Estimation of pentobarbital hydroxylase activity by gas-liquid chromatography

A method is presented for the estimation by gas-liquid chromatography of the in vitro metabolism of pentobarbital to its alcoholic metabolite, hydroxypentobarbital (5-ethyl-5-(1′-methyl-3′-hydroxybutyl) barbituric acid). The parent compound and metabolite are extracted from the incubation medium with ethyl acetate, the ethyl acetate is removed, and the trimethylsilyl derivatives are formed. These are separated and the metabolite quantitated on a gas-liquid chromatograph with a hydrogen flame ionization detector. As little as 1% metabolism can be readily determined. The identification of the products was confirmed by thin-layer chromatography and combination gas-liquid chromatography-mass spectrography. The present method offers a compromise between the sensitivity of the method employing radiolabeled pentobarbital and the convenience of measuring the disappearance of substrate.