Microassay for primidone and its metabolites phenylethylmalondiamide,phenobarbital and p-hydroxyphenobarbital in human serum,saliva, breast milk and tissues by gas chromatography—mass spectrometry using selected ion monitoring

A method for the quantitative determination of primidone and its metabolites phenobarbital, phenylethylmalondiamide (PEMA) and hydroxyphenobarbital (free and conjugated) in serum, urine, saliva, breast milk and tissue has been developed. Following the addition of the methyl analogues of primidone, phenobarbital and PEMA as internal standards and of saturated ammonium sulphate, the samples (5–100 μl) were extracted twice with ethyl acetate—benzene (20:80). The extracts were divided into two equal portions; one portion was ethylated by Greeley's method for the analysis of primidone, phenobarbital and hydroxyphenobarbital, while the other was trimethylsilylated for the analysis of primidone and PEMA. A gas chromatographic—mass spectrometric system was used for the analysis of the derivatized extracts. Linear calibration curves were obtained in the concentration range studied (between 100 ng/ml and 30 μg/ml). The recoveries of the drugs were between 80 and 93%. The relative standard deviations were between 3.2 and 5.9% (100-μl serum samples containing 1 μg/ml of the drugs). The lower detection limits were found to be between 1.4 and 3.7 ng/ml using serum samples of 100 μl.These methods have been applied to the study of the placental transfer and neonatal disposition of primidone and its metabolites in the human.