Rapid Metabolite Discovery, Identification, and Accurate Comparison

Article pubs.acs.org/JAFC

Rapid Metabolite Discovery, Identification, and Accurate Comparison of the Stereoselective Metabolism of Metalaxyl in Rat Hepatic Microsomes Xinru Wang,† Jing Qiu,‡ Peng Xu,§ Ping Zhang,† Yao Wang,† Zhiqiang Zhou,† and Wentao Zhu*,† †

Department of Applied Chemistry, China Agricultural University, Beijing 100193, China Institute of Quality Standards & Testing Technology for Agro-Products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of Agricultural Sciences, Beijing, China § Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China ‡

ABSTRACT: Metabolite identification and quantitation impose great challenges on risk assessment of agrochemicals, as many metabolite standards are generally unavailable. In this study, metalaxyl metabolites were identified by time-of-flight mass spectrometry and semiquantified by triple quadrupole tandem mass spectrometry with self-prepared 13C-labeled metalaxyl metabolites as internal standards. Such methodology was employed to characterize the stereoselective metabolism of metalaxyl in rat hepatic microsomes successfully. Metabolites derived from hydroxylation, demethylation, and didemethylation were identified and semiquantified. The results indicated that (+)-S-metalaxyl eliminated preferentially as the enantiomer fraction was 0.32 after 60 min incubation. The amounts of hydroxymetalaxyl and demethylmetalaxyl derived from (−)-R-metalaxyl were 1.76 and 1.82 times higher than that of (+)-S-metalaxyl, whereas didemethylmetalaxyl derived from (+)-S-metalaxyl was 1.44 times larger than that from (−)-R-metalaxyl. This study highlights a new quantitation approach for stereoselective metabolism of chiral agrochemicals and provides more knowledge on metalaxyl risk assessment. KEYWORDS: metabolic profile, LC-MS, rat hepatic microsome, stereoselective metabolism, metabolite identification

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INTRODUCTION To facilitate the elimination and/or utilization of exogenous chemicals, all living creatures perform a certain level of metabolism, which might result in the activation of drugs or even the production of new toxic species.1,2 In some cases, metabolites show better bioactivities than the parent compound, such as acetaminophen from acetanilide.3 In some other cases, metabolites exhibit more toxicities than the parent compound.4−6 For example, Mitchell proposed that a toxic metabolite of acetaminophen is the main cause of acetaminophen-induced hepatic necrosis.7 Also, metabolites are always important indicators of biological processes. Therefore, identification and quantitation of metabolites are very important in metabolism research as well as risk assessments of xenobiotics.8 As for chiral drugs, it is well-known that stereochemistry has a marked impact on biological activity as well as biological processes such as absorption, degradation, metabolism, and excretion behavior in organisms. Enantiomers of a chiral compound can interact with biomacromolecules stereoselectively, leading to entirely different and even contradictory biological effects.9 Consequently, stereoselective metabolic investigation is important for ecotoxicological and environmental risk assessment of current chiral pesticides. The main purpose of this study is to introduce a new way to facilitate the accurate comparison of the stereoselective metabolism of a chiral xenobiotic. Nowadays, regarding the analytical tools used for qualitative and quantitative analysis of metabolites, mass spectrometry (MS) coupled with liquid chromatography (LC) is considered to be the most preeminent and widely used tool,10−12 due © 2015 American Chemical Society

primarily to its superior sensitivity, selectivity, speed of analysis, cost effectiveness, and ability to separate, detect, and identify metabolites even in the presence of many endogenous interferences.13 Since molecular weight determination is always the first and basic step for qualitative analysis of metabolites, MS instruments with high-resolution mass measurement, such as time-of-flight (TOF) or Fourier transform (FT) mass spectrometers, are always preferred in this step. Then, for further structural confirmation, tandem mass spectrometry (MS/MS) is often used mainly because it covers a variety of scanning techniques including product ion, precursor ion, and neutral loss scanning. The results provide information on a series of characteristic fragment ion masses, all of which correspond to part of the unknown molecule. For metabolite quantitation, triple quadrupole MS coupled with LC operated in the multiple reaction monitoring (MRM) mode is considered as the gold standard.14 Traditionally, discovery of new metabolites from a single parent molecule is performed by tracking radioactive tracers that are catabolized from a radioactive parent compound.15,16 Another widely used approach is stable isotope labeling.17 For instance, Chowdhury et al. utilized stable isotope-labeled drugs to aid the detection and identification of ribavirin metabolites in rats.18 The advantage of stable isotope labeling is that the metabolites are labeled in complex biological matrixes, which Received: Revised: Accepted: Published: 754

May 27, 2014 January 6, 2015 January 12, 2015 January 12, 2015 DOI: 10.1021/jf5025104 J. Agric. Food Chem. 2015, 63, 754−760

Article

Journal of Agricultural and Food Chemistry

high-performance liquid chromatography (HPLC) with a preparative chiral column containing cellulose-tris(3,5-methylphenylcarbamate)based chiral stationary phase (CDMPC−CSP, provided by the Department of Applied Chemistry, China Agricultural University, Beijing). 13C6-MX and β-nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St Louis, MO). 13C6MX acid [N-(2, 6-dimethylphenyl)-N-(methoxyacetyl)-alanine] was synthesized in our own laboratory. HPLC grade solvents were purchased from Beijing Chemicals (Beijing, China). All other chemicals and solvents were analytical grade and purchased from standard commercial sources. Water was purified with a Milli-Q system. Stock solutions of rac-, (+)-S-, and (−)-R-MX were prepared in 2propanol and were stored at −20 °C. Working standard solutions were obtained by dilutions of the stock solution in 2-propanol. Animals. Adult male Sprague−Dawley rats (200−250 g, 5−6 weeks old) were purchased from the Vital River Laboratory Animal Company (Beijing, China) and were fed with a standard diet before use. All rats were handled in accordance with the standard guidelines for care and use of laboratory animals as laid down in the EU directive 2010/63/EU. Rat hepatic microsomes were prepared with a procedure as described by Zhu31 and were stored at −80 °C. Incubation of MX with Microsomes for Metabolite Identification. For the detection and identification of MX metabolites, rat hepatic microsomal incubations with MX of various time periods were carried out with a series of samples and controls in parallel. In particular, rac-MX (100 μM) was prepared in ethanol and added into the microsomal incubation system, which was made up of 1 mg of microsomal protein and 50 mM Tris-HCl buffer (pH7.4) with 5.0 mM MgCl2. The ethanol added to each incubate was less than 1.0% v/v. After preincubation in a water bath at 37 °C for 5 min, NADPH at a final reaction concentration of 1.0 mM was added to the mixtures to initiate the reaction, resulting in a final total reaction volume of 1.0 mL. By adding 1 mL of acetonitrile containing 0.1% formic acid(v/v), the reactions were terminated after incubation in the water bath (37 °C) for 5, 10, 20, 30, and 60 min. The samples were vortexed for 3 min. To obtain a clear extract for analysis, samples were frozen at −20 °C for 1 h for protein precipitation and then centrifuged at 12 000g at 4 °C for 4 min, and the supernatant was transferred to a new tube for one more centrifugation. Eventually the clear extract was collected for analysis. Qualitative Analysis of MX and Its Metabolites in Microsomes. Samples were first analyzed by Agilent 1290 UPLC/ microTOF Q II system (Bruker Co., DE); thus, the accurate molecular weights of the metabolites were acquired. A reversedphase Agilent UPLC C18 analytical column (50 × 2.1 mm i.d., 1.8 μm) was used. The electrospray ionization (ESI) source was operated at 4 kV and a desolvation temperature of 200 °C. The mobile phase was water/acetonitrile (90/10), and the flow rate was 0.1 mL/min. All MS spectra were obtained in the positive mode, and argon was used as collision gas for the MS/MS measurements. The instrument was calibrated in the m/z range 50−600. MS data were processed via Bruker Data Analysis software 4.1. The metabolites were further identified by an API 2000 LC-MS/MS System (AB Sciex Instruments) equipped with a turbo electrospray ionization probe in the MRM scan mode. Liquid chromatography was carried out using an Agilent 1200 (Santa Clara, CA) series HPLC equipped with a G1322 degasser, G1311A quatpump, G1316B column compartment, and G1329A autosampler (Wilmington, DE). A Waters Atlantis T3 column (150 × 2.1 mm i.d., 3 μm) (Eschborn, Germany) was used. The mobile phase system was 0.1% formic acid in water (v/ v):0.1% formic acid in acetonitrile (v/v) 50:50. A flow rate of 0.3 mL/ min and an injection volume of 40 μL were adopted in all steps. Positive ionization mode was used for detection of all analytes. The ion spray voltage was 4000 V, and the ion source temperature was 450 °C. The curtain gas was set at 10 psig, while ion source gas 1 and 2 were set at 45 and 50 psig, respectively. For data acquisition and analysis, Analyst v.1.4.2 (Applied Biosystems/MDS SCIEX) was used. Finally, rat hepatic microsomal incubation with 13C-labeled MX was performed to reconfirm metabolites as well as to produce the 13C-

can be distinguished from various interferences and possible laboratory contaminants.19 For the quantitative analysis of metabolites, the lack of metabolite standards always imposes a significant limitation. This is also the bottleneck for comprehensive metabolism research for chiral pesticides. Although synthesis of some potential metabolites can be achieved in theory, it would be a quite expensive and complex endeavor. Another possible approach to get metabolites might be incubation of substrates with in vivo methods20 or some in vitro systems.21,22 For example, in vivo methods often include administering fluorescent, isotopic, or radioactive labeled substrates to the living biological model, while potential in vitro systems often include cell cultures, enzyme mixtures, and liver microsomes. Simplicity and availability are the most obvious advantages of in vitro technologies. The relatively high concentration of substrate as well as metabolites in an in vitro system makes the metabolic study much easier than that with in vivo systems.23 Microsomal incubation is a valuable and the most widely used tool for metabolism research on xenobiotics,24 as it contains a significant amount of metabolic enzymes. Clements et al. developed a strategy of using liver microsomal incubation with drug candidates for the preparation of phase I drug metabolites, which could then be used as standards for in vivo experiments.25 This approach inspired us that the microsomal incubation could be a possible and convenient way to produce metabolite “standards” of a pesticide. Metalaxyl [N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-D,Lalanine methyl ester, MX, Figure 1] is a systemic fungicide

Figure 1. Chemical structure of metalaxyl enantiomers.

with protective and curative actions against oomycetes. Since introduction in 1977, it has been widely used in agriculture and horticulture.26 It has also been reported that metalaxyl has many hazardous effects on humans and other nontarget organisms.27,28 Recently, metalaxyl and its metabolites were detected in groundwater.29,30 For a comprehensive risk assessment of metalaxyl, data on the stereoselective metabolism are indispensable. Therefore, the present work involved the following four aspects: (1) TOF-MS was employed to acquire the accurate molecular mass information and preliminary structural presumption of MX metabolites; (2) 13C-labeled MX incubation and MS/MS detection were carried out for further structure identification; (3) 13C-labeled metabolites were prepared and used as internal standards for quantitation; (4) stereoselective metabolism of MX enantiomers were quantitively investigated in rat hepatic microsomes.

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MATERIALS AND METHODS

Chemicals. Racemic MX (rac-MX) standard (>99% purity) was provided by Institute for Control of Agrochemicals, China Ministry of Agriculture. (+)-S-MX and (−)-R-MX were prepared on an Agilent 755

DOI: 10.1021/jf5025104 J. Agric. Food Chem. 2015, 63, 754−760

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Journal of Agricultural and Food Chemistry labeled metabolites, which along with the synthesized 13C-labeled MX acid were used as internal standard in the subsequent stereoselective metabolism research of MX enantiomers. Quantitative Analysis for Stereoselective Metabolism of MX Enantiomers. (+)-S- and (−)-R-MX were incubated in the microsomal system described above, respectively. After 0, 5, 10, 20, 30, and 60 min, 10 μL of internal standard and 140 μL of acetonitrile containing 0.1% formic acid (v/v) were added to 50 μL of the incubation mixture to terminate the reaction. Then the samples were vortexed and centrifuged, and the supernatant was collected and stored at −20 °C until analysis. Triple quadrupole MS coupled with LC was used for quantitation. The LC solvents, column, and MS parameters used for the sample analysis were identical to those of the qualitative analysis described above.

fragmentation of MX and the detected metabolites. The fragment ions of these potential metabolites corresponded to the proposed structures. In the present study, the structure of the potential metabolites was presumed based on the LC-TOF-MS data and previously reported in vivo metabolic data.32 In general, the demethylation metabolite (m/z 266) should be N-(2,6dimethylphenyl)-N-(methoxyacetyl)alanine (M3), the MX acid. The didemethylation metabolite (m/z 252) should be N-(2,6-dimethylphenyl)-N-(hydroxyacetyl)alanine (M4). For the hydroxylation metabolite (m/z 296), the two possible positions of hydroxylation resulted in two possible structures: N-(2,6-dimethyl-5-hydroxyphenyl)-N-(methoxyacetyl)alanine methyl ester (M1) and N-(2-hydroxymethyl-6-methylphenyl)N-(methoxyacetyl)alanine methyl ester (M2). M1 and M2 are two isomers of hydroxymetalaxyl. Figure 3 summarizes the proposed biotransformation pathways of MX in rat hepatic microsomes. Much research has focused on the metabolism of MX in matrixes such as soil,33,34 plants,26 fungus35 and microsomes.36,37 In general, MX acid was the main metabolite in most soil matrixes.38,39 In human liver microsomes, as reported by Abass, hydroxylation, didemethylation, and lactone formation were the main biotransformation reactions.37 Since the metabolic profile of a compound always varies with the species used, the difference in metabolic behavior between human and rat liver microsomes may be partly attributed to different CYPs participating in MX metabolism. Quantitation Method for Metabolite Analysis. After preliminary confirmation of MX metabolites, 13C-labeled MX was incubated with rat hepatic microsomes (1) for further confirmation of the formation of the metabolites and (2) to obtain 13C-labeled metabolites, which were then used as the “internal standards” for identification and quantitative analysis of metabolites in the subsequent stereoselective metabolism study of MX. As a result, hydroxymetalaxyl (m/z 296), demethylmetalaxyl (m/z 266), and didemethylmetalaxyl (m/z 252) were all reconfirmed in this step. A 60 min incubation time was considered as optimal, giving the highest yield of 13Clabeled metabolites. Thus, besides a minor level of 13C6-MX acid, the demethylation metabolite (m/z 266), which was synthesized through a hydrolysis method, other labeled metabolites were successfully achieved with the incubation of 13 C6-MX for 60 min. Quantitative analysis of MX metabolites was carried out by LC-MS/MS. All analytes showed a strong signal of the quasimolecular ion [M + H]+ under positive electrospray ionization mode. The parameters of MRM mode for all analytes were optimized by selecting the most abundant product ion. Table 2 listed the MRM parameters for all analytes. Figure 4 illustrated a set of MRM chromatograms for the 60 min

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RESULTS AND DISCUSSION Identification of the Possible MX Metabolites by Rat Hepatic Microsomes. Data from LC-TOF-MS indicated that possible metabolites of hydroxylation, probably at two sites (M1 and M2), demethylation (M3), and didemethylation (M4) were detected after the microsomal incubation with 100 μM MX. Figure 2 shows the full-scan chromatogram and the

Figure 2. Total ion current chromatograms of UPLC-TOF-MS from the 60 min microsome incubations of (a) the control and (b) metalaxyl. Extracted ion chromatograms of (c) m/z 280, (d) m/z 296, (e) m/z 266, and (f) m/z 252.

extracted mass chromatograms of the metabolites formed by 60 min incubation. Table 1 lists the exact masses and

Table 1. Tentative Identification of Metalaxyl Metabolites Formed in Vitro by Rat Liver Microsomes, on the Basis of Exact Mass Determination by TOF-MS metabolite a

M1 M2b M3 M4 metalaxyl a

biotransformation

accurate mass

fragments

molecular formula

hydroxylation hydroxylation demethylation didemethylation −

296.1984 296.1984 266.1879 252.1666 280.1973

278.1853, 252.1662, 198.2208, 149.0540, 122.1127

C15H21NO5

234.1559, 198.2236, 149.0550, 122.1135 198.2208, 149.0537, 122.1118 248.1665, 220.1681, 192.1692, 122.1108

C14H19NO4 C13H17NO4 C15H21NO4

Hydroxylation of the phenyl ring. bHydroxylation of the aromatic methyl group. 756


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Figure 3. Proposed metabolic pathways of metalaxyl by rat hepatic microsomes. (M1 and M2) Hydroxymetalaxyl metabolite (m/z 296), (M3) demethylmetalaxyl metabolite (m/z 266), (M4) didemethylmetalaxyl metabolite (m/z 252).

Table 2. Mass Spectrometry Parameters for Determination of Metalaxyl and Its Metabolites by LC-MS/MS

a

compound

retention time (min)

Q1 mass (amu)

Q3 mass (amu)

DPa(V)

CEb(V)

CXPc(V)

metalaxyl hydroxymetalaxyl metabolites (m/z 296) demethylmetalaxyl metabolite (m/z 266) didemethylmetalaxyl metabolite (m/z 252)

5.83 2.43 2.96 2.32

280.2 296.1 266.2 252.0

220.0 146.4 220.0 206.0

61 46 71 10

15 29 17 15

8 6 4 4

DP, declustering potential. bCE, collision energy. cCXP, cell exit potential.

microsomal incubation of MX. Hydroxymetalaxyl (m/z 296), didemethylmetalaxyl (m/z 252), and demethylmetalaxyl (m/z 266) were detected. None of them were detected in the absence of rat hepatic microsomes. Stereoselective Metabolism of (+)-S- and (−)-R-MX. (+)-S- and (−)-R-MX at a concentration of 100 μM were incubated with rat hepatic microsomes, respectively. The degradation curves for MX enantiomers are shown in Figure 5a. (+)-S-MX degraded faster than its antipode through the incubation period with a t1/2 of 21.00 and 36.47 min for (+)-Sand (−)-R-MX, respectively. In addition, the enantiomer fraction (EF) was used to measure the stereoselective metabolism of MX (Figure 6). EF was calculated from the peak areas of (+)-S- and (−)-R-MX following the equation: EF = ( +)‐S ‐MX/[( +)‐S ‐MX + ( − )‐R ‐MX]

(1)

The EF was 0.32 after 60 min incubation, also indicating that degradation of MX in rat hepatic microsomes was stereoselective. This was consistent with the degradation aspect of rac-MX in rat hepatic microsomes in a previous study.36 In the present study, the peak area ratio of each metabolite and its corresponding 13C-labeled internal standard was used to

Figure 4. MRM chromatograms of LC-MS/MS from the 60 min microsome incubations of (a) m/z 280, (b) m/z 296, (c) m/z 266, and (d) m/z 252.

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cofactors and concentration of NADPH are also different. These might explain the difference in metabolite formation between the in vitro and in vivo systems. Except for a small quantity of demethylmetalaxyl metabolite (m/z 266), other metabolites were not detected in the control group. It seemed that MX underwent a slight hydrolysis in the absence of NADPH system. It is obvious that rat hepatic microsome exhibited significant stereoselectivity in the formation of all detected metabolites. (+)-S- and (−)-R-MX demonstrated quantitatively different metabolite patterns. Transformation of (−)-R-MX to its hydroxymetalaxyl (m/z 296) and demethylmetalaxyl metabolite (m/z 266) was markedly preponderant over its (+)-S-isomer, while the case of didemethylmetalaxyl metabolite (m/z 252), a secondary dimethyl metabolite of demethylmetalaxyl metabolite (m/z 266), was just the opposite. Specifically, after 60 min incubation, the amounts of hydroxymetalaxyl and demethylmetalaxyl derived from (−)-R-MX were 1.76 and 1.82 times higher than that of (+)-S-MX, whereas the production of didemethylmetalaxyl from (+)-S-MX was 1.44 times more than that from (−)-R-MX. Therefore, the minor amount of demethylmetalaxyl metabolite (m/z 266) from (+)-S-MX might well be attributed to the preferential transformation to its secondary dimethyl metabolite (m/z 252). As the fungicidal activity of metalaxyl was mainly attributed to the (−)-R-MX, it is interesting that the more active isomer (for target organism) also showed a higher metabolic activity in the in vitro system of the nontarget organism. Abundant reports suggested that two enantiomers of a chiral compound could always show differences in pharmacokinetic processes (absorption, distribution, metabolism, protein binding, and excretion).41 Optically active biological macromolecules might behave stereoselectivity when interacting with a chiral drug. In the present study, our results demonstrated that degradation of the parent MX as well as formation of the metabolites in rat hepatic microsomes were observably stereoselective, which should be highly attributed to the existence of several microsomal mono-oxygenases differing in their stereoselectivities and inducibilities for the enantiomers. In summary, we proposed a new approach using the peak area ratio of each metabolite and its corresponding stable isotope-labeled internal standard to quantitatively compare the stereoselective metabolic behavior of two enantiomers of a chiral compound. According to P. Schieberle,42 the quantitation method proposed in the present study should be categorized into the “Stable Isotope Dilution Assays (SIDA)”. As commercially unavailability of many labeled analytes was claimed as the main drawback of SIDA, our present study proposed a method using real biological samples to obtain the labeled standards. But the absolute quantities of the labeled metabolites were unclear, and it is difficult to establish the MRM method for some minor metabolites at very low concentrations. This should be regarded as the main limitation of our microsomal incubation method. With most of the main metabolites having been quantified, this approach could be of great value for its extensive application in the field of stereoselective metabolism research of a chiral compound, which is important for its comprehensive ecotoxicological and environmental risk assessment.

Figure 5. Concentration−time curves of (a) metalaxyl, (b) the hydroxymetalaxyl metabolites (m/z 296), (c) the demethylmetalaxyl metabolite (m/z 266), and (d) the didemethylmetalaxyl metabolite (m/z 252) after incubation of 100 μM (+)-S- and (−)-R-metalaxyl with rat hepatic microsomes, respectively.

Figure 6. Temporal variations of EFs of metalaxyl in the rat hepatic microsome in the 60 min time course after exposure to 100 μM metalaxyl enantiomers.

compare the differences of metabolic profile between the two enantiomers of MX. Therefore, with the ratio values at different incubation times figured out, metabolite formation from (+)-Sand (−)-R-MX by rat hepatic microsomes during the incubation is shown in Figure 5b−d. The amount of all metabolites increased over incubation time. For both enantiomers of MX, the hydroxymetalaxyl metabolites (m/z 296) were the major metabolites. The didemethylmetalaxyl metabolite (m/z 252) was at a moderate level, and demethylmetalaxyl metabolite (m/z 266) was found as the minor metabolite, probably because it could undergo further demethylation. A previous in vivo study indicated that metabolites resulted from didemethylation, hydroxylation of the aromatic methyl group, and demethylation of the ester group were major metabolites, while phenyl ring hydroxyl, ether group demethylated, and lactone metabolites were minor metabolites.40 Compared with in vitro systems, more complex metabolic pathways exist in vivo, and conditions such as 758


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acid metabolism using stable isotope labeling techniques. J. Lipid Res. 2006, 47, 1651−60. (18) Chowdhury, S. K.; Gopaul, V. S.; Blumenkrantz, N.; Zhong, R.; Kulmatycki, K. M.; Alton, K. B. Detection and characterization of highly polar metabolites by LC-MS: Proper selection of LC column and use of stable isotope-labeled drug to study metabolism of ribavirin in rats. Prog. Pharm. Biomed. Anal. 2005, 6, 277−293. (19) Birkemeyer, C.; Luedemann, A.; Wagner, C.; Erban, A.; Kopka, J. Metabolome analysis: the potential of in vivo labeling with stable isotopes for metabolite profiling. Trends Biotechnol. 2005, 23, 28−33. (20) Zhang, N. Y.; Fountain, S. T.; Bi, H. G.; Rossi, D. T. Quantification and rapid metabolite identification in drug discovery using API time-of-flight LC/MS. Anal. Chem. 2000, 72, 800−806. (21) Baranczewski, P.; Stanczak, A.; Kautiainen, A.; Sandin, P.; Edlund, P. O. Introduction to early in vitro identification of metabolites of new chemical entities in drug discovery and development. Pharmacol. Rep. 2006, 58, 341−52. (22) Holcapek, M.; Kolarova, L.; Nobilis, M. High-performance liquid chromatography-tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites. Anal. Bioanal. Chem. 2008, 391, 59−78. (23) Venkatakrishnan, K.; von Moltke, L. L.; Obach, R.; Greenblatt, D. J. Drug metabolism and drug interactions: application and clinical value of in vitro models. Curr. Drug Metab. 2003, 4, 423−459. (24) Tolleson, W. H.; Doerge, D. R.; Churchwell, M. I.; Marques, M. M.; Roberts, D. W. Metabolism of biochanin A and formononetin by human liver microsomes in vitro. J. Agric. Food Chem. 2002, 50, 4783− 90. (25) Clements, M.; Li, L. Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry. Anal. Chim. Acta 2011, 685, 36−44. (26) Zadra, C.; Marucchini, C.; Zazzerini, A. Behavior of metalaxyl and its pure R-enantiomer in sunflower plants (Helianthus annus). J. Agric. Food Chem. 2002, 50, 5373−7. (27) Demsia, G.; Vlastos, D.; Goumenou, M.; Matthopoulos, D. P. Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2007, 634, 32−39. (28) Al-Amoudi, W. M. Haematological and biochemical effects of metalaxyl fungicide on albino mice. Am. J. Biochem. 2012, 2, 62−66. (29) Hildebrandt, A.; Guillamón, M.; Lacorte, S.; Tauler, R.; Barceló, D. Impact of pesticides used in agriculture and vineyards to surface and groundwater quality (North Spain). Water Res. 2008, 42, 3315− 3326. (30) Reemtsma, T.; Alder, L.; Banasiak, U. A multimethod for the determination of 150 pesticide metabolites in surface water and groundwater using direct injection liquid chromatography−mass spectrometry. J. Chromatogr. A 2013, 1271, 95−104. (31) Zhu, W.; Dang, Z.; Qiu, J.; Liu, Y.; Lv, C.; Diao, J.; Zhou, Z. Species differences for stereoselective metabolism of ethofumesate and its enantiomers in vitro. Xenobiotica 2009, 39, 649−655. (32) Vleminckx, C. Pesticide residues in food-2002; Joint FAO/WHO Meeting on Pesticide Residues; Rome, Italy, September 16−25, 2002. (33) Droby, S.; Coffey, M. D. Biodegradation Process and the Nature of Metabolism of Metalaxyl in Soil. Ann. Appl. Biol. 1991, 118, 543− 553. (34) Sukul, P.; Spiteller, M. Persistence, fate, and metabolism of [14C]metalaxyl in typical Indian soils. J. Agric. Food Chem. 2001, 49, 2352−2358. (35) Zheng, Z.; Liu, S. Y.; Freyer, A. J.; Bollag, J. M. Transformation of Metalaxyl by the Fungus Syncephalastrum racemosum. Appl. Environ. Microbiol. 1989, 55, 66−71. (36) Zhang, P.; Shen, Z.; Xu, X.; Zhu, W.; Dang, Z.; Wang, X.; Liu, D.; Zhou, Z. Stereoselective degradation of metalaxyl and its enantiomers in rat and rabbit hepatic microsomes in vitro. Xenobiotica 2012, 42, 580−586.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +861062733547. Tel:+ 861062733547. Funding

We gratefully acknowledge financial support from the National Natural Science Foundation of China (contract grant numbers: 21207158 and 21021058) and Chinese Universities Scientific Fund (2012RC026). Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Chen, C.; Gonzalez, F. J.; Idle, J. R. LC-MS-based metabolomics in drug metabolism. Drug Metab Rev. 2007, 39, 581−97. (2) Clarke, N. J.; Rindgen, D.; Korfmacher, W. A.; Cox, K. A. Peer Reviewed: Systematic LC/MS Metabolite Identification in Drug Discovery. Anal. Chem. 2001, 73, 430 A−439 A. (3) Brodie, B. B.; Axelrod, J. The estimation of acetanilide and its metabolic products, aniline, N-acetyl p-aminophenol and p-aminophenol, free and total conjugated, in biological fluids and tissues. J. Pharmacol Exp Ther 1948, 94, 22−8. (4) Gillette, J. R.; Mitchell, J. R.; Brodie, B. B. Biochemical Mechanisms of Drug Toxicity. Annu. Rev. Pharmacol. Toxicol. 1974, 14, 271−288. (5) Knowles, S. R.; Uetrecht, J.; Shear, N. H. Idiosyncratic drug reactions: the reactive metabolite syndromes. Lancet 2000, 356, 1587− 91. (6) Liebler, D. C.; Guengerich, F. P. Elucidating mechanisms of druginduced toxicity. Nat. Rev. Drug Discovery 2005, 4, 410−20. (7) Mitchell, J. R.; Jollow, D. J.; Potter, W. Z.; Davis, D. C.; Gillette, J. R.; Brodie, B. B. Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J. Pharmacol. Exp. Ther. 1973, 187, 185−94. (8) Yang, W. C.; Regnier, F. E.; Jiang, Q.; Adamec, J. In vitro stable isotope labeling for discovery of novel metabolites by liquid chromatography-mass spectrometry: Confirmation of gamma-tocopherol metabolism in human A549 cell. J. Chromatogr. A 2010, 1217, 667−75. (9) Kurihara, N.; Miyamoto, J.; Paulson, G. D.; Zeeh, B.; Skidmore, M. W.; Hollingworth, R. M.; Kuiper, H. A. Chirality in synthetic agrochemicals: bioactivity and safety considerations. Pestic. Sci. 1999, 55, 219−219. (10) Lu, X.; Zhao, X.; Bai, C.; Zhao, C.; Lu, G.; Xu, G. LC-MS-based metabonomics analysis. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2008, 866, 64−76. (11) Lu, W.; Bennett, B. D.; Rabinowitz, J. D. Analytical strategies for LC-MS-based targeted metabolomics. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2008, 871, 236−42. (12) Ji, C.; Walton, J.; Su, Y.; Tella, M. Simultaneous determination of plasma epinephrine and norepinephrine using an integrated strategy of a fully automated protein precipitation technique, reductive ethylation labeling and UPLC-MS/MS. Anal. Chim. Acta 2010, 670, 84−91. (13) Lee, M. S.; Kerns, E. H. LC/MS applications in drug development. Mass Spectrom. Rev. 1999, 18, 187−279. (14) Chen, S.; Kong, H.; Lu, X.; Li, Y.; Yin, P.; Zeng, Z.; Xu, G. Pseudotargeted metabolomics method and its application in serum biomarker discovery for hepatocellular carcinoma based on ultra highperformance liquid chromatography/triple quadrupole mass spectrometry. Anal. Chem. 2013, 85, 8326−8333. (15) Chiku, S.; Hamamura, K.; Nakamura, T. Novel urinary metabolite of d-delta-tocopherol in rats. J. Lipid Res. 1984, 25, 40−8. (16) Simon, E. J.; Gross, C. S.; Milhorat, A. T. The Metabolism of Vitamin E: I. The Absorption and Excretion of D-α-Tocopheryl-5methyl-C14-succinate. J. Biol. Chem. 1956, 221, 797−805. (17) Parks, E. J.; Hellerstein, M. K. Thematic review series: Patientoriented research. Recent advances in liver triacylglycerol and fatty 759


Article

Journal of Agricultural and Food Chemistry (37) Abass, K.; Reponen, P.; Jalonen, J.; Pelkonen, O. In vitro metabolism and interactions of the fungicide metalaxyl in human liver preparations. Environ. Toxicol. Pharmacol. 2007, 23, 39−47. (38) Buser, H.-R.; Müller, M. D.; Poiger, T.; Balmer, M. E. Environmental Behavior of the Chiral Acetamide Pesticide Metalaxyl: Enantioselective Degradation and Chiral Stability in Soil. Environ. Sci. Technol. 2002, 36, 221−226. (39) Saha, T.; Sukul, P. Metalaxylits persistence and metabolism in soil. Toxicol. Environ. Chem. 2008, 58, 251−258. (40) Hambock, H. Metabolic pathways of CGA 48988 in the rat. Project unpublished report 31/81 from Ciba-Geigy Ltd., Basle, Switzerland, submitted to the World Health Organization by CibaGeigy, Ltd., 1981. (41) Brocks, D. R. Drug disposition in three dimensions: An update on stereoselectivity in pharmacokinetics. Biopharm. Drug Dispos. 2006, 27, 387−406. (42) Schieberle, P.; Molyneux, R. J. Quantitation of sensory-active and bioactive constituents of food: a journal of agricultural and food chemistry perspective. J. Agric. Food Chem. 2012, 60, 2404−2408.

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Rapid Metabolite Discovery, Identification, and Accurate Comparison

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