characterization of activation metabolism activity of indoxacarb in insects by liquid...

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 42, Issue 4, April 2014 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2014, 42(4), 463–468.

Received 24 September 2013; accepted 25 November 2013 * Corresponding author. Email: [email protected] This work was supported by the National Basic Research Program of China (No. 201203038). Copyright © 2014, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60720-3

RESEARCH PAPER

Characterization of Activation Metabolism Activity of Indoxacarb in Insects by Liquid Chromatography‐Triple Quadrupole Mass Spectrometry LI Fu-Gen1,2, AI Guo-Min3, ZOU Dong-Yun1, JI Ying2, GU Bao-Gen2, GAO Xi-Wu1,* 1 Department of Entomology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China 2 Institute for the Control of Agrochemicals, Ministry of Agriculture, Beijing 100125, China 3 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

Abstract: A triple-quadrupole LC-MS/MS method was developed for the investigation of indoxacarb N-decarbomethoxyllation activity catalyzed by insect crude enzymes. The metabolite, N-decarbomethoxyllated indoxacarb (DCJW), was determined using electrospray ionization in positive ion mode. The quantitative and qualitative analyses were performed in multiple reaction monitoring (MRM) mode. The relative standard deviation (RSD, n = 5) of DCJW was 2.1%, indicating the metabolite had a good chemical stability in 30 h after the sample preparation. Five injections of DCJW standard solution in acetonitrile, five injections of the same sample in one incubation (substrates plus enzyme samples), and five injections of 5 separate metabolic reactions with the whole body of a susceptible strain of Plutella xylostella (L.) were individually injected into LC-MS/MS system. The RSD was 0.7%, 1.1% and 2.7%, respectively. The average recoveries for 440, 880 and 2200 pg of DCJW added to incubation mixtures were 96.1%–102.9%, and the RSD of recoveries of three added levels ranged from 4.8% to 9.4%. The limits of quantification and detection were 0.1 and 0.01 pg, respectively. A good linearity was achieved in the range of 46–2310 pg (R2 = 0.9996). This method with high sensitivity and simplicity was applicable to the assay of indoxacarb N-decarbomethoxyllation activity of insect metabolic enzymes. A comparison of indoxacarb N-decarbomethoxyllation activity of an avermectin-resistant strain and a susceptible strain of Plutella xylostella (L.) was carried out using this novel LC-MS/MS method. The results showed that the indoxacarb N-decarbomethoxyllation activity in the resistant strain was 3.4-fold of that in the susceptible strain, implicating that negative cross-resistance might exist between indoxacarb and avermectin in Plutella xylostella (L.). Key Words: Liquid chromatography-triple quadrupole mass spectrometry; Indoxacarb; Activation metabolism; Plutella xylostella (L.)

1 Introduction

The metabolic enzymes that mediate direct insecticide metabolism in insects are mainly comprised of cytochrome P450s[1], hydrolases[2], glutathione-S-transferases[3], etc. The metabolism of insecticides catalyzed by metabolic enzymes in insects has great toxicological significance, performing at detoxification or/and activation metabolism, or the balance of the two metabolisms. The activity of the insecticide

metabolism affects the fortune in insects, action mechanism, biological activity, and resistance level of the insecticide[4]. In the study of entomology toxicology, especially for the resistance biochemical mechanism of insects resistant to insecticides, the classical spectrum, such as UV vis colorimetry and fluorometry, was commonly applied to the determination of the metabolism activity of insects to various model substrates or insecticide analogues[5]. Recently, the methods based on chromatography or chromatography/mass

LI Fu-Gen et al. / Chinese Journal of Analytical Chemistry, 2014, 42(4): 463–468

spectrometry have been described[6–9], however, few of them were used to characterize the direct metabolism activity of insects to insecticides[5]. As the determination of the metabolism activity of insects to insecticide itself is more conclusive than that to model substrates (one or more substrates) for the characterization of metabolism activity to insecticide [1], the development of an analytical method for the metabolism activity of insects to insecticides is clearly needed in the field of entomology toxicology.

Indoxacarb (DPX-JW062), a new oxadiazine insecticide developed by the DuPont Company, can efficiently control lepidopteran pests. The mechanism of action is that indoxacarb can be converted to the metabolite, N-decarbomethoxyllated indoxacarb (DCJW), in body fat and especially in the middle gut of lepidopteran insects. The formed DCJW can break the Na+ channel in the nerve cells of pests, leading to the loss of function to death of pests[10,11]. To date, few works have been reported on the determination of the activation metabolism activity of indoxacarb in insects. In this study, an accurate analytical method was developed using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) in multiple reaction monitoring (MRM) mode for the determination of the activation metabolism activity of indoxacarb in insects (Fig.1)[10,11]. The analytical parameters of MRM were optimized based the DCJW formed by the crude enzymes of the H. armigera larval midgut, and the novel assay method was ultimately established and validated using the DCJW produced by the crude enzymes of Plutella xylostella (L.). The quantitative and qualitative analyses of DCJW in MRM mode were performed at m/z 267.0 and m/z 207.0, 150.0, to characterize the hydrolysis activation metabolism activity. This method was applied to compare the activation metabolism activity between an avermectin-resistant strain and a susceptible strain of Plutella xylostella (L.) to insecticide indoxacarb.

2 Experimental 2.1 Instruments and reagents

An Agilent 1260/6460 high performance liquid

chromatograph/triple quadrupole mass spectrometer (Agilent, USA) was equipped with a MassHunter Workstation Software (Version B.04.00, Agilent, USA). A T6-Newcentury UV-VIS Spectrometer was purchased from PGENERAL Instruments

Co., Ltd., Beijing, China. A CR22E high-speed desktop centrifuge was from Hitachi Koki Co., Japan.

Indoxacarb (99.0% purity) was kindly supplied by the Institute for the Control of Agrochemicals, Ministry of Agriculture (ICAMA), Beijing, China. DCJW (99.0% purity) was supplied by Shenyang Research Institute of Chemical Industry, China. Formic acid (98.0% purity) was from Sigma-Aldrich Co., USA. HPLC-grade acetonitrile and acetone were purchased from Fisher Co., USA. Water was obtained using a Milli-Q water purification system. Bovine serum albumin (BSA) was from Beijing Tongzheng Biology Co., China. Analytical grade of ammonium acetate was purchased from Beijing Chemical Reagents Co., China. 2.2 Chromatographic conditions

The chromatographic separation was carried out using a

ZORBAX Eclipse XDB-C18 (100 mm × 2.1 mm, 3.5 μm, Agilent, USA) equipped with a pre-column (10 mm × 2.1 mm, 5 μm, the stationary phase was the same to the analytical column). The mobile phase in gradient mode consisted of acetonitrile and water (0.1% HCOOH). The gradient of acetonitrile was set at: 15%–80% in 0–15 min; 80%–95% in 15–25 min; 95%–100% in 25–26 min; 100% in 26–29 min; 100%–15% in 29–31 min; 15% in 31–37.5 min. The flow rate was set at 0.4 mL min–1, and the column temperature was set at 30 ºC. The injection volume was 15 μL. 2.3 MS/MS conditions

For the MS/MS analysis, ESI was performed in positive

ionization mode. The mass scan ranged from m/z 100 to 800, and the fragmentor voltage was 100 V. The capillary voltage and atomizing gas pressure were 4.0 kV and 275 kPa, respectively. The flow rate of drying gas was 10 mL min–1 and the temperature of solvent removal was 350 ºC. Nitrogen was used as collision gas. The triple quadrupole mass spectrometer was operated in MRM mode with m/z 470.1/267.0 (collision energy of 10 V, dwell time of 300 ms) as the quantitative ion and m/z 470.1/207.0, 150.0 (collision energy of 30 V and dwell time of 100 ms for each) as the qualitative ions.

2.4 The tested insects

Cotton bollworm population, Helicoverpa armigera (Hübner),

Fig.1 Bioactivation of indoxacarb in insects to N-decarbomethoxyllated metabolite (DCJW) by hydrolysis


maintained being cultivated by artificial diet, was collected from Handan, Hebei province in 1998. The insects were reared in the conditioned room maintained at (26 ± 1) ºC, 70%–80% relative humidity, with a 16:8 (light to dark) photoperiod. Adults were held under the same conditions and supplied with a 10% sugar solution.

Diamondback moth population, Plutella xylostella (L.), belongs to an avermectin-resistance strain (AVR) and a susceptible strain (Xuanhua Sensitive, XHS). Turnip sprouts method was used the method of feeding. The conditions were as followings: (25 ± 2) ºC, approximately 75% relative humidity, and a 16:8 (light to dark) photoperiod. The avermectin-susceptible strain (XHS) keeps being cultivated without exposing to insecticides after it was collected from Handan, Hebei province in 1997. The resistance strain (AVR) was selected from the congeneric avermectin-susceptible strain (XHS) and 100.5-fold resistance to avermectin was developed. 2.5 Preparation of enzyme 2.5.1 Preparation of the crude enzyme sample of H.

armigera larval midgut The midgut of the sixth instar larvae was obtained by

dissection on an ice tray. The midgut was gently shaken to free of its content and rinsed in an ice-cold 1.15% KCl (m/V) aqueous solution followed by drying using absorbent paper. The midguts were homogenized with ice bath in 0.1 M ammonium acetate solution. The homogenate was centrifuged at 4 ºC, 10000 × g for 20 min. The supernatant was filtered through the absorbent cotton and collected into a clean eppendorf tube, and then used as enzyme source for the determination of metabolism activity and protein concentration. 2.5.2 Preparation of the crude enzyme sample of

Plutella xylostella (L.) The third instar larvae of Plutella xylostella (L.) was

homogenized as mentioned in the preparation of the crude enzyme sample of cotton bollworm midguts. 2.6 Determination of protein concentration

Based on the previous method[12,13] and with BSA as the

standard, the protein concentration was determined within 5–15 min after adding Coomassie brilliant blue G-250. 2.7 Indoxacarb N-decarbomethoxyllation reaction

The incubation mixture in a volume of 1.5 mL consisted of

0.1 M ammonium acetate aqueous solution (pH 6.8) and 90 μM indoxacarb (the addition of 20-μL 6.75 mM indoxacarb

prepared in acetone). To initiate the reaction, 0.5 mL of enzyme solution was added. After incubation at 30 ºC in shaking water bath for 1 h, 1.5 mL ice-cold actonitrile was added to stop the metabolic reactions. The solution was centrifuged (10000 × g) for 10 min at 4 ºC. The supernatant was filtered through a 0.22-μm membrane and analyzed by LC-MS with MRM mode. The enzyme solution control (without substrate) and substrate control (without enzyme sample) were prepared to differentiate between the products originating from the enzyme sample and possible products from the incubation procedure.

The characterization of the metabolism activity of the enzyme is often performed based on the decrease of the substrate or the increase of the product. The latter is more accurate for the catalytic activity of insect metabolic enzymes for their low catalytic capacity with very low decrease of the substrate. In this study, the enzyme activity was expressed as pmol DCJW/(mg protein min).

The H. armigera midgut is characterized with high metabolism activity to xenobiotics, few interferences contained in the crude enzyme sample, and strong ability of activation metabolism to indoxacarb[10]. Therefore, the DCJW formed by H. armigera midguts was used to optimize MRM parameters. To get high concentration of DCJW, the incubation mixture in a total volume of 3 mL was used, and the equal volume of ice-cold ethyl acetate was added to stop the reaction. The lower water phase was extracted again with 2 mL,and 1 mL ethyl acetate. The ethyl acetate phase was combined and evaporated to dryness under weak nitrogen stream. The sample was then redissolved in 400 μL of acetonitrile for LC-MS analysis. 2.8 Comparison of N-decarbomethoxyllation activity

between avermectin-resistant strain and susceptible strain of Plutella xylostella (L.)

The difference of N-decarbomethoxyllation activity

between avermectin-resistant strain and indoxacarb- susceptible strain of Plutella xylostella (L.) was compared using the proposed LC-MS/MS method. The difference in metabolism activity was used to deduce whether there was negative cross-resistance between insecticide avermectin and indoxacarb in Plutella xylostella (L.). 3 Results and discussion 3.1 Development of metabolite DCJW assay method by

LC-MS 3.1.1 Structure identification of metabolite

The mixture obtained from the incubation reaction was

separated in the gradient elution of HPLC, and ESI was


performed in positive ionization mode. As shown in Fig.2, the retention time of the metabolite of indoxacarb by cotton bollworm midgut in the enzymatic reaction was 16.5 min by comparing with the incubation and substrate controls. As can be seen in Fig.3, based on the pseudo-molecular ion (a), fragmentation patterns (b and c), abundance ratio of chlorine isotope, the retention time of DCJW standard, and the toxicological significance of indoxacarb[10], the metabolite at the retention time of 16.5 min was identified as DCJW (Fig.3), implying indoxacarb N-decarbomethoxyllation activation

metabolism reaction mediated by the H. armigera midgut occurred. The pseudo-molecular ions with addition of [+H] and [+Na] were m/z 470.1 and 492.0, and the relative abundance ratio of m/z 472.1 to 470.1 was about 1/3 (Fig.3a). Additionally, m/z 267.0 arisen from the in-source dissociation of m/z 470.1 was observed under the mass spectrometric condition of fragmentor voltage of 100 V. In the body of lepidopteran insects, indoxacarb was prone to be metabolized by hydrolysis to produce DCJW, which may be catalyzed by esterases or amidases[10].

Fig.2 LC-MS chromatograms of blank incubations (without substrates) (a), control incubations (without enzyme samples) (b), and incubations

(substrates plus enzyme samples) (c) under gradient elution program (an injection of 15 μL)

Fig.3 Positive ion electrospray mass spectra of metabolite of indoxacarb in the midgut of H.armigera (a) Full scan; (b) product ion spectra of m/z 470.1 at 10–40 V; (c) collision induced dissociation reactions for DCJW


3.1.2 Optimization of MRM assay parameters

As shown in Fig.3b, the transition of m/z 470.1/267.0 was

chosen as the quantitative ion, and m/z 470.1/207.0 and 150.0 were chosen as the qualitative ions. The fragmentor voltage was optimized in a range of 60–120 V by using m/z 470.1 as the target and 100 V as the optimum fragmentor voltage. Also, the collision energy was optimized in a range of 2–20 V under the conditions of m/z 470.1/267.0 and fragmentor voltage of 100 V, and 10 V was selected as the optimum collision energy. As shown in Fig.3b, the collision energy of 30 eV was used for both the transition of m/z 470.1/207.0 and 150.0. With the developed conditions of MRM mode, the LC-MS/MS chromatogram is shown in Fig.4.

Because the DCJW standard was hard to be obtained, an alternative to using the DCJW formed by N-decarbomethoxyllation metabolism of indoxacarb mediated by the H. armigera midgut for the optimization of MRM parameters was practical and meaningful in the study of the metabolism activity of insects to indoxacarb, especially for the insects owing to their low metabolic activity. 3.1.3 Stability of incubation reaction sample

Due to a large amount of water presented in the prepared

samples and long analysis time required, the chemical stability of DCJW in samples should be evaluated. The same sample was analyzed for five times within 30 h at 6 ºC. The relative standard deviation (RSD) was 2.1% for the peak areas of m/z 470.1/267.0, which showed a good stability for DCJW within 30 h after the sample preparation. 3.1.4 Precision of analytical method

Five repeated injections of the acetonitrile solution of

DCJW, the same incubation reaction sample of Plutella xylostella (L.), and five separate incubation reaction samples

Fig.4 MRM analysis of N-decarbomethoxyllated metabolite of indoxacarb mediated by H.armigera midgut

(a) control incubations without enzyme samples; (b) blank incubations without substrates; (c) incubations (substrates plus enzyme samples)

were used for the investigation of the precision of analytical method. The RSDs were 0.7%, 1.1% and 2.7%, respectively, indicating the method had good stability and precision. 3.1.5 Limit of detection (LOD) and limit of quantification

(LOQ) The LOD of DCJW was 0.01 pg (S/N = 3) and the LOQ of

DCJW was 0.1 pg, indicating that this proposed method was sensitive and suitable for the relative study on indoxcarb metabolism especially for the insects that have low metabolism activity to indoxacarb. 3.1.6 Linearity

Standard stock solution of 3080 mg L–1 DCJW was

prepared in acetonitrile. The series of calibration solutions were prepared by diluting the stock solutions with crude enzyme sample of Plutella xylostella (L.) susceptible strain. Considering the metabolite concentration and recovery assessment, the calibration curve of injection amount (ordinate) in the range of 46–2310 pg and peak areas (abscissa) was obtained. The linear equation was y = 0.0089x + 6.2890 with a correlation coefficient (R2) of 0.9996. 3.1.7 Recovery experiment

The accuracy of the described method was estimated by

means of recovery experiments carried out at three added levels of 440, 880 and 2200 pg. The reaction was stopped by adding 1.5 mL of ice-cold acetonitrile after 1 h. The average recovery and the RSDs for the three added levels were in the range of 96.1%–102.9% and 4.8%–9.4% (Table 1), respectively, showing the developed method had good accuracy and precision. 3.2 Comparison of N-decarbomethoxyllation activity

between avermectin-resistant strain and susceptible strain of Plutella xylostella (L.)

The indoxacarb N-decarbomethoxyllation reaction

catalyzed by avermectin-resistant and susceptible strain of Plutella xylostella (L.) was carried out at the same time. The results showed that the indoxacarb N-decarbomethoxyllation activity of avermectin-resistant strain of Plutella xylostella (L.) increased significantly and was 3.4-fold compared with that of Table 1 Recovery of product N-decarbomethoxyllated indoxacarb

(DCJW) Added (pg) Recovery (%, n = 5) RSD (%, n = 5)

440 96.14 9.4 880 102.91 6.3 2200 98.86 4.8


the susceptible strain (Table 2). The increase of the activation metabolism indicated that the avermectin-resistant strain of Plutella xylostella (L.) might be more sensitive than that of the susceptible strain, which means that negative cross-resistance between avermectin and indoxacarb might be possible in Plutella xylostella (L.). The results could be confirmed by the bioassay of the resistant strain and susceptible strain of Plutella xylostella (L.), for instance, the pyrethroids-resistant strain of H. armigera in Australia was more sensitive to indoxacarb, which showed a negative cross-resistance between indoxcarb and pyrethroid insecticides[14]. Although the activation metabolism of indoxacarb was prone to occurrence in lepidopteran insects, different resistance levels to indoxacarb might be observed because of the presence of various resistance mechanism in Plutella xylostella (L.)[15–17]. Therefore, there is great toxicological significance for the study on the activation metabolism activity of indoxacarb in insects. 4 Conclusions

In the study of entomology toxicology, especially for the

biochemical mechanism of insecticide resistance, the characterization of insect metabolism activity by using model substrates requires substrates with various chemical structures or insecticide analog. However, this approach was considered of an indirect method. Therefore, the development of an analytical method for measuring direct insecticide metabolism by insects is clearly needed. In this work, a novel method for the characterization of indoxacarb N-decarbomethoxyllation activity by insects using triple-quadrupole LC-MS/MS in MRM mode was proposed. The established method has the advantages of simplicity, easy operation, and omission of the common procedures such as extraction with organic solvents and subsequent concentration and dissolution. Additionally, the method is suitable for the analysis of hydrolysis activation Table 2 Comparison of indoxacarb N-decarbomethoxyllation

activity of Avermectin Resistance (AVR) strain with Xuanhua Sensitive(XHS) strain of Plutella xylostella (L.)

Plutella xylostella (L.)

N-decarbomethoxyllation activity (pmol DCJW)/(mg protein min)

(mean ± S.E.)

Resistance ratio (R/S)

AVR strain 5.66 ± 0.18 3.43

XHS strain 1.65 ± 0.03

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