ISOLUTE® QuEChERS extraction of pesticides from spinach

Introduction

The diverse nature of the samples in food safety testing applications adds complexity making method standardisation challenging. However, default methodology for solid matrix pesticide analysis is firmly centred around ISOLUTE® QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) consisting of initial salt assisted solvent extraction and partitioning followed by dSPE (Dispersive Solid Phase Extraction) clean up.

This application brief describes Quechers extraction of a representative panel of 50 pesticides, incorporating pesticides, fungicides and insecticides from spinach samples.

Spinach extraction was performed using pre-packaged AOAC QuEChERS salts and a dSPE blend optimized for pigmented fruit and vegetables. Samples were homogenized and the initial salt assisted extraction performed using Biotage® Lysera bead mill homogenizer.

Following dSPE clean up, samples were analysed by GC-MS in SIM mode.

Experimental

Reagents:

Pesticide standards were purchased from Sigma-Aldrich (Dorset, UK). Spinach was sourced from local vendors. All solvents were HPLC grade from Fisher Scientific (Loughborough, UK) and Milli-Q (Merck Millipore, Germany) water used throughout.

Sample preparation

Matrix preparation:

15 g of sample was weighed into 50 mL Biotage® Lysera centrifuge tubes containing 15 g of ceramic beads.

Maceration procedure:

Simultaneous maceration of 3 spinach samples was achieved using a Biotage® Lysera equipped with 50 mL tube carriage kit as shown in Figure 1.: 4 x 40s cycles at 5 m/s with 20s dwell.

Figure 1. Biotage® Lysera with 50 mL tube carriage kit.

QuEChERS salt assisted extraction: 15 mL ACN was added to each sample followed by the standard AOAC QuEChERS salt mix (p/n Q0010-15V). Further mixing was performed on the Lysera using a single cycle of 60s at 0.8 m/s. Extracts were then centrifuged for 5 minutes at 4000 rpm to separate layers.

Figure 2 shows the homogenous spinach sample after maceration using Biotage® Lysera, and the sample resulting from the initial Quechers extraction.
Figure 2. Macerated spinach sample (Left) and Quechers extracted sample (after centrifugation) (Right).

dSPE: 2 mL aliquots of ACN extract (top layer) spiked with 24 µL of 10 ng/µL pesticide standard mix (equivalent to a 120 ppb concentration in raw matrix) were further cleaned up using 500 mg of ISOLUTE® Quechers AOAC Pigmented Fruit and Vegetable dSPE blend (from p/n Q0070-15V). Manual dSPE processing was performed by mixing the samples for 1 minute on a rotary mixer followed by centrifugation for 5 minutes at 4000 rpm.
Figure 3. Spinach extract during dSPE processing and after centrifugation.

Post extraction:

A 1 mL aliquot was removed and evaporated in high recovery GC vials at 40 °C for approximately 20 minutes using a TurboVap® 96 Dual with a 24 position adapter, followed by reconstitution in 30 μL of toluene prior to GC/MS analysis.

GC conditions

Instrument:

Agilent 7890A G (Agilent Technologies)

Column:

Restek Rxi-5ms, 30 m x 0.25 mm ID x 0.25 μm

Carrier gas:

Helium 1.2 mL/min (constant flow)

Inlet:

280 °C, Splitless, purge flow: 50 mL/min at 1.0 min

Injection volume:

1.3 μL

Oven conditions:

Initial temperature 90 °C

Ramp:

Available on request

Backflush:

3 void volumes (2.4 mins)

Transfer line:

300 °C

MS conditions

MS:

5975C MSD (Agilent Technologies Inc.).

Source temperature:

230 °C

Quadrupole temperature:

150 °C

Monitored ions:

EI signals were acquired using selected ion monitoring (SIM) mode. Monitored ions for each panel available on request.

Results

GC-MS SIM chromatograms for the 50 pesticide panel is shown in figure 4 (A = standard, B = Matrix blank, C = Spiked matrix).

ISOLUTE® Quechers provided high analyte recovery with the majority of analytes higher than 90% for a representative panel of 50 pesticides extracted from spinach (see figure 5) (spiked at 120 ppb equivalent in raw matrix). Good reproducibility (RSDs mostly <15%, n=7) was achieved (Figure 6).
A)
B)
C)
Figure 4. GC/MS SIM chromatograms (A = standard, B = Matrix blank, C = Spiked matrix).


Matrix removal using the AOAC Pigmented Fruit and Vegetable dSPE blend provided adequate sample clean up. Figure 7 shows the extracted spinach sample before and after dSPE clean up. Pigment is clearly reduced, allowing for robust analysis.
Figure 7. Extracted spinach sample before and after dSPE clean up.
Figure 8 shows the matrix factors, showing that matrix components are efficently removed.

Conclusion

Homogenization using Biotage® Lysera provided a homogenous sample suitable for subsequent salt assisted extraction. Use of the Lysera system for the salt assisted extraction step facilitated mixing sample, solvent and extraction salts in the same extraction tube, requiring no sample transfer step.

ISOLUTE® Quechers provided high analyte recovery with the majority of analytes higher than 90% for a representative panel of 50 pesticides extracted from spinach. Good reproducibility (RSDs mostly <15%, n=7) was achieved.

The AOAC Pigmented Fruit and Vegetable dSPE blend provided good removal of matrix components, allowing for robust and reliable analysis. 

Ordering information

Part Number: AN979