Quantitation of phenobarbital and its main metabolites in human urine

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A method for the quantitative determination of phenobarbital and free and conjugatedp-hydroxyphenobarbital in urine samples is described. The method includes initial extraction, purification on a small chromatographic column and finally determination by gas chromatography. The barbituric acids are methylated by trimethylanilinium hydroxide which serves as a “flash heater” methylating agent. The conjugate ofp-hydroxyphenobarbital, which appears to be a glucuronide, is hydrolysed with hydrochloric acid.