residues in Food by Acetonitrile Extraction and Partitioning
with Magnesium Sulfate. Gas Chromatography/Mass Spectrometry and Liquid Chromatography/
Tandem Mass Spectrometry, First Action 2007
2. European Committee for Standardization/Technical Committee CEN/TC275 (2008), Foods of plant
origin: Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile
extraction/ partitioning and cleanup by dispersive SPE QuEChERS-method
3. M. Anastassiades: QuEChERS a mini-multiresidue method for the analysis of pesticide residues in
low-fat products
Acknowledgments
Teledyne Tekmar wishes to thank Sigma Aldrich for supplying the QuEChERS extraction salts and dSPE
cleanup materials. Also, Tekmar would like to thank Restek for supplying the GC/MS consumables for
the analysis.

www.teledynetekmar.com 8 4736 Socialville Foster Rd

Mason, OH 45040 USA

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