Concurrentassayof Phenobarbital and Diphenyihydantoin in Plasmaby Vapor-phasechromatography Procedure Specimen Preparation

The weakly acidic phenobarbital and diphenylhydantoin, separated from interfering substances by simple solvent partition, are estimated concurrently by vapor.phase chromatography. Solid sample injection and internal standard (primidone) are used. The entire procedure can be completed within 2 h after venipuncture. Both drugs could be measured after therapeutic doses have been administered. SINCE THE pioneer work of Goldbaum (1,2), a profusion of methods for estimating phenobarbital in biological fluids have appeared. In 1956, Dill et al. (3) presented a colorimetric method for assay of diphenylhydantoin, and Plaa and Hine (4) published a spectrophotometric method for simultaneous determination of phenobarbital and di-phenyihydantoin. In 1963, Svensmark and Kris-tensen (5) improved the spectrophotometric method by modifying the solvent separation of these two drugs. Numerous authors have used paper and thin-layer chromatography to identify and roughly quantify them. Recently, Wallace (6) assayed diphenylhydantoin spectrophotometrically as the benzophenone, formed by permanganate oxidation of the drug. Sandberg et at. (7) meth-ylated diphenyihydantoin with diazomethane and estimated the methyl ether by vapor-phase chromatography (vpc). Chang and Glazko (8) estimate the p-hydroxylated metabolic product of diphenylhydantoin by vc of the trimethylsilyl derivative. Recently Sunshine et at. (9) evaluated methods for determination of barbiturates in biological materials. A rapid, specific assay of phenobarbital and diphenylhydantoin in body fluids is needed. This encouraged us to examine the possible use of vc for this purpose. The vi'c of drugs in crude extracts of biological specimens is subject to gross interference by both normal and drug constituents. By appropriate solvent partition this interference is eliminated. In our method, phenobarbital and diphenylhydantoin are extracted from plasma or serum that has been purified by partitioning between chloroform and neutral, alkaline, and acid water; the extract is assayed by vic. (a) Add 2.0 ml of plasma or serum to 1.0 ml of 1.OM phosphate buffer, pH 6.8, and 15 ml of freshly redistilled chloroform in a stoppered centrifuge tube. Gently agitate the mixture for 15 mm on a mechanical shaker. Centrifuge and separate the phases. Discard the aqueous phase. (b)Transfer 13 ml of the chloroform phase to 5 ml of 0.4M phosphate buffer, pH 11, in a stop-pered centrifuge tube. Shake mixture as before for 10 mm. Centrifuge and separate phases. The drugs being assayed here are in the aqueous phase; the extracted chloroform phase may be held for further examination of other drugs. (c) Adjust the alkaline extract to pH 1 with 0.5 ml of …