Enantiospecific quantification of hexobarbital and its metabolites in biological fluids by gas chromatography/electron capture negative ion chemical ionization mass spectrometry

Abstract

A highly sensitive and specific assay based on gas chromatography/electron capture negative ion chemical ionization mass spectrometry has been developed for the analysis of the enantiomers of hexobarbital and its major metabolites in human urine and plasma. S‐(+)‐(5‐²H₃)hexobarbital and R‐(−)‐(5‐²H₃)hexobarbital were synthesized for clinical studies along with (±)‐(1,5‐²H₆)hexobarbital and the deuterated major metabolites for use as internal and reference standards. Hexobarbital enantiomers and their metabolites were analyzed after pentafluorobenzyl and trimethylsilyl derivatization, following solid‐phase extraction from plasma and urine. Intense negative ion spectra were observed for all of the derivatives. The base peak in the spectra corresponded to the M ‐ penta‐fluorobenzyl anion [M  PFB]⁻ except for 1,5‐dimethylbarbituric acid, where M^−· was the most abundant ion. The applicability of the method was demonstrated by following the plasma concentration‐time profiles and urinary excretion in a male extensive metabolizer of mephenytoin who was given a pseudoracemic oral dose of hexobarbital containing equal 50 mg amounts of S‐(+)‐²(H₀)hexobarbital and R‐(−)‐(²H₃)hexobarbital. Marked stereoselective disposition was observed, with the R‐(−)‐enantiomer being more efficiently metabolized, primarily by alicyclic oxidation and ring cleavage.