Supercritical fluid chromatography separation of chiral pesticides: Unique capabilities to study cyhalothrin and metalaxyl as examples
Introduction
Separation of enantiomers is a well-known practice in pharmaceutical industry [1]. Enantioseparation of some pharmaceuticals is mandatory to launch enantiopure drugs into the market. However, enantioseparation of pesticides, despite the proven enantioselectivity present in nature -and unlike pharmaceutical compounds- is a complex field thoroughly. Different pesticides, belonging to varying chemical classes, can cause stronger or weaker interactions with the active site within an organism due to their different isomer spatial conformation [2]. As pesticides improve the productivity and profitability of agricultural products, their use has increased considerably in the United States and the European Union. Therefore, it is necessary to develop a proper analytical method to identify of chiral pesticides and to understand their degradation mechanism in food applications.
Many examples of pesticide enantioselectivity exist. Fipronil, for instance, demonstrates apparent enantioselective degradation in sediments. As a consequence, one isomer is bioaccumulated at higher concentrations in blackworm tissue than the other [3]. S-fipronil has also been found to affect more intensely the DNA methylation of genes related to neurological processes in zebrafish [4]. On the other hand, each paclobutrazol enantiomer presented effects of different magnitude in the photosynthetic pigments and transcriptions in C.vulgaris algae [5]. Benalaxyl and metalaxyl isomers have enantioselectivity effects that influence DNA methylation [6].
It has been demonstrated that the metalaxyl isomer metalaxyl-M (mefenoxam), is the most effective enantiomer [7]. This pesticide is of great interest and hence research on its enantioselectivity is abundant, for example, the enantioselective dissipation process in soil matrix [8]. different superoxide dismutase activities in tobacco plants [9]. enhanced metalaxyl-M persistence in olive-mill waste amended soils [10]. and the different influence of each enantiomer on metabolic pathway disturbance in rats [11]. Like most pesticides (except lambda-cyhalothrin), metalaxyl enantiomers have the same ARfD (0.5 mg/kg) [12]. Moreover, the United States Environmental Protection Agency (EPA) uses the same toxicological endpoints for metalaxyl and metalaxyl-M, and an analytical method for the enantioseparation of both enantiomers is also described in one of their reports [13].
Lambda-cyhalothrin is a pyrethroid (a pyrethrin derivate) and one of the diastereomeric pair of enantiomers in cyhalothrin [14]. The popularity of pyrethroids is increasing. Nonetheless, it is a known fact that these compounds can be toxic to mammals [15]. and there are several studies similar to those performed on metalaxyl, such as the one looking at their different ecotoxicological effects in zebrafish [16]. However, it is the different acute reference dose (ARfD) for each enantiomer of the lambda-cyhalothrin pair which makes it more significant. The ARfD for gamma-cyhalothrin (the S,R,R isomer) is 0.0025 mg/kg whereas the ARfD of its enantiomer (R,S,S) is 0.005 mg/kg. Therefore, the gamma-cyhalothrin enantiomer is twice as toxic as its enantiomer; accordingly, and it is essential to achieve good chromatographic separation and validation of both isomers in food analysis. All these factors are of potential relevance to European regulatory bodies given that continuous modifications to the maximum residue level for this compound are being implemented [17,18].
To obtain good separation, supercritical fluid chromatography (SFC) coupled to tandem mass spectrometry was applied in this study. Chiral analysis and purification are considered as one of the most dominant SFC applications in the pharmaceutical industry [19]. It is already known that SFC has many advantages, for instance, lower solvent consumption, shorter run times, lower matrix effects and improved ionization efficiency [20]. However, one of its many qualities is its ability to perform fast chiral separations with excellent resolution. The diffusivity in SFC is higher than in liquid chromatography (LC) so faster separations can be performed without compromising efficiency [21,22]. These attributes make SFC an excellent technique for preparative chromatography [23], [24], [25]. Regarding the mobile phase in LC, it is usually necessary modify it substantially when a chiral separation is performed with polysaccharide columns. However, in SFC, the same mobile phase is commonly used for chiral separations and achiral column multiresidue methods.
There are a wide variety of chiral stationary phases (CSP) classified depending on their chemical composition. Polysaccharide chiral stationary phases are the most widely used CSP across all the chromatographic fields [26,27]. Polysaccharide phases can to be derivatized at the active -OH groups, the carbamate form being the most popular. Cellulose-based and amylose-based chiral phases offer the possibility to interact with the analytes through the hydrogen bonds. Extensive research is being conducted on the structure of these stationary phases, since their mechanism of interaction with the analytes is still quite ambiguous. The high complexity of these stationary phases allows the separation of a considerable number of compounds [28].
The main objective of the present work is to achieve the maximum number of enantiomeric pesticide separations using SFC-ESI-MS/MS. In order to highlight the advantages of this approach, a multiresidue method was created containing 21 pesticides. Four matrices containing a broad complex range were analyzed (tomato, orange, leek, cayenne). Particular attention was given to lambda-cyhalothrin and metalaxyl isomers, including field tests inside a greenhouse.
Section snippets
Reagents and materials
Two different manufacturers provide the pesticide standards used in this study: Sigma Aldrich (Steinheim, Germany) and LGC (Teddington, United Kingdom). All the standards had a purity higher than 96% except for flucythrinate (87.5%). The analytical standards were stored at −30 °C. The standard-mix solution was prepared using individual stock solutions. Individual stock solutions (around 1000 mgL−1) were prepared from each standard and stored in the dark at −20 °C in amber glass vials.
Carbon
Enantiomeric separation of pesticides
Two polysaccharide-based CSP were tested: cellulose tris(3,5-dimethylphenylcarbamate) and amylose tris(3-chloro-5-methylphenylcarbamate). Both had the same length (250 × 4.6 mm) and the same particle size. The composition of the mobile phases and flows remain the same as those in our previous studies of pesticide residue analysis using SFC-ESI-MS/MS [29]. A gradient was created with the aim of separating the highest number of chiral pesticides. Twenty-one pesticides were analyzed: iprovalicarb,
Conclusions
Supercritical fluid chromatography in combination with cellulose polysaccharide columns, has been proven capable of performing fast, robust, and efficient separation of chiral pesticides. Following an examination of lambda-cyhalothrin and metalaxyl, it was found that their enantiomers could be identified at the 5 µg/kg concentration level. These pesticides were also evaluated in terms of linearity, reproducibility and matrix effects obtaining good results for each isomer. The greenhouse test
CRediT authorship contribution statement
Víctor Cutillas: Conceptualization, Methodology, Resources, Validation, Investigation, Writing - original draft, Writing - review & editing, Visualization. Mar García-Valverde: Methodology, Validation, Investigation. María del Mar Gómez-Ramos: Methodology, Validation, Investigation, Resources. Francisco José Díaz-Galiano: Methodology, Investigation, Writing - review & editing, Visualization. Carmen Ferrer: Conceptualization, Investigation, Writing - review & editing. Amadeo R. Fernández-Alba:
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.
Acknowledgments
The authors acknowledge the funding support provided by the European Commission, DG SANTE (Grant decision SI2.802063). The authors would also like to thank Shimadzu Corporation for providing the equipment and constant support of the work.
References (31)
- et al.
Enantioselective degradation and bioaccumulation of sediment-associated fipronil in Lumbriculus variegatus: toxicokinetic analysis
Sci. Total Environ.
(2019) - et al.
Exposure of low-dose fipronil enantioselectively induced anxiety-like behavior associated with DNA methylation changes in embryonic and larval zebrafish
Environ. Pollut.
(2019) - et al.
Enantioselective growth inhibition of the green algae (Chlorella vulgaris) induced by two paclobutrazol enantiomers
Environ. Pollut.
(2019) - et al.
Acylamino acid chiral fungicides on toxiciepigenetics in lambda DNA methylation
Food Chem. Toxicol.
(2017) - et al.
Environmental behavior of the enantiomers of the chiral fungicide metalaxyl in Mediterranean agricultural soils
Sci. Total Environ.
(2013) - et al.
Effects of metalaxyl enantiomers stress on root activity and leaf antioxidant enzyme activities in tobacco seedlings
Chirality
(2018) - et al.
Effect of olive-mill waste addition to agricultural soil on the enantioselective behavior of the chiral fungicide metalaxyl
J. Environ. Manage.
(2013) - et al.
Enantioselective biodegradation of the pyrethroid (±)-lambda-cyhalothrin by marine-derived fungi
Chemosphere
(2018) Chapter 1 - Milestones in supercritical fluid chromatography
- et al.
Evaluation of supercritical fluid chromatography coupled to tandem mass spectrometry for pesticide residues in food
J. Chromatogr. A
(2018)
Supercritical fluid chromatography for the enantioseparation of pharmaceuticals
J. Pharm. Biomed. Anal.
Supercritical fluid chromatography versus high performance liquid chromatography for enantiomeric and diastereoisomeric separations on coated polysaccharides-based stationary phases: application to dihydropyridone derivatives
J. Chromatogr. A
Preparative supercritical fluid chromatography: a powerful tool for chiral separations
J. Chromatogr. A
Preparative chromatographic resolution of racemates using HPLC and SFC in a pharmaceutical discovery environment
J. Chromatogr. B
Preparative enantioseparations using supercritical fluid chromatography
J. Chromatogr. A
Cited by (21)
Supercritical fluid chromatography time-of-flight mass spectrometry enantiomeric determination of basic drugs in sewage samples
2022, Journal of Chromatography ACitation Excerpt :Under final conditions described in Material and Methods section, compounds were separated in 15.5 min, with Rs values between 1.03 (STZ enantiomers) and 2.59 (FTZ enantiomers), Table 1. Total analysis time employed in this research was between twice [25] and four times [13] lower than those required in previous multianalyte methods using chiral LC as separation technique, under isocratic conditions; and in the range of values reported in other multianalyte SFC-MS applications [12]. Same SFC conditions were also tested to resolve the racemates of over the counter metabolites of TRA and VFX: N-desmethyl TRA and O-desmethyl VFX.
Enantioselective monitoring chiral fungicide mefentrifluconazole in tomato, cucumber, pepper and its pickled products by supercritical fluid chromatography tandem mass spectrometry
2022, Food ChemistryCitation Excerpt :The combined use of organic modifiers and CO2 can change the polarity, density, and solvating power of the mobile phase, and thus affecting the interaction between the target analyte and chiral stationary phase, which could improve the separation efficiency (Strubinger, Song, & Parcher, 1991; Tarafder, 2016). Alcohols are selected as organic modifier frequently (Cheng et al., 2017; Cutillas, Garcia-Valverde, Gomez-Ramos, Diaz-Galiano, Ferrer, & Fernandez-Alba, 2020; Jiang et al., 2019). So, four alcohols (proportion: 20%), including MeOH, EtOH, IPA, and BuOH, were employed to evaluate the separation effect of mefentrifluconazole enantiomers, respectively.
Current state and future perspectives of supercritical fluid chromatography
2022, TrAC - Trends in Analytical ChemistryCitation Excerpt :About 5 years ago, the concerns in the field focused on the contaminants coming from packaging materials, drugs and pesticides residues in foods [134]. Recent studies in the field involve in the analysis of plant protection products (e.g., lambda-cyhalothrin, metalaxyl, neonicotinoid dinotefuran, fosthiazate, triazole, oxathiapiprolin, triadimefon) [106,107,131,202–205], carcinogenic compounds (e.g., heterocyclic aromatic amines, biogenic amines) in meat and soy sauce [206,207], vitamin D and the related metabolites [199], pigments (e.g., chlorophyll) and food dyes (e.g., azo, triphenylmethane, xanthone, indigoid, quinoline and polyene classes) [51,208], triacylglycerol and fatty acids [126,209] in common food commodities (e.g., vegetables, fruits, meats, daily oily supplements), drinks (e.g., wine, milk). Several studies still focus on the fate of contaminants in food contact materials (e.g., acrylate monomers, plasticizers, antioxidants, and photoinitiators) [48,210,211].
Chiral polymeric membranes: Recent applications and trends
2022, Separation and Purification TechnologyCitation Excerpt :Nevertheless, LC presents same disadvantages as the need of large amount of organic solvents, the high cost of the CSPs for preparative scale and the difficulty to use on a large scale [31]. SFC is also emerged as a highly efficient technique for chiral separations, being associated to the advantage of reduced use of organic solvents [32-35]; however, it has the same limitations of LC concerning the cost of the CSPs and the large scale applications [27]. Chiral solid membranes can be a valuable alternative to preparative LC and SFC for industrial production due the simplicity in operation, the reduce use of solvents, which is the requirement for more sustainable methodologies, allied with the lower cost when compared to SFC and LC.