Enantioselective Separation and Dissipation of Prothioconazole and

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Enantioselective Separation and Dissipation of Prothioconazole and Its Major Metabolite ProthioconazoleDesthio Enantiomers in Tomato, Cucumber, and Pepper Duoduo Jiang, Fengshou Dong, Jun Xu, Xingang Liu, Xiaohu Wu, Xinglu Pan, Yan Tao, Runan Li, and Yongquan Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03607 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 25, 2019

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Journal of Agricultural and Food Chemistry

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Enantioselective Separation and Dissipation of Prothioconazole and Its Major

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Metabolite Prothioconazole-Desthio Enantiomers in Tomato, Cucumber, and

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Pepper

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Duoduo Jiang, Fengshou Dong*, Jun Xu, Xingang Liu, Xiaohu Wu, Xinglu Pan, Yan

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Tao, Runan Li, Yongquan Zheng

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State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant

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Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China

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

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Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of

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Agricultural Sciences, Beijing, 100193, P. R. China

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Tel.: +86 10 62815938; fax: +86 10 62815938.

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E-mail address: [email protected] (F. Dong).

Correspondence: Prof. Fengshou Dong, State Key Laboratory for Biology of Plant

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ACS Paragon Plus Environment


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ABSTRACT

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In this study, a simple and effective chiral analytical method was developed to

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monitor prothioconazole and prothioconazole-desthio at the enantiomeric level using

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supercritical fluid chromatography-tandem triple quadrupole mass spectrometry. The

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baseline enantioseparation for prothioconazole and prothioconazole-desthio was

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achieved within 2 min on a Chiralcel OD-3 column with CO2/0.2% acetic acid-5

30

mmol/L ammonium acetate 2-propanol (85:15, v/v) as mobile phase at a flow rate of

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1.5 mL/min and column temperature of 25 °C. The limit of quantitation (LOQ) for each

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enantiomer was 5 μg/kg, with baseline resolution of >3.0. The results of

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enantioselective dissipation showed R-(-)-prothioconazole was preferentially degraded

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in tomato, cucumber, and pepper under greenhouse conditions. S-(-)-prothioconazole-

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desthio was preferentially degraded in tomato and cucumber, however, R-(+)-

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prothioconazole-desthio was preferentially degraded in pepper. Results of this study

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may help to facilitate more accurate risk assessment of prothioconazole and its major

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metabolite in agricultural products.

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KEYWORDS: prothioconazole, prothioconazole-desthio, enantioselective separation,

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enantioselective dissipation, SFC-MS/MS

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INTRODUCTION

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Prothioconazole

(R,

S)-(2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-

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hydroxypropyl]-1,2-dihydro-3H-1,2,4-triazole-3-thione), belongs to triazole fungicides

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with outstanding protective and curative properties (Figure 1). Owing to the ability of

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inhibiting the C-14 α-demethylase enzyme involved in the biosynthesis of fungal sterols,

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prothioconazole, as a racemic mixture, has been used to control powdery mildew,

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Fusarium head blight, rusts, and leaf spot diseases on cereal, wheat, soybean, and other

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economic crops1-2. Prothioconazole-desthio, (R, S)-(2-(1-chlorocyclopropyl)-1-(2-

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chlorophenyl)-3(1,2,4-triazol-1-yl)-propan-2-ol),

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prothioconazole in plant and environment, has one chiral carbon atom and consists of

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a pair of enantiomers, similar to the case of prothioconazole (Figure 1). According to

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the toxicity data, prothioconazole-desthio is more toxic than prothioconazole in rats3,

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and has gradually received increasing attention. Nowadays, a few studies have been

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conducted on the analysis methods and degradation behaviors of racemic

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prothioconazole and its major metabolite in food and environment, including wheat3,

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peanut4, animal origin food5, and soil6, for food and environment safety purpose.

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However, there were limited research on the enantiomeric analysis methods of

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prothioconazole and prothioconazole-desthio, and no studies have reported the

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degradation of prothioconazole and prothioconazole-desthio in agricultural products at

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the enantiomeric level. Consequently, their actual effects are always underestimated or

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overestimated because enantiomers of many chiral pesticides differ in bioactivity,

the

3


major

metabolite

of


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metabolism, and environmental behaviors during the course of degradation, absorption

68

and accumulation7-14. Therefore, the study of the enantioselective degradation of

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prothioconazole and prothioconazole-desthio in agricultural products is crucial for the

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further accurate risk assessment.

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To date, few enantiomeric analysis methods of prothioconazole and its metabolites

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in agricultural food and environmental samples have been reported. Zhang et al.15

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presented a method for the simultaneous enantioselective determination of

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prothioconazole and prothioconazole-desthio in cucumber, pear, soil, and water by

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ultra-performance liquid chromatography−tandem mass spectrometry (UPLC-MS/MS),

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with a long retention time of 11.83-19.30 min, and the pretreatment was time-

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consuming. Jiang et al.16 developed a supercritical fluid chromatography (SFC) and

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vibrational circular dichroism spectroscopic method for the enantioselective

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determination of prothioconazole in soil and tomato. However, the limits of

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quantitation (LOQs) of prothioconazole enantiomers were in the range of 4.99-5.52

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mg/kg, which was lack of sensitivity. Hence, the development of a rapid and sensitive

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method is significant for the enantioselective determination of prothioconazole and

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prothioconazole-desthio in agricultural products.

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In China, tomato, cucumber, and pepper are widely cultivated, and powdery mildew,

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Fusarium head blight, and rusts happen frequently during the growth of these three

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vegetables. To date, few prothioconazole formulations were only registered on wheat

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in China. However, several studies have payed attention with foresight on the analysis

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methods of prothioconazole and its major metabolite in tomato and cucumber15-16, and 4


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it has possibility that prothioconazole would be registered on solanaceous and

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cucurbitaceous vegetables in the future. So, the establishment of analysis methods and

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study of dissipation of prothioconazole and prothioconazole-desthio enantiomers in

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solanaceous and cucurbitaceous vegetables are meaningful and important. In this study,

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a sensitive, effective, and rapid chiral analytical method was established to

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simultaneously determine prothioconazole and prothioconazole-desthio enantiomers in

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tomato, cucumber, and pepper by supercritical fluid chromatography-tandem triple

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quadrupole mass spectrometry (SFC-MS/MS). The pretreatment procedure for the

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target analytes were based on the QuEChERS (quick, easy, cheap, effective, rugged,

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and safe) method, which were simple and time-saving through the usage of butylated

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hydroxytoluene (BHT). Additionally, the proposed method was used to investigate the

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enantioselective degradation of prothioconazole and prothioconazole-desthio in tomato,

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cucumber, and pepper under greenhouse conditions. This study will provide significant

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information for the accurate risk assessment of the chiral fungicide, prothioconazole.

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MATERIALS AND METHODS

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Chemicals and Reagents. Racemic prothioconazole and prothioconazole-desthio

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standards (stereoisomer ratio=1:1, 99.0% purity) were obtained from the China

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Standard Material Center (Beijing, China). HPLC-grade acetonitrile (ACN), methanol

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(MeOH), ethanol (EtOH) and 2-propanol (IPA) were purchased from Sigma-Aldrich

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(Steinheim, Germany). Chromatographic grade formic acid and acetic acid (HAc) were

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obtained from Thermo Fisher Scientific (Waltham, MA, USA). Analytical grade

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sodium chloride (NaCl), butylated hydroxytoluene (BHT), anhydrous sodium sulfate 5



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(Na2SO4), ammonium acetate (NH4OAc) and acetonitrile were purchased from Beihua

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Fine-Chemicals Co. (Beijing, China). Ultrapure water was obtained from a Milli-Q

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reagent water system (Millipore, Bedford, MA, USA). The sorbents including

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primary/secondary amines (PSA, 40 μm), octadecylsilane (C18, 50 μm), and

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graphitized carbon black (GCB, 120-400 mesh) were purchased from Bonna-Agela

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Technologies (Tianjin, PRC).

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The standard stock solutions (100 mg/L) of rac-prothioconazole and rac-

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prothioconazole-desthio were prepared in LC-grade ACN. The standard working

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solutions

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prothioconazole-desthio at 10, 50, 100, 500, 1000, and 2000 μg/L (5, 25, 50, 250, 500,

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and 1000 μg/L of each enantiomer) were prepared in pure ACN from the stock solution

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by serial dilution. All the solutions were wrapped with aluminum foil and stored in a

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refrigerator at -20 ℃ prior to analysis.

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Field Trial and Sample Collection. The field trials were conducted according to the

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guidelines for pesticides residue trials (NY/T 788-2004)17. The greenhouse were

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located at the experimental base of the Institute of Plant Protection, Chinese Academy

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of Agricultural Sciences (Langfang, China, 116.4 °E, 39.3 °N). Each plot had an area

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of 50 m2 with a buffer area of one-meter. A 40% suspension concentrate (SC) of

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prothioconazole was sprayed on three plots. Because prothioconazole has not been

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registered on tomato, cucumber, or pepper in China, we referred to the dosage of 337.5

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g a.i/ha of 25% suspension concentrate (SC) of prothioconazole on wheat (1.5 times of

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the recommended high dosage)3. In addition, one plot was used as the control. Samples

and

matrix-matched

standard

solutions

6


of

prothioconazole

and

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(tomato, cucumber, and pepper) were collected from each treatment at 2 h, 1, 2, 3, 5, 7,

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and 14 days after spraying. All samples were mixed and stored at -20 ℃ until analysis.

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Sample Preparation. The extraction and purification procedures were based on

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QuEChERS method due to its simplicity and effectiveness18-19. 10 g of homogenized

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samples were weighed into a 50 mL plastic centrifuge tube. Then, 10 mL ACN and 1 g

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BHT were added before vortexing for 5 min. Next, 1 g of NaCl and 4 g of anhydrous

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Na2SO4 were added, and the tube was vortexed for another 3 min. Thereafter, the

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mixture was centrifuged for 5 min at 2588 g, and 1.5 mL of the upper layer was

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transferred into a single-use centrifuge tube containing 50 mg of C18. The tube was

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vortexed for 1 min and centrifuged at 2588 g for 5 min. Finally, the supernatant was

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filtered into an autosampler vial through a 0.22 μm nylon syringe filter for SFC-MS/MS

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analysis.

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Apparatus and Chromatographic Conditions. An ACQUITY UPC2 system (Waters,

146

Milford, MA, USA), equipped with an Acquity UPCC binary solvent manager, an

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Acquity UPCC convergence manager, an Acquity UPCC sample manager, a Waters

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compensation pump and an Acquity UPCC column manager was used for the

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chromatographic separation of the target analytes. Chiralcel OD-3 column (150 mm ×

150

3 mm, 3 μm particle size, Daicel, Japan), which was employed to separate the

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stereoisomers of prothioconazole and prothioconazole-desthio, was coated with

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cellulose tris (3,5-dimethylphenylcarbamate, 3 μm). Three additional chiral columns

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including a Chiralpak IA-3 [amylose tris (3,5-dimethylphenylcarbamate), 5 μm], Lux

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Cellulose-1 [cellulose tris (3,5-dimethylphenylcarbamate), 3 μm] and Lux Cellulose-2 7



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[cellulose tris (3-chloro-4-methylphenylcarbamate), 3 μm] were evaluated. The

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separation was conducted with the mobile phase (solvent A (CO2)/solvent B (0.2%

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HAc-5 mmol/L NH4OAc IPA ) = 85:15 (v/v), at a constant flow rate of 1.5 mL/min for

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5 min and an injection volume of 10 μL. The temperatures of chiral column and sample

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manager were maintained at 25 ℃, 20 ℃, respectively. ABPR pressure and rate of the

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compensation solvent (0.1% formic acid/MeOH) were 2000 psi, 0.2 mL/min,

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respectively. The detection of the target compounds was quantified using a Xevo triple-

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quadrupole (Xevo-TQD) mass spectrometer (Waters Corp., Milford, MA, USA),

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equipped with an electrospray ionization (ESI) source, operating in the positive mode

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for prothioconazole-desthio and negative mode for prothioconazole. The source

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parameters were set as follows: capillary voltage, 3.0 kV; source temperature, 150 ℃;

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and desolvation temperature, 500 °C. A 50 L/h cone gas flow and a 1000 L/h

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desolvation gas flow were employed. The nebulizer gas consisted of 99.95% nitrogen,

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and the collision gas, 99.99% argon at a pressure of 2 × 10-3 mbar in the T-Wave cell.

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Multiple reaction monitoring (MRM) was applied to the MS analysis of

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prothioconazole and prothioconazole-desthio with a dwell time of 163 ms per ion pair.

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The specific MS/MS parameters were optimized as follows: m/z 342.0 was selected as

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the precursor ion for prothioconazole with a cone voltage of 42 V; m/z 125.0 and 100.0

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were chosen as the quantitative and qualitative ion, respectively, when their collision

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energies were both set to 37 V. The cone voltage of prothioconazole-desthio was set to

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36 V, and m/z 312.1 was selected as the precursor ion. The m/z values of 70.0 and 125.0

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were selected as the quantitative ion and qualitative ion, respectively, and the 8


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corresponding collision energies were set to 34 and 48 V, respectively. Masslynx NT

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v.4.1 (Waters Corp.) software was used to collect and analyze the obtained data.

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Data Analysis. The separation parameters of prothioconazole and prothioconazole-

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desthio enantiomers, including the retention factor (k′), the selectivity factor (α) and the

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resolution (Rs), were calculated as follows20:

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k' = (t - t0)/t0

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α = k2/k1

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Rs = 1.177 × w1 + w2

(1) (2) t2 - t1

(3)

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where t0 is the void time at the conditions mentioned above (t0=0.73 min, determined

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using 1,3,5-tert-butylbenzene); t is the retention time; k is the retention factor; and w is

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the peak width at half height.

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The half-life (T1/2) of each enantiomer was calculated using the following equations:

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C = C0e -kt

(4)

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T1/2 = ln2/k

(5)

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Where C0 and C indicate the concentrations of the enantiomer at time 0 and time t,

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respectively. k is the degradation rate constant and ln2=0.693.

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The enantiomeric fraction (EF), describing the enantioselectivity of prothioconazole

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and prothioconazole-desthio degradation in three vegetable samples, was described

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using the following equation:

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EF =

197 198

A+ A

+

(6)

+ A-

Where A+ and A- represent the concentrations of the (+) and (-) enantiomers. RESULTS AND DISCUSSION 9



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Optimization of the Enantiomeric Separation. Selection of the Chiral Column. The

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interaction between chiral stationary phase (CSP) and mobile phase was regarded as

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the most important factor in the enantioseparation20. Given the wide use of cellulose-

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and amylose- based polysaccharide CSP in chiral separation, four chiral columns

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(Chiralcel OD-3, Chiralpak IA-3, Lux Cellulose-1, and Lux Cellulose-2) were selected

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to separate the target analyte under the same chromatographic condition: mobile phase

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of CO2/ 0.2% HAc-5 mmol/L NH4OAc IPA (85/15 v/v), the mobile phase flow rate of

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1.5 mL/min, and column temperature of 25 ℃. As Supplementary Figure S1 shows, the

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two pairs of enantiomers could not be completely separated when Chiralpak IA-3, Lux

208

Cellulose-1, and Lux Cellulose-2 were employed. Despite the fact that both Chiralcel

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OD-3 and Lux Cellulose-1 columns contained the same chiral selector, a perfect

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baseline separation of enantiomers was only achieved on Chiralcel OD-3. This could

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be due to the different silica forms employed in the manufacturing process, chain length,

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branching of the derivatized cellulose or the degree of substitution, carbon load, and

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coating solvent employed in the production process.

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Composition of the Mobile Phase. Considering that the composition of the mobile phase

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is crucial for the enantioseparation, different mobile phases (methanol, ethanol, IPA,

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0.2% HAc-IPA, and 0.2% HAc-5 mmol/L NH4OAc IPA) were investigated to achieve

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excellent chiral separation of the enantiomers on the Chiralcel OD-3 column. The

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results indicated that prothioconazole and prothioconazole-desthio enantiomers were

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both well separated only when 0.2% HAc-5 mmol/L NH4OAc IPA was employed as

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the modifier (Supplementary Figure S1). Moreover, high peak areas were obtained 10


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attributed to the ability of HAc to provide protons for the formation of cations and

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improve the ionization efficiency of the target analyte. In addition, the application of

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HAc and NH4OAc was beneficial to obtain symmetrical peak shapes.

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Another factor to consider was the different ratios of the modifier. As the proportion

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of the modifier increased, the retention time (RT) of the enantiomers decreased (Table

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1). However, when the proportion of the organic phase increased from 15% to 30%, the

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Rs decreased significantly from 3.12 to 1.51 for prothioconazole, and 3.73 to 2.21 for

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prothioconazole-desthio (Table 1). Although the Rs values were all acceptable (Rs >

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1.5), a high system pressure was noted. The instrument system pressure would be

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beyond its range when the modifier content was over 20%, which was owing to the

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high viscosity of IPA. This high pressure was not beneficial to the efficiency or the

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lifetime of the column either. Therefore, by comprehensively considering the analysis

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efficiency, resolution, and system pressure, 15% 0.2% HAc-5 mmol/L NH4OAc IPA

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was selected as the modifier for further study.

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Flow Rate and Column Temperature. Given the low viscosity and high diffusivity

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of the supercritical fluids, a high flow rate could be used to decrease the analysis time

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and increase the throughput capacity under the condition of good separation efficiency.

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Different flow rates (1.0, 1.5, and 2.0 mL/min) were evaluated with a mobile phase of

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0.2% HAc-5 mmol/L NH4OAc IPA (85:15, v/v). As shown in Table 1, the Rs values

240

and the RTs increased when the flow rate decreased in the selected range. All Rs values

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obtained from 1.0 to 2.0 mL/min were satisfactory. In addition, no noticeable

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differences in the Rs and RT values were found between the flow rate of 1.5 and 2.0 11



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mL/min. Considering the system pressure, the flow rate of 1.5 mL/min was selected for

244

further study.

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Noticeably, the column temperature could affect the adsorption-desorption

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equilibrium between CSPs and the analyte molecules via two pathways: enthalpy

247

driven separation and entropy driven separation21-22. In this study, the column

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temperature was evaluated from 25 ℃ to 40 ℃. From Table 1, the Rs values of the

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prothioconazole and prothioconazole-desthio enantiomers were decreased when the

250

temperature increased, which implied that the separation of the target enantiomers

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could be contributed to an enthalpically driven process and that a relatively low

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temperature was beneficial for obtaining better Rs values.

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Comprehensive consideration of RT, separation effect, and the system pressure,

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chiral separation of prothioconazole and prothioconazole-desthio was performed on

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Chiralcel OD-3 column using CO2/0.2% HAc-5 mmol/L NH4OAc IPA (85:15, v/v) as

256

the mobile phase of 1.5 mL/min and a column temperature of 25 °C. Comparing with

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the previous research, a noticeable improvement was achieved in the analysis time (

Enantioselective Separation and Dissipation of Prothioconazole and

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