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The steady-state 72 h urinary excretion of various phenytoin metabolites has been measured in 10 epileptic women, whose plasma phenytoin concentrations relative to the phenytoin dose fell during pregnancy and rose again post-partum. In later pregnancy and post partum, a mean of 61.3% and 48.9%, respectively, of the total daily phenytoin dose was eliminated as 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). Even though p-HPPH accounts for not much more than half the total daily phenytoin dose, increased excretion of this metabolite sufficed to account for the elimination of the entire increase in the dose of phenytoin required during pregnancy. There was no definite increase in the excretion of any other (minor) metabolite measured. Thus pregnancy seems not to enhance uniformly the capacity of the various metabolic pathways of phenytoin.

A high-performance liquid chromatographic method for the determination of the main phenytoin metabolite, hydroxyphenytoin, in the urine of epileptic patients is described. The use of an automated column-switching technique greatly simplifies the pretreatment steps. Thereby, both time and chemicals are saved. The possibility of error arising during the several pretreatment steps is considerably reduced. Following acid hydrolysis of the hydroxyphenytoin glucuronic acid conjugate the sample is diluted with water and after centrifugation is injected onto the pre-column. After washing for a short time with water, the substances which were absorbed on the head of the pre-column were backflushed with water--acetonitrile as eluent onto the analytical column. Separation is achieved by gradient elution using an ODS reversed-phase column with a particle size of 5 microns.

A method is described for measuring, without derivatization, the concentrations of phenobarbital, p-hydroxyphenobarbital, carbamazepine and phenytoin in biological fluids of epileptic patients undergoing long-term therapy. This method uses, at an isothermal temperature, a special column packing (GP-2% SP-2510-DA on 100-120 mesh Supelcoport). The lower limit of detection for all substances analyzed is 1 microgram/ml of biological material. The recovery of the compounds is about 95%, the reproducibility of the method is good (coefficient of variation, 4.3%). The mass spectra confirm the identity of substances eluting from the column. There is no interference from other commonly used antiepileptic drugs or endogenous substances. The method has the advantages of high specificity, sensitivity and rapidity and it appears to be suitable for the routine monitoring of blood and urine concentrations in patients receiving multi-drug therapy.

Simple and rapid quantitative analysis utilizing high-performance liquid chromatography was performed to determine the concentration of the antiepileptic agent phenytoin, and its main metabolite hydroxyphenytoin, in human urine. For the purposes of simple and rapid determination, the commercially available Extrelut columns were used with diethyl ether and chloroform as extraction solvents. High-performance liquid chromatography was performed on a LiChrosorb RP-8 column, with a mobile phase of methanol-0.02% ammonium acetate (1:1). The internal standard was 5-(4-methylphenyl)-5-phenylhydantoin. The method was applied to patients' urine, to examine the influence of concomitant drugs. Also, the results obtained using the commercially available enzymatic immunoassay method were compared with those from the present method, and it was concluded that a simple and rapid microanalysis is possible with a high extraction ratio.

A new metabolite of 5,5-diphenylhydantoin has been found in human urine and in rat bile. This new metabolite arises by direct glucuronidation of 5,5-diphenylhydantoin (i.e. no prior aryl hydroxylation occurs) and is characterized as the N-3 glucuronide. This structure assignment is based on (a) study of mass spectra of permethyl, perdeuteriomethyl, N-methyl-per-O-deuteriomethyl and deuteriomethyl ester permethyl derivatives and (b) comparison of the reactions of the new glucuronide metabolite and 5,5-diphenyl-3-methylhydantoin with diazomethane. Prominent ions at m/e 322 and 378 in the mass spectrum of the permethylated derivative of the glucuronide metabolite arise via retro Diels-Alder reactions of the glycone ring.

The effect of nonlinear kinetics on the steady-state ratio of serum phenytoin (PHT) to total 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) was studied in epileptic patients. In each patient, the ratio increased in a nonlinear fashion in relation to both the PHT dose and the PHT serum level. Patients with longer apparent PHT half-lives (t50%) had a higher PHT/p-HPPH ratio. The t50% was either measured directly and found to correlate well with the patient's calculated Michaelis-Menten parameters (Vmax and Km), or was calculated from Vmax and Km values. A close linear correlation was found between the steady-state PHT/p-HPPH ratio and t50% values, interindividually and intraindividually, i.e., as the t50% increased with increasing PHT concentration in a given patient, the PHT/p-HPPH ratio increased in the same proportion. This indicates a good correlation between the overall Michaelis-Menten kinetics of PHT elimination and the PHT/p-HPPH ratio. A close linear correlation between a wide range of PHT doses and steady-state serum total p-HPPH levels was found, suggesting that when the PHT dose is lower than Vmax, the fraction of PHT undergoing parahydroxylation is constant. It is concluded that if, in a given patient, the p-HPPH level corresponds to the value expected from the PHT dose, it can be assumed that compliance and bioavailability are adequate and that the dose does not exceed Vmax. In that case, the PHT/p-HPPH ratio will provide a good estimate of the t50% of PHT at that particular PHT level.

The ratio of the concentration of the phenytoin metabolite hydroxyphenyl phenylhydantoin (HPPH) to the creatinine concentration in morning urine samples was used as an index of compliance in adult epileptic patients on phenytoin therapy. For this purpose, the phenytoin dose was estimated from the urinary HPPH/creatinine ratio with the help of a regression equation that took into consideration body weight, height, age, and sex. A high correlation (r = 0.927 for men and r = 0.954 for women) between the prescribed and estimated dose was found for a reference group (127 hospitalized patients on supervised drug regimen). Compliance was judged from the discrepancy between the prescribed and the estimated dose for 45 patients immediately after their admittance to an epilepsy clinic, as well as for 98 outpatients, who had been treated by neurologists. Noncompliance was suspected in 6.7% of patients admitted to the clinic, as well as in 28.6% of the outpatients.

A gas-liquid chromatographic (GLC) assay utilizing an on-column methylation technique has been developed to assay urinary concentration of a phenytoin (5,5-diphenylhydantoin, PHT) metabolite, 5-(4-hydroxy-3-methoxyphenyl)-5-phenylhydantoin (MCAT). Assay of MCAT in 35 24-h urine samples from patients receiving chronic phenytoin (PHT) therapy indicated that 0.3-4.0% of the daily dose could be accounted for as MCAT. The GLC assay was specific for MCAT, and provided a minimal estimate of the fraction of the PHT dose being metabolized via the catechol/MCAT route. Multiple regression analyses indicated that the amount of urinary MCAT was dependent on the quantities of both dihydrodiol (DHD) and p-phenolic (p-HPPH) metabolites. Oxidative pathways involving both DHD and p-HPPH as substrates appear to be responsible for catechol/MCAT production.

An analytical method for determining phenytoin and its metabolites in the urine of epileptic patients is described. The analysis was performed for the m- and p-isomers of hydroxyphenytoin, the oxidative products of phenytoin, using gas chromatography. As an internal standard, 5-(4-methylphenyl)-5-phenylhydantoin was chosen; the extraction solvent from human urine was ether-chloroform (3:7). Phenytoin and its hydroxy isomers were satisfactorily determined by the modified on-column methylation technique on a 2% OV-17 column using temperature programming from 180 degrees C to 240 degrees C at 5 degrees C/min.