Gas chromatographic-mass spectrometric studies on the metabolic fate of ethotoin in man.

Unchanged ethotoin and 11 metabolic products of ethotoin were detected in the urine of subjects (2 men and 1 woman) receiving ethotoin. Nine of these products were identified by comparison of their retention times and mass spectra with those of authentic synthetic samples. Hydroxylation of the hydantoin ring at the 5-position produced 5-hydroxyethotoin and 5-hydroxy-5-phenylhydantoin. Aryl hydroxylation resulted in the formation of p-hydroxyethotoin, o-hydroxyethotoin, m-hydroxyethotoin, 3-methoxy-4-hydroxyethotoin, and 3,4-dihydroxyethotoin. Most of these were excreted as the glucuronide conjugate. A dihydrodiol of ethotoin and 3-ethyl-5-hydroxy-5-(4-hydroxyphenyl)hydantoin were isolated along with unchanged ethotoin and 5-phenylhydantoin. 2-Phenylhydantoic acid was also isolated and was shown to have the (R)(-)-configuration.

Integrated method for the gas chromatographic determination of antiepileptic drugs in human plasma.

The principles of three independent extraction methods were utilized to develop an integrated extraction scheme for use in routine therapeutic monitoring of seven antiepileptic agents. The final method, in which the three extraction methods were interfaced, permitted routine monitoring in a single 1 ml volume of human plasma of any one or combination of the following drugs: phenytoin (PHT), phenobarbital (PB), primidone (PD), 5-ethyl-5-phenylhydantoin (EPH), ethosuximide (ES), carbamazepine (CBZ), and valproic acid (VPA). An on-column methylation technique was used for simultaneous determination of PHT, PB, PD, EPH, and ES. CBZ and VPA were determined by independent methods as the underivatized compounds. Six appropriate internal standards were employed in the integrated method for quantitation of the drugs.

Determination of 5-(p-hydroxyphenyl)-5-phenylhydantoin and studies relating to the disposition of phenytoin in man.

A gas chromatographic on-column methylation technique was developed for the routine laboratory determination of 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), the principal urinary product of phenytoin )PHT) metabolism in man. 5-(p-Hydroxyphenyl)-5-(p-tolyl)hydantoin (HMPPH), a new internal standard, was synthesized and evaluated against 5-phenyl--5-(p-tolyl)hydantoin (MPPH), the compound normally used as the internal standard in p-HPPH assays. HMPPH withstood the challenges of intralaboratory quality control checks and tests of precision of p-HPPH values at times when MPPH provided erratic and unreliable values. Both an enzyme and acid treatment of urine were studied for the purpose of hydrolysis of p-HPPH-glucuronide, the form in which p-HPPH is excreted in urine. The use of both treatments in studies of three different patient urines pointed to the conclusion that acid-catalyzed decomposition of PHT dihydrodiol, a minor urinary metabolite of PHT in man, was unimportant from the analytical point of view, contributing little if anything to total urinary p-HPPH content. Some aspects of PHT disposition, as evidenced by studies of PHT plasma levels and p-HPPH urinary outputs in individual patients, are discussed.

Gas chromatographic on-column methylation technique for the simulataneous determination of antiepileptic drugs in blood.

An on-column methylation technique (OCMT) is described for the simultaneous, gas chromatographic determination in blood of ethosuximide (ES), phenobarbital (PB), primidone (PD), phenytoin (DPH), and 5-ethyl-5-phenylhydantoin (EPH). Multiple internal standards are employed in the OCMT, in order to eliminate or to minimize greatly error sources common to the technology of gas chromatography and to compensate for the different chemical dispositions of the antiepileptic drugs in an OCMT. The internal standards used in the OCMT were as follows: alpha,alpha,beta-trimethylsuccinimide (TMS) was used for the determination of ES; 5-ethyl-5-para-tolylbarbituric acid (MPB), for PB; 5-ethyl-5-(para-tolyl) hexahydropyrimidine-4,6-dione (MPD), for PD; and 5-(para-tolyl)-5-phenylhydantoin (MPPH), for EPH and DPH. The use of ether, the buffering of the plasma sample at pH 7.6, and the use of dilute (0.30-0.35 M) trimethyl-phenylammonium hydroxide (TMPAH) contributed to the specificity of the extraction scheme of the OCMT. The precision and accuracy of the OCMT was attributed to the use of appropriate, multiple internal standards. A method is described for the preparation of standard solutions of drugs in blank plasma (biological matrix) and for the use of the standards in calibration and daily intra-laboratory control of the OCMT.

Buffer catalysis of the racemization reaction of some 5-phenylhydantoins and its relation to the in vivo metabolism of ethotoin.

Evidence is presented to show that an optical isomer of 5-phenylhydantoin is subject to racemization (interconversion) in different buffer systems. With phosphate buffers in the pH range of 6.0-7.5, it appears that the buffer-catalyzed racemization reaction is due solely to catalysis by divalent phosphate (general base catalysis). Other buffers studied include arsenate, imidazole, triethanolamine, and pyrophosphate. When 5-phenylhydantoin, the N-de-ethylated metabolite of ethotoin, was administered to dogs in an earlier investigation, the observation was made that somewhat more than the theoretical quantity (50 mole percent of the dose) of the substances recovered from urine had the R-configuration. The principal metabolite was (R)-(-)-2-phenylhydantoic acid, formed stereo-specifically in a ring-opening reaction of (R)-5-phyenylhydantoin by dihydropyrimidinase (EC 3.5.2.2). The results of the present in vitro study support the hypothesis that in vivo the interconversion of the optical isomers of 5-phenylhydantoin can be catalyzed by buffering components of the mammalian physiological system, and that the catalytic activities of the endogenous buffer components can account for the racemization and ultimate metabolism of the (S)-isomer of 5-phenylhydantoin by dihydropyrimidinase.