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Enantioselective Metabolism of Flufiprole in Rat and Human Liver Microsomes Chunmian Lin, Yelong Miao, Mingrong Qian, Qiang Wang, and Hu Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05853 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 12, 2016

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Journal of Agricultural and Food Chemistry

1

Enantioselective Metabolism of Flufiprole in Rat and

2

Human Liver Microsomes

3

Chunmian Lin , Yelong Miao , Mingrong Qian , Qiang Wang , Hu Zhang

†

†

4 5

†

‡

‡

*,‡

College of Biological and Environmental Engineering, Zhejiang University of Technology,

Hangzhou 310014, China ‡

6

Institute of Quality and Standard for Agricultural Products, Zhejiang Academy of Agricultural

7

Sciences, Hangzhou 310021, China

8

ABSTRACT: The enantioselective metabolism of flufiprole in rat and human liver

9

microsomes in vitro was investigated in this study. The separation and determination

10

were performed using a liquid chromatography system equipped with a triple

11

quadrupole mass spectrometer and a Lux Cellulose-2 chiral column. The

12

enantioselective metabolism of rac-flufiprole was dramatically different in rat and

13

human liver microsomes in the presence of β-Nicotinamide adenine dinucleotide

14

phosphate regenerating system. The half-life (t1/2) of flufiprole in rat and human liver

15

microsomes were 7.22 and 21.00 min, respectively, for R-(+)-flufiprole, while the

16

value were 11.75 and 17.75 min, respectively, for S-(-)-flufiprole. In addition, the Vmax

17

of R-(+)-flufiprole was about 3-fold of S-(-)-flufiprole in rat liver microsomes, while

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its value in case of S-(-)-flufiprole was about 2-fold of R-(+)-flufiprole in human liver

19

microsomes. The CLint of rac-flufiprole also showed opposite enantioselectivy in rat

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and human liver microsomes. The different compositions and contents of

21

metabolizing enzyme in the two liver microsomes might be the reasons for the

22

difference in the metabolic behavior of the two enantiomers.

ACS Paragon Plus Environment


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KEYWORDS:

24

HPLC-MS/MS

flufiprole,

enantioselective

metabolism,


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liver

microsomes,

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INTRODUCTION

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Phenylpyrazole insecticides are widely used to protect a variety of crops, including

27

rice, fruits and vegetables, from insects such as ants, beetles, ticks and cockroaches1-3.

28

Another common phenylpyrazole insecticide, fipronil, has been banned in China in

29

2009, because of its high toxicity towards aquatic organisms, such as fish and

30

shrimp4-6. Flufiprole (or butene-fipronil), (R,S)-1-[2,6-dichloro-4-(trifluoromethyl)

31

phenyl]

32

sulfinyl]-pyrazole-3-carbonitrile (Figure 1), is a new kind of phenylprazole insecticide

33

introduced in 2002 by Dalian Raiser Pesticides Co., Ltd., China. It is a modified form

34

of fipronil with a similar effect against a broad spectrum of insect pests and has much

35

lower toxicity7-9. Both flufiprole and fipronil have a chiral sulfur atom consisting of

36

two enantiomers. In fact, although enantiomers have exactly the same physical and

37

chemical properties, they behave differently in bioactivity10-11, toxicity10,12 or

38

degradation10,13-14 when exposed to a chiral environment. In recent years, with the

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intensive study of chiral pesticides all over the world, many papers about the

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enantioselective degradation of chiral pesticides have been published15-18. For instance,

41

S-fipronil has been shown to degrade faster than R-fipronil in earthworms and natural

42

soil18. To our knowledge, there is no such report about the study on the metabolic

43

behavior of flufiprole at the enantiomer level, which may lead to potential

44

environmental risks.

-5-[N-(methallyl)

amino]-4-[(trifluoro-methyl)

45

Recently, it is popular to use in vitro models in pharmacology, toxicology and

46

toxicokinetic experiments, because there are fewer animals needed, fewer endogenous



47

interfering substrates and much shorter experiment time compared with in vivo

48

studies19-23. Liver is the major organ for the metabolism of many xenobiotics, where

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the majority of drug metabolic enzymes exist. In previous studies, many researchers

50

have reported the stereoselective degradation of many chiral pesticides in rat, rabbit

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and human liver microsomes (HLMs), including triazole fungicides24-25, benalaxyl26

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and fluroxypyr methylheotyl ester27. Taking benalaxyl enantiomers as an example, the

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t1/2 of R-benalaxyl in rat liver microsomes (RLMs) was about 2-fold of S-benalaxyl.

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However, R-benalaxyl degraded faster than S-benalaxyl in rabbit liver microsomes, at

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the same time there was no chiral inversion between R- and S-benalaxyl in both liver

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microsomes26. To evaluate the environmental risk of flufiprole comprehensively, it is

57

necessary to evaluate the enantioselective metabolism of flufiprole in different liver

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microsomes.

59

In this study, the metabolic behaviors of rac-flufiprole in RLMs and HLMs were

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investigated to determine that if similar enantioselectivity occurred. The

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determination of chiral flufiprole was achieved in high-performance liquid

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chromatography-tandem mass spectrometry (HPLC-MS/MS) equipped with a Lux

63

Cellulose-2 chiral column. The significant kinetic parameters of the enantiomers, such

64

as half-life (t1/2), maximum velocity of metabolism (Vmax) and Michaelis constant

65

(CLint), were also calculated to explore the enantioselective behaviors of flufiprole.

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The results might be helpful to understand the risks associated with flufiprole to

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humans, animals and the environment.

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MATERIALS AND METHODS


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Chemicals and Materials. Flufiprole with a purity of 96.6% was obtained from

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Dalian Raiser Pesticides Co., Ltd (Dalian, China). Two enantiomers of flufiproles

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with purity ≥98.0% were obtained from Daicel (Shanghai, China). HPLC-grade

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methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). A stock

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solution of racemic standard was prepared in methanol and stored at 4 °C. Working

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standard solutions were obtained by dilutions of the stock solution in methanol.

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Purified water was produced by a Milli-Q water purification system (Millipore

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Bedford,

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regenerating system, rat and human liver microsomes (20 mg/mL, 0.5 mL) were

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purchased from XenoTech (Lenexa, KS). Acetic acid (99.7% purity) was purchased

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from Sigma-Aldrich. All other chemicals and solvents were of analytical grade and

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purchased from commercial sources. The chiral analytical column, cellulose tris

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(3-chloro-4methylphenycarbamate)

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Phenomenex (Torrance, CA), and the column was sized 150 mm × 2.0 mm i.d. and

83

packed with 3 µm particles.

MA).

β-Nicotinamide

adenine

(Lux

dinucleotide

Cellulose-2),

phosphate

was

(NADPH)

purchased

from

84

Incubation in Liver Microsomes. Substrate-depletion studies in vitro were

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performed by incubation of rac-flufiprole (10 µM) for rat liver microsomes and

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rac-flufiprole (5 µM) for human liver microsomes with 0.5 mg of microsomal protein

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in 50 mM Tris-HCl buffer (pH 7.4) with 5.0 mM MgCl2. The concentrations of

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flufiprole were chosen based on pre-experiments. Flufiprole was prepared in methanol

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and added to the incubation media with the final methanol concentration not

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exceeding 1.0% v/v. All reaction mixtures were preincubated in a heated water bath at



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37 °C for 5 min before initiation of the reaction by addition of NADPH at a final

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reaction concentration of 1.0 mM. Incubation mixtures without NADPH served as

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controls (with deactivated microsomes). The final total reaction volume was 0.5 mL.

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After incubation in a water bath (37 °C) for 5-90 min, the reactions were terminated

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by adding 0.5 mL of ice-cold methanol and the sample was vortexed for 5 min. After

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centrifugating at 4000 rpm for 5 min, the supernatant was filtered through the

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membrane (PTFE, 0.22 µm) for enatioselective HPLC-MS/MS analysis. All

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experiments were performed as three independent trials.

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Kinetic metabolism Assays. The in vitro metabolic kinetics of flufiprole was

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studied by adding variable concentrations of flufiprole stock solution to the incubation

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mixtures. The final substrate concentrations were from 2 to 100 µM and the

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incubation time was 10 min with liver microsomes (0.5 mg protein/mL). Then, the

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sample preparation was performed according to the section above. The Vmax and Km

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values were calculated from nonlinear regression analysis of experimental data

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according to the Michaelis-Menten equation. Intrinsic clearance (CLint) was calculated

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as a ratio of the Vmax and to the Km. Nonlinear regression analysis was performed with

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Origin pro 8.5.

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Enantioselective HPLC-MS/MS Analysis. The determination of flufiprole was

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achieved on a Surveyor liquid chromatography (Thermo Fisher Scientific, Waltham,

110

MA, USA) equipped with a triple-quadrupole mass spectrometer. Xcalibur 2.0.7

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(Thermo Fisher Scientific) software was used to process the quantitative data obtained

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from calibration standards and samples. Complete chiral separation of flufiprole was


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achieved on a Lux Cellulose-2 chiral column28. The mobile phase consisted of 65%

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(v/v) (A) acetonitrile and 35% (v/v) (B) 2 mM ammonium acetate aqueous solution

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containing 0.1% formic acid. The flow rate was set to 0.3 mL/min, and the injection

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volume was 5 µL. The ESI-MS/MS interface (electrospray ionization coupled with

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tandem mass spectrometry) was operated in the negative ion mode. ESI source

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conditions were as follows: source temperature, 110 °C; desolvation temperature,

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500 °C; desolvation gas (N2) flow rate, 12 mL/min; cone gas (N2) flow rate, 40 mL/Hr;

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and collision gas (Ar) flow rate, 0.15 mL/min. Multiple reaction monitoring (MRM)

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was applied for flufiprole determination; transition m/z 488.9→288 was used for

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quantification, and m/z 488.9→249.9 was used for confirmation.

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Method validation. Calibration standards were prepared by adding a series of

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working standard solution of rac-flufiprole into the blank matrix (BM, the extracts

125

from the bovine serum protein). The preparing and extracting processes were the same

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as incubation samples. The calibration curves were generated by plotting peak areas

127

of quantification ion transition against the concentration of each enantiomer from 0.1

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to 50 µM with regression analysis. Linear regression analysis was performed with

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Microsoft Excel 2007. The precision and accuracy of the method were calculated by

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analyzing calibration standards at three concentration levels (0.5, 10 and 50 µM for

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each flufiprole enantiomer) and comparing the predicted concentration (obtained from

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the calibration curve) to the actual concentration of each enantiomer spiked in the BM.

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Both intraday and interday precisions are presented as relative standard deviation

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(RSD [SD/mean] × 100%). Limit of detection LOD (signal-to-noise ratio [S/N]=3)



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and limit of quantification LOQ (S/N=10) were determined with the method using the

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matrix-matched standards.

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RESULTS AND DISCUSSION

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Assay validation. Using m/z 488.9→288 as the quantification transition, linear

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calibration curves were obtained over the concentration range of 0.1-50 µM in blank

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matrix for R-flufiprole (y = 6.9227x × 10^4 - 29.9224, R2 = 0.9994) and S-flufiprole (y

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= 6.9938x × 10^4 – 14.3144, R2 = 0.9996). The RSD was less than 7% at all

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concentration.

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The precision and accuracy of intraday and interday, expressed with the RSD and

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the ratios of predicted concentration to acctural concentration, was shown in Table 1.

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For both enantiomers, the accuracies obtained were in the acceptable range of 90.3%-

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98.7% and the RSD ranged from 1.8% to 5.7%. The main recoveries ranged from

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97.5% to 100.8% with 2.4%-6.9% RSD (Table 2). The LODs for both enantiomers

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were estimated to be 0.005 µM, while the LOQs were 0.015 µM. In addition, the

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extraction procedure did not cause epimerize of flufiprole enantiomers as shown in

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Figure 2 F-G.

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Data Ananlysis. Under the HPLC-MS/MS condition, the enantiomers of flufiprole

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were separated completely on a Lux Cellulose-2 column (as shown in Figure 2A), and

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no endogenous interference peaks eluted at retention times in blank samples (without

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insecticides). The order elution was confirmed to be S-(-)-flufiprole for peak 1 and

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R-(+)-flufiprole for peak 2 based on published data28.

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The enantiomer fraction (EF) was used to measure the enantioselectivity of the


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flufiprole enantiomers during incubation in the liver microsomes. EF was calculated

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from the peak areas for the signals, defined by eq. 1. The EF values ranged from 0 to

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1 with EF=0.5 representing the racemic mixture according to the equation.

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EF= (S)-(-)-enantiomer / [(R)-(+)-enantiomer + (S)-(-)-enantiomer]

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The metabolism of rac-flufiprole and its enantiomers was assumed to follow the

162

first-order kinetics model, and the degradation constants (k) were calculated by eq. 2

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using regression analysis. The half-life (t1/2, min) was estimated from eq. 3. C = C0e-kt

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t1/2 = ln2/k = 0.693/k

165 166 167

(1)

(2) (3)

C and C0 are the concentrations of the R-(+)- or S-(-)-enantiomer at time t and time 0, respectively.

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Nonlinear regression of substrate concentration versus reaction velocity curves

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were analyzed using Origin 8.5 software by fitting the experimental data to the

170

Michaelis-Menten equation. The in vitro kinetic parameters were determined by

171

fitting the data to eq. 4, and the intrinsic metabolic clearance (CLint) was calculated by

172

eq. 5.

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V=Vmax×S/(Km＋S)

(4)

174

CLint=Vmax/Km

(5)

175 176

V, S, Vmax and Km represent the velocity of metabolism, substrate concentration, maximum velocity of metabolism and Michaelis constant, respectively.

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Metabolism of Racemic Flufiprole in RLMs and HLMs. The rac-flufiprole was

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dissipated rapidly in RLMs and HLMs with NADPH, and did not degradate obviously



179

in the absence of NADPH. Typical chromatograms of extracts from the samples after

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10 min incubation are displayed in Figures 2B-E. The concentrations of two

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enantiomers were different from each other after incubation in RLMs and HLMs. The

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R-(+)-flufiprole degraded faster than its antipode in RLMs, in contrast, the

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S-(-)-flufiprole degraded faster in HLMs.

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Concentration-time curves of R-(+)- and S-(-)-flufiprole after incubation in RLMs

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with rac-flufiprole at 10 µM and HLMs with rac-flufiprole at 5 µM were shown in

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Figure 3. The results demonstrated that the metabolisms of each enantiomer in RLMs

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and HLMs were in accordance with the first-order kinetics decay model. Rate

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constants (k), correlation coefficients (R2) and half-lives (t1/2) were calculated (Table

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3). It was shown that the half-life t1/2 of R-(+)-flufiprole was 7.22 and 21 min in

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RLMs and HLMs, while the half-life t1/2 of S-(-)-flufiprole was 11.75 and 15.75 min,

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respectively. Thus, metabolism of R-(+)- and S-(-)-flufiprole was much faster in

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RLMs, and indicated opposite enantioselectivity in RLMs and HLMs.

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The enantioselective metabolic behavior could also be conducted by EFs. In the

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experiment, the EF value of flufiprole increased from the initial 0.50 to 0.71 rapidly in

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RLMs because R-(+)-flufiprole degradated much faster than S-(-)-flufiprole under the

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same concentration. Then with the concentration of R-(+)-flufiprole decreasing

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quickly, the degradation rate of R-(+)-flufiprole also decreased until it became smaller

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than its value of S-(-)-flufiprole. As a result, the EF value decreased to 0.62

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(concentration RS) gradually in HLMs, indicating that there


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was significant enantioselectivity in the two kinds of liver microsomes.

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Enzyme Kinetics of Flufiprole Enantiomers. The Michaelis-Menten model was

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applied to different initial concentrations (5-100 µM) of rac-flufiprole using RLMs

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and HLMs as examples to elucidate the mechanism of stereoselective toxicokinetics

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of flufiprole. Metabolic rate constants (apparent Km and Vmax) were determined after a

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10 min incubation period in liver microsomes. The Michalis-Menten plots are shown

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in Figure 4. By nonlinear regression analysis, the values of Km, Vmax and CLint were

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calculated and are shown in table 4. The Vmax of R-(+)-flufiprole (3906.3±80.6

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µM/min/mg) was about 3-fold of S-(-)-flufiprole (1430.6±52.9 µM/min/mg) after

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incubation for 10 min in RLMs, however, the opposite trendency was found after

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incubation in HLMs. For the CLint, the ability of an organism to eliminate a particular

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chemical demonstrated diversity, the CLint of rac-flufiprole also showed opposite

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enantioselectivy in RLMs and HLMs. Besides, the CLint of R-(+)-flufiprole and

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S-(-)-flufiprole in RLMs was higher than that in HLMs. These enzyme kinetic results

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suggested that the rat liver microsomes in vitro had a stronger potency to eliminate

216

flufiprole than the human liver microsomes. Significant differences were found for the

217

stereoselective metabolism of rac-flufiprole in RLMs and HLMs.

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In conclusion, we investigated the enantioselective metabolism of rac-flufiprole in

219

RLMs and HLMs. The degradation velocity of flufiprole in HLMs was slower than

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that in RLMs. Moreover, there was significant opposite enantioselective behavior for

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the metabolism of flufiprole enantiomers in the two liver microsomes. Some

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studies24-27 verified that other fungicides, such as (2RS,3RS)-paclobutrazol,



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tebuconazole, benalaxyl and fluroxypyr methylheotyl ester, exhibited stereoselectivity

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of metabolism in liver microsomes. The reason for the difference in the metabolism

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behavior of the two enantiomers might be the different compositions and contents of

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metabolizing enzyme (especially cytochrome P450) in various species liver

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microsomes26, 29-30. Future studies should be focused on the isoforms of cytochrome

228

P-450 (CYP) that contribute to its stereoselectivy. These data should be important to

229

elucidate the mechanism of stereoselective toxicokinetics of flufiprole in RLMs and

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HLMs, and contribute to environmental and public health.

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AUTHOR INFORMATION

232

Corresponding Author *

233

E-mail [email protected]

234

Notes

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The authors declare no competing financial interest.

236

ACKNOWLEDGEMENT

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We acknowledge financial support from the National Natural Science Foundation of

238

China (21207118), the Project of Science and Technology Plan of Zhejiang Province,

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China (2014C37103), and the Natural Science Foundation of Zhejiang Province,

240

China (Y15B070017).

241

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FIGURE CAPTIONS NC

CN Cl

F 3C

N N HN Cl H3C

O

O S CF3 CH2

S F3C

H2C

N N NH Cl

Cl

CF3

CH3

mirror Figure 1. Chemical structure of flufiprole enantiomers (Asterisk indicated the chiral center).


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Figure 2. Representative HPLC-MS/MS chromatograms of (A) standard flufiprole, extract from (B) RLMs with 10 µM rac-flufiprole and (C) HLMs with 5 µM rac-flufiprole after incubation for 10min in the absence of NADPH, extract from (D) RLMs with 10 µM rac-flufiprole and (E) HLMs with 5 µM rac-flufiprole after incubation for 10min with NADPH, extract from RLMs with (F-G) individual flufiprole enantiomer.



Figure 3. Degradation of (A) rac-flufiprole (10 µM) in RLMs, (C) rac-flufiprole (5 µM) in HLMs for 90 min. The changes of EF values in (B) RLMs and (D) HLMs.

Figure 4. Michaelis-Menten kinetic analyses of rac-flufiprole (A) in RLMs and (B) in HLMs after 10min incubation.


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TABLE CAPTIONS Table 1．． Accuracy and Precision of Assay Method for Measurement of Flufiprole Enantiomers (n = 6) Intraday Concentration (µM) R-flufiprole 0.1 10 50 S-flufiprole 0.1 10 50

Interday

Accuracy (%)

RSD (%)

Accuracy (%)

RSD (%)

91.9 96.7 97.5

1.8 5.0 5.7

90.3 93.0 95.5

1.4 4.5 4.7

91.9 95.2 97.7

2.7 4.7 3.5

92.6 94.9 98.7

2.3 5.4 4.9

Table 2．． Method Recovery for Meassurement of Flufiprole Enantiomers (n = 3) R-flufiprole

S-flufiprole

Concentration (µM)

Accuracy (%)

RSD (%)

Accuracy (%)

RSD (%)

0.1 10 50

100.4 97.5 100.8

2.4 2.6 3.1

99.1 97.7 100.7

2.7 6.9 2.5

Table 3 ． Degradation Rate Constant (k), Half-Life (t1/2) and Correlation Coefficients (R2) Values for the Metabolism of rac-Flufiprole in Liver Microsomes Liver microsomes

spiked compd

RLMs

rac-flufiprole

HLMs

rac-flufiprole

detected compd

k (min-1)

t1/2(min)

R2

R-(＋)-flufiprole S-(－)-flufiprole R-(＋)-flufiprole S-(－)-flufiprole

0.096 0.059 0.033 0.044

7.22 11.75 21.00 15.75

0.885 0.889 0.991 0.915



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Table 4．． Metabolic Parameters of rac-Flufiprole Incubated in RLMs and HLMs Sample RLMs R-(＋)-flufiprole S-(－)-flufiprole HLMs R-(＋)-flufiprole S-(－)-flufiprole

Vmax (µM/min/mg)

Km (µM)

CLint (mL/min/mg protein)

R2

3906.3±80.6 1430.6±52.9

43.12±1.53 17.22±0.84

90.58 83.06

0.99 0.99

961.5±52.7 1785.7±86.3

27.12±2.49 35.18±2.34

35.45 50.76

0.98 0.99


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ABSTRACT GRAPHICS


Enantioselective Metabolism of Flufiprole in Rat ... - ACS Publications

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