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Analysis of 16 Pesticide Residues in Broccoli Using CarbonX dSPE




Conor Smith, Doug Fryer

United Science Corp.

15911 Furuby Rd.

Center City,MN55012

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 16 pesticide residues in broccoli. The pesticides are all amenable to GC/MS analysis. The method involves the extraction of the pesticides from homogenized broccoli 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), 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.

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. Two 15 g (±0.1 g) amounts of homogenized broccoli 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, 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 broccoli 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, 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)


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 broccoli sample extract processed with CarbonX dSPE method. Peak identification: 1. Dichlorvos 2. Phenylphenol 3. Diazinon 4. Chloropyrifos-methyl 5. Dichlorobenzophenone 6. Chloropyrifos 7. Heptepoxide 8. Folpet 9. ?-Chlordane 10. ?-Chlordane 11. Dieldrin 12. DDE 13. Ethion 14. Endosulfan Sulfate 15. Permethrin 16. Coumaphos, * Internal Standard Atrazine.



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


A .pdf of this app note can be downloaded here.


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