Optimization and validation of a single method for the determination of pesticide residues in Peumus boldus Molina leaves using GC-MSD, GC-MS/MS and LC-MS/MS

https://doi.org/10.1016/j.jarmap.2020.100254Get rights and content

Highlights

  • Pesticide multiresidue method based on Quechers template for the herbal Boldo developed.

  • TLC strategy for best method selection.

  • Evaluation of coextractives and matrix effects of the selected method.

  • 90 pesticide residues determined by GCMSMS, LCMSMS and GCMS.

Abstract

The QuEChERS methods, CEN 15662 and AOAC 2007.01, and different clean-up variations were applied to the determination of pesticide residues in Peumus boldus Molina leaves, using LC and GC coupled to single and tandem mass spectrometry. Boldo belongs to the class of medicinal and aromatic plants. Because of its complex chemical composition including terpene peroxides, alkaloids and phenolic acids at concentrations typically 100-fold higher than that of a possible contaminant, it is a very challenging matrix for pesticide residue determination. Thin layer chromatography was used to evaluate the efficacy of removal of matrix co-extractives using different clean-up sorbents, including neutral alumina, graphitized carbon black (GCB), octadecyl reverse phase silica gel (RP-C18) and pH modification in the extraction and clean-up steps. The most promising method was a combination of GCB, RP-C18 and primary-secondary amine (PSA) within the QuEChERS template. The final optimized method was validated according to the SANTE guidelines using LC–MS/MS and GC–MS/MS. The method scope includes 84 representative pesticide residues with recoveries ranging from 70 to 119% and within laboratory relative standard deviations below 15%. The method was initially validated for 25 representative pesticides using GC-MSD, making it applicable in those laboratories equipped only with a single mass detector. It is important to emphasize that the method complies with the requirements of international pharmacopoeias and food regulatory agencies, since herbal teas are found in both categories, as herbal remedies and foods.

Introduction

According to the World Health Organization, 70% of the world population uses non-conventional medicines, including medicinal plant treatments, at some point in their lives (Vogel et al., 2011; WHO, 2015). The market for medicinal and aromatic plants (MAPs) is expanding steadily, with a growing demand from consumers’ worlwide. The global market for botanical and plant-derived drugs is expected to grow from USD 29.4 billion in 2017 to around USD 39.6 billion by 2022 with an annual growth rate of 6.1% for the period 2017–2022 (BCC_Research, 2017). While MAPs were originally naturally growing in the environment and collected there, nowadays, due to their marketability, they are cultivated on a relatively extensive basis. This more extensive cultivation requires agricultural inputs, which depend on many factors, including the prevalence of insects and diseases in the field and during storage. As in any other crops, pesticides are used to ensure highe yields and adequate quality (Abhilash and Singh, 2008).

Much attention is paid to the development of analytical methodologies to assure the safe use of aromatic plants as medicines and food. The complex chemical composition of medicinal plants poses a challenge to the selective detection of contaminants present at trace levels. Some secondary metabolites have similar physicochemical properties to the pesticides under study, interfering with their determination. These metabolites are frequently present at higher concentrations than the pesticide residues that should be determined, forcing the analyst to implement complex purification processes. Such is the case with Peumus boldus, commonly known as boldo, a medicinal plant widely used in Latin America, the use of which is expanding to other parts of the world. The leaves of this Chilean tree are used for digestive and hepatobiliary disorders (Speisky and Cassels, 1994). The concentrations of terpene peroxides, alkaloids and phenolic acids in boldo leaves are higher than those of the suspected contaminants. The content of essential oils is approximately 2% w/w, mainly comprising ascaridol, a terpene peroxide. Boldo also typically contains alkaloids, mainly boldine, at approximately 0.5%, and polyphenols such as chlorogenic acid and shiquimates. This complex chemical composition makes analytical method development and validation rather challenging.

Different procedures such as matrix solid phase dispersion (MSPD), solid-liquid extraction (SLE) and solid phase extraction (SPE) have been applied for pesticide residue analysis in MAPs (Zuin et al., 2003; Pérez-Parada et al., 2011; Lozano et al., 2012; Pareja et al., 2015). With the advent of tandem mass spectrometry detectors, the sample preparation requirements have been reduced and are more straightforward, Nowadays, trends in pesticide residues analysis tend towards reduced sample size, solvent consumption and minimal clean-up steps. The QuEChERS method, introduced by Annastasiades & Lehotay (Anastassiades et al., 2003), is among the most widely employed methods for pesticide residues analysis due to its versatility. Variations of QuEChERS have been used for pesticide residue analysis in herbs, teas and spices, complex matrices that have in common high contents of pigments and secondary metabolites (Rajski et al., 2013). Modifications of this methodology have also been used for the determination of pesticide residues in camomile, medicinal plants from China and for a number of botanicals (Cajka et al., 2012; Lozano et al., 2012; Dzuman et al., 2015; Nie et al., 2015; Tripathy et al., 2017). The original QuEChERS method has been validated for the analysis of 24 pesticides in Calendula officinalis inflorescences (Besil et al., 2017). A citrate buffered version of the QuEChERS method was applied for the determination of pesticide residues in Cannabis sativa by LC–MS/MS (Pérez-Parada et al., 2016). Hayward et al. (2013) added a solid phase extraction cleanup step using PSA and GCB cartridges in tandem for the purification of acetonitrile extracts and applied this protocol to the analysis of various MAPs. Recently another unbuffered QuECHERS-based method was presented for the analysis of carbamate residues in different herbs without using PSA in the clean-up step (Nantia et al., 2017).

Many pharmacopoeias from all over the world include a special chapter for the determination of pesticide residues in MAPs. Within this context, the World Health Organization (WHO) advised that every country should have at least one official laboratory for pesticide residue analysis in MAPs and for quality control of those medicinal plants used as raw materials for phytopharmaceuticals (WHO, 2007). Pesticide residue analysis in MAPs according to pharmacopoeias such as the European (EU, 2019) or the MERCOSUR (GMC, 2016) Pharmacopoeias, are not performed following a fixed protocol. Instead, they require that the proposed methodology for the analysis of medicinal plants should be “fit for purpose” and validated according to the SANTE guidelines (DG-SANTE, 2015). Consequently, individual methods should be developed for the analysis of pesticide residues in MAPs due to their varied and different compositions. The presence of co-extractives in the extracts for pesticide residue analysis should be minimized to avoid excessive matrix effects and contamination of the analytical instrumentation. This is the case for boldo, an herb widely used for consumption as an infusion, and for which a pesticide residue analysis method is needed. As already described, it is an unsually complex analytical matrix because it contains high amounts of secondary metabolites from different chemical families. Variable amounts of chlorophylls and their degradation products can also be found in the primary extract for pesticide residue analysis. Boldo leaves are widely used and traded. In the nineteen-nineties more than 2000 tons per year of dried boldo leaves were collected from native trees and exported. Nowadays, due to its high consumption, cultivation systems have been developed and applied to domesticate boldo trees, which are native to South America but are now also cultivated in Africa, India and Europe. The agricultural practices and the technological package used depends on the region, therefore the pesticide residues can vary according to the geographical origin of the product (Bruneton, 1999). Due to the global trade of MAPs through complex networks, wide scope analytical methods for the determination of pesticide residues are required to cover the possible pesticide combinations used in different regions and ascertain the quality and safety of the herb. To the best of the authors’ knowledge there is, to date, no specific multiresidue method for the monitoring of pesticide residues in boldo leaves that can cover the broad scope of agrochemicals applied in the different regions of the world were the crop is grown.

Using the advantages of the flexibility of the QuEChERS sample preparation technique and the sensitivity of triple quadrupole detectors coupled to gas or liquid chromatography, the development of a multiresidue method for pesticide residues in boldo was explored. The aim of the work was to find a suitable method for routine analysis. The final selected methodology was fully validated.

Section snippets

Chemicals, materials, and standards

Acetonitrile (MeCN) and ethyl acetate (EtOAc), both HPLC grade, were supplied by J.T. Baker (USA). MeCN (ACS grade) was provided by Dorwil (Argentina). Deionized water was obtained using a Thermo Scientific (Marietta, OH, USA) EASY0 pure RoDi Ultrapure water purification system. Anhydrous magnesium sulphate extra pure (MgSO4), sodium citrate dibasic sesquihydrate, primary secondary amine (PSA), graphitized carbon black (GCB) and octadecyl reverse phase silica gel (RP-C18) were obtained from

Method selection

For the selection and evaluation of the analytical method to use on boldo leaves, a representative group of GC and LC amenable insecticides, herbicides and fungicides, belonging to different chemical families (organophosphates, carbamates, pyrethroids, neonicotinoids; chloroacetanilide, phenylamides; triazoles, strobilurins, and benzimidazoles), were chosen. The strategy was to test the QuEChERS CEN 15662 and AOAC 2007.01 methods and variations in the clean-up step using salts/materials with

Real samples analysis

Commercial samples were analyzed in order to investigate the applicability of the method. Twentysix commercial samples of Peumus boldus, purchased from local markets, were analysed. Four samples yielded positive results for carbendazim (230 μg kg−1, EU MRL 100 μg kg−1), acetamiprid (12 μg kg−1, EU MRL 3 mg kg−1), azoxystrobin (21 μg kg−1, Codex and EU MRL 70 mg kg−1) and pyraclostrobin (130 μg kg−1, EU MRL 2 mg kg−1). The MRLs showed correspond to the categories "Herbs” in Codex and the EU

Conclusions

A multiresidue method was developed for the determination of pesticide residues in boldo leaves, used as a representative matrix of a herb. The method was collaboratively developed and validated in two different laboratories, GACT-Uruguay and FEPL-Austria. The procedure showed satisfactory recoveries and precision (70–120%, RSD below 20%) at 50 μg kg−1 for 65 GC and 56 LC amenable pesticides. In addition, 51 of the pesticides could be analyzed at 10 μg kg-1. The final validated method, based on

Authors’ contributions

BM contributed to the design of the study, participated in the laboratory work in both labs, performed the treatment of the data recovered and drafted the paper. NB ran the laboratory work, adjusted the instrumental conditions, performed the data treatments and collaborated in the preparation of the manuscript. AB adjusted the sample preparation, TLC analysis. NG processed the real samples for analysis and helped in laboratory work and design the graphical abstract. VC coordinated the practical

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by Programa de Desarrollo de las Ciencias Básicas (PEDECIBA). Programa de Apoyo a la Investigación Estudiantil (PAIE). Agencia Nacional de Investigación e Innovación (ANII), Programa de Becas de Iniciación a la Investigación and Fundación para el Progreso de la Química (FUNDAQUIM). The RALACA-FAO/IAEA are acknowledged for the support with the standards, and the Joint FAO/IAEA Food and Environmental Protection Laboratory (FEPL) in Seibersdorf, Austria for the possibility

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