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Enantioselective metabolism of the chiral herbicide diclofop-methyl and diclofop by HPLC in loach (Misgurnus anguillicaudatus) liver microsomes in vitro
Accepted Manuscript Title: Enantioselective metabolism of the chiral herbicide diclofop-methyl and diclofop by HPLC in loach (Misgurnus anguillicaudatus) liver microsomes in vitro Author: Ruixue Ma Han Qu Xia Liu Donghui Liu Yiran Liang Peng Wang Zhiqiang Zhou PII: DOI: Reference:
S1570-0232(14)00523-6 http://dx.doi.org/doi:10.1016/j.jchromb.2014.08.014 CHROMB 19076
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
28-2-2014 7-8-2014 10-8-2014
Please cite this article as: R. Ma, Enantioselective metabolism of the chiral herbicide diclofop-methyl and diclofop by HPLC in loach (Misgurnus anguillicaudatus) liver microsomes in vitro, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.08.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enantioselective
metabolism
of
the
chiral
herbicide
diclofop-methyl and diclofop by HPLC in loach (Misgurnus
ip t
anguillicaudatus) liver microsomes in vitro
Authors:
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Ruixue Ma, email: [email protected]
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Han Qu, email: [email protected]
Donghui Liu, email: [email protected]
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Yiran Liang, email: [email protected]
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Xia Liu, email: [email protected]
Peng Wang, email: [email protected]
The corresponding author: email: [email protected] Tel/fax: +861062733547
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*
ed
Zhiqiang Zhou*,
Ac ce
Affiliation: Department of Applied Chemistry, China Agricultural University, Beijing 100193, PR China
Address: No. 2 Yuanmingyuan West Rd., Beijing 100193, PR China
1/1
Page 1 of 25
Abstract An efficient and reliable enantioseparation method by high-performance liquid chromatography (HPLC) for the chiral herbicide diclofop-methyl (DM) and its primary
ip t
metabolite diclofop (DC) was developed and validated, and the biological process of the enantiomers in loach liver microsomes (LLM) in vitro was investigated. The
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enantiomers of DM and DC were first separated on an immobilised-type stationary
us
phase of Chiralpak IC. The best resolutions were achieved under the chromatographic condition of n-hexane/IPA/TFA 96:4:0.1(v/v/v) at 20 ℃ with each Rs >2 and the two
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pairs of enantiomers could be eluted in about 10 mins. The extraction recoveries of the
M
analytes from LLM were 79.6%̶108.9% with RSD≤11.5%. The enzyme kinetics of DM enantiomers were different, with the Km value 320.7 for (S)-DM and 392.1 for
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(R)-DM. The metabolism experiment in vitro showed DM underwent a rapid phase-I metabolism with or without NADPH, indicating the esterases in liver played a
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dominant role. No interconversion between the two enantiomers was observed by
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single-enantiomer incubation. The preferential degradation of (S)-DM was confirmed with the half-time (t1/2) of (S)-DM 5.27 min and 21.2 min for (R)-DM. DC enantiomer was generated by its corresponding ester form and could not be further degraded during the incubation. It was the first study on biotransformation of DM and DC enantiomers in LLM in vitro. The results may help to evaluate the ecological risks of chiral pesticides.
Keywords: diclofop-methyl, diclofop, metabolism, enantioselective, Chiralpak IC, 2/2
Page 2 of 25
loach liver microsome
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1. Introduction Chemical pesticides have contributed substantially to increases agricultural
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production. Among the frequently used pesticides, more than one quarter have one or
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more asymmetric center(s) [1]. The two enantiomers may behave differently in bioactivity, toxicity, metabolism and degradation under certain environmental
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conditions [2-4]. It has been reported that the bioactivity of a given chiral pesticide
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often a result of the preferential reactivity of only one enantiomer while the other enantiomer might exert toxic effects on non-target organisms [1]. In most cases, the
ed
chiral pesticides are manufactured and employed in racemic form which means the enantioselective degradation may lead to an inaccurate environmental risk evaluation
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under traditional consideration [5]. To assess the environmental safety of chiral
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compounds, investigation on their stereochemistry is necessary [6]. Polysaccharide derivatives are the first and broadest choice of selectors for use as
chiral stationary phases (CSPs) for liquid chromatography [7, 8]. Chiralpak IC is a new generation
of
CSP
from
chemically
immobilised
cellulose
tris
(3,
5-dichlorophenylcarbamate) on silica gel, making these adsorbents robust and showing remarkable enantioselective performance [9]. Diclofop-methyl (DM), 2-[4-(2, 4-dichlorophenoxy) phenoxy]-propionate, is a widely used herbicide of the aryloxyphenoxy propionate (AOPP) class [10]. The total 3/3
Page 3 of 25
annual usage of diclofop-methyl in the USA was about 340,000 kg of active ingredient (a.i.) in the period 1987–1996 [11]. Its hydrolysate diclofop (DC) also has herbicidal activity. Both diclofop-methyl and diclofop are chiral and consist of a pair of
ip t
enantiomers, respectively (structures shown in Supplementary Information S1). In general, the (R)-enantiomers of AOPP herbicides show two times higher herbicidal
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activity than the racemic mixtures [12]. Although many herbicides are typically
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applied to the crop, substantial quantities of the active ingredient penetrate to the environmental, for example, up to 73% in the case of a controlled foliar application of
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the herbicide diclofop-methyl [13]. There are researches showing diclofop-methyl and
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diclofop are suspected to be endocrine disruptors and carcinogens, and thus are expected to cause a risk to some non-target terrestrial and aquatic plants [14]. Recent
ed
studies indicated that the (S)-enantiomers of DM and DC posed a stronger toxicity to three freshwater algae than the (R)-enantiomers [15].
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Biotransformation of xenobiotics in vertebrates is a multi-phase process, the first
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of which usually involves the cytochrome P450 (CYP) superfamily of enzymes [16, 17]. Fish are the most representative aquatic vertebrates. The oxidative metabolism of various xenobiotics in fish is catalyzed by cytochrome P450 (mainly in the liver), and various compounds can induce the activity of this enzyme [18]. It has been reported that fish are capable of metabolizing some typical mammalian CYP2 and CYP3 substrates [19]. However, studies of drug metabolism in fish are extremely limited. To our best knowledge, the metabolic fate and CYPs responsible for the metabolism of diclofop-methyl in fish are largely unknown, especially at the level of enantiomers. For 4/4
Page 4 of 25
this reason, research on diclofop-methyl environmental behaviors in fish is of the utmost importance. Loaches (Misgurnus anguillicaudatus) are small edible fish widely distributed in freshwater ecosystems and are adaptable to the ecological environment,
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which appear to be a proper biological model for studies on water pollutants. In vivo and in vitro metabolism studies are useful for understanding the fate of a xenobiotic in
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an organism. In vitro experiments using microsomal material from various organs (e.g.,
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liver) can shed light on such metabolism events with a minimum of expense and provide valuable information on toxicant exposure [20].
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In this work, for the first time, the simultaneous chiral analysis method of
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diclofop-methyl and diclofop enantiomers was established utilizing an immobilised CSP, and the metabolism of the racemic and optical pure isomer of diclofop-methyl in
ed
LLM in vitro was demonstrated. The results filled a vacancy of the stereoselective
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metabolic behavior of diclofop-methyl in aquatic species.
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2. Material & methods
2.1. Chemicals and reagents Racemic-diclofop-methyl (rac-DM, purity> 99.0%) was obtained from China
Ministry of Agriculture’s Institute for Control of Agrochemicals. Racemic-diclofop (rac-DC, purity > 98.0%) was synthesized in our laboratory. The enantiomers of DM (purity > 99.5%) were prepared with a preparatory chiral column (250 × 10 mm (I.D.), provided by Department of Applied Chemistry, China Agricultural University, Beijing). Water was purified by a Milli-Q system. Ethyl ether, n-hexane, 2-propanol (HPLC 5/5
Page 5 of 25
grade), and trifluoroacetic acid (TFA) were from Fisher Scientific (Fair Lawn, NJ, U.S.A.). β-Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Sigma-Aldrich (St Louis, MO, USA). All other chemicals and solvents were of
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analytical grade and purchased from commercial sources. The loaches were obtained from a local aquafarm. The fish employed were mixed-gender and were all adults and
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healthy, with a wet weight of 20 g to 25 g (15 cm to 20 cm) each.
2.2. Instrumentation and chromatographic conditions
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Analysis was performed using an Agilent 1260 Infinity HPLC (Agilent
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Technologies Palo Alto, CA) equipped with G1311C 1260 Quat pump VL, G1329B 1260 ALS, G1316A 1260 TCC, G1314F 1260 VWD. A Chiralyser–MP Optical
ed
Rotation Detector was purchased from IBZ Messtechnik (Germany). The Chiralpak IC column (250×4.6 mm i.d.) was supplied by Daicel Chemical Industries, Ltd. (Tokyo,
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Japan) with the CSPs [cellulose tris-(3, 5-dichlorophenylcarbamate)] polymer
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immobilized on silica. The mobile phase was composed of n-hexane and isopropanol with 0.1% trifluoroacetic acid (TFA). The flow rate was 1.0 mL/min. The configurations of the enantiomers were determined by the ORD signals.
2.3. Microsome Preparation Loaches were held for a week acclimation period in 50-L glass aquaria. The fish was killed and liver was quickly excised. Liver microsomes were prepared according to the method of differential ultracentrifugation [21]. The microsomal pellet was 6/6
Page 6 of 25
resuspended in 50 mmol/L Tris–HCl buffer (pH 7.4) containing 20% glycerol (v/v) and 0.1 mmol/L EDTA (ethylenediaminetetraacetic acid) and stored at -80℃ prior to use.
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Total protein content was determined using the Bradford method.
2.4. Enzyme kinetic assay
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To estimate enzyme kinetic parameters, a series of each enantiomer stock solutions
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were added to LLM (0.5 mg/mL) to achieve final substrate concentrations of 40–1920 µM. The substrate concentrations of each DM enantiomer were 40, 80, 160, 240, 480,
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960 and 1920 µM for R-DM and 40, 80, 160, 320, 640, 960 and 1920 µM for S-DM.
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Different incubation times were adopted for the enantiomers, 5 min for R-DM and 2 min for S-DM, respectively. The Vmax and Km values were calculated according to
ed
nonlinear regression Michaelis–Menten equation. Intrinsic clearance (CLint) was obtained as a ratio of Vmax to Km. Data analysis was performed using Graphpad Prism
Ac ce
pt
5 (GraphPad Software Inc. Press, San Diego CA, 2007).
2.5. Metabolism of DM and DC in LLM and the sample pretreatment The metabolism of DM by LLM was carried out in a reaction system of 1 mL
containing LLM (0.5 mg/mL), Tris–HCl buffer (50 mmol/L, pH 7.4), EDTA (1.0 mmol/L), NADPH (1.0 mmol/L) and rac-DM or single-enantiomer (40 μmol/L). Experiments were conducted at 19 ℃ to reflect temperatures most likely encountered by loaches under natural conditions. Two types of experimental blanks were included. One consisted of DM and heat-treated LLM (i.e., LLM heated at 100 ℃ for 60 mins 7/7
Page 7 of 25
in a water bath to render them inactive) plus all of the reaction components, called procedural blank. This would correct for any abiotic transformation of DM. The other blank (called negative control) contained LLM and all of the reaction components but
ip t
without NADPH. In addition, rac-DC metabolism in vitro was also conducted. All the incubations lasted for 3 hours.
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At each sampling point, pre-chilled ethyl acetate was introduced to terminate the
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reaction and 1 mL of 1M HCl solution was added to help extract diclofop. The sample was vortexed for 5 min and then centrifuged at 3500 rpm. The extraction and
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centrifugation were repeated with another 5 mL of ethyl acetate. The organic phase
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was combined and evaporated to dryness under a gentle stream of nitrogen at 30 ℃ and the resulting residue was redissolved in 500 µL of isopropanol for HPLC analysis.
ed
Statistical differences between two groups were assessed using a SPSS v15.0 software package (SPSS Inc., Chicago, IL). A p-value of less than 0.05 was considered
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statistically significant unless otherwise indicated.
2.6. Quality control
Stock solutions of DM standard were prepared in 2-propanol and stored at -20°C.
Stock solutions of R/S-DM standard were determined by external standard method. Working standard solutions of rac-DM, R-DM and S-DM were freshly prepared by dilutions of stock solution in 2-propanol. Calibration standards were prepared by adding a series of working standard solutions of rac-DM into the blank matrix (BM, the extracts from the prepared 8/8
Page 8 of 25
inactivated microsomes), and then stored at -20°C until analysis. Calibration curves were obtained over the range of 0.5-800 mg/L at 5 concentrations (0.50, 5.0, 20, 100, 800 mg/L) by plotting peak area versus the concentration of each enantiomer. The
ip t
linear regression analysis was performed using Microsoft Excel®. The extraction recovery was evaluated at three levels (0.50, 20 and 800 mg/L) by spiking mixed
cr
rac-DM and rac-DC standard into the prepared inactivated microsomes. Precision of
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the extraction recovery were performed in triplicate and expressed as RSD. Accuracy of the method was calculated by analyzing the calibration standard in 3 replicates
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consecutively and comparing the predicted concentration (obtained from the
M
calibration curve) to the actual concentration of each enantiomer spiked in the BM. Limit of quantification (LOQ) was defined as the lowest concentration in the
ed
calibration curve with acceptable precision and accuracy (less than 20% CV and within +/-20% RE). The parameters of method validation are listed in Table 1.
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The standard solutions in 2-propanol of DM and enantiomers were determined its
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optical purity and hydrolysis rate after 1, 7, 14 and 21 days of storage prior to use. There was no enantiomerization observed and the hydrolysis was within normal levels. The stability of calibration standard was assessed after 1, 8 h of preparation and 1, 4 and 10 d of storage at -20 °C. The RSD of storage stability was in the range of 4.6 %̶ 7.2 %, indicating the analysis of DM enantiomers in LLM sample was reliable under the experiment conditions.
3. Results & discussion 9/9
Page 9 of 25
3.1 Optimization of Enantioseparation Conditions There is scarcely any literature support of simultaneous chiral separation of DM and DC enantiomers on Chiralpak IC column. The influences of alcoholic modifier
ip t
concentration as well as temperature on the enantioseparation were investigated. The test temperature was varied from 10 ℃ to 30 ℃ and the concentration of alcoholic
cr
modifier IPA varied from 2% to 5%. For better separation and peak shapes, 0.1% (v:v)
us
TFA was added into the mobile phase. The resolution factor Rs was used to evaluate the separation and it was considered a complete separation when the Rs>1.5. Here the
an
four peaks on chromatograms were provisionally expressed as P1, P2, P3 and P4 based
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on the elution order. The capacity factors (k’) and the resolution factor of the adjacent two peaks (e.g. Rs12 for P1 and P2) were calculated (the Rs of DM and DC enantiomers
ed
affected by the IPA content and temperature listed in Supplementary Information S2). The retentions generally decreased with the IPA content increasing. At the ratio of 96/4
pt
(n-hexane/IPA, v/v), baseline separation was observed from 15 ℃ to 25 ℃ with
Ac ce
good resolution and reproducibility. The best enantioseparation was obtained at 20 ℃ with each Rs exceeding 2. Considering the two effect factors of the mobile phase and temperature, n-hexane/IPA (96/4) and 20 ℃
was adopted as the optimum
chromatographic condition. The four enantiomers could be eluted in about 10 mins. Chiral recognition was conducted using the ORD to confirm the elution orders and configuration. Fig.1 showed the chromatogram (a) and the ORD signal (b) of DM and DC enantiomers separated on Chiralpak IC column. The absolute configuration of DM and DC enantiomers was assigned by comparison of the ORD signals with those 10 / 10
Page 10 of 25
reported in the literature [10]. The ORD (+)-signal referred to R-enantiomer and the (-)-signal was S-enantiomer. The elution order was (+)-R-DC, (+)-R-DM, (-)-S-DC and (-)-S-DM on Chiralpak IC with n-hexane/IPA (96/4, v/v), which was opposite on
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cellulose-tris-(3, 5-dimethylphenylcarbamate) (CDMPC) CPS [22]. Previous work using the CDMPC as CPS took relatively long analysis time (more than 20 mins under
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the same optimum chromatographic condition) [10]. By contrast, the enantioseparation
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on IC column demonstrated its superiority of rapidness and solvents-saving with good performance and stability. The proposed chromatographic condition was allowed to
3.2 Enzyme kinetic analysis
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analyze the biological processes of the DM and DC enantiomers in LLM in vitro.
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This assay was applied to investigate the enzyme kinetics for DM metabolism in LLM. Substrate concentration versus reaction velocity curves of R- and S-enantiomer
pt
were obtained by fitting the experimental data to the Michaelis–Menten model (Fig.2),
Ac ce
and thus the parameters such as Km, Vmax and CLint were calculated for both enantiomers (Table 2). The enzyme kinetics fitted the Michaelis–Menten model well with R2>0.99. It was noteworthy that there existed quite differences of enzyme kinetics between the two enantiomers. The Km of (S)-DM was lower than that of the (R)-DM, which indicated the (S)-DM had greater affinity to the enzyme than its antipode. The intrinsic ability of hepatic enzymes to metabolize a compound is commonly referred to as “intrinsic clearance” (CLint). The CLint of (S)-DM was 2.23 times greater than that of the (R)-DM. The differences between the two enantiomers in enzyme kinetics 11 / 11
Page 11 of 25
suggest stereoselective degradation of DM in LLM.
3.3 Enantioselective metabolism of DM
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The degradation of DM in LLM followed the first-order kinetics, as shown in Fig. 3. The initial concentration of rac-DM was 40 µmol/L (13.6 µg/mL), and that was 20
cr
µmol/L (6.8 µg/mL) for each enantiomer. The (S)-DM was hydrolyzed much more
us
quickly than its antipode (P < 0.05). After 5 mins of incubation, the concentration of the (S)-DM quickly fell to 1.45 µg/mL, while the concentration of (R)-DM declined to
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5.37 µg/mL, just about 20% of the initial amount was hydrolyzed. By the incubation
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period of 30 mins, only a trace of (S)-DM was found but a level of 2.32 µg/mL of (R)-DM still could be detected. A quick formation of (S)-DC was observed in the first
ed
20 mins, and by contrast, the formation of (R)-DC was much slower. The kinetic parameters of metabolism are listed in Table 3. The single-enantiomer incubation
pt
showed a half-life (t1/2) was 5.27 mins for (S)-DM and 21.17 mins for (R)-DM. The
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degradation rate of (S)-DM was about 4 times as much as that of the (R)-DM. The enantiomer fraction (EF) values are calculated on the basis of the peak areas
of the respective enantiomers according to the equation below [23]. EF=
During the incubation, the EFs of DM increased from 0.81(at 5 mins) to 0.96, owing to the rapid dissipation of (S)-DM thus yielding a relative enantioenrichment of (R)-DM. The EFs of diclofop increased from 0.15 to 0.47 due to the preferential formation of (S)-DC. Along with the (R)-DC formation, the final concentrations of the DC 12 / 12
Page 12 of 25
enantiomers were similar (shown in Fig. 4). The hydrolysis of DM in the procedural blanks (deactivated LLM) was insignificant. The diclofop concentration changed inconspicuously (about 1/10 of DM
ip t
content), revealing the deactivated enzymes were incapable of catalyzing the DM hydrolysis. The formation of DC was dependent on the active enzyme in LLM.
cr
However, in the activated microsomes or negative control, the fast degradation of DM
us
and significant formation of DC were both observed, showing NADPH was unnecessary and the reaction was not driven by CYP450. The esterases in liver might
an
play a dominant role in the conversion processes. In a previous study, carp liver
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microsomal esterases had stronger ability to hydrolyze trans-permethrin than cis-permethrin and it was similar for rainbow trout liver microsomes [24]. Pyrethroids
ed
have inhibitory effect on the hydrolysis of ester bond of many species, thus enhanced the toxicity of these insecticides [25]. Our unpublished work showed that rac-DM
pt
posed high acute toxicity to aquatic organisms, but the toxicity of DC was much lower.
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Therefore, hydrolysis of ester bond is an important detoxification for DM. The DM toxicity depends on the activity of esterases, characteristics, the substrate specificity and the hydrolysis capability in target and non-target species. In the single-enantiomer experiments, the (R)-DM was hydrolyzed to form its
corresponding (R)-DC, and vice versa. Typical HPLC chromatograms of metabolism are shown in Fig. 5. There was no interconversion between the two enantiomers. DM and DC were both configurationally stable during the incubation. The metabolism of diclofop in vitro was also conducted. LLM treated with NADPH did not catalyze the 13 / 13
Page 13 of 25
degradation of diclofop, indicating diclofop might be persistent in fish. The mechanism of its metabolism in fish and enzymes involved needs further study. The AOPP herbicides, including DM and DC, act at acetyl-CoA carboxylase
ip t
(ACCase) that can be found in the non-target plants, animals, and humans [26]. Furthermore, other herbicides of the same group, e.g., quizalofop and haloxyfop inhibit
cr
ACCase [27], disrupt lipid metabolism [28], and interfere with membrane transport
us
[29]. In addition, research has shown that diclofop inhibits the biosynthesis of sex-pheromone in moths and thus precludes mating success, thereby proving to be a
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possible endocrine disruptor [30]. The toxicity of the two enantiomers of DM and DC
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was different and the herbicidally inactive S-enantiomers of both DM and DC may exert higher toxicity to three freshwater algae [15]. At present, research of DM and DC
ed
on aquatic ecological toxicology is scant, especially on the differences between enantiomers. Since the herbicidal activity of AOPP herbicides primarily exists in the
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R-enantiomer [11], the Netherlands and Switzerland have revoked registrations for
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racemic mixtures of chiral phenoxy herbicides, whilst approving registrations of single-isomer products [1]. For assessing the environmental safety of DM and DC, information on their stereochemistry is very important. Potential enantioselectivity in the chronic processes for diclofop should be further explored. Through the in-depth research on environmental behaviors of DM and DC enantiomers, it can help to reduce environmental loading and make the use of chiral pesticides more effective.
4. Conclusion 14 / 14
Page 14 of 25
An efficient and stable method for the simultaneous chiral analysis of DM and DC enantiomers on Chiralpak IC column was developed. The method was optimized by investigating the influence of mobile phase and temperature. The degradation of
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racemic DM and single enantiomer of DM as well as diclofop in LLM in vitro was conducted. The results suggested that the biotransformation process was dominated by
cr
microsomal esterases. The DM enantiomers were rapidly hydrolyzed to DC. The
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rac-DM degradation in LLM was enantioselective with the conversion rate of (S)-DM markedly greater than that of the R-enantiomer. Single-enantiomer experiments
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revealed that no enantiomerization was observed, suggesting the biotransformation
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was configurationally stable. In contrast, DC was not metabolized in LLM even with NADPH present. Further research on the mechanism of DC metabolism in loach and
ed
enzyme involved was needed. Resolving the capacity of aquatic vertebrates to metabolize chiral pesticides applied in the environment and their enantiomers will
Ac ce
pt
contribute to evaluate the ecological risks and thus its impact on human health.
Acknowledgement
The authors acknowledge the support of the Foundation for the Author of National
Excellent Doctoral Dissertation of PR China，Program for New Century Excellent Talents in University (NCET09-0738), the National Natural Science Foundation of China (21277171, 21337005), the New-Star of Science and Technology supported by Beijing Metropolis, Program for New Century Excellent Talents in University and Program for Changjiang Scholars and Innovative Research Team in University. 15 / 15
Page 15 of 25
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[14] F. Petit, L.P. Goff, J.P. Cravedi, Y. Valotaire, F. Pakdel, J. Mol. Endocrinol. 19 (1997) 321-335. [15] X. Cai, W. Liu, G. Sheng, J. Agric. Food Chem. 56 (2008) 2139-2146. [16] E.M. Smith, S. Chu, G. Paterson, C.D. Metcalfe, J.Y. Wilson, Chemosphere 79 (2010) 26-32. [17] T. Andersson, L. Förlin, Aquat. Toxicol. 24 (1992) 1-19. [18] S. Kitamura, T. Suzuki, T Kadota., M. Yoshida, K. Ohashi, S. Ohta, Drug Metab. Dispos. 31 (2003) 179-186. 16 / 16
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[23] T. Harner, K. Wiberg, R. Norstrom, Environ. Sci. Technol. 34 (2000) 218-220.
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(1983) 413-438.
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[26] D.L. Shaner, Pest Manag. Sci. 60 (2004) 17-24.
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[25] J.E. Casida, D.W. Gammon, A.H. Glickman, L.J. Lawrence, Annu. Rev. Pharmacol. Toxicol. 23
[27] A.C. Beynen, M.J.H. Geelen, Toxicology 24 (1982) 183-197.
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[28] E. Granzer, H. Nahm, Arzneim-Forsch 23 (1973) 1353. [29] G. Bettoni, F. Loiodice, V. Tortorella, D. Conte-Camerino, M. Mambrini, E. Ferrannini, S.H.
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[30] D. Eliyahu, S. Applebaum, A. Rafaeli, Pestic. Biochem. Physiol. 77 (2003) 75-81.
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Figure Captions
Fig. 1 Typical chromatogram of the enantioseparation of DM and DC (each
ip t
concentration of 20.0 mg/L) on Chiralpak IC with n-hexane/IPA/TFA (96/4/0.1, v/v/v;
cr
1 mL/min) (A) and the corresponding ORD signal (B)
Fig. 2 Effects of substrate concentration on the degradation rate of R-DM and S-DM in
us
loach liver microsomes
an
Fig. 3 Concentration-time curves of DM and DC enantiomers from metabolism assays
M
(n=3) incubated with rac-DM (A), R-DM (B) and S-DM (C)
value).
ed
Fig. 4 EFs of DM and the generated DC from rac-DM (40 µmol L-1) incubation (mean
pt
Fig. 5 Typical HPLC chromatograms of metabolism assays incubated with the R-DM
Ac ce
at 20 min (A) and S-DM at 5 min (B)
18 / 18
Page 18 of 25
*Highlights (for review)
Highlights ► Diclofop-methyl and diclofop were simultaneously enantioseparation on Chiralpak IC. ► Diclofop-methyl was rapidly degraded to diclofop by esterases from liver microsomes.
ip t
► The biotransformation was configurationally stable by single-enantiomer incubation. ► The degradation of diclofop-methyl in loach liver microsomes was enantioselective.
Ac
ce pt
ed
M
an
us
cr
► The (S)-diclofop-methyl degraded markedly faster than the (R)-enantiomer did.
1/1
Page 19 of 25
Ac ce p
te
d
M
an
us
cr
ip t
Fig.1
Page 20 of 25
Ac
ce
pt
ed
M
an
us
cr
i
Fig.2
Page 21 of 25
Ac
ce
pt
ed
M
an
us
cr
i
Fig.3
Page 22 of 25
Ac
ce
pt
ed
M
an
us
cr
i
Fig.4
Page 23 of 25
Ac ce p
te
d
M
an
us
cr
ip t
Fig.5
Page 24 of 25
Tables
Table 1 Method validation of the chiral HPLC method for analysis of DM and DC enantiomers (n=3) LOQ (mg L-1)
R2
Extraction Recovery (%)
RSD (%)
R-DM
y = 17.531x -8.3704
0.5
0.9999
79.6~92.3
5.6~8.2
S-DM
y = 17.497x -23.638
0.5
0.9999
80.9~96.4
4.8~7.9
R-DC
y = 21.097x - 15.338
0.5
0.9999
87.3~108.9
5.2~10.7
S-DC
y =21.183x - 45.136
0.5
0.9998
84.0~106.6
6.6~11.5
us
cr
ip t
Linear equation
substrate
Vmax(µmol min-1 mg-1protein)
Km(µmol L-1)
R2
R-DM
78.08
392.1
199.1
0.9942
S-DM
144.3
444.9
0.9953
an
CLint (ml min-1 mg-1protein)
M
Table 2. The parameters of enzyme kinetics
ed
320.7
Table 3. The parameters of metabolism of rac-DM and single-enantiomer
Ac
rac-DM
analyte
t1/2(min)
K(min-1)
R2
R-DM
20.04
0.0346
0.9274
S-DM
3.64
0.1904
0.8573
R-DC
37.61
0.0184
0.9340
S-DC
7.75
0.0894
0.8019
R-DM
21.17
0.0328
0.9802
R-DC
25.79
0.0269
0.9567
S-DM
5.27
0.1314
0.9288
S-DC
17.32
0.0400
0.7092
ce pt
incubation
R-DM
S-DM
Page 25 of 25
S1570-0232(14)00523-6 http://dx.doi.org/doi:10.1016/j.jchromb.2014.08.014 CHROMB 19076
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
28-2-2014 7-8-2014 10-8-2014
Please cite this article as: R. Ma, Enantioselective metabolism of the chiral herbicide diclofop-methyl and diclofop by HPLC in loach (Misgurnus anguillicaudatus) liver microsomes in vitro, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.08.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enantioselective
metabolism
of
the
chiral
herbicide
diclofop-methyl and diclofop by HPLC in loach (Misgurnus
ip t
anguillicaudatus) liver microsomes in vitro
Authors:
cr
Ruixue Ma, email: [email protected]
us
Han Qu, email: [email protected]
Donghui Liu, email: [email protected]
M
Yiran Liang, email: [email protected]
an
Xia Liu, email: [email protected]
Peng Wang, email: [email protected]
The corresponding author: email: [email protected] Tel/fax: +861062733547
pt
*
ed
Zhiqiang Zhou*,
Ac ce
Affiliation: Department of Applied Chemistry, China Agricultural University, Beijing 100193, PR China
Address: No. 2 Yuanmingyuan West Rd., Beijing 100193, PR China
1/1
Page 1 of 25
Abstract An efficient and reliable enantioseparation method by high-performance liquid chromatography (HPLC) for the chiral herbicide diclofop-methyl (DM) and its primary
ip t
metabolite diclofop (DC) was developed and validated, and the biological process of the enantiomers in loach liver microsomes (LLM) in vitro was investigated. The
cr
enantiomers of DM and DC were first separated on an immobilised-type stationary
us
phase of Chiralpak IC. The best resolutions were achieved under the chromatographic condition of n-hexane/IPA/TFA 96:4:0.1(v/v/v) at 20 ℃ with each Rs >2 and the two
an
pairs of enantiomers could be eluted in about 10 mins. The extraction recoveries of the
M
analytes from LLM were 79.6%̶108.9% with RSD≤11.5%. The enzyme kinetics of DM enantiomers were different, with the Km value 320.7 for (S)-DM and 392.1 for
ed
(R)-DM. The metabolism experiment in vitro showed DM underwent a rapid phase-I metabolism with or without NADPH, indicating the esterases in liver played a
pt
dominant role. No interconversion between the two enantiomers was observed by
Ac ce
single-enantiomer incubation. The preferential degradation of (S)-DM was confirmed with the half-time (t1/2) of (S)-DM 5.27 min and 21.2 min for (R)-DM. DC enantiomer was generated by its corresponding ester form and could not be further degraded during the incubation. It was the first study on biotransformation of DM and DC enantiomers in LLM in vitro. The results may help to evaluate the ecological risks of chiral pesticides.
Keywords: diclofop-methyl, diclofop, metabolism, enantioselective, Chiralpak IC, 2/2
Page 2 of 25
loach liver microsome
ip t
1. Introduction Chemical pesticides have contributed substantially to increases agricultural
cr
production. Among the frequently used pesticides, more than one quarter have one or
us
more asymmetric center(s) [1]. The two enantiomers may behave differently in bioactivity, toxicity, metabolism and degradation under certain environmental
an
conditions [2-4]. It has been reported that the bioactivity of a given chiral pesticide
M
often a result of the preferential reactivity of only one enantiomer while the other enantiomer might exert toxic effects on non-target organisms [1]. In most cases, the
ed
chiral pesticides are manufactured and employed in racemic form which means the enantioselective degradation may lead to an inaccurate environmental risk evaluation
pt
under traditional consideration [5]. To assess the environmental safety of chiral
Ac ce
compounds, investigation on their stereochemistry is necessary [6]. Polysaccharide derivatives are the first and broadest choice of selectors for use as
chiral stationary phases (CSPs) for liquid chromatography [7, 8]. Chiralpak IC is a new generation
of
CSP
from
chemically
immobilised
cellulose
tris
(3,
5-dichlorophenylcarbamate) on silica gel, making these adsorbents robust and showing remarkable enantioselective performance [9]. Diclofop-methyl (DM), 2-[4-(2, 4-dichlorophenoxy) phenoxy]-propionate, is a widely used herbicide of the aryloxyphenoxy propionate (AOPP) class [10]. The total 3/3
Page 3 of 25
annual usage of diclofop-methyl in the USA was about 340,000 kg of active ingredient (a.i.) in the period 1987–1996 [11]. Its hydrolysate diclofop (DC) also has herbicidal activity. Both diclofop-methyl and diclofop are chiral and consist of a pair of
ip t
enantiomers, respectively (structures shown in Supplementary Information S1). In general, the (R)-enantiomers of AOPP herbicides show two times higher herbicidal
cr
activity than the racemic mixtures [12]. Although many herbicides are typically
us
applied to the crop, substantial quantities of the active ingredient penetrate to the environmental, for example, up to 73% in the case of a controlled foliar application of
an
the herbicide diclofop-methyl [13]. There are researches showing diclofop-methyl and
M
diclofop are suspected to be endocrine disruptors and carcinogens, and thus are expected to cause a risk to some non-target terrestrial and aquatic plants [14]. Recent
ed
studies indicated that the (S)-enantiomers of DM and DC posed a stronger toxicity to three freshwater algae than the (R)-enantiomers [15].
pt
Biotransformation of xenobiotics in vertebrates is a multi-phase process, the first
Ac ce
of which usually involves the cytochrome P450 (CYP) superfamily of enzymes [16, 17]. Fish are the most representative aquatic vertebrates. The oxidative metabolism of various xenobiotics in fish is catalyzed by cytochrome P450 (mainly in the liver), and various compounds can induce the activity of this enzyme [18]. It has been reported that fish are capable of metabolizing some typical mammalian CYP2 and CYP3 substrates [19]. However, studies of drug metabolism in fish are extremely limited. To our best knowledge, the metabolic fate and CYPs responsible for the metabolism of diclofop-methyl in fish are largely unknown, especially at the level of enantiomers. For 4/4
Page 4 of 25
this reason, research on diclofop-methyl environmental behaviors in fish is of the utmost importance. Loaches (Misgurnus anguillicaudatus) are small edible fish widely distributed in freshwater ecosystems and are adaptable to the ecological environment,
ip t
which appear to be a proper biological model for studies on water pollutants. In vivo and in vitro metabolism studies are useful for understanding the fate of a xenobiotic in
cr
an organism. In vitro experiments using microsomal material from various organs (e.g.,
us
liver) can shed light on such metabolism events with a minimum of expense and provide valuable information on toxicant exposure [20].
an
In this work, for the first time, the simultaneous chiral analysis method of
M
diclofop-methyl and diclofop enantiomers was established utilizing an immobilised CSP, and the metabolism of the racemic and optical pure isomer of diclofop-methyl in
ed
LLM in vitro was demonstrated. The results filled a vacancy of the stereoselective
pt
metabolic behavior of diclofop-methyl in aquatic species.
Ac ce
2. Material & methods
2.1. Chemicals and reagents Racemic-diclofop-methyl (rac-DM, purity> 99.0%) was obtained from China
Ministry of Agriculture’s Institute for Control of Agrochemicals. Racemic-diclofop (rac-DC, purity > 98.0%) was synthesized in our laboratory. The enantiomers of DM (purity > 99.5%) were prepared with a preparatory chiral column (250 × 10 mm (I.D.), provided by Department of Applied Chemistry, China Agricultural University, Beijing). Water was purified by a Milli-Q system. Ethyl ether, n-hexane, 2-propanol (HPLC 5/5
Page 5 of 25
grade), and trifluoroacetic acid (TFA) were from Fisher Scientific (Fair Lawn, NJ, U.S.A.). β-Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Sigma-Aldrich (St Louis, MO, USA). All other chemicals and solvents were of
ip t
analytical grade and purchased from commercial sources. The loaches were obtained from a local aquafarm. The fish employed were mixed-gender and were all adults and
us
cr
healthy, with a wet weight of 20 g to 25 g (15 cm to 20 cm) each.
2.2. Instrumentation and chromatographic conditions
an
Analysis was performed using an Agilent 1260 Infinity HPLC (Agilent
M
Technologies Palo Alto, CA) equipped with G1311C 1260 Quat pump VL, G1329B 1260 ALS, G1316A 1260 TCC, G1314F 1260 VWD. A Chiralyser–MP Optical
ed
Rotation Detector was purchased from IBZ Messtechnik (Germany). The Chiralpak IC column (250×4.6 mm i.d.) was supplied by Daicel Chemical Industries, Ltd. (Tokyo,
pt
Japan) with the CSPs [cellulose tris-(3, 5-dichlorophenylcarbamate)] polymer
Ac ce
immobilized on silica. The mobile phase was composed of n-hexane and isopropanol with 0.1% trifluoroacetic acid (TFA). The flow rate was 1.0 mL/min. The configurations of the enantiomers were determined by the ORD signals.
2.3. Microsome Preparation Loaches were held for a week acclimation period in 50-L glass aquaria. The fish was killed and liver was quickly excised. Liver microsomes were prepared according to the method of differential ultracentrifugation [21]. The microsomal pellet was 6/6
Page 6 of 25
resuspended in 50 mmol/L Tris–HCl buffer (pH 7.4) containing 20% glycerol (v/v) and 0.1 mmol/L EDTA (ethylenediaminetetraacetic acid) and stored at -80℃ prior to use.
ip t
Total protein content was determined using the Bradford method.
2.4. Enzyme kinetic assay
cr
To estimate enzyme kinetic parameters, a series of each enantiomer stock solutions
us
were added to LLM (0.5 mg/mL) to achieve final substrate concentrations of 40–1920 µM. The substrate concentrations of each DM enantiomer were 40, 80, 160, 240, 480,
an
960 and 1920 µM for R-DM and 40, 80, 160, 320, 640, 960 and 1920 µM for S-DM.
M
Different incubation times were adopted for the enantiomers, 5 min for R-DM and 2 min for S-DM, respectively. The Vmax and Km values were calculated according to
ed
nonlinear regression Michaelis–Menten equation. Intrinsic clearance (CLint) was obtained as a ratio of Vmax to Km. Data analysis was performed using Graphpad Prism
Ac ce
pt
5 (GraphPad Software Inc. Press, San Diego CA, 2007).
2.5. Metabolism of DM and DC in LLM and the sample pretreatment The metabolism of DM by LLM was carried out in a reaction system of 1 mL
containing LLM (0.5 mg/mL), Tris–HCl buffer (50 mmol/L, pH 7.4), EDTA (1.0 mmol/L), NADPH (1.0 mmol/L) and rac-DM or single-enantiomer (40 μmol/L). Experiments were conducted at 19 ℃ to reflect temperatures most likely encountered by loaches under natural conditions. Two types of experimental blanks were included. One consisted of DM and heat-treated LLM (i.e., LLM heated at 100 ℃ for 60 mins 7/7
Page 7 of 25
in a water bath to render them inactive) plus all of the reaction components, called procedural blank. This would correct for any abiotic transformation of DM. The other blank (called negative control) contained LLM and all of the reaction components but
ip t
without NADPH. In addition, rac-DC metabolism in vitro was also conducted. All the incubations lasted for 3 hours.
cr
At each sampling point, pre-chilled ethyl acetate was introduced to terminate the
us
reaction and 1 mL of 1M HCl solution was added to help extract diclofop. The sample was vortexed for 5 min and then centrifuged at 3500 rpm. The extraction and
an
centrifugation were repeated with another 5 mL of ethyl acetate. The organic phase
M
was combined and evaporated to dryness under a gentle stream of nitrogen at 30 ℃ and the resulting residue was redissolved in 500 µL of isopropanol for HPLC analysis.
ed
Statistical differences between two groups were assessed using a SPSS v15.0 software package (SPSS Inc., Chicago, IL). A p-value of less than 0.05 was considered
Ac ce
pt
statistically significant unless otherwise indicated.
2.6. Quality control
Stock solutions of DM standard were prepared in 2-propanol and stored at -20°C.
Stock solutions of R/S-DM standard were determined by external standard method. Working standard solutions of rac-DM, R-DM and S-DM were freshly prepared by dilutions of stock solution in 2-propanol. Calibration standards were prepared by adding a series of working standard solutions of rac-DM into the blank matrix (BM, the extracts from the prepared 8/8
Page 8 of 25
inactivated microsomes), and then stored at -20°C until analysis. Calibration curves were obtained over the range of 0.5-800 mg/L at 5 concentrations (0.50, 5.0, 20, 100, 800 mg/L) by plotting peak area versus the concentration of each enantiomer. The
ip t
linear regression analysis was performed using Microsoft Excel®. The extraction recovery was evaluated at three levels (0.50, 20 and 800 mg/L) by spiking mixed
cr
rac-DM and rac-DC standard into the prepared inactivated microsomes. Precision of
us
the extraction recovery were performed in triplicate and expressed as RSD. Accuracy of the method was calculated by analyzing the calibration standard in 3 replicates
an
consecutively and comparing the predicted concentration (obtained from the
M
calibration curve) to the actual concentration of each enantiomer spiked in the BM. Limit of quantification (LOQ) was defined as the lowest concentration in the
ed
calibration curve with acceptable precision and accuracy (less than 20% CV and within +/-20% RE). The parameters of method validation are listed in Table 1.
pt
The standard solutions in 2-propanol of DM and enantiomers were determined its
Ac ce
optical purity and hydrolysis rate after 1, 7, 14 and 21 days of storage prior to use. There was no enantiomerization observed and the hydrolysis was within normal levels. The stability of calibration standard was assessed after 1, 8 h of preparation and 1, 4 and 10 d of storage at -20 °C. The RSD of storage stability was in the range of 4.6 %̶ 7.2 %, indicating the analysis of DM enantiomers in LLM sample was reliable under the experiment conditions.
3. Results & discussion 9/9
Page 9 of 25
3.1 Optimization of Enantioseparation Conditions There is scarcely any literature support of simultaneous chiral separation of DM and DC enantiomers on Chiralpak IC column. The influences of alcoholic modifier
ip t
concentration as well as temperature on the enantioseparation were investigated. The test temperature was varied from 10 ℃ to 30 ℃ and the concentration of alcoholic
cr
modifier IPA varied from 2% to 5%. For better separation and peak shapes, 0.1% (v:v)
us
TFA was added into the mobile phase. The resolution factor Rs was used to evaluate the separation and it was considered a complete separation when the Rs>1.5. Here the
an
four peaks on chromatograms were provisionally expressed as P1, P2, P3 and P4 based
M
on the elution order. The capacity factors (k’) and the resolution factor of the adjacent two peaks (e.g. Rs12 for P1 and P2) were calculated (the Rs of DM and DC enantiomers
ed
affected by the IPA content and temperature listed in Supplementary Information S2). The retentions generally decreased with the IPA content increasing. At the ratio of 96/4
pt
(n-hexane/IPA, v/v), baseline separation was observed from 15 ℃ to 25 ℃ with
Ac ce
good resolution and reproducibility. The best enantioseparation was obtained at 20 ℃ with each Rs exceeding 2. Considering the two effect factors of the mobile phase and temperature, n-hexane/IPA (96/4) and 20 ℃
was adopted as the optimum
chromatographic condition. The four enantiomers could be eluted in about 10 mins. Chiral recognition was conducted using the ORD to confirm the elution orders and configuration. Fig.1 showed the chromatogram (a) and the ORD signal (b) of DM and DC enantiomers separated on Chiralpak IC column. The absolute configuration of DM and DC enantiomers was assigned by comparison of the ORD signals with those 10 / 10
Page 10 of 25
reported in the literature [10]. The ORD (+)-signal referred to R-enantiomer and the (-)-signal was S-enantiomer. The elution order was (+)-R-DC, (+)-R-DM, (-)-S-DC and (-)-S-DM on Chiralpak IC with n-hexane/IPA (96/4, v/v), which was opposite on
ip t
cellulose-tris-(3, 5-dimethylphenylcarbamate) (CDMPC) CPS [22]. Previous work using the CDMPC as CPS took relatively long analysis time (more than 20 mins under
cr
the same optimum chromatographic condition) [10]. By contrast, the enantioseparation
us
on IC column demonstrated its superiority of rapidness and solvents-saving with good performance and stability. The proposed chromatographic condition was allowed to
3.2 Enzyme kinetic analysis
M
an
analyze the biological processes of the DM and DC enantiomers in LLM in vitro.
ed
This assay was applied to investigate the enzyme kinetics for DM metabolism in LLM. Substrate concentration versus reaction velocity curves of R- and S-enantiomer
pt
were obtained by fitting the experimental data to the Michaelis–Menten model (Fig.2),
Ac ce
and thus the parameters such as Km, Vmax and CLint were calculated for both enantiomers (Table 2). The enzyme kinetics fitted the Michaelis–Menten model well with R2>0.99. It was noteworthy that there existed quite differences of enzyme kinetics between the two enantiomers. The Km of (S)-DM was lower than that of the (R)-DM, which indicated the (S)-DM had greater affinity to the enzyme than its antipode. The intrinsic ability of hepatic enzymes to metabolize a compound is commonly referred to as “intrinsic clearance” (CLint). The CLint of (S)-DM was 2.23 times greater than that of the (R)-DM. The differences between the two enantiomers in enzyme kinetics 11 / 11
Page 11 of 25
suggest stereoselective degradation of DM in LLM.
3.3 Enantioselective metabolism of DM
ip t
The degradation of DM in LLM followed the first-order kinetics, as shown in Fig. 3. The initial concentration of rac-DM was 40 µmol/L (13.6 µg/mL), and that was 20
cr
µmol/L (6.8 µg/mL) for each enantiomer. The (S)-DM was hydrolyzed much more
us
quickly than its antipode (P < 0.05). After 5 mins of incubation, the concentration of the (S)-DM quickly fell to 1.45 µg/mL, while the concentration of (R)-DM declined to
an
5.37 µg/mL, just about 20% of the initial amount was hydrolyzed. By the incubation
M
period of 30 mins, only a trace of (S)-DM was found but a level of 2.32 µg/mL of (R)-DM still could be detected. A quick formation of (S)-DC was observed in the first
ed
20 mins, and by contrast, the formation of (R)-DC was much slower. The kinetic parameters of metabolism are listed in Table 3. The single-enantiomer incubation
pt
showed a half-life (t1/2) was 5.27 mins for (S)-DM and 21.17 mins for (R)-DM. The
Ac ce
degradation rate of (S)-DM was about 4 times as much as that of the (R)-DM. The enantiomer fraction (EF) values are calculated on the basis of the peak areas
of the respective enantiomers according to the equation below [23]. EF=
During the incubation, the EFs of DM increased from 0.81(at 5 mins) to 0.96, owing to the rapid dissipation of (S)-DM thus yielding a relative enantioenrichment of (R)-DM. The EFs of diclofop increased from 0.15 to 0.47 due to the preferential formation of (S)-DC. Along with the (R)-DC formation, the final concentrations of the DC 12 / 12
Page 12 of 25
enantiomers were similar (shown in Fig. 4). The hydrolysis of DM in the procedural blanks (deactivated LLM) was insignificant. The diclofop concentration changed inconspicuously (about 1/10 of DM
ip t
content), revealing the deactivated enzymes were incapable of catalyzing the DM hydrolysis. The formation of DC was dependent on the active enzyme in LLM.
cr
However, in the activated microsomes or negative control, the fast degradation of DM
us
and significant formation of DC were both observed, showing NADPH was unnecessary and the reaction was not driven by CYP450. The esterases in liver might
an
play a dominant role in the conversion processes. In a previous study, carp liver
M
microsomal esterases had stronger ability to hydrolyze trans-permethrin than cis-permethrin and it was similar for rainbow trout liver microsomes [24]. Pyrethroids
ed
have inhibitory effect on the hydrolysis of ester bond of many species, thus enhanced the toxicity of these insecticides [25]. Our unpublished work showed that rac-DM
pt
posed high acute toxicity to aquatic organisms, but the toxicity of DC was much lower.
Ac ce
Therefore, hydrolysis of ester bond is an important detoxification for DM. The DM toxicity depends on the activity of esterases, characteristics, the substrate specificity and the hydrolysis capability in target and non-target species. In the single-enantiomer experiments, the (R)-DM was hydrolyzed to form its
corresponding (R)-DC, and vice versa. Typical HPLC chromatograms of metabolism are shown in Fig. 5. There was no interconversion between the two enantiomers. DM and DC were both configurationally stable during the incubation. The metabolism of diclofop in vitro was also conducted. LLM treated with NADPH did not catalyze the 13 / 13
Page 13 of 25
degradation of diclofop, indicating diclofop might be persistent in fish. The mechanism of its metabolism in fish and enzymes involved needs further study. The AOPP herbicides, including DM and DC, act at acetyl-CoA carboxylase
ip t
(ACCase) that can be found in the non-target plants, animals, and humans [26]. Furthermore, other herbicides of the same group, e.g., quizalofop and haloxyfop inhibit
cr
ACCase [27], disrupt lipid metabolism [28], and interfere with membrane transport
us
[29]. In addition, research has shown that diclofop inhibits the biosynthesis of sex-pheromone in moths and thus precludes mating success, thereby proving to be a
an
possible endocrine disruptor [30]. The toxicity of the two enantiomers of DM and DC
M
was different and the herbicidally inactive S-enantiomers of both DM and DC may exert higher toxicity to three freshwater algae [15]. At present, research of DM and DC
ed
on aquatic ecological toxicology is scant, especially on the differences between enantiomers. Since the herbicidal activity of AOPP herbicides primarily exists in the
pt
R-enantiomer [11], the Netherlands and Switzerland have revoked registrations for
Ac ce
racemic mixtures of chiral phenoxy herbicides, whilst approving registrations of single-isomer products [1]. For assessing the environmental safety of DM and DC, information on their stereochemistry is very important. Potential enantioselectivity in the chronic processes for diclofop should be further explored. Through the in-depth research on environmental behaviors of DM and DC enantiomers, it can help to reduce environmental loading and make the use of chiral pesticides more effective.
4. Conclusion 14 / 14
Page 14 of 25
An efficient and stable method for the simultaneous chiral analysis of DM and DC enantiomers on Chiralpak IC column was developed. The method was optimized by investigating the influence of mobile phase and temperature. The degradation of
ip t
racemic DM and single enantiomer of DM as well as diclofop in LLM in vitro was conducted. The results suggested that the biotransformation process was dominated by
cr
microsomal esterases. The DM enantiomers were rapidly hydrolyzed to DC. The
us
rac-DM degradation in LLM was enantioselective with the conversion rate of (S)-DM markedly greater than that of the R-enantiomer. Single-enantiomer experiments
an
revealed that no enantiomerization was observed, suggesting the biotransformation
M
was configurationally stable. In contrast, DC was not metabolized in LLM even with NADPH present. Further research on the mechanism of DC metabolism in loach and
ed
enzyme involved was needed. Resolving the capacity of aquatic vertebrates to metabolize chiral pesticides applied in the environment and their enantiomers will
Ac ce
pt
contribute to evaluate the ecological risks and thus its impact on human health.
Acknowledgement
The authors acknowledge the support of the Foundation for the Author of National
Excellent Doctoral Dissertation of PR China，Program for New Century Excellent Talents in University (NCET09-0738), the National Natural Science Foundation of China (21277171, 21337005), the New-Star of Science and Technology supported by Beijing Metropolis, Program for New Century Excellent Talents in University and Program for Changjiang Scholars and Innovative Research Team in University. 15 / 15
Page 15 of 25
References [1] A. Williams, Pestic. Sci. 46 (1996) 3-9. [2] N.M. Maier, P. Franco, W. Lindner, J. Chromatogr. A 906 (2001) 3-33.
ip t
[3] H.R. Buser, M.D. Müller, T. Poiger, M.E. Balmer, Environ. Sci. Technol. 36 (2002) 221-226 [4] A.W. Garrison, P. Schmitt, D. Martens, A. Kettrup, Environ. Sci. Technol. 30 (1996) 2449-2455.
cr
[5] W. Liu, J. Gan, D. Schlenk, W.A. Jury, Proc. Natl. Acad. Sci. USA 102 (2005) 701-706.
[7] E. Yashima, J. Chromatogr. A 906 (2001) 105-125
us
[6] H.P.E. Kohler, W. Angst, W. Giger, C. Kanz, S. Muller, M.J. Suter, Chimia 51 (1997) 947-951.
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[8] P. Franco, A. Senso, L. Oliveros, C. Minguillón, J. Chromatogr. A 906 (2001) 155-170
M
[9] P. Franco, T. Zhang, J. Chromatogr. B 875 (2008) 48-56
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[11] J. Ye, J. Wu, W. Liu, TRAC-Trend. Anal. Chem. 28 (2009) 1148-1163. [12] N. Kurihara, J. Miyamoto, G.D. Paulson, B. Zeeh, M.W. Skidmore, R.M. Hollingworth, H.A.
pt
Kuiper, Pure Appl. Chem. 69 (1997) 2007-2025.
Ac ce
[13] A.E. Smith, R. Grover, A.J. Cessna, S.R. Shewchuk, J.H. Hunter, J. Environ. Qual. 15 (1986) 234-238.
[14] F. Petit, L.P. Goff, J.P. Cravedi, Y. Valotaire, F. Pakdel, J. Mol. Endocrinol. 19 (1997) 321-335. [15] X. Cai, W. Liu, G. Sheng, J. Agric. Food Chem. 56 (2008) 2139-2146. [16] E.M. Smith, S. Chu, G. Paterson, C.D. Metcalfe, J.Y. Wilson, Chemosphere 79 (2010) 26-32. [17] T. Andersson, L. Förlin, Aquat. Toxicol. 24 (1992) 1-19. [18] S. Kitamura, T. Suzuki, T Kadota., M. Yoshida, K. Ohashi, S. Ohta, Drug Metab. Dispos. 31 (2003) 179-186. 16 / 16
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[19] D.R. Buhler, J.L. Wang-Buhler, Comp. Biochem. Physiol. 121 (1998) 107-137. [20] C.S. Mazur, J.F. Kenneke, Environ. Sci. Technol. 42 (2007) 947-954. [21] J.F. Kenneke, D.R. Ekman, C.S. Mazur, B.J. Konwick, A.T. Fisk, J.K. Avants, A.W. Garrison,
ip t
Chirality 22 (2010) 183-192. [22] X. Gu, Y. Lu, P. Wang, Z. Dang, Z. Zhou, Food Chem. 121 (2010) 264-267.
cr
[23] T. Harner, K. Wiberg, R. Norstrom, Environ. Sci. Technol. 34 (2000) 218-220.
us
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M
[26] D.L. Shaner, Pest Manag. Sci. 60 (2004) 17-24.
an
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[28] E. Granzer, H. Nahm, Arzneim-Forsch 23 (1973) 1353. [29] G. Bettoni, F. Loiodice, V. Tortorella, D. Conte-Camerino, M. Mambrini, E. Ferrannini, S.H.
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Ac ce
[30] D. Eliyahu, S. Applebaum, A. Rafaeli, Pestic. Biochem. Physiol. 77 (2003) 75-81.
17 / 17
Page 17 of 25
Figure Captions
Fig. 1 Typical chromatogram of the enantioseparation of DM and DC (each
ip t
concentration of 20.0 mg/L) on Chiralpak IC with n-hexane/IPA/TFA (96/4/0.1, v/v/v;
cr
1 mL/min) (A) and the corresponding ORD signal (B)
Fig. 2 Effects of substrate concentration on the degradation rate of R-DM and S-DM in
us
loach liver microsomes
an
Fig. 3 Concentration-time curves of DM and DC enantiomers from metabolism assays
M
(n=3) incubated with rac-DM (A), R-DM (B) and S-DM (C)
value).
ed
Fig. 4 EFs of DM and the generated DC from rac-DM (40 µmol L-1) incubation (mean
pt
Fig. 5 Typical HPLC chromatograms of metabolism assays incubated with the R-DM
Ac ce
at 20 min (A) and S-DM at 5 min (B)
18 / 18
Page 18 of 25
*Highlights (for review)
Highlights ► Diclofop-methyl and diclofop were simultaneously enantioseparation on Chiralpak IC. ► Diclofop-methyl was rapidly degraded to diclofop by esterases from liver microsomes.
ip t
► The biotransformation was configurationally stable by single-enantiomer incubation. ► The degradation of diclofop-methyl in loach liver microsomes was enantioselective.
Ac
ce pt
ed
M
an
us
cr
► The (S)-diclofop-methyl degraded markedly faster than the (R)-enantiomer did.
1/1
Page 19 of 25
Ac ce p
te
d
M
an
us
cr
ip t
Fig.1
Page 20 of 25
Ac
ce
pt
ed
M
an
us
cr
i
Fig.2
Page 21 of 25
Ac
ce
pt
ed
M
an
us
cr
i
Fig.3
Page 22 of 25
Ac
ce
pt
ed
M
an
us
cr
i
Fig.4
Page 23 of 25
Ac ce p
te
d
M
an
us
cr
ip t
Fig.5
Page 24 of 25
Tables
Table 1 Method validation of the chiral HPLC method for analysis of DM and DC enantiomers (n=3) LOQ (mg L-1)
R2
Extraction Recovery (%)
RSD (%)
R-DM
y = 17.531x -8.3704
0.5
0.9999
79.6~92.3
5.6~8.2
S-DM
y = 17.497x -23.638
0.5
0.9999
80.9~96.4
4.8~7.9
R-DC
y = 21.097x - 15.338
0.5
0.9999
87.3~108.9
5.2~10.7
S-DC
y =21.183x - 45.136
0.5
0.9998
84.0~106.6
6.6~11.5
us
cr
ip t
Linear equation
substrate
Vmax(µmol min-1 mg-1protein)
Km(µmol L-1)
R2
R-DM
78.08
392.1
199.1
0.9942
S-DM
144.3
444.9
0.9953
an
CLint (ml min-1 mg-1protein)
M
Table 2. The parameters of enzyme kinetics
ed
320.7
Table 3. The parameters of metabolism of rac-DM and single-enantiomer
Ac
rac-DM
analyte
t1/2(min)
K(min-1)
R2
R-DM
20.04
0.0346
0.9274
S-DM
3.64
0.1904
0.8573
R-DC
37.61
0.0184
0.9340
S-DC
7.75
0.0894
0.8019
R-DM
21.17
0.0328
0.9802
R-DC
25.79
0.0269
0.9567
S-DM
5.27
0.1314
0.9288
S-DC
17.32
0.0400
0.7092
ce pt
incubation
R-DM
S-DM
Page 25 of 25