Automated clean-up of pesticides in broccoli using ISOLUTE® cSPE for QuEChERS

QuEChERS is a sample preparation technique commonly used in food safety testing for pesticide residue analysis. This two-stage extraction and clean-up technique was developed to streamline sample processing, making it Quick, Easy, Cheap, Effective, Rugged and Safe. ISOLUTE® cSPE for QuEChERS are pre-packed cartridges designed to further improve the efficiency of the QuEChERS clean-up procedure.

Traditionally, clean-up of QuEChERS extracts for pesticide residue analysis relies on dispersive solid phase extraction (dSPE). The dSPE workflow requires that the sample is shaken with the dSPE media and then centrifuged to separate the clean sample extract. Alternatively, ISOLUTE® cSPE for QuEChERS cartridges are packed with QuEChERS ‘dispersive SPE’ sorbent blends allowing convenient flow-through sample processing using column solid phase extraction (cSPE). In addition, the cSPE workflow can be automated on a Biotage® Extrahera™ sample preparation workstation leading to increased throughput, reduced errors, and improved data quality.

Figure 1. Example of pesticide structures.

Introduction

This application note describes the extraction of a panel of 81 commonly analysed pesticides from broccoli using a two-step QuEChERS workflow. Samples are first homogenized and extracted with ISOLUTE® EN QuEChERS extraction salts using the Biotage® Lysera™ bead mill homogenizer. Following centrifu- gation, an aliquot of extract is cleaned up using ISOLUTE® cSPE for QuEChERS cartridges, processed using a Biotage® Extrahera™ automated sample preparation workstation. Extracts are evaporated using TurboVap™ Dual 96 and analysis is performed using LC-MS/MS.

This application note includes optimised conditions for homogenization, extraction, extract clean-up, evaporation, and analysis.

Analytes

To demonstrate the versatility of the sample preparation approach, a panel of 81 analytes were selected to reflect a broad range of pesticide classes and chemical characteristics. The pesticides analysed in this application note are listed below.

Thiabendazole, Carbendazim, Propamocarb, Acephate, Methomyl, Methamidophos, Pentachloronitrobenzene, Thiamethoxam, Flonicamid, Flupyradifurone, Sulfoxaflor, Imidacloprid, Thiacloprid, Clothianidin, Dimethoate, Phosdrin, Acetamiprid, Tricyclazole, Cyantraniliprole, Spiroxamine, Imazalil, Metalaxyl, Carbofuran, Dichlorvos, Oxamyl, Atrazine, Propoxur, Carbaryl, Spinosad, Chlorantraniliprole, Dimethomorph, Pyriclaben, Spirotetramet, Phosmet, Paclobutrazol, Methiocarb, Spinetoram, Mandipropamid, Azoxystrobin, Fluopyram, Fluopicolide, Fluxapyroxad, Methoxyfenozide, Penthiopyrad, Boscalid, Propiconazole, Fenbuconazole, Malathion, Iprodione, Kresoxim methyl, Isoprothiolane, Fenamidone, Diflubenzuron, Tebuconazole, Fenoxycarb, Prallethrin, Bifenazate, Captan, Systhane, Ametoctradin, Ethoprophos, Novaluron, Cyflufenamid, Trifloxystrobin, Pyraclostrobin, Coumaphos, Buprofezin, Pirimiphos methyl, Diazinon, Clofentezine, Dibrom, Cyflumetofen, Bifenthrin, Fenpyroximate, Sprodiclofen, Spiromesifen, Etoxazole, Piperonyl butoxide, Chloropyrifos, Pyriproxyfen, Quinoxyfen.

Sample preparation procedure

Format

Step 1: QuEChERS Extraction: ISOLUTE® EN QuEChERS extrac- tion salts (4 g MgSO4 + 1 g sodium citrate + 0.5 g sodium citrate sesquihydrate+ 1 g sodium chloride) p/n Q0020-15V

Step 2: cSPE Clean-up: ISOLUTE® EN Pigment 300 mg/3 mL (Tabless), p/n Q0080-0030-BG

Homogenization

10 g of manually cut broccoli was weighed into a 50 mL centri- fuge tube with 3.3 mL of H2O and frozen. 3.3 g of metal beads were added to the centrifuge tube and three samples were homogenized simultaneously using the Biotage® Lysera and 50 mL tube carriage with the following settings:

• Speed: 6 m/s
• Processing Time: 40 seconds
• Number of Cycles: 4
• Dwell Time: 15 seconds
  
  

Note: Freezing the sample aids in a more successful homogeni- sation and reduces the risk of increased temperatures. Many pesticides are thermally sensitive to any heat generated during sample processing which may result in loss of recoveries for these compounds. Use of the Biotage® Lysera with optional Cryo Cooling Unit can prevent increase of sample temperature and may protect heat sensitive samples during homogenization.

Step 1: QuEChERS extraction

10 mL of acetonitrile and the ISOLUTE® EN QuEChERS extraction salts were added to the homogenized broccoli and shaken using the Lysera with the following settings:

• Speed: 2.4 m/s
• Processing Time: 15 seconds
• Number of Cycles: 1
• Dwell Time: N/A

The samples were then centrifuged for 5 minutes at 5000 RCF. To evaluate the performance of ISOLUTE® cSPE for QuEChERS clean-up, the supernatant was spiked at a concentration of 30 ppb with a mixture of 81 pesticides (see Additional Information). No internal standards were used.

Step 2: Automated clean-up procedure

An excess of 1 mL of supernatant from step 1 was transferred into 16 x 100 mm test tubes and placed into the upper processing shelf of the Biotage® Extrahera™ HV-5000, (position 4, sample rack 16 x 100 mm, 24 positions). ISOLUTE® EN Pigment cSPE cartridges (p/n Q0080-0030-BG) were placed onto the upper processing shelf of the Biotage® Extrahera™ HV-5000 (position 3, column rack 24 x 3 mL), while collection vials were placed in the lower collection carousel (position B, collection rack 12 x 75 mm). Using the Extrahera™ HV-5000, 1 mL of QuEChERS extract was loaded onto each column. The columns were processed by applying 0.7 bar for 210 seconds, followed by 5 bar for 15 seconds and a 15 second plate dry. Extracts were collected directly into LC vials.

For options to increase batch size and throughput with minimal sample transfer steps, see Appendix 2.

Post extraction

Samples were evaporated using the TurboVap™ 96 Dual with the following parameters:

• 24 Configuration, Single Mode
• Gas Temp: 25 °C
• Plate Temp: 25 °C
• Gas Flow: 25 L/min
• Plate Height: 56 mm
• Time: 30 minutes

Extracts were reconstituted with 20 µL of Methanol plus 380 µL of H2O and vortexed prior to analysis.

Analytical conditions

UHPLC conditions

Instrument

Waters Acquity UPLC

Column

ACE EXCEL C18 (100 x 2.1mm, 1.7 µm) equipped with security guard.

Mobile phase

A: 0.1 % Formic acid in Water B: Acetonitrile

Flow rate

0.3 mL/min

Column temperature

40 °C

Autosampler temperature

10 °C

Injection volume

10 μL (Partial Loop with Needle Overfill)

Table 1. UHPLC gradient.

Curve settings: 1; 6 linear gradient; 11.

Table 2. MS/MS conditions

The monitored ions for each compound are listed in table 3 below. Ions were acquired using Multiple-reaction monitoring (MRM) in positive ion mode.

Table 3. Ions monitored for each analyte.

Results

Using the automated cSPE procedure described in this applica- tion note, a panel of 81 pesticides spiked into broccoli extracts were cleaned up using cSPE. Of the 81 pesticides the majority demonstrated recovery between the acceptable limits of 70-130%.

Figure 2 shows recoveries obtained from broccoli extract spiked prior to cSPE clean-up at a concentration equivalent to 30 ppb in raw broccoli (n=7). Excellent reproducibility was achieved, 81 analytes demonstrating relative standard deviation (RSD) lower than 20% (n=7) with most being lower than 5%.

Figure 3 shows %RSD obtained from broccoli extract spiked prior to cSPE clean-up at a concentration equivalent to 30 ppb in raw broccoli. Automation of the method ensures variation is kept to a minimum.

Note: The effect of adding an additional 50 µL aliquot of aceto- nitrile to each cSPE column following flow through of the extract was evaluated. This was combined with the cleaned-up extract prior to evaporation. This led to a small increase in recovery of 0-10% for most analytes. The data shown in this application note does not include this additional step.

Figure 2. Average analyte % recoveries obtained from broccoli extract spiked at a concentration equivalent to 30 ppb in raw broccoli (n=7). Analytes shown in order of chromatographic elution.

Figure 3. % RSDs obtained from broccoli extract spiked at a concentration equivalent to 30 ppb in raw broccoli (n=7).

Linearity and LOQ 

Calibration curves were produced for broccoli extract spiked with pesticides, within the concentration range 0.38 ppb to 75 ppb (equivalent in raw broccoli). 

To ensure that calibration samples reflect real samples as closely as possible, samples were prepared using pooled broccoli extract (from QuEChERS step 1). The highest-level
spiked sample was prepared at a concentration of 75 ppb. The remaining spiked samples we re prepared by a serial dilution of the highest-level spiked sample using additional blank broccoli extract. 1 mL of each concentration of calibration samples were then cleaned up using cSPE columns. 

Calibration curves were gene rated down to 0 38 ppb. Where analyte sensitivity was below this level, an estimate of LOQ has been determined based o n signal to noise ratios (S/N) > 10:1, see table 4 below. Good linearity was observed for most analytes, typically r2 values greater than 0.99.

Table 4. Linearity and LOQ

*Data generated without the use of internal standards, see additional information.

Calibration curves

Calibration curves for selected pesticides are shown in figures 4-7 below.
Figure 4. Methamidophos, calibration range 0.38 – 75 ppb
Figure 5. Carbofuran, calibration range 0.38 – 75 ppb
Figure 6. Methiocarb, calibration range 0.38 – 75 ppb
Figure 7. Malathion, calibration range 0.38 – 75 ppb

Discussion and conclusion

The use of ISOLUTE® cSPE for QuEChERS cartridges provides a simple, robust flow-through clean-up of QuEChERS extracts from broccoli samples for pesticide analysis. The cartridges deliver high recoveries, low RSDs, and exceptionally clean extracts.

Excellent linearity was achieved across a wide calibration range for all analytes, demonstrating that the flow-through cSPE approach provides consistent analyte recovery and matrix removal at all concentration levels, with LOQ typically < 1 ppb.

When automated using the Biotage® Extrahera™ HV-5000, a batch of 24 samples can be processed in 9.2 minutes (13.75 minutes for 48 samples). ISOLUTE® cSPE for QuEChERS cartridges provide a simplified extract clean-up workflow when compared to the dSPE equivalent (see figure 8, page 9). This results in fewer manual transfer steps and higher throughput. In comparison, the equivalent dSPE clean-up procedure takes 23.5 minutes for a batch of 24 samples.

Traditionally QuEChERS using dSPE for clean-up does not utilize a concentration step. However, standard QuEChERS methods (AOAC, EN and mini-multiresidue) allow for an evaporative concentration step where solvent exchange from acetonitrile to one that is both beneficial to the LC system and the pesticides. Typically, a concentration factor of x4 is utilized.

To improve our working sample concentration range we incorporated a post clean-up evaporation and reconstitution step. After cSPE clean-up the extract was evaporated to dryness under highly controlled ambient conditions using the TurboVap® 96 Dual and reconstituted in a total volume of 400 µL of water: methanol (95:5 v/v), from which 10 µL was injected. No significant evaporative losses were observed. This represents a 2.5 x increase in analyte concentration injected into the LC-MS/MS system, allowing for a more appropriate injection solvent for analysis.

Figure 8. Comparison of dSPE vs cSPE clean-up workflow

Chemicals and reagents

• All pesticide stock solutions were purchased from LGC Ltd. (Middlesex, UK) and stored at -20°C.
• California Pesticide Class 1, Class 2A, Class 2B mixes were purchased at 100 µg/mL. All other compounds were purchased as individual stocks at 1 mg/mL
  and diluted to 100 µg/mL with acetonitrile.
• A spiking solution was prepared weekly in methanol at a concentration of 4 µg/mL and stored at -20°C.
• Methanol was purchased from Rathburn Chemicals Ltd. (Walkerburn, UK).
• Acetonitrile was purchased from Rathburn Chemicals Ltd. (Walkerburn, UK).

Additional information

Preparation of pooled sample for cSPE evaluation

The focus of this application note is evaluation of the automated clean-up step using cSPE columns. Therefore, to provide homogenous samples for comparison purposes, supernatants prepared in step 1 were pooled and spiked appropriately with pesticide standards. 1 mL aliquots of the pooled supernatants were used for evaluation of the cSPE clean-up step.

For recovery testing, the pooled sample was spiked at a concentration of 40 ng/mL which equates to 30 ppb in raw broccoli matrix. This is calculated with the assumption that 10 g of broccoli matrix with QuEChERS salts results in 7.5 mL of supernatant for cSPE clean up. Supernatant volumes will be dependent on matrix type and initial QuEChERS salt used.

Additional 50 µL volume of acetonitrile can be loaded onto each column to improve recoveries if struggling for sensitivity. For determination of linearity and LOQ, the highest-level spiked sample was prepared at a concentration of 75 ppb. The remaining spiked samples were prepared by a serial dilution of the highest-level spiked sample with the pooled blank broccoli extract.

Use of internal standards

No internal standardisation was used in this application note, however if used, we would recommend that appropriate internal standards were added to the homogenised matrix along with acetonitrile and QuEChERS salts during step 1, prior to mixing and subsequent centrifugation.

Evaporation and reconstitution

The use of LC vials (Greyhound p/n 545100-406) for extract collection, evaporation and reconstitution reduces variability by eliminating any transfer steps. Pre- and post-evaporation sample vials are illustrated in figure 9 below. Pre-evaporation sample (left) consists of 1 mL of broccoli extract in acetonitrile after passing through the cSPE column (Q0080-0030-BG). Post-evaporation sample (right) is the same extract after evaporation to dryness and reconstitution in 400 µL H2O: MeOH 95:5 v/v (2.5 x concentration factor). Direct to LC vial elution eliminates the need for sample transfer for evaporation, and further reduces the risk of analyte loss.

Figure 9. Pre (left)- and post (right)-evaporation and reconstitution samples in LC vials.

Appendix 1: Biotage® Extrahera™ HV-5000 method

The cSPE clean-up method described in this application note was automated on the Biotage® Extrahera™ HV-5000. This appendix contains the software settings required to configure the Extrahera™ HV-5000 to run this method for a 1 mL sample aliquot.

Appendix 2: Options for streamlining the automated QuEChERS process

Homogenization & Extraction

As part of the QuEChERS workflow, use of the Biotage® Lysera for initial sample pre-treatment and extraction (QuEChERS step 1) reduces the need for manual steps, saving time and reducing the possibility of manual handling errors. The need to clean a blending device in-between each sample is eliminated and an even homogenisation between each sample is ensured.

In this application note, ISOLUTE® EN QuEChERS extraction salts (p/n Q0020-15V) optimized for extraction of 10 g of sample matrix were used to prepare samples in 50 mL tubes, for the initial extraction step.

In place of manually shaking samples by hand, 3 x 50 mL tubes can be shaken simultaneously in 15 seconds using the Biotage Lysera.

Clean-up

Using the Biotage® Extrahera™ HV-5000 platform for automated clean-up, 50 mL extraction tubes from the initial homogeniza- tion and extraction step can be transferred directly to the Extrahera™ HV-5000 after centrifugation, eliminating a manual sample transfer step. The supernatant can then be aspirated directly from the 50 mL centrifuge tube to the cSPE column utilizing the Extrahera’s ‘Smart Pipetting’ function, ensuring no particulate is transferred. A maximum of 12 samples can be processed using 50 mL tubes.

Increasing batch size and sample throughput with minimal manual sample transfer

As an alternative to 50 mL tubes for homogenization and extraction, Biotage® Lysera can be used to homogenize samples in smaller tubes (30 mL, 7 mL) simultaneously processing batch sizes of up to 12 samples.

Reduction of the starting mass of sample, for example from 15 g to 5 g or 2 g, extracted with an appropriately reduced weight of extraction salt, and utilizing smaller extraction tubes can lead to increased batch size and further reduction in overall processing time.

After centrifuging these can be transferred directly to the Extrahera™ HV-5000 for cSPE clean-up, with no additional manual sample transfer steps. These options are summarized in the table below.

Options for increasing batch size with minimal manual sample transfer

Alternatively, a manual transfer of the supernatant into test tubes can be included after QuEChERS extraction, which can then be processed in a batch size of up to 48.

Manual processing options

Clean-up of pre-extracted samples using ISOLUTE cSPE for QuEChERS cartridges can be performed manually using the Biotage® Pressure+48 Positive Pressure Manifold or Biotage® VacMaster™-20. Processing parameters are available on request.

Literature Number: AN1009