Enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4′‐hydroxymephenytoin in human plasma and urine by liquid chromatography/tandem mass spectrometry

Abstract

A sensitive method using enantiospecific liquid chromatography/tandem mass spectrometry detection for the quantitation of S- and R-mephenytoin as well as its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma and urine has been developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile, while urine samples were diluted twice with the mobile phase before injection. The analytes were then separated on a chiral alpha(1)-acid glycoprotein (AGP) column and thereafter detected, using electrospray ionization tandem mass spectrometry. In plasma, the lower limit of quantification (LLOQ) was 1 ng/mL for S- and R-4'-hydroxymephenytoin and S-nirvanol and 3 ng/mL for R-nirvanol and S- and R-mephenytoin. In urine, the LLOQ was 3 ng/mL for all compounds. Resulting plasma and urine intra-day precision values (CV) were <12.4% and <6.4%, respectively, while plasma and urine accuracy values were 87.2-108.3% and 98.9-104.8% of the nominal values, respectively. The method was validated for plasma in the concentration ranges 1-500 ng/mL for S- and R-4'-hydroxymephenytoin, 1-1000 ng/mL for S-nirvanol, and 3-1500 ng/mL for R-nirvanol and S- and R-mephenytoin. The validated concentration range in urine was 3-5000 ng/mL for all compounds. By using this method, the metabolic activities of two human drug-metabolizing enzymes, cytochrome P450 (CYP) 2C19 and CYP2B6, were simultaneously characterized.

Artemisinin antimalarials moderately affect cytochrome P450 enzyme activity in healthy subjects

“…The plasma concentrations of S ‐mephenytoin and S ‐4′‐OH‐mephenytoin were determined by a separate LC/MS/MS method [18]. The enantiomers were separated on a chiral α 1 ‐acid glycoprotein column, 150 × 4 mm (Chromtech, Hägersten, Sweden).…”

Prediction of In Vivo Clearance and Associated Variability of CYP2C19 Substrates by Genotypes in Populations Utilizing a Pharmacogenetics-Based Mechanistic Model

“…Of note, CYP2C9 was chosen over the SimCYP default setting of CYP2B6 in mediating N-demethylation because CYP2C9 is more clinically relevant at physiologic concentrations (Ko et al, 1998). Identical parameters were assumed in R-mephenytoin modeling, except that CYP2C9-mediated N-demethylation was assumed to be the only metabolic clearance route because formation of R-49-hydroxymephenytoin by CYP2C19 was negligible (Jansson et al, 2006).…”

Chiral chromatographic resolution of antiepileptic drugs and their metabolites: a challenge from the optimization to the application

“…Nevertheless, owing to the co‐elution of R ‐nirvanol and S ‐mephenytoin on the AGP column, a Nucleosil 100 C 2 column was coupled in series to retain the S ‐ and R ‐mephenytoin and avoid compound overlap. The same group overcame this problem by developing an LC‐MS/MS method that did not require the C 2 column (Jansson et al ., ). This allowed not only the application of a unique column (AGP) but also simpler and faster sample preparation protocols that consisted of plasma protein precipitation with acetonitrile or diluting the urine samples instead of the cumbersome liquid–liquid extractions.…”

“…Thus, Jansson et al . () investigated different ammonium acetate buffers (5 and 10 m m ), with or without pH adjustment to 7.0, and different acetonitrile contents (2–5%). The selected mobile phase, 2% acetonitrile in 5 m m ammonium acetate, gave a good separation between the enantiomers and low ion suppression.…”

“…The results found during method validation, particularly regarding the intra‐ and inter‐day accuracy and precision for LLOQ and QC samples (Table ), clearly corroborate this affirmation because they fulfill all criteria stipulated by international regulatory guidelines for validation. Moreover both calibration curves in plasma and urine were linear at concentration ranges (Table ) that included those found in human plasma and urine after racemic mephenytoin administration to 75 healthy volunteers (Jansson et al ., ).…”