LABTips: Hi-Tech Techniques for Pesticide Analysis

Pesticide analysis is an increasingly important analytical field requiring continuing technological advancements to keep up with the pace of evolving industries and regulations. Significant increases in global agriculture yield are needed to meet the food demands of a growing population, translating to extensive use of pesticides in support of more abundant crop outputs.¹ Additionally, the increasing legalization of both medical and recreational cannabis products comes with a surge in need for pesticide testing to comply with varying local and state regulations. With the safety of consumers at the forefront of expanding testing efforts, improving, emerging and hi-tech methods provide the sensitivity, accuracy and speed labs need to conquer this critical mission. This article covers some of the advanced techniques used in pesticide testing and how analysts can make the most out of state-of-the-art technologies:

1. Optimize Proven Technology, like QuEChERS

Since it was first published in 2003, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction method has become a staple in many pesticide testing labs; as the name suggests, the method minimizes both the time and materials needed while providing adequate recovery from a range of matrices. Over the years, several modifications have been made to the original QuEChERS protocol, including in the official AOAC and EN QuEChERS methods that use an acetic acid and citrate buffer, respectively, to maintain a stable pH range.

While many labs take advantage of the straightforward protocols outlined in the original and buffered QuEChERS methods, which are generally applicable to a wide range of samples, the true beauty of QuEChERS is that it is easily customizable and provides a versatile framework to tackle countless complex matrices. Modified QuEChERS methods have been developed and published for a range of foods, beverages, cannabis matrices, clinical samples and even textiles. Familiarizing oneself with the functions of different QuEChERS solvents, salts and sorbents allows for tuning and testing of different parameters, and a path toward developing one’s own optimized method based on the matrices they encounter in their day-to-day work.²

For example, those analyzing bright, sweet foods like berries may experiment with different levels of primary and secondary amines (PSAs) for removing sugars and graphitized carbon black (GCB) for cleanup of pigments in the dispersive solid phase extraction (dSPE) step of QuEChERS. In another example, QuEChERS is applied to dry cannabis plants using different mixtures of PSA, GCB, C18 and zirconia sorbents.³ While GCB is effective to facilitate the removal of chlorophyll, care must be taken to minimize the retention of pesticides with planar structures, such as quinoxyfen. Additionally, relatively new sorbents commercialized in the last few years, such as those based on zirconium dioxide or size exclusion, can allow for more effective clean-up of especially challenging fatty matrices.⁴

Another advancement in the application of QuEChERS is the automation of sample preparation steps, adapting the approach for high-throughput operations and improving consistency by reducing manual steps. QuEChERS can be automated by varying degrees ranging from automation of shaking steps,to automated, online cleanup,to full automation and online configuration of extraction and clean-up for continuous preparations and analysis.⁵ Automating your QuEChERS workflow can not only free up analysts from hands-on prep time, but also reduce variations from manual handling, leading to more reliable and reproducible results.

2. Make the Upgrade to UHPLC

Liquid chromatography-mass spectrometry (LC-MS) is one of the most advanced analytical techniques available to pesticide analysts, with the improved speed of high-performance liquid chromatography (HPLC) and greater specificity of tandem mass spectrometry (MS/MS) opening up new opportunities to analyze dozens of pesticides rapidly and simultaneously. Ultra-high-performance liquid chromatography (UHPLC) goes a step further to maximize both speed and separation efficiency in the pesticide lab. Transferring an HPLC method to UHPLC conditions has the potential to cut analysis time by orders of magnitude,and improved peak resolution over HPLC makes it an excellent method for multi-residue screening.⁶ Additionally, the greater separation efficiency afforded by UHPLC can help overcome some matrix effects from complex samples.

Method transfer from HPLC to UHPLC involves many factors to consider. This includes converting your current column dimensions to the smaller dimensions of UHPLC columns, which are smaller both in terms of particle size, and length and diameter.⁷ UHPLC uses much higher pressure pumps in the range of 12,000 to 18,000 psi, requiring hardware suited to work at these high pressures and making it especially important to avoid pressure fluctuations, which can easily shorten column lifetime. New flow rates, isocratic step times, gradient slopes and step times, and injection volumes also need to be calculated relative to the changes in column dimension. In addition to faster separations, better peak resolution and lower detection limits, UHPLC also has the added benefit of lower solvent consumption than HPLC.

Tandem mass spectrometry detectors are the best complement to UHPLC separation due to their high efficiency, selectivity and sensitivity.⁸ Additionally, electrospray ionization (ESI) is the most popular ionization method for analyzing pesticides in food, as it is applicable to a wider range of pesticides than atmospheric pressure chemical ionization (APCI). Triple quadrupole (QQQ) tandem mass spectrometers are the most commonly-used analyzers in this application; although less popular, quadrupole time-of-flight (QTOF) offer improved efficiency and dynamic range over QQQ analyzers making them applicable to non-targeted analysis of an nearly unlimited number of pesticide residues.⁹ Quadrupole-linear ion trap (Q-Trap) systems similarly offer enhanced sensitivity for higher confidence and confirmation of pesticide identification and quantification.

3. Consider Enhanced GC Technologies

Gas chromatography-mass spectrometry (GC-MS) is also widely used for the analysis of non-polar pesticides. While LC-MS has become more important due to the increasing use of polar pesticides,¹ GC methods have continued to evolve and advance, finding important applications in the analysis of food, cannabis, water and other environmental samples. One of these advancements is comprehensive two-dimensional gas chromatography (GCxGC), in which two columns with different separation mechanisms are used to achieve greater separation and peak resolution, with the effluent from the first column being transferred to the second column through the use of a modulator. GCxGC is especially useful for separating coextracts from difficult matrices and resolving coeluting analytes due to its superior separation power.¹⁰ In one study comparing five MS methods, including GCxGC-TOF-MS, GC-MS/MS and UHPLC-MS/MS, GCxGC enabled the deconvolution of 342 pesticides in ginseng in a single 32-minute run.¹¹ However, the GCxGC-TOF-MS method does has its limitations, as it had a higher false negative rate than other methods at the maximum residue levels for pesticides in both ginseng and spinach. Considering that GCxGC separation is not compatible with more sensitive triple quadruple and Q-Trap detectors, a balance should be struck considering the exact nature and needs of your application.

Another advanced GC technique that has gained momentum in the last couple decades is low pressure gas chromatography-mass spectrometry (LPGC-MS), which can speed up analysis as much as three-fold by using the MS vacuum and a wide-bore column to achieve a low pressure within the column.¹² As this method is relatively easy to incorporate with an existing GC system, it offers a promising option for labs already using GC-MS for their pesticide analysis to increase throughput without significant hardware changes. However, thoughtful and thorough method development is required to adapt flow rate, temperature and data acquisition conditions to the new column system, and this method also involves a trade-off between speed and resolution that should be carefully weighed. LPGC produces the best results when paired with a powerful MS detector, such as a QQQ-MS/MS system, that can adequately compensate for the moderately lower separation efficiency of the low-pressure system.¹³

4. Look Forward to a Portable Future

The methods already mentioned provide comprehensive identification and quantification for safety, compliance and research purposes, and require significant time, resources and training to achieve within a controlled lab setting. However, the increasing scope of pesticide use has contributed to a higher demand for rapid, on-site screening capabilities, giving rise to cheaper and more portable technologies that require little or no sample preparation. Portable GC-MS instruments have offered one opportunity to bridge the gap between field and lab, using rapid sample prep strategies like solid-phase microextraction (SPME), needle trap, purge-and-trap, thermal desorption and heated headspace sampling to make on-site analysis more feasible.¹⁴ While not miniaturized to the degree seen in handheld, “point-and-shoot” optical instruments used in other applications, and with lower peak capacity than full-sized instruments, relatively lightweight and ruggedly-built GC-MS systems offer novel opportunities for rapid on-site detections.

Optical methods are also emerging as a possible avenue for portable pesticide screening. Bioassays such as enzyme-linked immunosorbent assays (ELISA) have been used to detect organophosphates, neonicotinoids and fungicides in food samples and fluorescence detection methods have also grown thanks to recent advancements in nanomaterials.¹ Portable surface-enhanced Raman spectroscopy (SERS) instruments have also been proposed as potential screening instruments, and further research has been focused on integration of optical screening methods with smartphones. While most of these technologies remain theoretical or with limited application, the ongoing work to improve optical testing methods could bring new possibilities in the future of pesticide testing.

References

1. Wahab, S.; Muzammil, K.; Nasir, N.; Khan, M.S.; Ahmad, M.F.; Khalid, M.; Ahmad, W.; Dawria, A.; Reddy, L.K.V.; Busayli, A.M. Advancement and New Trends in Analysis of Pesticide Residues in Food: A Comprehensive Review. Plants 2022, 11, 1106. https://doi.org/10.3390/plants11091106
2. "Quechers dSPE selection-which one is best?," Blog Post by Nancy Schwartz, Restek (2020). https://www.restek.com/en/chromablography/chromablography/quechers-dspe-selection--which-one-is-best/ 
3. Stenerson, K.; Halpenny, M. Analysis of Pesticide Residues in Cannabis Using QuEChERS Extraction and Cleanup Followed by GC/MS/MS Analysis. Reporter US 2016, 34.1, 17-19. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/analytical-chemistry/quechers/pesticide-cannabis-quechers-gcms-ms 
4. Belarbi, S.; Vivier, M.; Zaghouani, W.; De Sloovere, A.; Agasse, V.; Cardinael, P. Comparison of Different d-SPE Sorbent Performances Based on Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) Methodology for Multiresidue Pesticide Analyses in Rapeseeds. Molecules 2021, 26, 6727. https://doi.org/10.3390/molecules26216727
5. Chong, C.M.; Hubschmann, H. Fully Automated QuEChERS Extraction and Cleanup of Organophosphate Pesticides in Orange Juice. LCGC Supplements: Advances in Food and Beverage Analysis 2021, 39, 12-16. https://www.chromatographyonline.com/view/fully-automated-quechers-extraction-and-cleanup-of-organophosphate-pesticides-in-orange-juice 
6. "New Technologies for the Simultaneous Analysis of Multiple Pesticide Residues in Agricultural Produce," Application Note by Catherine Ryan and Gordon Kearney, Waters Corporation (2007). https://www.waters.com/webassets/cms/library/docs/720001172en.pdf 
7. "Best Practices in HPLC to UHPLC Method Transfer," Article by Jackie Trudell, Labcompare (2021). https://www.labcompare.com/10-Featured-Articles/575516-Best-Practices-in-HPLC-to-UHPLC-Method-Transfer/ 
8. Stachniuk, A., Fornal, E. Liquid Chromatography-Mass Spectrometry in the Analysis of Pesticide Residues in Food. Food Anal. Methods 9, 1654–1665 (2016). https://doi.org/10.1007/s12161-015-0342-0
9. "Q-TOF MS and Residue Analysis," Application Note by Jinchuan Yang, Kenneth J. Rosnack and Joseph Romano, Waters Corporation (2011). https://www.waters.com/nextgen/en/library/application-notes/2011/Q-TOF-MS-and-Residue-Analysis.html 
10. Tuzimski, T. Multidimensional Chromatography in Pesticides Analysis. In Pesticides - Strategies for Pesticides Analysis; Stoytcheva, M.; IntechOpen: London, 2011; pp 155 170. https://www.intechopen.com/chapters/12953
11. Douglas G Hayward, Jon W Wong, Kai Zhang, James Chang, Feng Shi, Kaushik Banerjee, Paul Yang, Multiresidue Pesticide Analysis in Ginseng and Spinach by Nontargeted and Targeted Screening Procedures, Journal of AOAC INTERNATIONAL, Volume 94, Issue 6, 1 November 2011, Pages 1741–1751, https://doi.org/10.5740/jaoacint.SGEHayward
12. "An Introduction to Low-Pressure GC-MS (LPGC-MS)," Restek Technical Literature Library (2021). https://www.restek.com/en/technical-literature-library/articles/an-introduction-to-low-pressure-GC-MS-LPGC-MS/ 
13. "A New (Faster) Method for Pesticide Analysis: LPGC And Short Collision Cell Technology," Blog Post, JEOL USA (2022). https://www.jeolusa.com/BLOG/a-new-faster-method-for-pesticide-analysis-lpgc-and-short-collision-cell-technology 
14. Thomas, R.J.; Lee, E.; Truong, T.; Porter, N. The Applicability of Field-Portable GC-MS for the Rapid Sampling and Measurement of High-Boiling-Point Semivolatile Organic Compounds in Environmental Samples. LCGC Supplements: Special Issues 2016, 14, 20-26. https://www.chromatographyonline.com/view/applicability-field-portable-gc-ms-rapid-sampling-and-measurement-high-boiling-point-semivolatile-or