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Analysis of 18 Pesticide Residues in Spinach Using CarbonX for QuEChERS AOAC dSPE Kits by GC/MS 

 

Authors: 

Conor Smith Ph.D., Doug Fryer

United Science Corp. 15911 Furuby Rd.

Center City, MN55012 USA

 

 Abstract

 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 spinach. The pesticides are all amenable to GC/MS analysis. The method involves the extraction of the pesticides from homogenized spinach 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 CoA (to remove pigments and sterols), CarbonX PSA (to remove sugars, fatty acids, organic acids) 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. The limit of quantitation (LOQ) for most pesticides, excluding Folpet, was 10 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.

 

Reagents and Chemicals

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. A 15 g (±0.1 g) amount of homogenized spinach sample was placed into a 50 mL centrifuge tube along with an extract packet containing 6g of MgSO4 and 1.5 g NaAcetate. (United Science P/N 1605165160) Next, 15 mL of 1% Acetic Acid in ACN is added to each tube. Sample tubes are capped tightly, and hand-shaken vigorously for 1 min. Tubes are centrifuged at 4000 rpm for 5 min. Next, the ACN extract is decanted into a separate container. The volume of ACN extracts (about 10 mL) will be enough for multiple sample tests when using 2 mL dispersive SPE tubes (United Science P/N 1605040110).

For preparation of the spiked solutions the ACN extract is added to the 2 mL tube followed quickly by the corresponding amount of spiking solution. The 2 mL tube contains 50 mg of CarbonX PSA, 50 mg of CarbonX CoA and 150 mg of anhydrous MgSO4. For the 200 ng/g tube, 960 µL of ACN extract is added followed by 40 µL of spiking solution. For the 50 ng/g tube, 990 µL of ACN extract is added followed by 10 µL of spiking solution. For the 10 ng/g tube, 998 µL of the ACN extract are added followed by 2 µL of the spiking solution.

The tubes are tightly capped and vortexed for 1 min. We suggest vortexing the tubes for a few seconds before adding the sample, to prevent possible agglomerates. The 2 mL tubes are centrifuged with a micro-centrifuge at 13,000 rpm for 2 min. The samples are then transferred directly into autosampler vials for GC/MS injection.

The standards are prepared via the same method except that no spiking occurs before removal of the pigment and extra tubes must be used to result in more than 3 mL of de-pigmented matrix. This de-pigmented matrix is then spiked in a similar manner described above for 200, 50, and 10 ng/g standards. These standards are then transferred directly into autosampler vials for GC/MS injection.

 

Recovery and Reproducibility

The recovery and reproducibility were evaluated by spiking pesticides standards in comminuted spinach sample at levels of 10, 50 and 200 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, PSA, MgSO4 tube, followed by spiking of the resulting matrix blank. The analysis was performed in replicates of three (n = 3) at each level.

 

Instrument Conditions

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 200 ng/g fortified spinach 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.

Figure 2. Pesticide recovery data for CarbonX at 10, 50, and 200 ng/g.  Note good recovery of planar pesticides Chlorothalonil, Dichlorobenzophenone, Folpet, and Coumaphos without the need for additional solvents.

PDF version available here.

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