Quantitative determination of phenobarbital derivatives by GC-MS

Analytical methods based on stable isotope labeling in conjunction with gas chromatography-mass spectrometry have been developed for the new anticonvulsant 1,3-bis(methoxymethyl)phenobarbital and two of its metabolites, 1-methoxymethylphenobarbital and phenobarbital. Deuterium-labeled analogs of the three compounds were synthesized for use as internal standards in measuring the concentration of the unlabeled drug and its metabolites in biological fluids. The compounds were extracted from the acidified biological fluid, and the parent drug was separated from its two metabolites. The latter were methylated with diazomethane before GC-MS analysis. Selected ions in the spectra of the three compounds and their respective internal standards were monitored using a quadrupole mass spectrometer controlled by a digital selected ion monitor. The sensitivity of the assay was 50 pmoles/ml for each compound. The method was applied to the study of the plasma and brain levels of the three compounds in the rat after intravenous administration of the parent drug (6 mg/kg). Rapid formation of the two metabolites was observed, and accumulation of phenobarbital in the rat brain was found. The plasma levels of the three compounds were also measured in man after oral doses. It was concluded that a significant aspect of the anticonvulsant action of 1,3-bis(methoxymethyl) phenobarbital is its rapidconversion in vivo to phenobarbital