KEY WORDS: enantioseparation; chiral pesticide; chiral stationary phase; amylose; HPLC; elution order

Transcription

1 CHIRALITY (212) Direct Enantioseparation of Nitrogen-heterocyclic Pesticides on Amylose-tris-(5-chloro-2-methylphenylcarbamate) by Reversed-Phase High-Performance Liquid Chromatography WENWEN YANG, 1,2,3 JING QIU, 1,2 * TIANJIN CHEN, 1,2 SHUMING YANG, 1,2 AND SHICONG HOU 3 1 Institute of Quality Standards & Testing Technology for Agro-Products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of Agricultural Sciences, Beijing 181, China 2 Key Laboratory of Agri-Food Quality and Safety, Ministry of Agriculture, Beijing 181, China 3 College of Science, China Agricultural University, Beijing 1193, China ABSTRACT In this study, 11 nitrogen-heterocyclic pesticides were stereoselectively separated on amylose-tris-(5-chloro-2-methylphenylcarbamate) chiral stationary phase, using reversed-phase high-performance liquid chromatography with diode array detector and optical rotation detector at 426 nm. The effects of mobile phase composition and column temperature (5 4 C) on separation were investigated. When acetonitrile and water were used as mobile phase, satisfactory separations were obtained on amylose-tris-(5-chloro-2-methylphenylcarbamate) for four pesticides with elution orders of (+)/( )-simeconazole (1), ( )/(+)-nuarimol (3), ( )/(+)-carfentrazone-ethyl (4), and ( )/(+)/( )/(+)-bromuconazole (9) and part separations for three with elution orders of ( )/(+)-famoxadone (6), (+)/( )-fenbuconazole (1), and ( )/(+)-triapenthenol (11). Only two chromatographic peaks on diode array detector were obtained for diclobutrazol (2), cyproconazole (5), etaconazole (7), and metconazole (8), although they should have four stereoisomers in theory because of presences of two chiral centers in molecules. The stereoisomeric optical signals of all pesticides did not reverse with temperature changes but would reverse with different solvent types for some pesticides. These results will be useful to prepare and analyze individual enantiomers of chiral pesticides. Chirality :, Wiley Periodicals, Inc. KEY WORDS: enantioseparation; chiral pesticide; chiral stationary phase; amylose; HPLC; elution order INTRODUCTION Chiral pesticides have become a hot research target in recent years, because more than 25% of the commercialized pesticides are chiral 1 and composed of enantiomers/stereoisomers with different bioactivity and toxicity, but most of them are produced and applied in the form of racemates in agriculture. 2 For instance, triadimenol has two chiral centers and presents four stereoisomers with different biological responses. 3 Thereinto, the (1S, 2R)-isomer is the highest in fungicidal activity (up to 1-fold more active than the other three). 4 So, it is necessary to separate enantiomers of chiral pesticides for further researches on activity, toxicity, environmental fates, and more efficient agricultural applications. Enantiomeric separation by high-performance liquid chromatography (HPLC) using chiral stationary phase (CSP) has significant advances compared with gas chromatography and capillary electrophoresis, which is rapid, nondestructive, and little possible in epimerization 5 and has been a powerful method for preparing individual enantiomers and stereoselectively analyzing chiral pesticides. 6 Polysaccharide-based CSPs including cellulose and amylose derivatives such as cellulose-tris-(3,5-dimethylphenylcarbamate) (CDMPC) and amylose-tris-(3,5-dimethylphenylcarbamate) are the most used CSPs in the past three decades due to their excellent and wide stereoselective discrimination abilities. 7 These derivatives can be used in HPLC both on normal phase (NP-HPLC) and on reversed phase (RP-HPLC) modes, 8,9 and generally, NP-HPLC was used more widely because it could get better resolution than RP-HPLC. 1 However, some reports tried to apply them on RP-HPLC because it has good solubility for polar 212 Wiley Periodicals, Inc. compounds and easier sample preparation methods 11 and also obtained good separation results by choosing suitable mobile phase. 12 Additionally, RP-HPLC was more compatible to electron spray ionization-mass spectrometry than NP-HPLC. 13,14 For example, previously, we used CDMPC on RP-HPLC to successfully separate nine chiral triazole fungicides and compared the effects of column particles, mobile phases, and temperature on resolution. 15 Tian also used CDMPC to separate 2 chiral pesticides, and seven pesticides can obtain separations. 16 At present, few reports were involved in using amylose-based CSP on RP-HPLC to stereoselectively separate chiral pesticides. In this work, stereoselective separations of 11 nitrogen-heterocyclic chiral fungicides including simeconazole (1), diclobutrazol (2), nuarimol (3), carfentrazone-ethyl (4), cyproconazole (5), famoxadone (6), etaconazole (7), metconazole (8), bromuconazole (9), fenbuconazole (1), and triapenthenol (11) (Fig. 1) were performed by RP-HPLC on CSP of amylose-tris-(5-chloro-2-methylphenylcarbamate) (ACMPC). The influences of mobile phase and temperature Contract grant sponsor: National Natural Science Foundation of China; Contract grant numbers: , Contract grant sponsor: National High Technology Research and Development Program of China; Contract grant number: 211AA186, *Correspondence to: Jing Qiu; Institute of Quality Standards and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 181, China. jinn.qiu@hotmail.com Received for publication 15 April 212; Accepted 29 May 212 DOI: 1.12/chir.2292 Published online in Wiley Online Library (wileyonlinelibrary.com).

2 YANG ET AL. Fig. 1. Molecular structures of 11 chiral pesticides. on separation were investigated, and stereoisomeric elution orders were identified by optical rotation detector (ORD). EXPERIMENTAL Chemicals and Reagents Racemic simeconazole (1), diclobutrazol (2), nuarimol (3), carfentrazone-ethyl (4), cyproconazole (5), famoxadone (6), etaconazole (7), metconazole (8), bromuconazole (9), fenbuconazole (1), and triapenthenol (11) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany), and the purities were higher than 95%. The stock solutions of all compounds were prepared at 1 mg l 1 in mobile phases. NaNO 2 (1 mg l 1 )was used to determine the void time (t ). Acetonitrile (ACN) and methanol (MET) (HPLC grade) were obtained from Fisher (Fair Lawn, NJ, USA). Water was Wahaha pure water and purchased from Wahaha Group Co. Ltd. (Hangzhou, China). Instruments Chromatographic separation was carried out with an Agilent 12 series HPLC equipped with G1322A degasser, G1311A quatpump, G1316B column compartment, G1315C diode array detector (DAD), G1329A autosampler, and a 2-ml sample loop (Wilmington, DE, USA). The UV signals were acquired and manipulated by an Agilent Chemstation. The stereoisomeric rotation signals of chiral pesticides were identified by CHIRALYSER MP ORD at 426 nm, produced by IBZMESSTECHNIK Company (Hannover, Germany) and provided by Beijing Separation Science & Technology Development Co., Ltd (Beijing, China). The optical signals were acquired and processed by an Agilent Chemstation through signal transformation with an Agilent 359E A/D converter. Chromatographic Conditions Stereoisomers were separated on Lux Amylose-2 with CSP of ACMPC, produced by Phenomenex Incorporation (Torrance, CA, USA) and obtained from Guangzhou FLM Scientific Instrument Co., Ltd. (Guangzhou, China). The column was mm (i.d., inside diameter) and packed with 5-mm particles. The mobile phases were different proportions of ACN or MET and water. The flow rate was.8 ml min 1. Enantioseparations were investigated in 5-4 C. The UV (ultraviolet) detection wavelength was set at 22 nm. The injection volume was 1 ml.

3 DIRECT ENANTIOSEPARATION OF NITROGEN-HETEROCYCLIC PESTICIDES ON AMYLOSE-BASED CSP BY REVERSED-PHASE HPLC Chromatographic parameters are calculated with follow equations. Capacity factor (k )=(t R -t )/t, t R is retention time, t is void time; separation factor (a)=k 2 /k 1, k 1 and k 2 are capacity factors of first and second eluting stereoisomers, respectively; resolution factor (R s )=2 (t 2 -t 1 )/(W 2 + W 1 ), W 1 and W 2 are peak widths of first and second eluting stereoisomers, respectively. RESULTS AND DISCUSSION Effects of Different Modifiers on Enantioseparation As shown in Figure 1, all 11 pesticides contain nitrogenheterocyclic ring in their molecules. Thereinto, simeconazole (1), diclobutrazol (2), cyproconazole (5), etaconazole (7), TABLE 1. Stereoselective separations of chiral pesticides with different mobile phases Pesticide Mobile Elution phase v/v k 1 k 2 a R s order Simeconazole (1) ACN/water 5/ (+)/( ) 7/ / MET/water 7/ (+)/( ) 9/ Diclobutrazol (2) ACN/water 5/ (+)/( ) 7/ / MET/water 7/3 (+)/( ) 8/ / Nuarimol (3) ACN/water 5/ ( )/(+) 7/ / MET/water 7/3 (+)/( ) Carfentrazone-ethyl (4) ACN/water 5/ ( )/(+) 7/ / /1 MET/water 7/3 ( )/(+) 8/ / Cyproconzole (5) ACN/water 5/ (+)/( ) 7/ / MET/water 7/3 (+)/( ) 8/ / Famoxadone (6) ACN/water 5/ ( )/(+) 7/ /2 MET/water 7/3 (+)/( ) Etaconzole (7) ACN/water 5/ (+)/( ) 7/ /2 MET/water 7/3 ( )/(+) Metconazole (8) ACN/water 5/ ( )/(+) 7/ / MET/water 7/3 (+)/( ) Fenbuconazole (1) ACN/water 5/ (+)/( ) 6/4 MET/water 7/3 Triapenthenol (11) ACN/water 4/ (+)/( ) 5/5 MET/water 7/3 ( )/(+) Pesticide Mobile phase v/v k 1 k 2 k 3 k 4 a 12 a 23 a 34 R s12 R s23 R s34 Elution order Bromuconazole (9) ACN/water 5/ ( )/(+)/ ( )/(+) 7/ / MET/water 7/ ( )/(+)/ ( )/(+) 8/ /

4 YANG ET AL. metconazole (8), bromuconazole (9), fenbuconazole (1), and triapenthenol (11) belong to triazole pesticides with the same structural moiety of the 1,2,4-triazole ring. Triapenthenol (11) is mainly used as plant growth regulator and others as fungicides. Diclobutrazol (2), cyproconazole (5), etaconazole (7), metconazole (8), and bromuconazole (9) contain two chiral carbons and consist of four stereoisomers in the theory; the others only consisted of a pair of enantiomers. The enantiomers/ stereoisomers of these pesticides were respectively separated by using ACN or MET and water as mobile phase on ACMPC, and all results were summarized in Table 1. When ACN and water were used as mobile phase, simeconazole (1), nuarimol (3), carfentrazone-ethyl (4), and famoxadone (6) got good enantiomeric separation except for fenbuconazole (1) and triapenthenol (11). The resolution decreased with increase of ACN from 5 to 9% in mobile phase, and the maximum R s was from nuarimol (3) at percentage of 5/ 5 (v/v). But when MET and water were used as mobile phase, simeconazole (1) and carfentrazone-ethyl (4) just can be partly separated; nuarimol (3), famoxadone (6), etaconazole (7), metconazole (8), fenbuconazole (1), and triapenthenol (11) cannot be separated; diclobutrazol (2), cyproconazole (5), and bromuconazole (9) were separated as two peaks. It is significant that the separation effects using ACN/water on ACMPC were better than those using MET/water for all above mentioned pesticides, and the typical separation chromatograms were shown in Figure 2. It was worth mentioning that diclobutrazol (2), cyproconazole (5), etaconazole (7), and metconazole (8) were separated as two peaks on DAD, although they have four stereoisomers and should show four chromatographic peaks, and only bromuconazole (9) got full separation. Dong et al. used amylose-tris-[(s)-a-methyphenylcarbamate] to separate simeconazole (1), and the best resolutions were 1.39 and 3.75 when using 2% of isopropanol or ethanol in n-hexane as the mobile phase, respectively. 17 This result was better than present separation on ACMPC with R s of Previous report showed that the separation of carfentrazone-ethyl (4) enantiomers on CDMPC was not satisfactory, 18 but it obtained separation on Chiralpak AS column from Daicel Chemical Industries (Tokyo, Japan) using n-hexane/ethanol (98/2) with the best Rs of In this article, it also got good separation with R s of on ACMPC. Baseline enantioseparation of famoxadone (6) was performed on CDMPC(Lux Cellulose-1 column) by LC-MS/MS (Liquid chromatography - tandem mass spectrometry), and the mobile phase was 85% of methanol and 15% of 5 mmol L -1 ammonium acetate solution added to.1% formic acid. 2 Cyproconazole (5) showedtwo chromatographic peaks on DAD in present separation, but it can be separated as four peaks by supercritical fluid chromatography on Chiralpak AD with maximum R s of 2.83 between third and fourth eluted stereoisomers. 21 Additionally, cyproconazole (5) and bromuconazole (9) also can get good separations by cyclodextrin-modified micellar electrokinetic capillary chromatography. 22 In the report of B.J. Konwick et al., cyproconazole (5), metconazole (8), and bromuconazole (9) were separated using the Chiralsel-Dex column on gas chromatography with electron capture detector. The stereoisomers of cyproconazole (5) could be separated as three peaks, metconazole (8) obtained baseline enantioseparation and bromuconazole (9) exhibited four of its expected peaks. 23 In previous reports, fenbuconazole (1) got very satisfactory separation on CDMPC with R s of using ACN/water and MET/water 14,15 and was significantly better than separation on ACMPC with R s of.85. To our knowledge, the stereoselective separations of other chiral pesticides have not been reported until now. Effects of Column Temperature on Enantioseparation The change of column temperature has an effect on chiral separation for the impact on discrimination actions between chiral compounds and CSP. Because fenbuconazole (1) and triapenthenol (11) cannot get good separation using ACN/water and most pesticides cannot obtain baseline separations using MET/water, the column temperatures from 5 to 4 C on enantioseparation were investigated only on pesticides (1) (9) using ACN/water, and the results were summarized in Table 2. As raising temperature, most capacity factors (k ) and separation factors (a) decreased, and resolution factors (R s ) firstly increased and then decreased except famoxadone (6) that has continually decreased. Most results were not similar to previous reports that the separation effects were better for chiral triazole fungicides with decrease of column temperature on CDMPC using ACN/water or Fig. 2. The typical UV and OR chromatograms of chiral pesticides: simeconazole (1), diclobutrazol (2), nuarimol (3), carfentrazone-ethyl (4), cyproconazole (5), famoxadone (6), etaconazole (7), metconazole (8), bromuconazole (9), fenbuconazole (1), triapenthenol (11). Chromatographic conditions: ACN/water (6/4) at 2 C for (1), (2) and (7), at3 C for (4), (6), (8) and (9), at4 C for (3); ACN/water (5/5) at 3 C for (5), (1) and (11).

5 DIRECT ENANTIOSEPARATION OF NITROGEN-HETEROCYCLIC PESTICIDES ON AMYLOSE-BASED CSP BY REVERSED-PHASE HPLC TABLE 2. Effects of column temperatures on separation Pesticide Temperature ( C) k 1 k 2 a R s Elution order Simeconazole (1) 4 (+)/( ) Diclobutrazol (2) (+)/( ) Nuarimol (3) ( )/(+) Carfentrazone-ethyl (4) ( )/(+) Cyproconzole (5) (+)/( ) Famoxadone (6) ( )/(+) Etaconzole (7) (+)/( ) Metconazole (8) ( )/(+) Pesticide Temperature ( C) k 1 k 2 k 3 k 4 a 1 a 2 a 3 R s12 R s23 R s34 Elution order Bromuconazole (9) ( )/(+)/ ( )/(+) The mobile phase was ACN and water (6/4, v/v). MET/water as mobile phases. 9 The reason may be the different chiral discrimination actions from amylose- and cellulosebased CSPs. The best resolutions were obtained at 2 C for simeconazole (1), carfentrazone-ethyl (4), cyproconazole (5), and metconazole (8); 3 C for diclobutrazol (2) and nuarimol (3); and 4 C for famoxadone (6) and etaconzole (7). Most compounds cannot get the linear relations between ln(a) or ln(r s ) and 1/T on amylose-based CSPs, but those can be done on cellulose-based CSPs. 24,25 Elution Order HPLC with ORD is often applied to detect elution orders according to positive (+) or negative ( ) optical rotation signals of enantiomers. 15,26 Generally, the change of solvent percentage in mobile phase would not change enantiomeric optical rotation signals, but the change of solvent types would lead to reversed signals. For example, previous report showed that signals of triadimefon enantiomers reversed when solvents changed from CCl 4 to ACN/water or MET/water. 13 In this article, the stereoisomeric optical rotation signals of all pesticides were identified with ORD at 426 nm in different mobile phase systems, and all results were summarized in Tables 1 and 2. The column temperature from 5 to 4 C, the percentage of ACN from 5 to 9%, and MET from 7 to 9% did not respectively lead to changes of stereoisomeric rotation signals for all investigated chiral pesticides.

6 YANG ET AL. The (+)-stereoisomers of simeconazole (1), diclobutrazol (2), cyproconazole (5), fenbuconazole (1), and ( )-enantiomer of carfentrazone-ethyl (4) were firstly eluted from ACMPC using ACN/water or MET/water as mobile phase. According to peak areas from separation chromatograms of bromuconazole (9), peaks 1 and 2 and peaks 3 and 4, respectively, were a pair of enantiomers. The rotation signals of four stereoisomers orderly were ( ), (+), ( ), and (+). These pesticides showed same stereoisomeric rotation signals with change of solvent types. But for other chiral pesticides, the signals of first eluting stereoisomers changed from ( ) to (+) for nuarimol (3), famoxadone (6), and metconazole (8) and from (+) to ( ) for etaconzole (7) and triapenthenol (11) with the change of mobile phases from ACN/water to MET/ water. The elution orders of fenbuconazole (1) on ACMPC using ACN/water were same to those on CDMPC using ACN/ water or MET/water with (+)-enantiomer firstly eluted from CSP. Additionally, it is interesting that obvious (+) and ( )- rotation signals were obtained for some chiral pesticides like fenbuconazole (1) and triapenthenol (11), although they just got a little separations. Diclobutrazol (2), cyproconazole (5), and etaconazole (7) showed two peaks of optical rotation and UV signals, but etaconazole (7) showed four peaks of optical rotation, although it only had two peaks of UV signals employing ACN/water as mobile phase. This means the ORD can give optical signals, although the enantiomers/stereoisomers cannot get obvious separations on UV detectors. Further studies using other CSPs need to be investigated for obtaining effective separations and clearly understanding the elution orders of four stereoisomers of diclobutrazol (2), cyproconazole (5), etaconazole (7), and metconazole (8) onthesecsps. CONCLUSION Present study investigated the content and type of solvent in mobile phase and column temperature on enantioseparations of 11 nitrogen-heterocyclic chiral pesticides on ACMPC. The results showed that better separations for all pesticides were obtained with ACN/water as the mobile phase than MET/water. Three pesticides can get satisfactory separations with (+)-simeconazole (1) and ( )-enantiomers of nuarimol (3), and carfentrazone-ethyl (4) firstly eluted from ACMPC. Three pesticides only got part separations with (+)-fenbuconazole (1) and ( )-enantiomers of famoxadone (6) and triapenthenol (11) firstly eluted. The other five pesticides contain four stereoisomers in the theory; only bromuconazole (9) was fully separated as four peaks on DAD, and diclobutrazol (2), cyproconazole (5), etaconazole (7), and metconazole (8) just showed two peaks. The results from ORD showed that the temperature changes from 5 to 4 C did not reverse the stereoisomeric optical signals of all pesticides, but the change of solvent types from ACN/water to MET/water can reverse some. These results may be helpful for preparation of single enantiomer from racemic pesticides and establishing effective analytical method to study stereoselective behaviors of chiral pesticides in environment. LITERATURE CITED 1. Maier NM, Franco P, Lindner W. Separation of enantiomers: needs, challenges, perspectives. J Chromatogr A 21;96: Ali I, Aboul-Enein HY. Chiral pollutants: distribution, toxicity and analysis by chromatography and capillary electrophoresis. Chichester: Wiley; 24. p Deas AHB, Carter GA, Clark T, Clifford DR, James CS. The enantiomeric composition of triadimenol produced during metabolism of triadimefon by fungi: III. Relationship with sensitivity to triadimefon. Pestic Biochem Physiol 1986;26: Kurihara N, Miyamoto J, Paulson G, Zeeh B, Skidmore MW, Hollingworth R, Kuiper H. Chirality in synthetic agrochemicals: bioactivity and safety considerations. Pestic Sci 1999;55: Li ZY, Zhang ZC, Zhou QL, Wang QM, Gao RY, Wang QS. Stereo- and enantioselective determination of pesticides in soil by using a chiral liquid chromatography in combination with matrix solid-phase dispersion. J AOAC Int 23;86: Haginaka J. Pharmaceutical and biomedical applications of enantioseparations using liquid chromatographic techniques. J Pharmaceut Biomed 22;27: Yashima E. Polysaccharide-based chiral stationary phases for highperformance liquid chromatographic enantioseparation. J Chromatogr A 21;96: Perrin C, Vu VA, Matthijs N, Maftouh M, Massrt DL, Vander HY. Screening approach for chiral separation of pharmaceuticals. Part 1, Normal-phase liquid chromatography. J Chromatogr A 22;947: Perin C, Matthijs N, Mangelins D, Granier-Loyaux C, Maftouh M, Massart DL, Vander HY. Screening approach for chiral separation of pharmaceuticals. Part 2. Reversed phase liquid chromatography. J Chromatogr A 22;966: ZhouZQ,WangP,JiangSR,WangP.Chiralseparationof2-allyl-4-hydroxy-3- Methyl-2- cyclopenten-1-one on coated amylpectin chiral stationary phase in high performance liquid chromatography. Chin J Chrom 23;21: Tian Q. Study on the analysis and separation of chiral pesticide enantiomers on polysaccharide-type chiral stationary phases under reversed phase conditions. Ph.D Dissertation, China Agriculture University. 12. Ishikawa A, Shibata T. Cellulosic chiral stationary phase under reversedphase condition. J Liq Chromatogr 1993;16: LiangHW,QiuJ,LiL,LiW,ZhouZQ,LiuFM,QiuLH.Stereoselectiveseparation and determination of triadimefon and triadimenol in wheat, straw and soil by liquid chromatography-tandem mass spectrometry. J Sep Sci 211;34: Li YB, Dong FS, Liu XG, Xu J, Li J, Kong ZQ, Chen X, Liang XY, Zheng YQ. Simultaneous enantioselective determination of triazole fungicides in soil and water by chiral liquid chromatography/tandem mass spectrometry. J Chromatogr A 212;1224: Qiu J, Dai SH, Zheng CM, Yang SM, Chai TT, Bie M. Enantiomeric separation of triazole fungicides with 3-mm and 5-mm particle chiral columns by reversephase high-performance liquid chromatography. Chirality 211;23: Tian Q, Lv CG, Wang P, Ren LP, Qiu J, Li L, Zhou ZQ. Enantiomeric separation of chiral pesticides by high performance liquid chromatography on cellulose tris-3,5-dimethyl carbamate stationary phase under reversed phase conditions. J Sep Sci 27;3: Dong FS, Cao Q, Liu XG, Zheng YQ, Li CJ. Enantiomeric separation of simeconazole by HPLC with amylose chiral column. Chin J Appl Chem 28;25: Wang P, Jiang SR, Liu DH, Jia GF, Wang QX, Wang P, Zhou ZQ. Effect of alcohols and temperature on the directchiral resolutions of fipronil, isocarbophos and carfentrazone-ethyl. Biomed Chromatogr 25;19: Li YB, Dong FS, Liu XG, Xu J, Chen WY, Cheng L, Ning P, Li J, Wang YH, Zheng YQ. Enantioselective separation of the carfentrazone-ethyl enantiomers in soil, water and wheat by HPLC. J Sep Sci 21;33: Qian MR, Wu LQ, Zhang JW, Li R, Wang XY, Chen ZM. Stereoselective determination of famoxadone enantiomers with HPLC-MS/MS and evaluation of their dissipation process in spinach. J Sep Sci 211;34: Toribio L, Nozal MJ, Bernal JL, Jiménez JJ, Alonso C. Chiral separation of some triazole pesticides by supercritical fluid chromatography. J Chromatogr A 24;146: Ibrahim WAW, Warno SA, Aboul-Enein HY, Hermawan D, Sanagi MM. Simultaneous enantioseparation of cyproconazole, bromuconazole, and diniconazole enantiomers by CD-modified MEKC. Electrophoresis 29;3: Konwick BJ, Garrison AW, Avants JK, Fisk AT. Bioaccumulation and biotransformation of chiral triazole fungicides in rainbow trout(oncorhynchus mykiss). Aquat Toxicol 26;8: Tian Q, Ren LP, Lv CG, Zhou ZQ. Chiral resolution of eight triazole pesticides by high performance liquid chromatography under reversed condition. Chin J Anal Chem 21;38: Bobbitt DR, Linder SW. Recent advances in chiral detection for high performance liquid chromatography. Trends Anal Chem 21;2: Tian Q, Bi CL, Ren LP, Wang LP, Zhou ZQ. The application of high performance liquid chromatography with circular dichroism detector in the chiral compounds. Chin J Anal Chem 26;34: