Gas chromatography/negative ion chemical ionization mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry quantitative profiling ofN-acetylcysteine conjugates of valproic acid in urine: application in drug metabolism studies in humans

Abstract

We report a GC/NICI-MS assay and a LC/ESI-MS/MS assay for the analysis of N-acetylcysteine (NAC) conjugates of (E)-2,4-diene VPA (NAC I and NAC II) identified in humans. The assay also includes the analysis of the NAC conjugate of 4,5-epoxy VPA (NAC III), an identified metabolite in rats treated with 4-ene VPA for its use in metabolic studies in animals. The highly sensitive GC/MS assay was designed to monitor selectively the diagnostic and most abundant [M - 181](-) fragment anion of the di-PFB derivatives of NAC I, NAC II, and NAC IV, the internal standard (IS) and the PFB derivative of NAC III. The higher selectivity of LC/MS/MS methodology was the basis for an assay which could identify and quantitate the underivatized conjugates simultaneously using MRM of the diagnostic ions m/z 130 and 123 arising from the CID of their protonated molecular ions [MH](+). The GC/MS assay employed liquid-liquid extraction whereas the LC/MS/MS assay used a solid-phase extraction procedure. Linearity ranges of the calibration curves were 0.10-5.0microg ml(-1) by GC/MS and 0.10-1.0microg ml(-1) by LC/MS/MS for NAC I, NAC II and NAC III (r(2) = 0.999 or better). Both assays were validated for NAC I and NAC II and provided good inter- and intra-assay precision and accuracy for NAC I and NAC II. The LOQ by LC/MS/MS was 0.1microg ml(-1), representing 1 ng of NAC I and NAC II. The same LOQ (0.1microg ml(-1)) was observed by GC/MS and was equivalent to 100 pg of each metabolite. NAC III was detected at concentrations as low as 0.01 microg ml(-1) by both methods. The total urinary excretion of the NAC conjugates in four patients on VPA therapy was determined to be 0.004-0.088% of a VPA dose by GC/MS and 0.004-0. 109% of a VPA dose by LC/MS/MS.

Identifying Genomic and Developmental Causes of Adverse Drug Reactions in Children

“…In mitochondria, 4-ene-VPA-CoA undergoes β-oxidation to (E)-2-propyl-2,4-pentadienoic acid-CoA (2,4-diene-VPA-CoA), a chemically reactive metabolite that is subject to glutathione conjugation [28,29]. Identification of various N -acetylcysteine conjugates in the urine of VPA-treated patients indicates that this bioactivation-detoxification pathway occurs in vivo in humans [28,29,74,75], but its role in the pathogenesis of VPA hepatotoxicity is unknown.…”

LC–MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

“…Several methods for quantification of VPA in plasma have been described, and most of them have been based on immunological methods [10] and gas chromatographic procedures with the flame ionization detector (FID) [11,12] or mass spectrometric (MS) detection [13][14][15]. Although desired sensitivity has been achieved using GC-MS/MS, prior derivatization limits its suitability for routine sample analysis.…”

Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the [U-13C6]-d-glucose tracer in mice

“…Valproic acid (2-propyl-pentanoic acid, VPA) has clinical applications in the treatment of a variety of neuropsychiatric illnesses such as epilepsy and bipolar disorder (Simon and Penry 1975 ; Keane et al 1982 ); however, it has been shown to act as a hepatotoxin in some patients (Eadie et al 1988 ). While hepatotoxicity of VPA is not well-understood, numerous studies have identified several metabolites that are potentially responsible for the observed toxicity (Tang and Abbott 1996 , 1997 ; Silva et al 2004 ; Gopaul et al 2000 ; 2003 ). Liver toxicity occurs with an overall incidence of 1 in 20,000, but a frequency as high as one in 600 or one in 800 is also common in high-risk groups such as infants below 2 years of age receiving anticonvulsant polytherapy (Perucca 2002 ).…”