Chiral Pesticides with Asymmetric Sulfur: Extraction, Separation, and Determination in Different Environmental Matrices

  Citation: López-Cabeza, R.; Francioso, A. Chiral Pesticides with Asymmetric Sulfur: Extraction, Separation, and Determination in Different Environmental Matrices. Separations 2022,9, 29. https:// doi.org/10.3390/separations9020029 Academic Editor: Marcello Locatelli Received: 27 December 2021 Accepted: 16 January 2022 Published: 26 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). separations Review Chiral Pesticides with Asymmetric Sulfur: Extraction, Separation, and Determination in Different Environmental Matrices Rocío López-Cabeza 1,2 and Antonio Francioso 1,3,* 1Departamento de Química Orgánica, Instituto Universitario de Bio-Orgánica Antonio González, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain; [email protected] 2Instituto de Recursos Naturales y Agrobiología de Sevilla, (IRNAS), CSIC, Avenida de Reina Mercedes 10, 41012 Sevilla, Spain 3Dipartimento di Chimica Biologica “A. Rossi Fanelli”, Sapienza Universitàdi Roma, P.le Aldo Moro 5, 00185 Roma, Italy *Correspondence: [email protected] Abstract: Chiral pesticides with S atoms as asymmetric centers are gaining great importance in the search for new pesticides with new modes of action. As for the rest of the chiral pesticides, the determination of the stereoisomers separately has become crucial in the environmental risks assessment of these pesticides. Therefore, the development of suitable extraction and clean-up methods as well as efficient stereoselective analytical techniques for stereoisomers determination in environmental samples is essential. Currently, liquid/solid phase extraction, microextraction, and QuEChERS-based methods are most commonly used to obtain chiral pesticides from environmental samples. Gas, liquid, and supercritical fluid chromatography together with capillary electrophoresis techniques are the most important for the determination of the stereoisomers of chiral pesticides containing S atoms in its structure. In this study, all these techniques are briefly reviewed, and the advantages and disadvantages of each are discussed. Keywords: chirality; asymmetric sulfur; enantioselectivity and stereoselectivity; pesticides; extraction methods; chromatographic techniques; capillary electrophoresis; mass spectrometry 1. Introduction The development of more effective, selective, eco-friendly, and profitable agrochemicals has led to the design of pesticides with increasingly complex structures, many of them chiral [ 1 , 2 ]. Chiral pesticides present at least one asymmetrical atom (or chiral center) in theirs structure, resulting in a pair of enantiomers that are non-superimposable mirror images of each other [ 1 ]. If the compound has more than one chiral center, for instance n centers, a maximum number of 2 n stereoisomers is possible [ 1 ]. The enantiomers of a chiral pesticide have identical physicochemical properties, so they behave in the same way in achiral media. However, in chiral media such as soils or organisms, the behavior of each enantiomer is usually different [ 3 ]. Therefore, chiral pesticides can undergo enantioselective transformation processes (degradation, isomerization, etc.) in soils, which could lead to a different concentration of each enantiomer in the environment. Furthermore, only one enantiomer is generally active against the target organism, while the other may be inactive, have a different active function, or even be toxic to non-target organisms [ 4 ]. For these reasons, determining the concentration of each enantiomer/stereoisomer separately in environmental samples (soil, water, and plant samples, among others) has become crucial for the environmental risk assessment of chiral pesticides. Overall, the asymmetric center of a chiral compound is a carbon atom attached to four different groups, although the chirality is also possible due to the presence of an Separations 2022,9, 29. https://doi.org/10.3390/separations9020029 https://www.mdpi.com/journal/separations Separations 2022,9, 29 2 of 19 asymmetric nitrogen, phosphorus, or sulfur atom (Figure 1) [ 5 ]. Sulfur has a lone pair of electrons that can act as a fourth “group,” which results in a chiral center when combined with three other functional groups that are different from each other [1]. Separations 2022, 9, 29 2 of 18 water, and plant samples, among others) has become crucial for the environmental risk assessment of chiral pesticides. Overall, the asymmetric center of a chiral compound is a carbon atom attached to four different groups, although the chirality is also possible due to the presence of an asymmetric nitrogen, phosphorus, or sulfur atom (Figure 1) [5]. Sulfur has a lone pair of electrons that can act as a fourth “group,” which results in a chiral center when combined with three other functional groups that are different from each other [1]. Figure 1. Stereogenic centers on different (C, N, P, and S) atoms tetrahedral configuration. The search for new functional groups in the structure of pesticides that can avoid cross resistance has led to the development of new groups based on sulfur such as the sulfoximine moiety [6] and sulfiliminyl moiety [7]. Therefore, the presence of asymmetric sulfur atoms in new chiral pesticides is becoming increasingly important. In general, the analysis of the enantiomers of a chiral compound represents a significant analytical challenger since, as mentioned above, the physicochemical properties of the enantiomers are identical, which makes their individual determination considerably difficult. In this review, the most used techniques for the extraction and determination of pesticide enantiomers from environmental samples are described, emphasizing the analysis of chiral pesticides with an asymmetrical sulfur atom in their structure. 2. Extraction and Clean-Up Methods Used in the Determination of Chiral Pesticides. Extraction methods should not be stereoselective; however, they must provide suitable recovery (and reproducibility) of stereoisomers from complex environmental samples, as well as minimize the matrix interferences (i.e., co-extracted/co-eluting compounds and detector signal suppression) [8]. Several extraction and clean-up methods have been proposed to obtain chiral pesticides from the environmental samples such as liquid–solid extractions with organic solvents, solid-phase extraction modes, microextraction methods, and QuEChERS process (quick, easy, cheap, effective, rugged, and safe) [8]. As can be seen in Table 1, QuEChERS and liquid–solid extraction with organic solvents combined with some solid-phase extraction technique are the most frequently reported procedures for the extraction and clean-up of chiral pesticides with an asymmetric sulfur. QuEChERS was firstly proposed in 2003 [9], and its first application in soil analysis was performed by Lesueur et al. [10]. The original QuEChERS procedure consists of an initial solid–liquid extraction of the sample with acetonitrile (1/1, v/w ratio), R1 C R4 R2 R3 N R3 R2 R1 N R3 R2 R1R4 P OR2 O R1O R3O S R2 O R1 S R3 R1 R2 sp3carbon trisubstituted amines quaternary ammonium salts phosphate triesters sulfoxides sulfonium ions Figure 1. Stereogenic centers on different (C, N, P, and S) atoms tetrahedral configuration. The search for new functional groups in the structure of pesticides that can avoid cross resistance has led to the development of new groups based on sulfur such as the sulfoximine moiety [6] and sulfiliminyl moiety [7]. Therefore, the presence of asymmetric sulfur atoms in new chiral pesticides is becoming increasingly important. In general, the analysis of the enantiomers of a chiral compound represents a significant analytical challenger since, as mentioned above, the physicochemical properties of the enantiomers are identical, which makes their individual determination considerably difficult. In this review, the most used techniques for the extraction and determination of pesticide enantiomers from environmental samples are described, emphasizing the analysis of chiral pesticides with an asymmetrical sulfur atom in their structure. 2. Extraction and Clean-Up Methods Used in the Determination of Chiral Pesticides Extraction methods should not be stereoselective; however, they must provide suitable recovery (and reproducibility) of stereoisomers from complex environmental samples, as well as minimize the matrix interferences (i.e., co-extracted/co-eluting compounds and detector signal suppression) [ 8 ]. Several extraction and clean-up methods have been proposed to obtain chiral pesticides from the environmental samples such as liquid–solid extractions with organic solvents, solid-phase extraction modes, microextraction methods, and QuEChERS process (quick, easy, cheap, effective, rugged, and safe) [ 8 ]. As can be seen in Table 1, QuEChERS and liquid–solid extraction with organic solvents combined with some solid-phase extraction technique are the most frequently reported procedures for the extraction and clean-up of chiral pesticides with an asymmetric sulfur. QuEChERS was firstly proposed in 2003 [ 9 ], and its first application in soil analysis was performed by Lesueur et al. [ 10 ]. The original QuEChERS procedure consists of an initial solid–liquid Separations 2022,9, 29 3 of 19 extraction of the sample with acetonitrile (1/1, v/w ratio), which is followed by a saltingout step with anhydrous MgSO 4 and NaCl to promote the water partition from the organic phase and its dehydration. Then, an aliquot of the acetonitrile supernatant is cleaned up by dispersive solid-phase extraction using the sorbent “primary secondary amine” (PSA) and anhydrous MgSO 4 . PSA, which is a weak anion exchanger, removes co-extracted acidic compounds (e.g., fatty acids and organic acids), and the MgSO 4 removes the water content from the acetonitrile phase [ 11 ]. After centrifuging and filtering, a clean extract is obtained for analysis [ 9 ]. Different changes have been made to the original QuEChERS procedure to improve the performance of this method based on the type of analyte and matrix, such as pH control and the use of alternative clean-up methods [11]. Several techniques based on solid phase extraction (SPE) have also been used for the extraction of chiral pesticides, as well as for cleaning up extracts from environmental samples. Some examples are dispersive solid phase extraction (DSPE), matrix-solid phase dispersion (MSPD), solid-phase microextraction (SPME), and magnetic solid-phase extraction (MSPE) [ 12 ]. The main sorbents used in these solid phase extractions are primary secondary amine (PSA), hydrophilic sorbents (i.e., Florisil, U.S. Silica Company, Katy, TX, USA), lipophilic sorbents (i.e., reverse phase C18), and multi-walled carbon nanotubes (MWCNTs). The latter sorbent has a large specific surface that makes it an excellent SPE sorbent for pesticides. However, when carbon nanotubes are used in SPE cartridge, the high back-pressure of the nanoparticles-packed columns results in resistance to sample flow [ 13 ]. This limitation has been overcome by incorporating nanoparticles with magnetic properties into carbons nanotubes, obtaining magnetic multi-walled carbon nanotubes (MMWCNTs). This new sorbent can be used in DSPE for pesticides due to the possibility of collecting it easily by applying an external magnetic field [12]. Separations 2022,9, 29 4 of 19 Table 1. Extraction and analytical techniques used in the chiral analysis of the pesticides described in this review. Group Pesticide Matrix Extraction and Clean-Up Analytical Determination Chiral Stationary Phase (Chiral Column) or Chiral Selector Analysis Conditions Ref. Organophosphorus compounds Fensulfothion Standard solutions HPLC-UV Amylose tris(3,5dimethylphenylcarbamate) (Chiralpak®AD column, Daicel Corporation, Tokyo, Japan), 250 mm ×4.6 i.d. and 10 µm particle size Heptane:ethanol (90:10) at a flow rate of 1 mL/min and Tcolumn: 25 ◦C [14] MEKC-ABS (detection at 200 nm) Sodium dodecylsulfate/carboxymethylβ-CD/hydroxypropyl-β-CD BGE: sodium borate buffer (pH 8.7), T: 25 ◦C and voltage of 10–30 kV [15] Fenamiphos sulfoxide Soils QuEChERS and clean-up by DSPE using MgSO 4 and PSA as sorbents HPLC-DAD (detection at 225 nm) Amylose tris(3,5dimethylphenylcarbamate) (Chiralpak®AD-H column, Daicel Corporation, Tokyo, Japan), 250 mm ×4.6 i.d. and 5µm particle size N-hexane:2-propanol (87:13) at a flow rate of 0.8 mL/min, Vinjection: 20 µL and Tcolumn: 20 ◦C [16] - Accelerated solvent extraction - Solid–liquid extraction with organic solvents CE-UV (detection at 214 nm) Carboxymethyl-βCD/hydroxypropyl-β-CD BGE: acetic acid/ammonia buffer (pH 5), T: 25 ◦C and voltage of 25 kV [17] Phenylpyrazoles Fipronil Standard solutions HPLC-DAD (detection at 230 nm) Cellulose tris(3,5 dimethylphenylcarbamate) (Chiralpak®IB column, Daicel Corporation, Tokyo, Japan), 250 mm × 4.6 mm i.d. and 5 µ m particle size N-hexane:2-propanol (95:5) at a flow rate of 1 mL/min, Vinjection: 20 µL, and Tcolumn: 30 ◦C [18] SFC-UV/Vis (detection at 230 nm) Cellulose tris(3,5dimethylphenylcarbamate) (Lux 3 µ Cellulose-1 column), 250 mm ×4.6 mm i.d. and 3 µm particle size ScCO2:methanol (95:5) at a flow rate of 2 mL/min, Vinjection: 10 µL, and Tcolumn: 35 ◦C [19] Separations 2022,9, 29 5 of 19 Table 1. Cont. Group Pesticide Matrix Extraction and Clean-Up Analytical Determination Chiral Stationary Phase (Chiral Column) or Chiral Selector Analysis Conditions Ref. Water and sediments Water: liquid–liquid extraction with an organic solvent Sediments: solid–liquid extraction with organic solvents and clean-up by SPE using Alltech silica cartridge GC-ECD Tert-butyldimethylsilyl-β -cyclodextrin) dissolved in 15% diphenyl and 85% dimethyl polysiloxane (BGB-172 column), 30 m ×0.24 mm i.d. and 0.25 µm film Detector temperature: 325 ◦C, detector gas: nitrogen (60 mL/min), and inlet T: 260 ◦C [20] Extraction of samples not detailed GC-MS Tert-butyldimethylsilyl-β -cyclodextrin) dissolved in 15% diphenyl and 85% dimethyl polysiloxane (BGB-172 column), 30 m ×0.24 mm i.d. and 0.25 µm film MS source and the quadrupoles temperature: 230 ◦C and 150 ◦C, carrier gas: helium (25 psi) and inlet T: 230 ◦C [21] Sediments and aquatic organisms (L. minor and A. woodiana) Extraction with an organic solvent and clean-up of the extracts from organisms by SPE using a silica cartridge for L. minor and a Florisil cartridge for A. woodiana GC-ECD Tert-butyldimethylsilyl-β -cyclodextrin) dissolved in 15% diphenyl and 85% dimethyl polysiloxane (BGB-172 column), 30 m ×0.24 mm and 0.25 µm film Detector temperature: 350 ◦C Inlet T: 250 ◦C [22] Soils and water Water: extraction with MMWCNTs-NH2 Soils: extraction with an organic solvent and MSPE using MMWCNTs-NH2as sorbent UPLC-MS/MS (API mass spectrometer) Amylose tris(3-chloro-5methylphenylcarbamate) (Chiralpak®IG column, Daicel Corporation, Tokyo, Japan), 250 mm ×4.6 mm i.d. and 5 µ m particle size Acetonitrile:water (5 mM ammonium acetate and 0.05% formic acid) (53:47) at a flow rate of 0.6 mL/min and Tcolumn: 30 ◦C [13] Separations 2022,9, 29 6 of 19 Table 1. Cont. Group Pesticide Matrix Extraction and Clean-Up Analytical Determination Chiral Stationary Phase (Chiral Column) or Chiral Selector Analysis Conditions Ref. Paddy soils Solid–liquid extraction with organic solvents and clean-up by glass chromatography column using active carbon, Al2O3, and anhydrous Na2SO4as sorbents HPLC-DAD (detection at 280 nm) Cellulose tris(3,5dimethylphenylcarbamate) (Chiralpak®OD-H column, Daicel Corporation, Tokyo, Japan), 250 mm ×4.6 mm i.d. and 5 µm particle size N-hexane:2-propanol (90:10) at a flow rate of 1 mL/min, Vinjection: 20 µL and Tcolumn: 22 ◦C [23] Vegetables Solid–liquid extraction with an organic solvent and clean-up with glass chromatography column using active carbon, Al2O3, and Na2SO4as sorbents HPLC-UV (detection at 225 nm) 1-(3,5-dinitrobenzamido)- 1,2,3,4-tetrahydrophenanthrene (Whelk-O1®column, Regis Technologies, Morton Grove, IL, USA) and 250 mm × 4.6 mm i.d. N-hexane:isopropanol (95:5) at a flow rate of 1 mL/min, Vinjection: 20 µL and Tcolumn: 10 ◦C [24] Plant samples Extraction with an organic solvent and clean-up by DSPE using PSA, C18, and carbon nanotubes as sorbent. UPLC-Q-Exactive Orbitrap MS Amylose tris(3,5dimethylphenylcarbamate) (Chiralpak®AD-RH column, Daicel Corporation, Tokyo, Japan), 150 mm ×4.6 mm i.d. and 5 µm particle size Water:acetonitrile (50:50) at a flow rate of 0.3 mL/min [25] QuEChERS and clean-up by DSPE using MMWCNTs as sorbent UHPLC-MS/Qtrap Amylose tris(3-chloro-5methylphenylcarbamate) (Chiralpak®IG column, Daicel Corporation, Tokyo, Japan), 250 mm ×4.6 mm i.d. and 5 µ m particle size Water (0.1% formic acid):acetonitrile (gradient condition) at a flow rate of 0.4 mL/min, Vinjection: 2 µL and Tcolumn: 35 ◦C [12] Separations 2022,9, 29 7 of 19 Table 1. Cont. Group Pesticide Matrix Extraction and Clean-Up Analytical Determination Chiral Stationary Phase (Chiral Column) or Chiral Selector Analysis Conditions Ref. Flufiprole Standard solutions HPLC-DAD (detection at 230 nm) Cellulose tris(3,5 dimethylphenylcarbamate) (Chiralpak®IB column, Daicel Corporation, Tokyo, Japan), 250 mm ×4.6 mm i.d. and 5 µ m particle size N-hexane:ethanol (95:5) at a flow rate of 1 mL/min, Vinjection: 20 µL and Tcolumn: 30 ◦C [18] SFC-UV/Vis (detection at 230 nm) Cellulose tris(3,5dimethylphenylcarbamate) (Chiralpak®OD-H column, Daicel Corporation, Tokyo, Japan), 250 mm ×4.6 mm i.d. and 5 µm particle size ScCO2:ethanol (91:9) at a flow rate of 2 mL/min, Vinjection: 10 µL and Tcolumn: 35 ◦C [19] Soils, vegetables, and fruits QuEChERS and clean-up by SPE using Alumina-N-SPE cartridge HPLC-UV (detection at 230 nm) Cellulose tris(3-chloro-4methylphenylcarbamate) (Lux Cellulose-2 column), 250 mm ×4.6 mm i.d. and 5 µ m particle size Acetonitrile:water (55:45) at a flow rate of 0.7 mL/min, Vinjection: 20 µL and Tcolumn: 30 ◦C [26] Paddy fields, rice straw, and rice QuEChERS and clean-up by SPE using Cleanert PestiCarb/PSA cartridge UPLC-MS/MS Cellulose tris(4-chloro-3methylphenylcarbamate) (Lux Cellulose-4 column), 150 mm ×2.0 mm i.d. and 3 µ m particle size Acetonitrile:water (0.1% acid formic) (65:35) at a flow rate of 0.25 mL/min, Vinjection: 1µL and Tcolumn: 25 ◦C [27] Plant samples QuEChERS and clean-up by DSPE using MMWCNTs as sorbent UHPLC-MS/Qtrap Amylose tris(3-chloro-5methylphenylcarbamate) (Chiralpak®IG column, Daicel Corporation, Tokyo, Japan), 250 mm × 4.6 mm i.d. and 5 µ m particle size Water (0.1% formic acid):acetonitrile (gradient condition) at a flow rate of 0.4 mL/min, Vinjection: 2 µL and Tcolumn: 30 ◦C [12] Separations 2022,9, 29 8 of 19 Table 1. Cont. Group Pesticide Matrix Extraction and Clean-Up Analytical Determination Chiral Stationary Phase (Chiral Column) or Chiral Selector Analysis Conditions Ref. Ethiprole Standard solutions SFC-UV/Vis (detection at 230 nm) Amylose tris(S)-α-(3,5dimethylphenylcarbamate) (Chiralpak®AS-H column, Daicel Corporation, Tokyo, Japan), 250 mm ×4.6 mm i.d. and 5 µm particle size ScCO2:methanol (91:9) at a flow rate of 2 mL/min, Vinjection: 10 µL and Tcolumn: 35 ◦C [19] Soils, paddy soils, vegetables, and fruits QuEChERS and clean-up by SPE using Florisil cartridge HPLC-UV (detection at 225 nm) Cellulose tris(3-chloro-4methylphenylcarbamate) (Lux Cellulose-2 column), 250 mm ×4.6 mm i.d. and 3 µ m particle size Methanol:water (65:35) at a flow rate of 0.7 mL/min, Vinjection: 20 µL and Tcolumn: 35 ◦C [28,29] Sulfoxamines Sulfoxaflor Rice, cucumber and apple samples QuEChERS and clean-up by SPE using Cleanert PestiCarb/PSA cartridge HPLC-DAD (detection at 220 nm) Amylose tris(3,5-dimethylphenyl carbamate) (Chromega Chiral ®, ES Industries, West Berlin, USA CCA), 250 mm ×4.6 mm i.d. and 5 µm particle size N-hexane:ethanol:methanol (90:2:8) at a flow rate of 1 mL/min, Vinjection: 20 µL and Tcolumn: 20 ◦C [30] Plant samples QuEChERS and clean-up by DSPE using MMWCNTs UHPLC-MS/Qtrap Amylose tris(3-chloro-5-methylphenyl carbamate) (Chiralpak®IG column, Daicel Corporation, Tokyo, Japan), 250 mm ×4.6 mm i.d. and 5 µ m particle size Water (0.1% formic acid):acetonitrile (gradient condition) at a flow rate of 0.4 mL/min, Vinjection: 2µL and Tcolumn: 30 ◦C [12] Plant samples (tea leaves) MSPD using Florisil and C18 as sorbents UHPLC-HRMS Cellulose tris-(4-methylbenzoate) (Chiral Cel®OJ-3R, Daicel Corporation, Tokyo, Japan), 150 mm ×4.6 mm i.d. and 3 µ m particle size Water (0.1% formic acid):acetonitrile (48:52) at a flow rate of 0.4 mL/min, Vinjection: 1 µL and Tcolumn: 30 ◦C [31] Separations 2022,9, 29 9 of 19 Table 1. Cont. Group Pesticide Matrix Extraction and Clean-Up Analytical Determination Chiral Stationary Phase (Chiral Column) or Chiral Selector Analysis Conditions Ref. DSPE using PSA as sorbent UHPLC-MS/MS Cellulose tris-(4-methylbenzoate) (Chiral Cel OJ-3R), 150 ×4.6 mm i.d. and 3 µm particle size Water:acetonitrile (80:20) at a flow rate of 0.3 mL/min, Vinjection:1 µL and Tcolumn: 30 ◦C [32] Soils and vegetables QuEChERS and clean-up by DSPE using MWCNTs and anhydrous MgSO4as sobents UPC2-MS-MS Amylose tris(3,5-dimethylphenyl carbamate) (Chiralpak®IA-3, Daicel Corporation, Tokyo, Japan), 150 mm ×4.6 mm i.d. and 3 µm particle size scCO2::2propanol:acetonitrile (95:3:2) at a flow rate of 2.2 mL/min, Vinjection: 1 µL and Tcolumn: 40 ◦C [33] Vegetables QuEChERS and clean-up by DSPE with MWCNTs UHPSFC-MS/MS Amylose tris(3,5-dimethylphenyl carbamate) (Chiralpak®IA-3, Daicel Corporation, Tokyo, Japan), 150 mm ×4.6 mm i.d. and 3 µm particle size scCO2::2propanol:acetonitrile (95:3:2) at a flow rate of 2.2 mL/min, Vinjection: 1 µL and Tcolumn: 40 ◦C [34] Marine and freshwater media - EKC-DAD Succinyl-β-CD BGE: borate buffer (pH 9.0), T: 15 ◦ C and voltage of 20 kV [35] Separations 2022,9, 29 16 of 19 Orbitrap mass analyzer have been used to determine the sulfoxaflor stereoisomers in plant samples. SFC-MS/MS is a great chromatography system for the chiral determination of sulfoxaflor in environmental samples due to the combination of the high efficiency and fast separation of SFC and the excellent specificity of MS detector. Thus, sulfoxaflor stereoisomers have been successfully separated and analyzed in environmental samples by ultraperformance convergence chromatography/tandem triple quadruple mass spectroscopy (UPC 2 -MS/MS) [ 33 ] and ultrahigh-performance supercritical fluid system coupled with a triple-quadrupole mass spectrometer (UHPSFC-MS/MS) [34]. Jiménez-Jiménez et al. [ 35 ] evaluated the stability of sulfoxaflor stereoisomers in marine and fresh water. For that, the authors used EKC-UV and tested 14 different CSs to find the one that provide the best separation. This method has the advantage that the CS and its concentration can be easily changed, which facilitates the screening of several CSs to obtain the most optimal one for a given chiral pesticide. Finally, the authors found that 15 mM Succinylβ -CD in 100 mM borate buffer (pH = 9.0) was the best CS conditions for the separation of sulfoxaflor stereoisomers. 5. Conclusions and Future Perspective In the previous sections, the main extraction and analytical separation techniques used for the stereoselective determination of chiral pesticides have been discussed, with emphasis on the methodology used for pesticides that contain an asymmetric S atom in their structure. Table 1summarizes the extraction and clean-up methods along with the analytical methodology used for the chiral pesticides with a chiral sulfur described in this review. Overall, QuEChERS is the most widely method used for the extraction of these chiral pesticides from environmental samples. In the case of analytical techniques, liquid chromatography has proven to be the most used for the resolution of all the pesticides reviewed. Absorbance detectors (UV, UV/Vis, and DAD) are widely used coupled to an HPLC system. However, the most recent studies on the stereoselective determination of new sulfur pesticides used preferably MS detection due to the high sensitivity and selectivity of this technique, which is essential in complex environmental samples with a high matrix effect. The search for pesticides with new modes of actions that prevent cross-resistance has led, in some cases, to the development of compounds with novel moieties based on S such as the aforementioned group of sulfoxamines insecticides. Another example is the N-cyano sulfilimines that present a high insecticidal activity [ 7 ] and whose commercialization could be promising. All of these compounds have an asymmetric S in its structure. For this reason, the following would be recommended: - Carry out the environmental risks assessment of the stereoisomers separately due to the possible enantioselectivity of its bioactivity against target pest and its toxicity against non-target organisms. - Take into account the possible stereoselective behavior of pesticide metabolites that have chiral sulfur in their structure. - Develop new CS and CSP to improve the resolution of chiral pesticides with asymmetric S atoms. - Optimize the instrumentation of promising techniques such as SFC or CE techniques for inclusion in the routine analysis of these pesticides. Author Contributions: Conceptualization, A.F. and R.L.-C.; software, A.F. and R.L.-C.; validation, A.F. and R.L.-C.; formal analysis, A.F. and R.L.-C.; investigation, A.F. and R.L.-C.; resources, A.F.; data curation, R.L.-C.; writing—original draft preparation, A.F. and R.L.-C.; writing—review and editing, A.F. and R.L.-C.; visualization, A.F. and R.L.-C.; project administration, A.F.; funding acquisition, A.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: This study was supported by an EMBO and a FEBS grant to A.F. Separations 2022,9, 29 17 of 19 Conflicts of Interest: The authors declare no conflict of interest. References 1. Ulrich, E.M.; Morrison, C.N.; Goldsmith, M.R.; Foreman, W.T. Chiral Pesticides: Identification, Description, and Environmental Implications. 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