Simultaneous Determination of Valproic Acid and its Toxic Metabolites, 4-ene-VPA and 2,4-diene-VPA in Rat Plasma using a Gas Chromatographic-mass Spectrometric Method

Abstract

− A gas chromatographic-mass spectrometric (GC-MS) method was developed for the simultaneous determination of valproic acid (VPA) and its toxic metabolites, 4-ene-VPA and 2,4-diene-VPA in rat plasma. Extraction was performed in weak acidic condition (pH 5.2) to avoid degradation of 4-ene-VPA and 2,4-diene-VPA. The recoveries for 4-eneVPA and 2,4-diene-VPA were more than 70% and that for VPA was 33-42%. R value for each compounds exceeded 0.998 in calibration curve during all the analysis. Accuracy and precision ranged from 88.3 to 113.2% and from 2.16 to 14.2%, respectively The method was successfully applied to monitor plasma concentrations of VPA, 4-ene-VPA and 2,4-diene-VPA after intravenous administration of VPA at the dose of 100 mg/kg, suggesting that these toxic metabolites may involved in the hepatotoxicity induced by VPA. Key words− valproic acid (VPA), 4-ene-VPA, 2,4-diene-VPA, quantitative analysis, GC-MS (gas chromatographic-mass spectrometry), pharmacokinetics Valproic acid (VPA, 2-propylpentanoic acid, Fig. 1) is a broad-spectrum antiepileptic agent that has a branched-chain carboxylic acid structure. VPA has been used to treat bipolar disorder, migraine, and neuropathic pain (Loscher et al., 1993; Murakami et al., 1992; Sugimoto et al., 1983), but it has been demonstrated to be associated with fatal liver toxicity (Perucca, 2002; Zimmerman et al., 1982; Zafrani et al., 1982; Bryant et al., 1986). Although the mechanism by which VPA leads to liver toxicity has not been fully elucidated, a large body of evidence suggests that reactive VPA metabolites (4ene-VPA; 2-propyl-4-pentanoic acid and its subsequent metabolite, 2,4-diene-VPA; 2-propyl-2,4-pentanoic acid) may mediate the hepatotoxicity by inhibiting mitochondrial fatty acid β-oxidation (Zimmerman et al., 1982; Gerber et al., 1979; Kassahun et al., 1991). Therefore it is important to monitor the plasma concentration of 4-ene-VPA and 2,4diene-VPA as well as VPA itself to evaluate effect and toxicity of VPA. In order to monitor plasma concentration of VPA and its toxic metabolites, it is necessary to have reliable and sensitive method for the simultaneous quantitative determination of those compounds. Because of structural similarity between fatty acid (non polar compound) and VPA, most of analytical method for VPA and/or its metabolite has been used GC-MS (gas chromatography-mass spectrometry) (Fisher et al., 1992; Yu et al., 1995; Leis et al., 2003). But those reports did not focus on the toxic metabolites, 4-ene-VPA and 2,4-diene-VPA, so it is very hard to evaluate pharmacokinetics of VPA and its toxic metabolites. Recently the method using HPLC-MS/MS (high performance liquid chromatography-tandem mass spectrometry) was reported to analyze VPA, however this method was developed only for the quantification of VPA and 4-eneVPA. Thus it is also not adequate to monitor plasma concentrations of VPA and its toxic metabolites (Cheng et al., 2007). Moreover, sample extraction with organic solvent was performed in the pH condition less than pH 3, which was not favorable condition for 4-ene-VPA and 2,4-diene-VPA since they could be degraded in acidic condition. In this study we developed adequate method for the quantitative analysis of VPA, 4-ene-VPA and 2,4-diene-VPA in rat plasma. We used mild pH condition to prevent degradation of the metabolites. This analytical method was validated and applied to evaluate pharmacokinetics of VPA and its metabolites after intravenous administration to rats. Corresponding Author : Tel : +82-2-958-5062, E-mail : jbhluck@kist.re.kr DOI : 10.4333/KPS.2010.40.3.155 156 Min Sun Lee, Young-Joo Lee, Bong Chul Chung and Byung Hwa Jung J. Pharm. Invest., Vol. 40, No. 3 (2010) Experimental