Pesticide testing is vital for evaluating the safety of fruits and vegetables for consumption. The conventional extraction method for pesticides from produce is known as the QuEChERS method. This method is labor intensive, time consuming and wasteful. Thus, there is a call for better methods of extraction.
Image Credit: CEM Corporation
This application details how the EDGE®, in combination with Q-Matrix® Hydra, a water-removing sorbent, was utilized for the extraction of a panel of pesticides from 10 g of homogenized cucumber, tomato and green pepper in under nine minutes.
Additionally, other matrices, either due to their nature or their economic value, can present a significant challenge when attempting extraction with just the QuEChERS method alone. Spices are among such challenging matrices, due to the dehydrated state in which they are obtained.
Image Credit: CEM Corporation
The EDGE® offers a workable alternative to the QuEChERS method for the extraction of pesticides from spices, with the capacity to extract pesticide residues in under seven minutes, in just a single automated step.
The recoveries of the pesticide panel exceeded 70% for each matrix tested, and these results were extremely reproducible. The EDGE offers a fully automated extraction that unites both the sample and sorbent into a single sample vessel, easing the human burden of extraction and the need for several sample transfers.
Introduction
When purchasing produce, consumers demand to know that their vegetables and fruits are free from harmful levels of toxic compounds, such as pesticides. Recently, consumers have also demonstrated a rising concern for contaminants and additives in their foods.
Pesticide testing is crucial because it is known that they are harmful to humans and wildlife at specific concentrations.
The present industry standard method employed for the extraction of pesticides from fruits, vegetables and spices is the QuEChERS method. This method of extraction demands laborious weighing of salts, inaccurate manual-shaking and numerous sample transfers, which is very time consuming.
All in all, one extraction can take between 20 and 60 minutes to complete. Additionally, the multi-step transfers produce substantial waste.
However, due to these obstacles, food manufacturers desire an innovative, high-speed method that is preferably automated with the same high recoveries and reproducibility. So, while the QuEChERS method has been a generally accepted method to meet some of these challenges, it is far from ideal.
Moreover, due to the large number of pesticides under observation and the reduction in the maximum residue limits, sample preparation and analysis can present major challenges.
The manual, multi-step process of the QuEChERS method necessitates numerous sample transfers and produces a lot of consumable waste. Alternative methods can assist in streamlining this workflow, producing enhanced recoveries for challenging matrices, such as spices, with simple and fast methods.
The EDGE automated extraction system executes extraction and clean-up in a single automated step as and when necessary. The extract collected is automatically filtered, cooled and prepared for analysis. Both the rinsing and the washing of the sample are factored into the run time which ensures no carryover.
The EDGE supplies the fastest automated pesticide extraction possible in just a simple, single method. A panel of pesticides were extracted in less than 9 minutes from 10 g of homogenized cucumber, tomato and green pepper utilizing the EDGE’s new bubbling feature and Q-Matrix® Hydra, a water removing sorbent.
The EDGE streamlines the extraction process by utilizing Q-Cup® technology, which includes both the sample and Q-Matrix Hydra in a single sample cell, thus easing the need for numerous transfers and the production of waste.
Also, the EDGE uses its automated bubbling feature to acquire thorough sample mixing, while easing the burden of manual shaking. The recoveries of each pesticide exceeded 70%, with RSD values less than 15%, demonstrating a thorough extraction with excellent reproducibility.
Materials and Methods
Reagents for Produce
Cucumbers, tomatoes and green peppers were bought from a local grocery store. Q-Matrix Hydra is a product supplied by CEM Corporation. ACS Grade acetonitrile and acetic acid were acquired via Sigma-Aldrich.
A custom mix that contained 1000 µg/ ml of methomyl, aldicarb sulfoxide, dimethoate, etoxazole, monocrotophos, paclobutrazol, pirimicarb, prochloraz, triticonazole and spirodiclofen in acetonitrile was supplied by SPEX CertiPrep.
Sample Preparation
A Magic Bullet blender was used to homogenize the fruits and vegetables and a Q-Cup was set up to hold the S1 Q-Disc® stack (C9 + G1+ C9 sandwich).
Ten grams of homogenized fruit/vegetable was layered on top of the sorbent, a portion of 2.5 g of Q-Matrix Hydra that was weighed in a Q-Cup. Five hundred nanograms of the custom pesticide mix was used to spike the sample.
A Q-Screen® was positioned on top of each sample by means of a Q-Screen tool. The Q-Cups were slid on to a rack, along with 50 mL polypropylene conical tubes, and extracted on the EDGE utilizing Acetonitrile w/ 1.0% Acetic Acid (v/v).
The extract was relocated to a Q-Dry™ solvent evaporator for evaporation to <5 mL. Extracts were subsequently diluted to 5 mL with acetonitrile with 1% acetic acid. All samples and blanks were prepared in triplicate. The extracts were relocated to vials for analysis.
EDGE Method for Pesticides in Wet Foods
Q-Disc: S1 Q-Disc stack (C9 + G1+ C9 sandwich)
Cycle 1:
- Extraction Solvent: Acetonitrile w/ 1.0% Acetic Acid (v/v)
- Agitation: 02:00 (mm:ss)
- Top Add: 25 mL
- Bottom Add: 0 mL
- Rinse: 5 mL
- Temperature: 40 °C
- Hold Time: 01:00 (mm:ss)
Cycle 2: (Rinse only)
- Extraction Solvent: Acetonitrile w/ 1.0% Acetic Acid (v/v)
- Top Add: 0 mL
- Bottom Add: 0 mL
- Rinse: 10 mL
- Temperature: - - -
- Hold Time: - -:- -
Wash 1:
- Wash Solvent: Acetonitrile w/ 1.0% Acetic Acid (v/v)
- Wash Volume: 10 mL
- Temperature: 40 °C
- Hold: 0:03 (mm:ss)
Reagents for Spices
Black pepper, cinnamon, oregano and paprika were purchased at a local grocery store. All samples were injected with 10 µL of 100 µg/mL stock solution of Restek LC multi-residue pesticide standards 1, 6, 7 and 9.
Prior to analysis, the spiked samples were put aside for 30 minutes. Sodium acetate, magnesium sulfate and primary secondary amine were obtained in bulk from Silicycle.
Extraction of the spiked samples was conducted via the EDGE and/or QuEChERS method. Acetonitrile with 1% acetic acid was utilized for the extraction, rinse and wash solvent.
Sample Preparation
A 2 g portion of each spice was weighed into a Q-Cup that was assembled containing a S1 Q-Disc® stack (C9+G1+C9 sandwich). A Q-Screen® was positioned on top of each sample utilizing a Q-Screen tool.
The Q-Cups were set in the removable rack of the EDGE, along with 50 mL polypropylene conical tubes. The rack was placed into position on the EDGE. The CEM-approved EDGE method for pesticide residues was carried out. The extract was then relocated to a Q-Dry™ evaporator for evaporation to <5 mL.
Extracts were then diluted to 5 mL using acetonitrile with 1% acetic acid. All samples and blanks were prepared in triplicate. The extracts were relocated to vials for analysis.
EDGE Method for Pesticide Residues
Q-Disc: S1 Q-Disc stack (C9+G1+C9 sandwich)
Cycle 1:
- Extraction Solvent: 1% Acetic Acid in Acetonitrile
- Top Add: 25 mL
- Bottom Add: 0 mL
- Rinse: 5 mL
- Temperature: 40 ºC
- Hold Time: 03:00 (mm:ss)
Cycle 2: (Rinse only)
- Extraction Solvent: 1% Acetic Acid in Acetonitrile
- Top Add: 0 mL
- Bottom Add: 0 mL
- Rinse: 5 mL
- Temperature: - - -
- Hold Time: - -:- -
Wash 1:
- Wash Solvent: 1% Acetic Acid in Acetonitrile
- Wash Volume: 10 mL
- Temperature: 40 ºC
- Hold Time: 00:03 (mm:ss)
QuEChERS Method
A 2 g portion of each spice was weighed out and placed into a 50 mL centrifuge tube. Ten milliliters of DI water was introduced to the tubes and then the tubes were shaken manually for 30 seconds and allowed to stand for 30 minutes, per modified QuEChERS methodology for dry samples.
Subsequently, a volume of 10 mL of acetonitrile with 1% acetic acid was introduced to the tubes and vortexed for 1 minute using the VWR Analog Vortex Mixer. Then, 1.5 g of sodium acetate and 6 g of magnesium sulfate were introduced into each tube and the resealed tubes were shaken for 1 minute and then centrifuged at 6000 rpm for 5 minutes using the Thermo CL2 Centrifuge.
A volume of 1 mL of the acetonitrile layer was inserted into a 50 mL centrifuge tube that contained 150 mg of magnesium sulfate and 50 mg of primary secondary amine. The tubes were centrifuged at 9000 rpm for 10 minutes after being shaken for 1 minute. The supernatant was relocated to a vial for analysis. All samples and blanks were prepared in triplicate.
Analysis
For the fruit and vegetables, a volume of 2 µL of each extract that had been evaporated was inserted onto a Restek ARC-18 column (2.7 μm, 100 x 2.1 mm) and separated utilizing a Waters Acquity UPLC.
The UPLC program used a gradient of A: water that contained 10 mM ammonium acetate with 0.2% formic acid and B: methanol that contained 10 mM ammonium acetate with 0.2% formic acid.
The gradient exceeded 7 minutes and started at 95% in A and ended at 100% in B. The MRM transitions for each pesticide were analyzed using an Xevo TQD mass spectrometer that was attached.
For the spices, a volume of 10 µL of each extract was inserted into a Waters Acquity UPLC utilizing a Xevo TQD triple quad mass spectrometer as well as a Restek ARC-18, 2.7 µm, 100 x 2.1 mm column with a flow of 0.4 mL/min.
An elution program with a 7-minute ramp from 95% in A (water that contained 2 mM ammonium formate and 0.2% formic acid) and 5% in B (methanol that contained 2 mM ammonium formate and 0.2% formic acid) to 100% B and one MSD transition was used for quantifying each pesticide. Analysis of each sample was conducted in triplicate.
Results
Table 1 (page 3) illustrates the recoveries for the pesticide mix from cucumber, tomato and green pepper. The EDGE effectively extracted the pesticides successfully in under 9 minutes, including cooling, filtering and system washing.
Table 1. Recovery Data from 10 g of Homogenized Cucumber, Tomato, and Green Pepper (extracted via the EDGE). Source: CEM Corporation
| Pesticide |
Cucumber |
RSD |
Tomato |
RSD |
Green Pepper |
RSD |
| Methomyl |
80.64% |
10.52% |
86.79% |
5.34% |
84.56% |
1.83% |
| Aldicarb Sulfoxide |
88.41% |
9.11% |
92.89% |
4.05% |
87.50% |
9.16% |
| Monocrotophos |
94.65% |
13.17% |
91.89% |
3.61% |
93.92% |
13.88% |
| Dimethoate |
80.31% |
3.80% |
89.54% |
3.54% |
92.60% |
10.26% |
| Pirimicarb |
74.80% |
11.75% |
87.87% |
6.82% |
76.23% |
9.61% |
| Paclobutrazol |
84.34% |
4.20% |
83.51% |
6.81% |
72.90% |
5.08% |
| Triticonazole |
83.23% |
5.56% |
84.68% |
5.43% |
78.45% |
5.79% |
| Etoxazole |
81.53% |
7.14% |
83.23% |
3.66% |
73.30% |
11.86% |
| Prochloraz |
89.44% |
6.10% |
92.95% |
4.65% |
77.57% |
8.82% |
| Spirodiclofen |
87.52% |
4.87% |
89.65% |
9.31% |
77.74% |
7.74% |
The pesticide recoveries generated exceeded 70% for all matrices, and the comparative standard deviations were less than 15%, indicating excellent recoveries and reproducibility, in only one simple step. Therefore, the automated EDGE is a rapid and efficient alternative to the QuEChERS method.
Additionally, the EDGE successfully extracted the pesticides from the spices efficiently in less than seven minutes, including filtration, cooling and system washing. Table 1 exhibits the EDGE recovery data of numerous pesticides from cinnamon, oregano, black pepper and paprika via UPLC MS/MS analysis.
Table 1. EDGE Recovery of Multiple Pesticides from Spices. Source: CEM Corporation
| |
Cinnamon |
Oregano |
Black Pepper |
Paprika |
| Compound |
Average |
STDEV |
Average |
STDEV |
Average |
STDEV |
Average |
STDEV |
| Acephate |
84.72% |
4.26% |
91.35% |
7.22% |
91.04% |
2.60% |
82.25% |
4.01% |
| Fuberidazole |
78.44% |
4.13% |
86.11% |
8.32% |
94.44% |
0.61% |
86.54% |
2.41% |
| Omethoate |
94.08% |
9.95% |
95.73% |
11.25% |
87.61% |
4.01% |
95.09% |
1.17% |
| Monocrotophos |
104.36% |
18.67% |
92.35% |
8.35% |
87.44% |
1.65% |
86.50% |
4.53% |
| Dimethoate |
100.60% |
2.41% |
86.46% |
8.19% |
99.31% |
5.35% |
95.10% |
1.47% |
| Dicrotophos |
85.50% |
5.89% |
90.75% |
8.32% |
95.19% |
1.83% |
97.87% |
4.14% |
| Trichlorfon |
91.67% |
5.14% |
105.09% |
11.05% |
86.39% |
3.71% |
89.92% |
11.11% |
| Penconazole |
95.01% |
2.88% |
88.55% |
9.06% |
84.43% |
10.70% |
91.79% |
1.16% |
| Vamidothon |
86.76% |
5.49% |
103.66% |
9.46% |
92.33% |
3.83% |
110.78% |
3.85% |
Cyproconazole
Isomers |
90.22% |
9.71% |
83.68% |
11.35% |
77.50% |
13.45% |
94.35% |
1.19% |
| Paclobutrazol |
81.70% |
3.25% |
89.88% |
3.35% |
72.91% |
4.44% |
89.04% |
1.33% |
| Flutriafol |
95.07% |
3.03% |
88.68% |
8.44% |
82.41% |
10.26% |
93.55% |
0.50% |
| Tebuconazole |
95.39% |
3.07% |
82.62% |
9.22% |
102.21% |
2.47% |
95.36% |
2.42% |
| Hexaconazole |
92.42% |
3.51% |
85.36% |
9.58% |
80.71% |
2.73% |
90.35% |
2.02% |
| Flusilazole |
95.01% |
3.00% |
85.84% |
9.60% |
91.01% |
2.48% |
92.69% |
2.80% |
| Desmedipham |
100.60% |
2.50% |
78.61% |
0.42% |
93.37% |
3.71% |
96.15% |
1.03% |
| Metconazole |
94.16% |
1.68% |
87.39% |
8.88% |
80.56% |
3.75% |
89.49% |
5.55% |
| Diniconazole |
93.24% |
0.62% |
90.20% |
9.20% |
92.54% |
4.98% |
99.08% |
7.16% |
| Etaconazole |
95.57% |
4.49% |
83.72% |
8.19% |
77.00% |
13.32% |
88.62% |
7.69% |
| Pencycuron |
90.21% |
1.13% |
78.60% |
3.31% |
93.17% |
7.80% |
95.83% |
6.67% |
| Epoxiconazole |
97.40% |
2.95% |
84.60% |
8.70% |
96.25% |
18.69% |
87.85% |
8.81% |
| Fenarimol |
96.35% |
4.89% |
84.28% |
8.07% |
95.35% |
15.98% |
91.11% |
6.32% |
| Ipconazole |
93.17% |
3.39% |
84.65% |
11.38% |
95.66% |
2.95% |
86.64% |
8.96% |
| Fenbuconazole |
95.34% |
3.57% |
85.35% |
9.60% |
98.21% |
18.14% |
89.09% |
6.32% |
| Bitertanol |
100.38% |
2.82% |
84.08% |
7.74% |
107.76% |
24.04% |
89.18% |
5.81% |
| Triflumizole |
91.61% |
5.92% |
88.40% |
11.70% |
85.86% |
4.56% |
98.02% |
0.25% |
| Etoxazole |
91.55% |
2.74% |
80.78% |
9.46% |
99.28% |
4.13% |
83.67% |
1.45% |
| Spirotetramat |
97.21% |
3.59% |
66.52% |
13.86% |
110.81% |
5.49% |
89.56% |
9.22% |
| Dimethomorph |
92.18% |
2.39% |
86.63% |
7.22% |
88.89% |
9.58% |
87.09% |
10.41% |
| Difenoconazole |
76.53% |
0.35% |
84.50% |
9.94% |
103.31% |
1.20% |
90.39% |
7.73% |
| Spirodiclofen |
91.96% |
1.97% |
79.72% |
8.37% |
100.64% |
7.70% |
97.18% |
4.97% |
| Spinosyn A |
37.81% |
5.79% |
65.99% |
11.58% |
85.75% |
12.91% |
94.45% |
0.06% |
Table 2 displays the QuEChERS data of a number of pesticides recovered from cinnamon, oregano, black pepper and paprika via UPLC MS/MS analysis. All-in-all, improved recoveries and RSD values were acquired via the EDGE extraction in contrast to QuEChERS.
Table 2. QuEChERS Recovery of Multiple Pesticides from Spices. Source: CEM Corporation
| |
Cinnamon |
Oregano |
Black Pepper |
Paprika |
| Compound |
Average |
STDEV |
Average |
STDEV |
Average |
STDEV |
Average |
STDEV |
| Acephate |
41.03% |
5.69% |
34.37% |
3.76% |
60.37% |
14.35% |
63.47% |
10.03% |
| Fuberidazole |
32.18% |
7.50% |
35.02% |
5.05% |
55.43% |
14.21% |
44.89% |
6.87% |
| Omethoate |
44.14% |
6.39% |
33.80% |
3.77% |
61.83% |
14.84% |
54.09% |
9.42% |
| Monocrotophos |
46.38% |
7.09% |
39.30% |
3.00% |
62.78% |
15.95% |
50.20% |
10.09% |
| Dimethoate |
46.29% |
6.65% |
37.31% |
3.72% |
66.40% |
15.68% |
72.41% |
15.89% |
| Dicrotophos |
40.27% |
5.72% |
40.15% |
8.04% |
64.60% |
16.38% |
64.35% |
11.88% |
| Trichlorfon |
48.40% |
7.42% |
40.69% |
3.76% |
64.16% |
16.61% |
85.83% |
6.76% |
| Penconazole |
46.23% |
7.64% |
43.47% |
5.08% |
73.62% |
13.18% |
62.49% |
14.94% |
| Vamidothon |
46.91% |
5.89% |
49.49% |
14.82% |
63.00% |
16.45% |
61.80% |
11.91% |
Cyproconazole
Isomers |
48.91% |
5.94% |
44.67% |
4.91% |
58.74% |
10.65% |
64.40% |
23.31% |
| Paclobutrazol |
67.31% |
10.41% |
63.46% |
26.63% |
91.60% |
28.59% |
65.17% |
17.44% |
| Flutriafol |
49.11% |
7.36% |
46.34% |
5.98% |
62.54% |
16.31% |
65.82% |
16.58% |
| Tebuconazole |
42.98% |
7.26% |
44.82% |
2.06% |
32.21% |
8.12% |
63.29% |
15.71% |
| Hexaconazole |
49.65% |
8.04% |
46.91% |
6.38% |
60.41% |
17.70% |
65.28% |
14.21% |
| Flusilazole |
48.47% |
7.87% |
79.82% |
6.42% |
53.40% |
13.24% |
65.96% |
14.00% |
| Desmedipham |
46.35% |
8.19% |
39.58% |
1.83% |
55.06% |
14.74% |
59.42% |
14.59% |
| Metconazole |
42.44% |
6.29% |
41.88% |
1.53% |
51.96% |
16.46% |
60.00% |
17.65% |
| Diniconazole |
34.67% |
6.89% |
35.34% |
3.90% |
100.95% |
2.15% |
55.66% |
11.24% |
| Etaconazole |
45.34% |
8.88% |
45.76% |
4.12% |
0.00% |
0.00% |
63.22% |
16.31% |
| Pencycuron |
48.55% |
7.08% |
42.83% |
4.94% |
64.29% |
14.55% |
62.59% |
15.71% |
| Epoxiconazole |
46.96% |
7.29% |
44.93% |
2.76% |
54.25% |
18.33% |
61.03% |
13.97% |
| Fenarimol |
48.90% |
7.19% |
31.04% |
26.17% |
74.14% |
16.39% |
65.97% |
17.11% |
| Ipconazole |
42.00% |
7.04% |
42.62% |
4.06% |
56.87% |
15.33% |
60.08% |
14.11% |
| Fenbuconazole |
47.38% |
6.95% |
45.35% |
2.62% |
62.10% |
15.75% |
63.10% |
15.32% |
| Bitertanol |
46.32% |
5.72% |
43.67% |
4.82% |
69.58% |
44.82% |
66.25% |
17.46% |
| Triflumizole |
43.67% |
7.73% |
44.22% |
4.75% |
60.27% |
15.65% |
63.98% |
16.18% |
| Etoxazole |
43.37% |
5.30% |
41.48% |
4.54% |
56.78% |
14.12% |
64.12% |
19.35% |
| Spirotetramat |
60.77% |
12.71% |
54.61% |
5.03% |
52.93% |
13.78% |
64.65% |
15.96% |
| Dimethomorph |
43.67% |
8.68% |
39.25% |
20.25% |
57.71% |
16.30% |
80.20% |
32.04% |
| Difenoconazole |
43.02% |
6.65% |
42.76% |
2.93% |
59.50% |
13.79% |
61.36% |
15.05% |
| Spirodiclofen |
55.05% |
10.94% |
52.77% |
3.31% |
52.60% |
12.58% |
63.48% |
17.80% |
| Spinosyn A |
48.81% |
36.19% |
34.28% |
3.93% |
32.76% |
25.95% |
29.16% |
8.62% |
Additionally, the adjustments necessary to make the QuEChERS method applicable to the dry nature of the spices resulted in a long and tedious process. With QuEChERS already being a labor-intensive manual process, the EDGE delivers the added benefit of a simple, automated method, along with enhanced recoveries.
Conclusion
The EDGE extracted the pesticides in quick succession from 10 g samples of homogenized cucumber, tomato and green pepper, utilizing an innovative bubbling feature and Q-Matrix Hydra. Recoveries were achieved with more than 70% and exceptional reproducibility (RSDs less than 15%).
The EDGE builds upon a number of disadvantages that hold the QuEChERS method back. These include, waste generation and the necessity to make several sample transfers, by using Q-Cup Technology and automation. Predicated on the variety of matrices tested, the method as detailed above can be applied to other fruits and vegetables.
Furthermore, the extraction process built into the EDGE automated extraction system facilitated the effective extraction from a complex matrix of spices. A single CEM-approved extraction method was used for all spices which simplified the process significantly, in comparison to QuEChERS.
With a single automated method, pesticides were extracted more efficiently than with the conventional QuEChERS process.
Some pesticides are known to be vulnerable to heat. For samples where temperature control is a concern, a room temperature extraction can be programmed on the EDGE.
The EDGE, with its effective pesticide extraction method, is the optimal choice for testing labs that want just one automated method with good repeatable results for all samples.
This information has been sourced, reviewed and adapted from materials provided by CEM Corporation.
For more information on this source, please visit CEM Corporation.