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Clozapine and its two major metabolites, N-desmethylclozapine and clozapine N-oxide were quantified using a high-performance liquid chromatographic method with UV detection in dog plasma following a single dose of clozapine. The analysis was performed on a 5-micrometer Hypersil CN (CPS-1; 250x4.6 mm) column. The mobile phase consisted of acetonitrile-water-1 M ammonium acetate (50:49:1, v/v/v), which was adjusted to pH 5.0 with acetic acid. The detection wavelength was 254 nm. A liquid-liquid extraction technique was used to extract clozapine and its metabolites from dog plasma. The recovery rates for clozapine, N-desmethylclozapine, and the internal standard (I.S.) were close to 100% using this method. The recovery rate for clozapine N-oxide (62-66%) was lower as expected because it is more polar. The quantitation limits for clozapine, clozapine N-oxide, and N-desmethylclozapine were 0.11, 0.05 and 0.05 microM, respectively. Intra-day reproducibility for concentrations of 0.1, 1.0 and 5.0 microM were 10.0, 4.4 and 4.2%, respectively, for N-oxide; 11.2, 4.3 and 4.9%, respectively, for N-desmethylclozapine; and 10.8, 2.2 and 4.9%, respectively, for clozapine. Inter-day reproducibility was15% for clozapine N-oxide,8% for N-desmethylclozapine and19% for clozapine. This simple method was applied to determine the plasma concentration profiles of clozapine, N-desmethylclozapine and clozapine N-oxide in dog following administration of a 10 mg/kg oral dose of clozapine.

Retrospective analyses were carried out on 22 analytes involved in nine bioequivalence studies on conventional release products, in which 90% confidence intervals were calculated for partial areas under the plasma concentration versus time curves (AUCs) from the first sampling time to AUC last , The use of partial areas up to 2× the maximum t max among all subjects in any given study gave the same bioequivalence decisions as AUC last .

A bioequivalence study was carried out to compare the bioavailabilities of two formulations containing racemic hydroxychloroquine. The study was based on a single phase parallel design because the enantiomers of hydroxychloroquine have very long terminal phase half lives. A group of n = 72 healthy young men were randomly assigned to receive a single dose of either test ( n = 36 subjects) or the reference ( n = 36 subjects) formulation after which serial blood samples were harvested over 77 days. The two products were found to be bioequivalent in that 90% confidence intervals fell entirely within bioequivalence limits of 80–125% for the log transformed parameters C max and AUC 4 whether calculated from non-chiral or enantioselective blood concentrations. Similarly, 90% confidence intervals for the partial areas from AUC 4 through AUC Inst all fell within 80–125%. These data suggest that enantioselective assays were not required and that blood samples need only have been harvested up to 12 h.

There are few archival data on the impact of stereoisomerism on bioequivalence studies. A bioequivalence study was carried out on two formulations of doxepin containing 15% of the active cis isomer and 85% of the less active trans isomer. Plasma concentrations of cis N-desmethyldoxepin remained at approximately 15% of total in only 2 of 30 healthy volunteers who completed the study. In the remaining 28 subjects, however, the proportion of cis N-desmethyldoxepin steadily increased with time such that by 72 h post dose, cis N-desmethyldoxepin comprised approximately 90% of the metabolite present in plasma. Nevertheless, the 90% confidence intervals (In AUClast, In Cmax and In Cmax/AUClast) for N-desmethyldoxepin fell entirely within bioequivalence li whether stereoselective or non-stereoselective data were analyzed. Thus the results of this study did not support arguments in favor of the use of stereoselective methods in bioequivalence studies.

Sprague Dawley rats (n = 16) were injected intramuscularly with fluphenazine decanoate (78 mg/kg) in sesame oil. Groups of four rats were surgically prepared on days 1, 7, 15 and 32 post-injection and samples of lymph obtained from the thoracic duct. Fluphenazine was found in similar concentrations in lymph and plasma, although the decanoate was not detected in either body fluid. These data suggest that the lymphatic system plays a role in the presystemic absorption of fluphenazine after i.m. administration of fluphenazine decanoate in the rat. In vitro experiments showed that fluphenazine decanoate was very rapidly hydrolyzed when incubated with rat plasma ( t 1 2 0.6 h ) or muscle homogenates ( t 1 2 1.0 h ). Rates of hydrolysis of fluphenazine decanoate were lower in dog muscle homogenates ( t 1 2 4.3 h ), and very much lower when incubated with plasma from dog 42.9 h) or humans ( t 1 2 50.9 h).

A bioequivalence study on perphenazine/amitriptyline combination compared three formulations (TST-1, TST-2 and REF) containing the same nominal dose of perphenazine, while TST-2 contained 20% less amitriptyline than the other two formulations. ANOVA-SNK on log-transformed partial areas AUC024 through AUC0∞ showed the intra-subject variabilities of perphenazine to be consistently higher than those of amitriptyline or nortriptyline. There was also a marked increase in intra-subject variability for perphenazine on extrapolating AUC0last to infinity. Thus, whhile the 1–2α confidence intervals (90% CIs) and ratios of geometric means for perphenazine fell within preset limits of 80–125% in all pairwise comparisons, the CIs for AUC0∞ violated the upper limit in comparisons of TST-2 and REF (or TST-2 and TST-1). This outcome could be attributed to high intra-subject variability on AUC0∞. ANOVA-SNK of amitriptyline identified a significant difference in extent of amitriptyline absorbed from TST-2 compared with TST-1 and REF. Thus, in pairwise comparisons between TST-1 and REF, all 90% CIs for amitriptyline partial areas AUC04 through AUC0∞ fell within 80–125% limits. In corresponding comparisons between TST-2 and REF (or TST-2 and TST-1), however, the lower limit of the 90% CIs for AUC01 through AUC0last consistently fell below the 80% cut off. By contrast, 90% CIs associated with the ratios Cmax/AUC04 through Cmax/AUC0∞ all fell within preset limits indicating no differences in absorption rate of amitriptyline, consistent with the theoretical argument that Cmax/AUC is influenced by rate but not extent of absorption. Thus the partial area method is applicable to the evaluation of both relative rate and extent of absorption from conventional release products. Similarly, analyses of nortriptyline data showed that the partial area method is also effective in the evaluation of rate and extent of formation of a metabolite in a bioequivalence study. The partial area method permits a greater number of plasma samples to be devoted to accurate estimation of tmax and Cmax since the necessity to estimate AUC0∞ is obviated.

Chlorpromazine N-oxide, fluphenazine N 4 ’-oxide, prochlor-perazine N 4 ’-oxide, sulforidazine N -oxide, and trifluoperazine N 4 ’-oxide were synthesized by oxidation of the designated nitrogen atom in the N- 10 side chain of the respective parent drug with 3-chloroperoxybenzoic acid. In the case of trifluoperazine, a stepwise increase in the amount of oxidant yielded the N 1 ,N 4 -dioxide and N 1 ’, N 4 ’,S-trioxide. The N’,S- dioxides of chlorpromazine and sulforidazine were obtained by hydrogen peroxide oxidation of the appropriate parent drug.

1. Two pilot studies were carried out to investigate the enantioselective pharmacokinetics of methylphenidate (MPH) in children with attention deficit-hyperactivity disorder (ADHD). A more definitive study, which included administration of an intravenous dose, was carried out in healthy young men. 2. Serial plasma samples were harvested from predose to 8 hours in the first pilot study, predose to 12 hours in the second pilot study and predose to 16 hours in the definitive study. Plasma levels of the separate isomers d-MPH and 1-MPH were determined by an enantioselective gas chromatographic method. 3. In the first pilot study, 6 boys with ADHD each received his regular dose of MPH (10mg n = 5, 5mg n = 1), which contained equal proportions of d-MPH and 1-MPH in an immediate release formulation (MPH-IR). No MPH was detectable in the predose plasma. Thereafter, plasma levels of the more active d-MPH were 4 to 10 fold higher than those of 1-MPH. Plasma levels of 1-MPH were so low that it was not possible to monitor them beyond 4 hours in some children. 4. In the second pilot study, 5 boys and 1 girl with ADHD each received their regular dose (20mg) of a slow release formulation (MPH-SR). No MPH was detectable in the predose plasma. Thereafter, plasma levels of the more active d-MPH were 5 to 10 fold higher than those of 1-MPH. It was possible to monitor plasma levels of 1-MPH over the full 12 hour period of study in 5 of the 6 children.(ABSTRACT TRUNCATED AT 250 WORDS)

The comparative bioavailability of a new tablet formulation of fluphenazine dihydrochloride (5 mg) and a reference product (fluphenazine dihydrochloride, Prolixin, 5 mg) was assessed in drug-free psychiatric patients. Twenty-six patients were initially entered in the study, of whom 22 completed the protocol. Each patient received the test (T) and the reference formulation (R) in a balanced two-way crossover design. Plasma concentrations of fluphenazine were monitored over a period of 48 h after drug administration using a sensitive HPLC method. One patient did not show any measurable plasma concentration for one formulation at any sampling time and, therefore, bioavailability was assessed in the remaining 21 patients. All pharmacokinetic parameters showed wide intersubject variation. The maximum plasma concentration (Cmax), time to Cmax, and area under the curve up to the last measurable concentration (AUClast0), infinity (AUCinfinity0), or truncated areas (such as AUC16(0), AUC24(0) were compared by analyses of variance and found not to be significantly different in each case across the formulations. Except for AUC24(0), AUC32(0), and AUC48(0), ANOVA of all other parameters showed a high power (greater than 80%) to detect a 20% difference in the mean value of each bioequivalence parameter between T and R. The two formulations were found to be bioequivalent in that confidence intervals of the mean values of AUCinfinity0, AUClast0, truncated AUCs, or Cmax for T:R ratios were, in each case, well within the acceptable range of 100 +/- 20%.

To the Editor: Attention-deficit hyperactivity disorder (ADHD) in children is characterized by varying degrees of inattention, impulsiveness, and hyperactivity. Many children with ADHD respond well to treatment with the amphetamine derivative dl-threo-methylphenidate (dl-MPH). The drug is marketed as a racemic mixture, although d-MPH has been reported to be more active than l-MPH. Moreover, recent studies have shown that plasma levels of d-MPH were higher than those of the l-antipode in children with ADHD under medication with dl-MPH in the form of either instantaneous-release (Ritalin), or sustained-release (Ritalin SR) dosage forms.

Dose proportionality in some pharmacokinetic parameters for thioridazine and its two active metabolites (mesoridazine and sulforidazine) was investigated in 11 healthy human subjects following oral administration of three single doses (25, 50, and 100 mg) of thioridazine hydrochloride separated in each case by an interval of two weeks. Also, after a further two weeks, another 100-mg dose of thioridazine (divided as 5 mg every 0.5 h) was administered to all the volunteers to investigate the effect of a slow rate of dosage input on the pharmacokinetic parameters of this drug. An HPLC method was used to measure concentrations of thioridazine, mesoridazine, and sulforidazine in plasma samples collected up to 72 h following each dose. Dose proportionality for the three single doses of thioridazine was observed for all three analytes in the area under the plasma concentration versus time curves (AUC infinity 0 or AUCt0) and the maximum plasma concentration (Cmax) in that the relationships between the dose and these parameters were each describable by an equation for a straight line (r2 greater than or equal to 0.8). However, the mean apparent distribution and elimination rate constants for thioridazine and mesoridazine and the mean apparent oral clearance for thioridazine decreased significantly with increasing dose. This suggests nonlinear trends in the elimination kinetics at high doses of thioridazine. When a 100-mg divided oral dose of thioridazine was administered, no statistically significant differences between single and divided doses were observed in the mean AUC infinity 0 or AUCt0 for thioridazine or sulforidazine. A significant decrease in the mean AUC infinity 0 or AUCt0 was observed for mesoridazine after the administration of the divided dose.(ABSTRACT TRUNCATED AT 250 WORDS)

The individual enantiomers of chiral drugs may have profound differences in their pharmacologic and toxicologic properties. Their metabolism and pharmacokinetics may also differ markedly, and the presence of one enantiomer may inhibit the metabolism of the other. Stereospecificity is required in analytical methods, whether these be chromatography or radioimmunoassay.

Sensitive and enantioselective gas chromatographic assays have been developed and applied to the quantitation in human urine of the enantiomers of methoxyphenamine and its three primary oxidative metabolites, namely, N-desmethylmethoxyphenamine, O-desmethylmethoxyphenamine and 5-hydroxymethoxyphenamine. The separation of the various analytes was achieved through the combined use of high-resolution gas chromatography coupled with electron-capture detection and employing a capillary OV-225 column. The formation of diastereometric derivatives involved the chiral acylating reagent N-heptafluorobutyryl-L-prolyl chloride. The assays for methoxyphenamine and O-desmethylmethoxyphenamine were linear over the range 0.25-2.0 micrograms/ml for each analytes' enantiomers, while in the case of the enantiomers for N-desmethylmethoxyphenamine and 5-hydroxymethoxyphenamine linearity was shown over the ranges 0.094-0.75 and 0.188-1.5 micrograms/ml, respectively. The mean coefficients of variation in all cases were less than 4%.

Haptens were prepared by the reaction of d-ephedrine or l -ephedrine with methyl acrylate and subsequent alkaline hydrolysis of the methyl ester groups. The haptens were coupled to bovine serum albumin by a mixed anhydride method, and the resulting drug-protein conjugates were used to immunize rabbits. Antisera raised to these conjugates were highly stereospecific. Neither antiserum cross-reacted with the optical antipode of its substrate nor with racemic pseudoephedrine. Separate radioimmunoassays (RIAs), developed for d-ephedrine and 1-ephedrine, were used to measure the concentrations of the enantiomers of ephedrine in the blood of two volunteers dosed with racemic ephedrine. The RIAs were validated by comparing the sum of the concentrations of the enantiomers, determined by RIA, with total ephedrine concentrations determined by a nonstereoselective GLC-ECD method.

It has been shown previously that immediate-release dl-threo-methylphenidate (Ritalin) undergoes stereoselective disposition in human adults as well as in children with attention deficit-hyperactivity disorder. Although the sustained-release formulation of dl-threo-methylphenidate (Ritalin-SR) has been demonstrated to be effective in sustaining the attention of the children with attention deficit-hyperactivity disorder, there are no data on plasma levels of methylphenidate after administration of the sustained-release formulation. The purpose of this present investigation was twofold: (1) to determine whether the levels of methylphenidate were sustained for over a time period of 8 h, and (2) to examine enantioselective aspects of the pharmacokinetics following the ingestion of the sustained-release formulation. Six children with attention deficit-hyperactivity disorder were given 20 mg of sustained-release dl-threo methylphenidate. Plasma samples were harvested for a period up to 12 h following ingestion of the drug. The levels of both the enantiomers were sustained for a period of 8 h (the plasma levels of d-methylphenidate were 8- to 10-fold higher than those of the l-enantiomer in the profiles of all six children). Mean areas under the plasma level time curves were 132.78 +/- 92.47 ng.h/mL for d-methylphenidate and 12.73 +/- 7.37 ng.h/mL for l-methylphenidate. The values of oral clearance and apparent volume of distribution calculated for l-enantiomer were higher than the corresponding values for the d-antipode.

An enantioselective gas chromatographic quantitation assay was developed for the enantiomers of dl-threo -methylphenidate in plasma and urine. dl-threo -Methylphenidate and the internal standard were acylated with N-heptafluorobutyryl-1-prolylchloride under Schotten—Baumann conditions prior to gas chromatographic separation on achiral mixed stationary phases. The derivatives were detected by means of a nitrogen—phosphorus detector. Linear and reproducible calibration curves were obtained over the concentration ranges 0.43–43.25 and 2.16–216.25 ng/ml enantiomer in plasma or urine, respectively. This enantioselective gas chromatographic quantitation assay was applied in a single oral dose disposition study of dl-threo -methylphenidate in a healthy adult volunteer. Stereoselective differences were observed in the plasma concentration—time profiles and cumulative urinary excretion profiles following oral doses of 20 and 40 mg of dl-threo -methylphenidate hydrochloride. Only d-threo -methylphenidate was detectable in plasma after 4 h.

The binding of sulfadimethoxine to selected human blood protein fractions and to fresh serum has been examined by means of a new equilibrium dialysis technique which minimizes experimental error and permits the evaluation of low-level binding. Certain alpha-globulin fractions, containing mixtures of proteins, were found to bind the drug. Scatchard analysis of the binding of sulfadimethoxine to fresh serum, calculated as though all of the binding is due to albumin, gives a different result from that obtained with isolated albumin. This may be a reflection of the contribution of the alpha-globulins to the overall binding of sulfadimethoxine in fresh serum. Although sulfadimethoxine is amphoteric, it did not bind to the alpha 1-acid glycoprotein. The drug behaves as an acidic compound when binding to the blood proteins.

The bioavailability of a new tablet formulation (5 mg) of haloperidol was estimated relative to two lots of a reference product. Twenty-eight healthy male volunteers completed all three phases in that they received the test (T) and the two reference formulations (R1 and R2) in a balanced three-way crossover design. Using a sensitive HPLC method, plasma concentrations of haloperidol and reduced haloperidol were monitored over a period of 96 h following administration of each formulation. Haloperidol was measurable in the plasma of all the volunteers, whereas reduced haloperidol was measurable in only 6 out of 28 volunteers following each administration. Therefore, the assessment of bioequivalence in this study is based on haloperidol data only. The maximum plasma concentration (Cmax), time to Cmax (tmax), and area under the curve up to the last measurable concentration (AUCot) or infinity (AUCo infinity) were compared by analyses of variance and found not to be significantly different across the formulations. The relative bioavailability based on T:R1 or T:R2 ratios of AUCo infinity, AUCot, and Cmax was, in each case, within the acceptable range of 100 +/- 20%. Also, the relative bioavailability of R1 compared with R2 was within 100 +/- 20% in terms of the above bioavailability parameters. Except for tmax, all other pharmacokinetic parameters showed wide intersubject variation.