Simultaneous Enantioseparation of Three Chiral Antifungal Pesticides by Hydroxypropyl-γ-CD-Modified Micellar Electrokinetic Chromatography

Simultaneous enantioseparation of three commonly used chiral antifungal pesticides (diniconazole, hexaconazole, and imazalil) was first studied based on micellar electrokinetic chromatography (MEKC) with hydroxypropyl-γ-CD (HP-γ-CD) as chiral selector. In this study, the importance of experimental parameters such as chiral selector type and concentration, sodium dodecyl sulfate (SDS) concentration, the ratio of methanol, and separation voltage in optimizing were investigated. The simultaneous enantioseparation of diniconazole, hexaconazole, and imazalil was successfully achieved in 30 mM borate buffer (pH 9.0) containing 10 mM HP-γ-CD and 20 mM SDS with methanol (8%) added as organic modifiers. The resolution of diniconazole, hexaconazole, and imazalil was 15.2, 2.12, and 2.78, respectively, and the peak efficiency (N) was over 566,825 plates/m. This study provides an alternative way to systematically separate chiral antifungal pesticides with high efficiency.


Introduction
Nowadays, developing green pesticides [1] has become a hotspot in the feld of chemistry and environmental research since traditional pesticides have heavily afected environmental safety.Chiral pesticide [2] is one of the most important green pesticides because of its high efcacy, small dosage, and safety for the environment.While the R-enantiomer of chiral pesticides showed great diference compared to S-enantiomers in biological activity, environmental behavior, and biological toxicity [3], for example, the efciency of Salithion's Senantiomer was higher than that of R-enantiomer for mosquito, armyworm, and mouse; however, the result was contrary to that of housefy [4].When exposed to 100 and 200 μgL −1 of hexaconazole, the (−)-enantiomer was found to accumulate more readily in zebrafsh than the (+)-enantiomer, suggesting that hexaconazole has signifcant enantioselective bioaccumulation [5].Terefore, the preparation of enantiomeric purity compounds is essential.However, most chiral pesticides were used in their racemic form because of the restriction of technology and cost, which resulted in low efcacy.Te key factor in developing chiral pesticides is to establish an efective way to separate and analyze chiral compounds.
Numerous chromatographic methods have been used for enantiomeric separations of pesticides including highperformance liquid chromatography (HPLC) [6,7], gas chromatography (GC), and capillary electrophoresis (CE) [8].Te chiral column is the fatal factor in separating enantiomers for HPLC and GC, but it is expensive and difcult to separate diferent kinds of chiral compounds simultaneously.In contrast, MEKC, a new powerful way for enantiomeric separation, has been broadly recognized due to its advantages including simple operation, quick speed, high efcacy, and low consumption, especially for the usage of pseudostationary phases that provide more selectivity for chiral separation compared to traditional CE [9,10].
Diferent kinds of chiral pesticides have been produced, including triazole fungicides, pyrethroid insecticides, and phenoxy herbicides.Diniconazole, hexaconazole, and imazalil are common fungicides used for crop sterilization (Figure 1).Many ways have been performed previously to separate them, but most methods using HPLC or GC could only separate one kind of fungicide with chiral columns [11,12].For example, Li et al. [13] successfully resolved imazalil with cellulose tris(3-chloro-4-methylphenylcarbamate) (CCMPC) as a chiral stationary phase, and Chai et al. [14] used cellulose-tris(4-methylbenzoate) (CTMB) as a chiral stationary phase to obtain better resolution.Electrophoresis methods also have been reported to separate chiral substances, for example, the separation of diniconazole by MEKC with both HP-c-CD and hydroxypropyl-β-cyclodextrin (HP-β-CD) [15,16] and enantiomeric separation of diniconazole and hexaconazole using sulfated-β-CD (S-β-CD).Besides, hexaconazole was separated using supercritical fuid chromatography (SFC) [17].However, although the chiral stationary phase method of high-performance liquid chromatography has high sensitivity, it also has the defects of complex operation and high cost.Te methods using SFC are costly and difcult to operate, and electrophoresis methods sufer from poor peak and small resolution.Terefore, it is of practical signifcance to develop a rapid, simple, high peak value, and highresolution method for the chiral resolution of chiral antifungal pesticides.To our knowledge, the method using CD-MEKC for the simultaneous separation of diniconazole, hexaconazole, and imazalil has never been reported yet.
In this study, we aimed to establish a simultaneous enantioseparation method of diniconazole, hexaconazole, and imazalil (Figure 1) by using CD-MEKC for the frst time.Te method could be a technical protocol for the separation of enantiomeric purity compounds.Furthermore, it could reduce the pollution of the environment and harm human beings due to the use of pesticides.Background electrolyte was prepared by dissolving Na 2 B 4 O 7 .10H 2 O in ultrapure water to 100 mM.Te running bufer was prepared by dissolving CD and SDS in the background bufer and adjusting the pH of butter with NaOH or H 3 PO 4 .All bufers were fltered through a 0.22 μm flter before use.Stocking solutions of signal chiral antifungal pesticides were prepared in methanol at a concentration of 2000 μg/mL and stored at 4 °C.Working solutions were prepared by diluting the stocking solutions with a background bufer to a suitable concentration.All samples were fltered before sampling.
Preconditioning capillary: Te capillary was fushed with 0.1 mol/L NaOH for 5 min followed by fushing with ultrapure water for 5 min and running bufer for 10 min before use.Te capillary was fushed with 0.1 mol/L NaOH, ultrapure water, and running bufer for 3 min between injections to confrm the stability of the system and the reproducibility of results.At the end of the experiment, the capillary was rinsed for 3 min with ultrapure water to protect the column and UV detector.

Results and Discussion
3.1.Selection of Cyclodextrins.CD, as an additive, is commonly used for chiral separation, especially for α-CD, β-CD, and c-CD.To expand the application range of CD, chemical modifcations were introduced by using the derivatization of CD to enhance the enantioselectivity.Many modifed CDs have been performed previously, for instance, hydroxypropyl-CD, hydroxymethyl-CD, and sulfobutyl-CD.
Te separation abilities of β-CD, HP-β-CD, and HPc-CD were studied in the 15 mM sodium borate containing 20 mM SDS with pH from 2 to 9, and all concentrations of CDs were 20 mM.Results showed that neither diniconazole nor hexaconazole could be enantiomerically separated from β-CD and HP-β-CD.In addition, β-CD was barely dissolved in phosphate bufer and turned into crystals after a night.Te baseline was abnormal with high noise when HP-β-CD was used.We found that all samples were separated efectively by using HP-c-CD, with constant baseline and low noise.Terefore, HP-c-CD was used as a chiral additive for the following experiments.

Te Efect of Cyclodextrin Concentration.
Te concentration is an important parameter to control in enantiomeric separation.Migration times and resolution would be changed by the variation in the CD concentration, which impacts the enantiomeric separation.Te chiral separation of all chiral antifungal pesticides was studied at 5, 10, 15, and 20 mM HPc-CD in the 15 mM sodium borate containing 20 mM SDS at pH 9. Figure 2 shows that the chiral resolution was increased with the HP-c-CD concentration in the range from 5 mM to 10 mM and the chiral resolution was best at 10 mM HP-c-CD, while the chiral resolution of samples was decreased with the HP-c-CD concentration since the concentration was higher than 10 mM.Te possible reason is that HP-c-CD has no electric charges, so the moving speed is equal to μ eo .Te content of the sample in CD was increased with the increase of 2 Journal of Analytical Methods in Chemistry HP-c-CD concentration; accordingly, the content of the sample in SDS was decreased.As a result, the sample's μ app was increased, so the retention time of the sample was shorter than before.Te inclusion complexes formed by cyclodextrins and chiral substances were partitioned between the pseudostationary phase and aqueous solution.Eventually, the good enantioresolution of the three chiral antifungal pesticides was achieved at 10 mM HP-c-CD, so 10 mM was the optimal concentration for HP-c-CD.

Te Efect of SDS Concentration.
Te concentration of SDS was also an important parameter in chiral separation.In this study, the efect of varying SDS concentration from 15 to 30 mM in the 15 mM sodium borate containing 10 mM HPc-CD at pH 9 was also performed to obtain better enantiomeric separation of all chiral antifungal pesticides (Figure 3).It can be seen from Figure 3 that the analysis time of diniconazole, hexaconazole, and imazalil increased from 6 to 12 min when the SDS concentration increased from 15 to 30 mM.Te reason was that SDS, as a pseudostationary phase, was negatively charged which led to its migratory direction contrary to electroosmotic fow (EOF).Terefore, the sample was partitioned between SDS micelles and aqueous solution, and the moving speed of SDS was enhanced with the increase of negative charge, while the μ app of the sample was reduced when the concentration of SDS was increased.As a result, the retention time of the chiral antifungal fungicide became longer.Tere was no chiral resolution for all of them at 15 mM SDS, and partial separation of the three enantiomers appeared when the concentration of SDS increased to 20 mM.However, the chiral resolution decreased when the concentration of SDS decreased from 20 to 30 mM.Terefore, the optimal concentration of SDS was 20 mM.

Te Efect of Methanol Concentration.
To achieve high enantioresolution, the ratio of methanol was also investigated.Te chiral separation of all chiral antifungal pesticides was studied in the 15 mM sodium borate containing 10 mM HPc-CD, and 20 mM SDS at pH 9 when the ratio of methanol was 1%, 5%, 8%, 10%, 15%, 20%, 25%, and 30%.As shown in Figure 4, the migration time of the three chiral enantiomers increased with the increase in the methanol ratio.Te reason was that the organic solvent reduced the EOF and the  sample's μ app , which led to an increase in analysis time.Te chiral resolution of six peaks was changed regularly along with the amount of methanol, and the Rs of imazalil II and hexaconazole I were increased with an increase in the methanol ratio, while the Rs of hexaconazole II and diniconazole II were decreased.Te Rs of diniconazole I and imazalil I were good when the ratio of methanol was 8%, 10%, and 15%, and 10% of methanol was the best choice for the following experiment based on the comprehensive consideration of the migration time and chiral resolution.

Te Efect of Borate Concentration.
Te concentration of sodium borate also had a great infuence on the chiral resolution; therefore, the enantioresolution of the three chiral antifungal pesticides was studied at 15, 30, and 45 mM sodium borate in sodium borate containing 10 mM HPc-CD and 20 mM SDS with pH 9 (Figure 5).Baseline separation was successfully achieved for all samples except for hexaconazole II and diniconazole II at 30 mM sodium borate.Te current intensity was so high that it may cause joule heat when the concentration of sodium borate increased to 45 mM.In comprehensive consideration, the optimal concentration of sodium borate was 30 mM.

Te Efect of Voltage.
Te efect of diferent voltages on the chiral separation was also studied because the separation voltage was the main factor of EOF.Te voltage was optimized from 10 to 20 kV.It can be learned from Figure 6 that the separation condition was improved with the reduction of voltage due to the decrease of EOF. 15 kV was chosen preliminarily for chiral separation based on comprehensive consideration of resolution and migration time.

Optimization Considerations for MEKC Chiral Separation
Conditions.Not all enantiomers had been successfully separated yet after a series of optimization experiments, and the separation of hexaconazole II and diniconazole II remained the main problem.From the results of methanol optimization, the separation resolution increased with the reduction of methanol, so the ratio of methanol was reduced from 10% to 8%.Meanwhile, considering that the reduction of EOF might improve the resolution and a low capillary inside diameter (ID) could improve the peak profle and reduce the EOF and Joule heat, the capillary column was changed from 75 μm ID with an efective length of 40 cm (80 cm total length) to 50 μm ID with an efective length of 60 cm (80 cm total length), and the separation voltage was decreased to 13 kV.Te three pairs of chiral antifungal pesticides were ideally separated, and the resolution between two adjacent peaks was all more than 1.80.Te chromatogram in optimal conditions can be seen in Figure 7.

Peak Resolution and Column
Efciency.Te infuence of several experimental conditions including the type and concentration of chiral selector, the concentration of SDS, the ratio of methanol, the concentration of sodium borate, and the separation voltage was investigated.Te optimal conditions were as follows: capillary column: 50 μm × 80 cm, 60 cm efective length; gravity injection: 10 s; voltage: +13 kV; bufer: 30 mM sodium borate, 20 mM SDS, 10 mM HP-c-CD, 8% methanol; wavelength: 220 nm; at room temperature; sample concentration: 0.1 mg/mL   diniconazole, 0.05 mg/mL hexaconazole, and 0.05 mg/mL imazalil.Meanwhile, the peak efciency (N) and resolution peaks were analyzed based on the chromatogram.It can be seen from Table 1 that an HP-c-CD-MEKC method for the simultaneous enantioseparation of diniconazole, hexaconazole, and imazalil had good efciency (N ˃ 560000) and resolutions between two adjacent peaks.Te resolutions of diniconazole, hexaconazole, and imazalil were 15.2, 2.12, and 2.78, respectively, which showed that the method met the requirement of enantiomeric separation for the three kinds of chiral antifungal pesticides.

Conclusions
Based on micellar electrokinetic chromatography (MEKC) including HP-c-CD as chiral selector, chiral separation for three kinds of chiral antifungal pesticides (diniconazole, hexaconazole, and imazalil) was successfully achieved for the frst time; the minimum chiral resolution for a pair of enantiomers was 2.12, and the maximum resolution was 15.2.Tis study provides an alternative way to systematically separate chiral antifungal pesticides with simplicity and practicability, high efciency, and good resolution, and chiral columns and coat capillaries are not needed to be prepared.It can provide help for the study of enantiomers of diniconazole, hexaconazole, and imazalil in environmental behavior, biotoxicity, and so on.However, this method also has some shortcomings because the traditional capillary electrophoresis sample injection method has some disadvantages, which leads to the poor repeatability of the experimental method.It is expected to use quantitative capillary electrophoresis to improve repeatability.

Figure 2 :
Figure 2: Chromatogram of three chiral antifungal pesticide enantiomers with diferent HP-c-CD concentrations.

Figure 3 :
Figure 3: Chromatogram of three chiral antifungal pesticide enantiomers in diferent SDS concentrations.

Table 1 :
Peak efciencies (N) and resolution between two adjacent peaks.