A Rapid Method for the Screening and Confirmation of Over 400 Pesticide Residues in Food

To utilize the power of UltraPerformance Liquid Chromatography (UPLC) combined with fast MS acquisition rates, to give a rapid method for the screening of 402 pesticide residues in a single 10 minute run. A second injection, for confirmatory purposes, will meet SANCO Analytical Quality Control procedures for pesticide residue analysis (SANCO/2007/3131¹).

Pesticides are widely used in the production of foodstuffs to meet consumer demand for plentiful food at reasonable prices, all year round. However, continued growth in the use of pesticides, poor agricultural practices, and illegal use can pose significant risks to human health through the presence of pesticide and metabolite residues in food products. Most countries have strict regulations governing pesticides. Legislation imposes Maximum Residue Limits² (MRLs) for pesticide residues in food products requiring analytical techniques that are sensitive, selective, and robust.

Multi-residue pesticide analysis is challenging due to the low levels present, the wide variety of pesticides, and the very different chemical classes they represent. As there are currently well over 1,000 pesticides in use, laboratories are under increasing pressure to broaden the range of pesticides determined in a single analysis over a shortened run time.

The need to meet mandated detection limits, develop generic sample preparation techniques for complex matrices, and the desire to increase sample throughput are the main challenges facing food safety testing laboratories today. The use of a single multi-residue method per instrument can dramatically improve return on investment by removing the need to change method parameters. This is often the case when analyzing a wide variety of commodities with differing lists of legislated pesticides.

Advances in chromatographic separation and detection technologies have enabled analysts to increase the number of analytes determined in a single run. Tandem quadrupole mass spectrometry offers a highly specific and selective detection technique that has become the technique of choice within the laboratory.³

The following method describes a solution for the rapid analysis of pesticides in mango, avocado, and fruit-based baby food extracts that is able to exceed current worldwide legislation.

Dispersive SPE, commonly referred to as “QuEChERS”, is a simple and straightforward sample preparation technique suitable for multi-residue pesticide analysis in a wide variety of food and agricultural products.⁴ The homogenized food samples were extracted with organic solvent using Waters DisQuE dispersive sample preparation tubes. Once mixed, the pesticide residues were partitioned into the organic solvent, which was then subjected to further clean-up. The supernatant was collected, diluted, and injected onto the LC-MS/MS system as described below:

Extraction Procedure⁴:

1. Add 15 g homogenized sample to a 50-mL DisQuE extraction tube containing 1.5 g sodium acetate and 6 g magnesium sulfate. Add 15 mL 1% acetic acid in acetonitrile.
2. Add any pre-extraction internal standards.
3. Shake vigorously for one minute and centrifuge > 1500 rcf for one minute.
4. Transfer 1 mL of the acetonitrile extract in to the 2-mL DisQuE extraction tube containing 50 mg PSA and 150 mg of magnesium sulphate.
5. Shake for 30 seconds and centrifuge >1500 rcf for one minute.
6. Transfer 100 μL of final extract into an autosampler vial. Add any post-extraction internal standards. Dilute with 900 μL water.

Acquisition and Processing methods

The data were acquired using Waters MassLynx Software, v. 4.1. Incorporated into MassLynx, the IntelliStart technology automates optimization of MS parameters for the sample and also monitors the health of the MS system, reducing the time for operator-intensive troubleshooting and upkeep.

This data was processed using TargetLynx Application Manager. This quantification package enables automated data acquisition, processing, and reporting for quantitative data, incorporating a range of confirmatory checks that identify samples that fall outside user-specified or regulatory thresholds.

The analysis of 402 pesticide residues (Appendix 1) in mango, avocado, and fruit-based baby food was achieved using ACQUITY TQD: liquid chromatography combined with tandem quadrupole mass spectrometry (UPLC-MS/MS) operated in Multiple Reaction Monitoring (MRM) mode.

The rapid determination and confirmation method was achieved in two parts. Part one was a single injection with one MRM transition per pesticide, ideal for screening purposes. Part 2, where compounds of interest can then be confirmed, was achieved by two separate injections with two MRM transitions per pesticide.

Figure 1 shows all 402 pesticide residues in one 10 minute run, fully utilizing the enhanced speed and resolution of UPLC.

For all injections, the same UPLC conditions were used saving analytical time and costs, thus maximizing return on investment. This single setup will allow analysts with less experience to run the method as the need for changes to be made in between batches is removed.

The IntelliStart technology provides simple instrument setup and MS method development and therefore easy access even for the most inexperienced MS user.

Part 2, where compounds of interest can then be confirmed, was achieved by two separate injections with two MRM transitions per pesticide. Figures 2 and 3 show the separation of 201 pesticide residues across two run times of 10 minutes each.

The selectivity given using a tandem quadrupole mass spectrometer (ACQUITY TQD) shows an advantage over a single quadrupole instrument as it allows co-eluting compounds to be identified and quantified with confidence.

The enhanced speed and resolution of UPLC enabled all peaks to elute within eight minutes. Dwell times of 5 ms were used to achieve at least 12 data points across each peak for both quantification and confirmatory ions.

A calibration curve was prepared in the injection solvent (water:methanol, 90:10 v/v) and injected. Excellent linearity was achieved using a weighting factor of 1/x with a high coefficient of determination achieved. This is shown in Figure 4.

The 402 pesticide mix was spiked into the three matrices and the extracts analyzed. Figures 5, 6, and 7 show pesticides at 10 μg/kg, equivalent to the lowest worldwide (EU) legislation, in mango, avocado, and fruit-based baby food extracts respectively.

The advantage of using ACQUITY TQD is that ion ratio confirmation is also possible. This is used to confirm the identity of any pesticide that was presumptive positive from the screening method. Within the EU, ion ratio confirmation is important for pesticide analysis as documented in SANCO/2007/3131¹.

In Part 2, the confirmatory runs, all 402 pesticides were chromatographed with both primary (for quantitation) and secondary (for confirmation) MRM transitions present. Figure 8 shows three more compounds in the three matrices with both MRM transitions.

A rapid multi-residue method was developed for the screening of over 400 pesticides in one 10 minute run with one MRM transition per pesticide. For confirmation, two 10 minute runs were required with two MRM transitions per pesticide. The analysis of pesticides in mango, avocado and fruit-based baby food extracts was able to exceed current worldwide legislated limits.

Improved efficiency and increased sample throughput were realized through the combination of powerful UPLC and fast MS acquisition technologies. The Waters ACQUITY TQD as shown in Figure 9 offers:

• Enhanced chromatographic resolution and short analysis times
• Incorporation of confirmatory MRM traces
• Compliance with legislative regulations such as SANCO
• IntelliStart technology is designed to reduce the burden of complicated operation, training new users, time-intensive troubleshooting, and upkeep
• The small footprint of the ACQUITY TQD will give any laboratory an advantage as it removes the need for larger instrumentation. 

The benefits of this Waters UPLC-MS/MS solution for a revenue conscious laboratory can be realized through increased efficiency through analytical time savings and decreased need for sample retesting, resulting in increased lab productivity. Cost savings can be made by lowering the use of lab consumables with the environmental impact of solvent usage also being reduced.

The sensitivity achieved for a large number of pesticide residues in real food matrices indicates this UPLC-MS/MS method is an ideal basis for the rapid analysis of pesticides in a wide range of food samples.

1. Website: http://ec.europa.eu/food/plant/protection/resources/qualcontrol_en.pdf
2. Commission of the European Communities EC 396/2005, OJ 2005; L70:1.
3. Leandro C.C., Hancock P., Fussell R.J., Keely B.J., J. Chrom A 2007; 1144:161.
4. Lehotay, J.AOAC Int. 90(2) 2007, 485-520.

Acknowledgements

The authors would like to thank Central Science Laboratory (CSL), Sand Hutton, York, UK and VWA, Amsterdam, The Netherlands for kindly supplying MRM transitions and standard solution mixes that were analyzed in this project. Furthermore, University of Jaume I Castellon, Spain, Waters UK, Waters US and Nihon Waters, Japan demonstration laboratories are all thanked for their contributions in supplying MRM transitions.