This application note describes the use of a quick, easy, cheap, effective, rugged, and safe (QuEChERS) AOAC sample preparation technique used in the extraction and cleanup of 18 pesticide residues in red pepper. The pesticides are all amenable to GC/MS analysis. The method involves the extraction of the pesticides from homogenized red pepper with a buffered aqueous/acetonitrile solvent system with partitioning using added salt. This is followed by cleanup using dispersive solid phase extraction (dSPE) with CarbonX for QuEChERS (to remove pigments and sterols), PSA (to remove sugars, fatty acids, and organic acids), C18 functionalized silica (to remove fats and waxes), and MgSO₄ (to remove leftover water). The pesticides are then analyzed by GC/MS operating in selective ion monitoring (SIM) mode. The method was validated in terms of recovery and reproducibility. All pesticides were evaluated at 50 ng/g. The application produced excellent recoveries for all pesticides, including planar pesticides, without the need for the addition of toxic solvents such as toluene.

All reagents and solvents were high-performance liquid chromatography (HPLC) grade. Methanol (MeOH) was from sigma Aldrich, Acetonitrile (ACN) was from J&W, and glacial acetic acid (HAc) was from Ampresco. All pesticide standards were purchased from Sigma-Aldrich.

 Solutions and Standards

A 1% acetic acid in ACN solution was prepared by adding 10 mL of HAc to 1 L of ACN.

Standard stock solution (2 mg/mL) was made in MeOH and stored at -20 °C. This solution was diluted to a spiking solution (5 µg/mL in MeOH) for use in the method.

 Sample Preparation

The sample preparation procedure includes sample comminution, extraction and partitioning, and dispersive SPE clean-up. Two 15 g (±0.1 g) amounts of homogenized red pepper sample are placed into two 50 mL centrifuge tubes. For the spiked sample, 150 µL of spiking solution is added, and then the tube is vortexed for 30 seconds. Next, 15 mL of 1% Acetic Acid in ACN is added to each tube. Finally, an extract packet containing 6g of MgSO₄ and 1.5 g NaAcetate is added. (United Science P/N 1605165160) is added. Sample tubes are capped tightly, and hand-shaken vigorously for 1 min. Tubes are centrifuged at 4000 rpm for 5 min. Next, the ACN extracts are decanted into separate containers. The volume of ACN extracts (about 10 mL per extract) will be enough for multiple sample tests when using 2 mL dispersive SPE tubes (United Science P/N 1605040114).

The 2 mL tubes contain 50 mg of PSA, 50 mg of CarbonX for QuEChERS, 50 mg of C18 functionalized silica, and 150 mg of anhydrous MgSO₄. We suggest vortexing the tubes for a few seconds before adding the sample, to prevent possible agglomerates. For both the spiked and unspiked extract, 1 mL is added to the 2 mL dSPE tubes. At least three tubes are used for the unspiked extract so that there is sufficient cleaned up matrix for the matrix standard. The tubes are tightly capped and vortexed for 1 min. The 2 mL tubes are centrifuged with a micro-centrifuge at 13,000 rpm for 2 min. The spiked samples are then transferred directly into autosampler vials for GC/MS injection.

For the matrix standard, 990 µL of cleaned up extract is added to a vial, followed by 10 µL of spiking solution. This mixture is then added into an autosampler vial for GC/MS injection.

Recovery and Reproducibility

The recovery and reproducibility are evaluated by spiking pesticide standards in comminuted red pepper sample at 50 ng/g. These samples were quantitated against a matrix standard. The ACN extract is spiked with microliter amounts of the pesticide mixture in MeOH. The standard is prepared by first removing the pigment through dSPE with the CarbonX for QuEChERS, PSA, C18, and MgSO₄ tube, followed by spiking of the resulting matrix blank. The analysis was performed in replicates of three (n = 3) at each level.

Instrument Condition

An Agilent 5890 GC/MS was used for this study.

GC conditions

Inlet:                                                    Splitless

Inlet liner:                                         Agilent Ultra Inert splitless single taper wool p/n 51903163

Carrier gas:                                        Helium

Inlet pressure:                                 19.6 psi (constant pressure mode) during run

Inlet temperature:                         250 ºC

Injection volume:                           1.0 µL

Purge flow to split vent:               30 mL/min at 0.75 min

Oven temperature program:      70 ºC (1 min), 50 ºC/min to 150 ºC (0 min), 6 ºC/min to 200 ºC (0 min), 16 ºC/min to 280 ºC (6 min)

Column:                                               Agilent J&W HP-5MS Ultra Inert 15 m × 0.25 mm, 0.25 µm (p/n 19091S-431UI)

MSD

MS conditions

Tune file:                                            Atune.u

Mode:                                                  SIM (refer to Table 1 for settings in detail)

Source, quad, transfer

line temperature:                           230 ºC, 150 ºC and 280 ºC respectively

Solvent delay:                                  2.30 min

Multiplier voltage:                         Autotune voltage

 

Table 1 – SIM acquisition parameters used for the analysis of 18 pesticides by GC/MS

Figure 1. GC/MS Chromatogram of 50 ng/g fortified red pepper sample extract processed with CarbonX dSPE method. Peak identification: 1. Dichlorvos 2. Phenylphenol 3. Lindane 4. Diazinon 5. Chlorothalonil 6. Chloropyrifos-methyl 7. Dichlorobenzophenone 8. Chloropyrifos 9. Heptepoxide 10. Folpet 11. ?-Chlordane 12. ?-Chlordane 13. Dieldrin 14. DDE 15. Ethion 16. Endosulfan Sulfate 17. Permethrin 18. Coumaphos,              * Internal Standard Atrazine.

Figure 2. Pesticide recovery data for CarbonX at 50 ng/g.  Note good recovery of planar pesticides Chlorothalonil, Dichlorobenzophenone, Folpet, and Coumaphos without the need for additional solvents.

 

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