Gas chromatography/negative ion chemical ionization mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry quantitative profiling of N-acetylcysteine conjugates of valproic acid in urine: application in drug metabolism studies in humans.
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.
Gopaul
S V
SV
Faculty of Pharmaceutical Sciences, 214 East Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Farrell
K
K
Abbott
F S
FS
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000378:metabolism / Q000652:urine; D000293:Adolescent; D000704:Analysis of Variance; D000818:Animals; D000927:Anticonvulsants / Q000378:metabolism / Q000652:urine*; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid / Q000379:methods; D004827:Epilepsy / Q000188:drug therapy / Q000378:metabolism / Q000652:urine; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods* / Q000706:statistics & numerical data; D051381:Rats; D014635:Valproic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000652:urine*
D000927:Anticonvulsants; D014635:Valproic Acid; D000111:Acetylcysteine