Analysis of Pesticides in Foods Using GC–MS/MS: An Interview with José Fernando Huertas-Pérez

1. Why are improved methods for analyzing pesticides in food important?

Food safety is of utmost importance, especially in today’s global market where raw materials and products are traded internationally. Intensive farming practices, driven by the growing global population and high food demand, rely on pesticides to control pests, diseases, and weeds, helping farmers to increase yield and quality of goods. However, to protect consumers´ health and ensure food safety, regulatory authorities worldwide have set stringent maximum residue limits (MRLs) for pesticide levels in different food commodities.

In my opinion, given the complexity of food matrices, the need to cover a wide and diverse range of pesticides, and the low concentration levels required, the development of improved selective and sensitive analytical methodsis critical, as they are essential tools for safeguarding consumer health and promoting the trade of high-quality products.

2. How does the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method enhance the extraction and clean-up process when analyzing multi-residue pesticides in complex food matrices using gas chromatography-tandem mass spectrometry (GC–MS/MS)?

The QuEChERS is based on water-acetonitrile extraction and salting out for phase partitioning, followed by clean-up of the organic phase by dispersive solid phase extraction (d-SPE). This “generic” sample preparation procedure offers the compromises needed to handle a wide scope of pesticides, with diverse chemical and physical properties. Initially, it was developed and optimized for pesticides in fruits and vegetables by Michelangelo Anastasiades and Steven Lehotay a couple of decades ago. Since then, QuEChERS has been frequently modified to extend its effectiveness to different matrices and analytes.

The advantages of QuEChERS, as indicated by its name, make it an ideal sample preparation procedure for routine environments where high throughput and the ability to cover a wide scope of analytes and matrices simultaneously are of utmost importance. Our primary goal was to develop a method for GC-amenable pesticides in complex matrices, that could afterwards be easily implemented in routine, and this objective guided our selection of common QuEChERS chemicals during the method development phase.

3. What are the primary challenges in analyzing complex food matrices such as roasted coffee, green tea, and curry with GC–MS/MS, and how can these be effectively addressed?

Those are very complex food matrices and contain substantial amounts of natural compounds, which can often hamper the extraction efficiency, interfere with the analytes of interest and generate matrix effect. Therefore, sample preparation, and particularly the clean-up phase, is a critical step in the analytical process.

To improve the method performance in those matrices, it is also very important to increase the GC–MS/MS selectivity and sensitivity, which can be achieved by carefully selecting precursor/product ions and applying an efficient method calibration approach able to correct efficiently the method bias.

4. How does the selection of precursor and product (daughter) ions improve selectivity and accuracy in the confirmation of pesticides using GC–MS/MS?

The selection of appropriate precursor and product ions is crucial for improving the selectivity and accuracy of pesticide confirmation using GC–MS/MS. For certain pesticides, choosing the right ions based on the specific matrix is essential to avoid matrix interferences, ensuring accurate quantification and reliable confirmation. Additionally, selecting optimal precursor and product ions can significantly lower the limit of quantification for some pesticides, enhancing the method's sensitivity and overall performance. This careful ion selection is fundamental to achieving precise and accurate results in complex food matrices.

5. What techniques, such as the standard addition method, are most effective for minimizing matrix effects during pesticide quantification, and why is this method preferred over matrix-matched calibration?

Different method calibration approaches can be followed to mitigate matrix effect on the analysis of pesticides, and other contaminants, when using chromatographic techniques. The CEN/TS 17061 European guidelines provide valuable insights into all those approaches. To be clear, while CEN/TS 17061 does not directly cover chromatography, its principles can enhance the efficiency, reliability, and compatibility of spectrometry- and spectroscopy-based workflows with chromatographic techniques.

Matrix-matched calibration and standard addition are both very efficient techniques for reducing matrix effects. Standard addition requires more measurements than matrix matched calibration, and sometimes repetition of the analysis when the (a priori unknown) sample concentration is out of the working range initially targeted. Ultimately, we made the choice to quantify by standard addition for responding at best to the need to cover a wide range of matrices without compromising on the reliability of the results. Additionally, standard addition does not rely on having an appropriate representative matrix sometimes difficult to obtain.

6. Why is adhering to European Union (EU) SANTE guidelines critical during the validation of analytical methods, particularly regarding recovery rates, precision, and ion ratio requirements?

The EU SANTE guidelines for quality control and method validation are widely recognized and applied not only for pesticide determination but also for various other food contaminants (2). These guidelines provide specific validation criteria and guidance for designing a systematic and comprehensive set of experiments to evaluate method performance.

By following these guidelines, laboratories ensure that the analytical methods they apply are accurate for the reliable quantification and confirmation of contaminants in food, which is essential to ensure food safety, regulatory compliance, and consistency of results across different laboratories.

7. What modifications to the traditional QuEChERS procedure are most effective for analyzing complex matrices like spices and herbs, and why are these changes necessary?

Dealing with such a complex and dry matrices usually requires decreasing the sample intake. Additionally, optimization of the dispersive solid-phase extraction (d-SPE) clean-up is needed, by adjusting the quantities of chemicals to be used and/or employing emerging efficient ones.

In our study, we used QuEChERS common chemicals, similar to those used for fruits, vegetables and cereals, with the goal of developing a method that could be easily implemented in routine laboratories, already performing pesticides analysis. We tested different combinations of those chemicals to maximize the number of pesticides with acceptable validation results across the three matrices simultaneously.

It is important to note that optimal conditions might differ if the objective were to optimize method performance for specific compound/matrix combinations. The advantages and limitations of traditional and emerging d-SPE adsorbents used for QuEChERS sample preparation on spices and herbs was nicely summarized in a review from the group of Prof. Fernández-Alba (3).

8. How does the chemical and physical diversity of the 172 pesticides studied influence the method development and validation process for GC–MS/MS?

Dealing with such a diverse scope of pesticides and complex matrices requires assuming compromises by applying “generic” conditions, which should be optimized to foster good method performance for as many compounds as possible. In preliminary stages, we chose to apply QuEChERS as sample preparation procedure, applying existing chromatographic conditions and standard addition as the method calibration approach. During the development phase, we focused on testing different d-SPE clean-up conditions and exploring the selection of specific precursor/product ions for certain compounds.

During the validation process, we adhered to a typical design and acceptance criteria as described in the EU SANTE guidelines, with a short commodity check to assess the method's performance in other complex matrices. The extensive amount of data and information generated with a wide scope multiresidue method is substantial. Therefore, leveraging supplier software for data revision/interpretation and having an additional tool to automate calculations has been of great help.

9. What are the advantages of using GC–MS/MS for multi-residue pesticide analysis in complex food matrices compared to other analytical techniques?

Today, GC–MS/MS platforms provide excellent sensitivity and selectivity, which are essential for the determination of multiresidue contaminants in complex food matrices at low concentration levels. GC–MS/MS is suitable for GC-amenable pesticides (that is, semi- and volatile compounds) and should be used alongside LC–MS/MS, which is suitable for thermolabile, polar and non-volatile compounds. These two techniques are complementary and offer a comprehensive approach for pesticide analysis.

As regards of mass analysers, triple quadrupoles offer excellent sensitivity, selectivity, dynamic ranges and quantitation accuracy. They are relatively more affordable and easier to use than high resolution mass analysers such as the orbitrap or time of flight. Because of those reasons, GC– and LC–MS/MS have become the instrumentation generally employed for food control in routine labs over the last couple of decades.

Having said that, high-resolution MS is gaining popularity especially for monitoring LC-amenable compounds, with a wider target scope than GC-amenable ones. It enables the simultaneous monitoring of a higher number of compounds than triple quadrupole, offers higher selectivity, and allows for non-targeted screening and retrospective analysis.

10. Were your results as expected?

Yes, overall, the results were as expected. We acknowledged that achieving satisfactory results for the entire scope of such a diverse list of compounds and complex matrices was not feasible. The method performed within criteria or slightly out of them for at least three-quarters of the compounds on the matrices fully validated (target limit of quantification [LOQ] of 0.01 mg/kg).

Today, we have a method that allows us to quantify a significant number of compounds in these complex matrices. For other compounds the method did not perform according to validation criteria, but it still provides valuable information about their occurrence. Ultimately, for compounds that responded very poorly, the study highlighted the need for specific conditions or single-residue methods to achieve satisfactory results.

11. What has been the feedback from other researchers regarding this work?

The feedback has been very positive. The method has been well received by the community, and we have been contacted by other researchers and analysts to compliment us on our work, seeking further information, and proposing suggestions for future research. These interactions have led to engaging discussions that have helped us foster current and new collaborations.

12. Do you plan to continue developing methods for other pesticides or analytes in food based on this work?

Absolutely. One of the key responsibilities of my team is to continuously explore the potential of current and emerging procedures and technologies for multi- and single-residue analytical methods, not only for pesticides but also for any other food contaminant. In line with this work, we have evaluated the performance of QuEChERS for 470 LC-amenable pesticides in complex matrices using LC–MS/MS. Additionally, I recommend having a look at a recent study published by scientists from my group, where they assessed the capabilities of LC coupled to high-resolution mass spectrometry in combination with QuEChERS, for a total scope of 1,113 compounds, including pesticides, plant toxins, and mycotoxins, with excellent validation results in cereals, fruit and vegetables (4).

I wouldn´t like to finish without thanking my whole team for the invaluable support and inputs they always provide. Deep thanks go to my co-authors for their passion and dedication, which was instrumental for the success of this project.

References

(1) Huertas-Pérez, J. F.; Baslé, Q.; Dubois, M.; Theurillat, X. Multi-residue Pesticides Determination in Complex Food Matrices by Gas Chromatography Tandem Mass Spectrometry. Food Chem. 2024, 436, 137687. DOI: 10.1016/j.foodchem.2023.137687

(2)SANTE/11813/2017 Home Page: https://www.eurl-pesticides.eu/userfiles/file/EurlALL/SANTE_11813_2017-fin.pdf (accessed 2024-11-19).

(3) Parrilla-Vázquez, P.; Ferrer, C.; Martínez Bueno, M. J.; Fernández-Alba, A. R. Pesticide Residues in Spices and Herbs: Sample Preparation Methods and Determination by Chromatographic Techniques. Trends Anal. Chem. 2019, 15, 13-22. DOI: 10.1016/j.trac.2019.03.022

(4) Bessaire, T.; Savoy, M. C.; Ernest, M.; Christinat, N.; Badoud, F.; et al. Enhanced Surveillance of > 1100 Pesticides and Natural Toxins in Food: Harnessing the Capabilities of LC-HRMS for Reliable Identification and Quantification. Foods 2024, 13 (19), 3040.DOI: https://doi.org/10.3390/foods13193040

José Fernando Huertas-Pérez is Specialist in Chemical Contaminants analytics and mitigation at the Nestlé Institute for Food Safety and Analytical Sciences, Nestlé Research Switzerland. He received his Ph.D. in Analytical Chemistry in 2008 from the University of Granada, Spain, and is specialized in liquid and gas chromatography coupled to mass spectrometry. His team focuses on the development and validation of methodologies for the determination of food contaminants, including agrochemicals and veterinary drugs, natural toxins, environmental pollutants and food process contaminants, and their further implementation on Nestlé Quality Assurance Centers. He has co-authored over 50 papers and book chapters.