Simultaneous determination of phenobarbital and p-hydroxyphenobarbital and their stable isotope labeled analogs by gas chromatography mass spectrometry.

A method suitable for pulse dosing studies is described for the quantitation by gas chromatography mass spectrometry of phenobarbital (5-ethyl-5-phenylbarbituric acid), p-hydroxyphenobarbital (5-ethyl-5(4-hydroxyphenyl)barbituric acid) and, simultaneously, their (13C15N2)-labeled analogs in serum and urine. Differently labeled analogs are used as internal standards (5-ethyl-5(2,3,4,5-tetradeuterophenyl)-2-(13C)barbituric acid for quantitation of phenobarbital, and 5-ethyl-5(4-hydroxy-3,5-dideuterophenyl)2-(13C)-1,3-(15N2)barbituric acid for p-hydroxyphenobarbital). In the procedure, the chemical work-up of the samples (1.0 ml for serum, 0.5 ml for urine) is based on an extractive methylation technique for the generation of the permethylated derivatives. The mass spectrometric measurement technique consists of repetitive scanning over a preselected mass region of the permethylated derivatives of the analytes as they elute from the gas chromatograph. The method is evaluated for serum concentrations ranging from 0.1-30.0 micrograms ml-1 for phenobarbital and 0.1-10.0 micrograms ml-1 for p-hydroxyphenobarbital, and for urine concentrations of both phenobarbital and p-hydroxyphenobarbital of 1.0-50.0 micrograms ml-1. Application of the method to determination of the pharmacological equivalence of phenobarbital and (13C15N2)-labeled phenobarbital in man is also demonstrated.