molecules Article Chiral Separation and Determination of Etoxazole Enantiomers in Vegetables by Normal-Phase and Reverse-Phase High Performance Liquid Chromatography 1,2,3, , 1,2, 1,2 1,2 1,2 Ping Zhang * y, Yuhan He y, Sheng Wang , Dongmei Shi , Yangyang Xu , Furong Yang 1,2, Jianhao Wang 1,2 and Lin He 1,2,3,* 1 Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400715, China; [email protected] (Y.H.); [email protected] (S.W.); [email protected] (D.S.); [email protected] (Y.X.); [email protected] (F.Y.); [email protected] (J.W.) 2 Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China 3 State Cultivation Base of Crop Stress Biology for Southern Mountainous Land of Southwest University, Southwest University, Chongqing 400715, China * Correspondence: [email protected] (P.Z.); [email protected] (L.H.); Tel.: +86-23-68251514 (P.Z.); +86-23-68254105 (L.H.) These authors contributed equally to this paper. y Received: 17 June 2020; Accepted: 6 July 2020; Published: 9 July 2020 Abstract: The chiral separation of etoxazole enantiomers on Lux Cellulose-1, Lux Cellulose-3, Chiralpak IC, and Chiralpak AD chiral columns was carefully investigated by normal-phase high performance liquid chromatography and reverse-phase high performance liquid chromatography (HPLC). Hexane/isopropanol, hexane/n-butanol, methanol/water, and acetonitrile/water were used as mobile phase at a flow rate of 0.8 mL/min. The effects of chiral stationary phase, mobile phase component, mobile phase ratio, and temperature on etoxazole separation were also studied. Etoxazole enantiomers were baseline separated on Lux Cellulose-1, Chiralpak IC, and Chiralpak AD chiral columns, and partially separated on Lux Cellulose-3 chiral column under normal-phase HPLC. However, the complete separation on Lux Cellulose-1, Chiralpak IC, and partial separation on Chiralpak AD were obtained under reverse-phase HPLC. Normal-phase HPLC presented better resolution for etoxazole enantiomers than reverse-phase HPLC. Thermodynamic parameters, including DH and DS, were also calculated based on column temperature changes from 10 ◦C to 40 ◦C, and the maximum resolutions (Rs) were not always acquired at the lowest temperature. Furthermore, the optimized method was successfully applied to determine etoxazole enantiomers in cucumber, cabbage, tomato, and soil. The results of chiral separation efficiency of etoxazole enantiomers under normal-phase and reverse-phase HPLC were compared, and contribute to the comprehensive environmental risk assessment of etoxazole at the enantiomer level. Keywords: etoxazole; chiral separation; enantiomers; risk assessment; HPLC 1. Introduction Chiral pesticides have become a hotspot in the field of pesticide research. About 30% of commercial pesticides are chiral [1,2]. However, most commercial chiral pesticides are sold in the racemate form. Enantiomers of chiral pesticide have almost the same physical and chemical properties in achiral environments and generally different properties in chiral environments, including biological activity, metabolism, degradation, and toxicity [3–9]. With the increasing number of synthesized and registered Molecules 2020, 25, 3134; doi:10.3390/molecules25143134 www.mdpi.com/journal/molecules Molecules 2020, 25, x 2 of 17 Molecules 2020, 25, 3134 2 of 13 number of synthesized and registered chiral pesticides introduced to market, it is urgent and necessary to investigate the environmental fates and toxicological risks of chiral pesticides at the enantiomerchiral pesticides level. introduced to market, it is urgent and necessary to investigate the environmental fates and toxicologicalEtoxazole risks[(RS) of-5- chiraltert-butyl pesticides-2-[2-(2,6 at- thedifluorophenyl enantiomer level.)-4,5-dihydro-1,3-oxazol-4-yl]phenetole, CAS Etoxazole153233-91-1 [((RSFigure)-5-tert 1)-butyl-2-[2-(2,6-difluorophenyl)-4,5-dihydro-1,3-oxazol-4-yl]phenetole,] is a diphenyloxazole acaricide used to control various mites on vegetables,CAS 153233-91-1 flowers, (Figure fruits,1)] isgrass a diphenyloxazole, etc. in agriculture. acaricide The used mode to controlof action various (MoA mites) of etoxazole on vegetables, is to affectflowers, adult fruits, egg grass, develo etc.pment in agriculture. by inhibiting The chitin mode biosynthesis, of action (MoA) thus of it etoxazoleis used against is to a ffnymphs,ect adult mite egg larvae,development eggs, and by inhibiting is safe for chitin adult biosynthesis, mites [3,10– thus13]. Etoxazole it is used against has an nymphs, asymmetric mite chiral larvae, carbon eggs, andatom is insafe its for chemical adult mites structure [3,10– and13]. consists Etoxazole of has two an enantiomers asymmetric ( chiralR-etoxazole carbon and atom S in-etoxazole, its chemical at a structure ratio of 1:1and) consists[3]. In recent of two years, enantiomers etoxazole (R -etoxazoleresidue has and beenS-etoxazole, reported atin a soil, ratio berry, of 1:1) apple, [3]. In orange recent years,, and vegetableetoxazoles residue [13–16], has which been pose reporteds a high in soil, risk berry,for human apple, and orange, environmental and vegetables health. [13 –16], which poses a high risk for human and environmental health. Figure 1. Chemical structure and chromatogram of etoxazole enantiomers on a Lux cellulose-3 column Figurewith an 1. ACN Chemical/H2O ratio structure of 70/30 and at 40chromatogram◦C under reverse-phase of etoxazole conditions. enantiomers on a Lux cellulose-3 column with an ACN/H2O ratio of 70/30 at 40 °C under reverse-phase conditions. Enantiomer separation of chiral pesticides is the first step in enantiomer risk assessment. ManyEnantiomer methods have separation been reported of chiral for pesticide chiral separation,s is the firstincluding step in enantiomer normal-phase risk assessment. high performance Many methodsliquid chromatography have been reported (NP-HPLC) for chiral [17 separation], reverse-phase, including high normal performance-phase high liquid performance chromatography liquid chromatography(RP-HPLC) [18,19 ],(NP gas-HPLC chromatography) [17], reverse (GC)-phase [20], capillary high performance electrophoresis liquid (CE) [chromatography21], supercritical (fluidRP-HPLC chromatography) [18,19], gas (SFC)chromatography [22,23], thin-layer (GC) [20], chromatography capillary electrophoresis (TLC) [24], ( micellarCE) [21], electrokinetic supercritical fluidchromatography chromatography (MEKC) (SFC [25) ,26[22,23], capillary], thin-layer electro-chromatography chromatography (TLC (CEC)) [24], [27], micellar and ultraperformance electrokinetic chromatographyconvergence chromatography (MEKC) [25,26], (UPCC) capillary [28 electro]. HPLC,-chromatography SFC, and (CEC UPCC) [27] have, and become ultraperformance the most convergenceeffective and chromatography widely used approaches (UPCC) [28]. for HPLC chiral, SFC separation, and UPCC and h enantiomerave become detectionthe most effective because andnumerous widely chiralused approach stationaryes phasesfor chiral (CSPs) separation can be and used enantiomer [29], especially detection polysaccharide-based because numerous chiral CSPs, stationaryincluding amylose-tris-(3,5-dimethylphenylcarbamate),phases (CSPs) can be used [29], especially cellulose-tris-(3,5-dimethylphenylcarbamate), polysaccharide-based CSPs, including amylosecellulose-tris-(3,5-dichlorophenylcarbamate),-tris-(3,5-dimethylphenylcarbamate), cellulose-tris-(4-chloro-3-methylphenylcarbamate),cellulose-tris-(3,5-dimethylphenylcarbamate etc.,), celluloseand cyclodextrin-based-tris-(3,5-dichlorophenylcarbamate CSPs, including β),-CD, celluloseγ-CD,-tris and-(4- otherchloro derivatives-3-methylphenylcarbamate such as β-OH,)α, -PM,etc., andβ-PM, cyclodextrin and γ-PM- [based27]. CSPs, including β-CD, γ-CD, and other derivatives such as β-OH, α-PM, β-PM,The and separation γ-PM [27]. methods of etoxazole enantiomers include reverse-phase HPLC [30], normal-phase HPLCThe [ 31separation], ultraperformance methods of liquid etoxazole chromatography enantiomers (UPLC) include [32 ],reverse ultraperformance-phase HPLC liquid[30], normalchromatography-phase HPLC tandem [31],mass ultraperformance spectrometry liquid (UPLC-MS chromatography/MS) [3], and ( gasUPLC chromatography) [32], ultraperformance (GC) [15]. liquidHowever, chromatography the systematic comparisontandem mass of chiralspectrometry separation (UPLC of etoxazole-MS/MS enantiomers) [3], and gas under chromatography normal-phase (HPLCGC) [15]. and However, reverse-phase the systematic HPLC has comparison not been investigated.of chiral separation In our of study,etoxazole etoxazole enantiomers enantiomers under normalwere separated-phase HPLC on Luxand Cellulose-1,reverse-phase Lux HPLC Cellulose-3, has not been Chiralpak investigated. IC, and In Chiralpakour study, ADetoxazole chiral enantiomerscolumns under were normal-phase separated on and Lux reverse-phase Cellulose-1, conditions.Lux Cellulose Baseline-3, Chiralpak separation IC, onand Lux Chiralpak Cellulose-1, AD chiral columns under normal-phase and reverse-phase conditions. Baseline separation on Lux Molecules 2020, 25, 3134 3 of 13 Chiralpak IC, and Chiralpak AD columns, and partial separation on Lux Cellulose-3 were achieved under normal-phase HPLC. Furthermore, etoxazole enantiomers were completely separated on Lux Cellulose-1 and Chiralpak IC, and partially separated on the Chiralpak AD column under reverse-phase conditions. The effects of the chiral stationary phase, mobile phase component,