The bioequivalence of highly variable drugs and drug products.

'Highly variable drugs' have been defined as those drugs for which the within-subject variability (WSV) equals or exceeds 30% of the maximum concentration (Cmax) and/or the area under the concentration versus time curve (AUC). Despite the fact that highly variable drugs are generally safe with flat dose response curves, the bioequivalence of their formulations is a problem because the high variability means that large numbers of subjects are required to give adequate statistical power. Highly variable drug products are poor quality formulations where high within-formulation variability (e.g. tablet to tablet variability) poses a problem rather than high innate WSV of the drug itself. A further problem caused by high variability is that a subset of the population may respond differently to the two formulations producing a significant subject x formulation interaction. Practical examples are shown using replicate designs. The methods proposed to deal with the problems posed by highly variable drugs include: (i) Drug regulatory jurisdictions states that the 90% confidence interval (90% CI) around the test to reference geometric mean ratio (GMR) is required to fit with bioequivalence acceptance limits of 0.8 - 1.25 for both Cmax and AUC. The WSV for single point estimation of Cmax is often greater than that for AUC. One strategy therefore is not to require a 90% CI for Cmax of drugs that do not exhibit a toxicity associated with Cmax and merely require the GMR to fall within the acceptance limits. (ii) To arbitrarily broaden the bioequivalence acceptance limits. For example, to permit a sponsor to justify the use of wider limits e.g the 90% CI around the GMR of Cmax values might be required to fit within acceptance limits of 0.75 - 1.33 or even 0.70 - 1.42. (iii) A more systematic approach would be to broaden the acceptance limits by scaling to either the residual variance from a 2-period design or to the WSV of the reference product in a replicate design. Subsequent evaluations of scaling procedures have demonstrated that smaller numbers of subjects are required for bioequivalence studies on formulations of highly variable drugs. A disadvantage of scaling is that the method is less sensitive to differences between the means compared with unscaled treatment, such that the GMR may prove to be unacceptably low or high. This possibility has let to a suggestion that the GMR must fall within acceptance limits of 0.8 - 1.25 in scaled treatments. (iv) A similar method is to scale the metric rather than the acceptance limits. This method was proposed by the United States' Food and Drug Administration in the context of Individual bioequivalence, but may also be applied (v) to average bioequivalence. (vi) To carry out bioequivalence studies at steady state whenever a multiple dose regimen is ethically acceptable for healthy volunteers. This solution is based on the observation that high variability in a single dose study tends to be dampened at steady state, thus increasing statistical power. Drug regulators have not favored this approach on the grounds that bioequivalence testing should be based on the most discriminating test possible. (vii) Finally the use of metabolite data has been proposed since in many (but by no means all) cases, metabolite is less highly variable than that of the parent drug. This subject remains controversial except when the administered substance is a prodrug which converted by metabolism into the active drug.

Exposure measures applied to the bioequivalence of two sustained release formulations of bupropion.

OBJECTIVE: To examine the effect of the exposure measures C(max) (peak exposure), AUC(E) (early exposure) and AUC (total exposure) on the bioequivalence of two sustained release formulations of bupropion (i) in the fasted state and (ii), after a high fat meal. The ratio C(max)/AUC (sensitive to rate of absorption) was also evaluated. METHODS: A two-formulation, two-sequence, four-period replicate design study was performed in 29 healthy men and women after an overnight fast. Similarly, a two-period study was performed in 20 healthy men and women after ingestion of a high fat breakfast. Plasma concentrations of bupropion were measured by HPLC/MS/MS and the data were analyzed (SAS PROC MIXED) by the Schuirmann-Sattersthwaite procedure (four-period study) and by the two one-sided test procedure (SAS PROC GLM) (two-period study). Standard bioequivalence limits of 80 - 125% were applied to all measures including AUC(E) and C(max)/AUC. RESULTS: In the fasting study, the mean plasma concentration vs. time curves from (including over the first 24 hours) following the two administrations of each formulation were similar although there was a significant difference in median t(max) between formulations. This may have contributed to a low estimate of geometric mean ratio (GMR) for AUC(E) (69%) which was judged to have failed bioequivalence. There was also rather low GMRs for Cmax (88%) and C(max)/AUC (89%) but these measure passed because the within-subject variabilities (WSV) were relatively low (19.6% and 11.2%, respectively). Total exposure (AUC(last)) met standard bioequivalence limits of 80 - 125% easily. The raw data from the two-period fed also showed differences in the shapes of the plasma concentration vs. time curves around C(max) although there was no difference in median t(max). The WSV at median t(max) was high (34%) as was the GMR (117%) for AUCE which failed, as did C(max) (GMR 112%). The WSV was very high at early time points before settling into a "plateau" at about 11%. DISCUSSION: There was no "spike" in the plasma concentration vs. time profiles up to median t(max) or beyond and therefore there was no evidence of dose dumping of the test formulation in either fasted or fed states. No bioequivalence limits have been set for AUC(E) but the application of standard BE limits of 80 to 125% meant that the fed study was clearly underpowered given the high WSV at early time points. CONCLUSIONS: More research is needed on the interesting concept of early exposure. The WSV is often high at median t(max) which means that standard bioequivalence limits of 80 - 125% may be inappropriate. Despite the lack of dose dumping, application of AUC(E) to the fasting study, would have resulted in failure to declare bioequivalence since the GMR for this measure was only 69.5%. Application of a 90% confidence interval to AUC(E) to the fed study would have required powering to cope with the fact that this measure was highly variable.

Absolute bioavailability and stereoselective pharmacokinetics of doxepin.

1. Commercial doxepin contains geometric isomers in the proportions Z:E = 15:85. Z-doxepin and its metabolite Z-N-desmethyldoxepin are both active antidepressants, whereas the corresponding E-isomers are less active therapeutically. 2. The present pharmacokinetic study was a balanced, randomized, two-treatment, two-period, two-sequence crossover design in which 12 healthy male volunteers were given single doses of commercial doxepin intravenously and orally on two occasions separated by a washout period. 3. A two-compartment model with no lag time and first-order elimination fitted the plasma concentration-time curves after intravenous dosing. Pharmacokinetic parameters estimated from the model were comparable with those estimated by non-compartmental methods. 4. All pharmacokinetic parameters displayed a wide between-subject variability. Both isomers of doxepin showed large volumes of distribution and relatively short half-lives in plasma, suggestive of extensive distribution and/or tissue binding. The mean fraction absorbed after oral administration was 0.29 for each isomer. Renal clearances of each isomer were very low after either oral or intravenous dosing, although all four analytes were quantifiable in the urine for prolonged periods. 5. After oral dosing, plasma concentrations of the doxepin isomers remained roughly in the ratio Z:E = 15:85, whereas those of N-desmethyldoxepin were closer to 1:1 in all but two outliers, who had high levels E-N-desmethyldoxepin.

Effects of food on the pharmacokinetics of methylphenidate.

PURPOSE: To test the hypothesis that the pharmacokinetics of d-methylphenidate (d-MPH) would be altered by food ingested before administration of an immediate release formulation (dl-MPH- IR) but not when food is ingested before a slow release formulation (dl-MPH-SR). METHODS: A randomized, four-phase, open label, crossover design was conducted in 24 healthy men who each received, on separate occasions, dl-MPH-IR and dl-MPH-SR taken after an overnight fast and 15 min after a standardized breakfast (20% protein, 21% fat, 59% carbohydrate). Plasma MPH levels were monitored by a validated, stereoselective. GLC-ECD method. RESULTS: For plasma d-MPH, there were significant differences (ANOVA) between dl-MPH-IR and dl-MPH-SR in tmax, Cmax (peak exposure), and Cmax/AUC (sensitive to rate of absorption). Dl-MPH-SR on average delayed tmax from 2.3 to 3.7 h and lowered Cmax 34%. There was no significant difference between the formulations in AUC (extent of absorption). For dl-MPH-IR, food significantly increased Cmax (23%) and AUC (15%) and for dl-MPH-SR the corresponding increases were Cmax (17%) and AUC (14%). After dl-MPH-IR, food delayed average tmax from 2.0 to 2.5 but had no effect on tmax after dl-MPH-SR. There was no effect of food on Cmax/AUC (rate of absorption). CONCLUSIONS: Food caused a significant increase in extent of absorption but had no effect on rate of absorption of d-MPH after either dl-MPHIR or dl-MPH-SR.

Evaluation of the bioequivalence of highly-variable drugs and drug products.

PURPOSE: To establish procedures for the effective evaluation of bioequivalence (BE) for highly-variable drugs and drug products (HVD/P). METHODS: 2- and 4-period crossover BE studies with 24 subjects were simulated which generally assumed within-subject coefficients of variation of 40%. The relationship between the fraction of studies in which BE was accepted (the statistical power) and the ratio of geometric means (GMR) of the two formulations was evaluated for various methods of analysis. These included, primarily, scaled average BE (ABE), the corresponding approach of expanding BE limits (BEL), and, for comparison, unscaled ABE and scaled individual BE (IBE). RESULTS: Scaled ABE and expanding BEL showed very similar properties in both 2- and 4-period studies. They had steeper power curves than scaled IBE. Unscaled ABE had very low statistical power. The acceptance of BE by unscaled and scaled ABE and expanding BEL was almost independent of subject-by-formulation interaction and the ratio of within-subject variations of the two formulations. By contrast, the conclusions reached by scaled IBE were strongly affected by these parameters. CONCLUSIONS: Scaled ABE and expanding BEL evaluate BE effectively for HVD/P in both 2- and 4-period investigations. However, additional, useful information can be obtained from 4-period studies.

Dose staggering as a strategy to reduce drug--drug interactions due to reversible enzyme inhibition between orally administered drugs with high first pass effect: a computer simulation study.

A physiological computer model was designed to simulate the metabolic drug-drug interactions between two orally co-administered drugs due to reversible enzyme inhibition using drug concentrations in the portal vein. The extent of interactions was compared at steady-state for the effects of a delay in time between the administration of the substrate and the inhibitor. It was demonstrated that the extent of the interactions can be strongly affected by a time interval between the two drug administrations. By delaying the administration of the inhibitor until after the absorption phase of the substrate, one can significantly reduce the extent of the drug--drug interactions. This is because drug concentrations in the portal vein and the liver are much higher than that in the systemic circulation during the absorption phase. The model also showed that interactions involving substrates with a high extraction ratio (E(H)), i.e., drugs with higher first-pass effect, can be more strongly affected by dose staggering. Substrates with a low absorption rate constant (k(a)) require a longer interval with the inhibitor in order to reduce the extent of the interactions. This observation suggests dose staggering as a simple and cost-effective way to reduce the extent of unwanted drug--drug interactions in clinical practice.

Prescribability and switchability of highly variable drugs and drug products.

At the AAPS/FDS Workshop (Crystal City, Arlington, VA, March 6-8, 1995), it was agreed that some form of scaling should be permitted for highly variable drugs, although there was no agreement on a method. Currently, much emphasis is focused on developing a practical methodology for individual bioequivalence (IBE) and population bioequivalence (PBE) to replace or complement average bioequivalence (ABE). The latter requires only the mean bioavailabilities of two formulations to be sufficiently similar, whereas PBE also considers their distributions. IBE, on the other hand, is a comparison of the individual responses to the two formulations within subjects and is therefore concerned with switchability (interchangeability) between two multisource formulations. Multisource formulations refer (i) to generic copies of an innovator's formulation or (ii) to different formulations used in stages leading up to the final marked formulation. Evaluation of both PBE and IBE require replicate design studies. The FDA Working Group on IBE has been experimenting with methods in which a one-sided 95% confidence interval is computed based on the Bootstrap technique which ensures that the consumer risk is maintained at 5%. The IBE metric can then be scaled according to the within subject variance of the reference formulation. Thus if the variability of the test formulation (T) is higher than that of the reference (R), the formulation may fail IBE but not ABE. Conversely, if R is more variable than T, then the formulations may be considered to be IBE, even with a difference in means of more than 20%. Experimentation with existing data on our files shows that scaling has a considerable effect on the IBE decision for highly variable drugs. Evidence will also be presented to show that scaling makes the conditions more conservative for potent drugs with steep dose response curves reducing the risk of two generic formulations being BE with the same reference product but not BE with each other. On the other hand, broadening the BE limits for safe, highly variable drugs increases statistical power and reduces the number of subjects required. Even with the introduction of scaling, however, it is clearly difficult to obtain a single IBE criterion suitable to be applied to all drug products/studies.

The role of metabolites in a bioequivalence study II: amoxapine, 7-hydroxyamoxapine, and 8-hydroxyamoxapine.

BACKGROUND: Amoxapine is a dibenzoxazepine type tricyclic antidepressant. The mechanism of clinical action in patients is not well understood. In animals, amoxapine blocks the reuptake of norepinephrine and, to a lesser extent serotonin, into their respective neurons and blocks the response of dopaminergic receptors to dopamine. The major metabolite, 8-hydroxyamoxapine, has similar norepinephrine uptake inhibitory action to the parent drug, but has a more pronounced inhibitory action on serotonin uptake. Another major metabolite, 7-hydroxyamoxapine blocks post-synaptic dopamine receptors. SUBJECTS AND METHODS: The present study was a traditional two-treatment, two-period, two-sequence crossover design with the primary objective to investigate the average bioequivalence of two formulations of amoxapine. Secondary objectives were to explore the potential roles of metabolites and truncated (partial) areas in bioequivalence studies. Serial plasma samples were harvested from immediately pre dose to 96 hours post dose. The parent drug and the two hydroxy metabolites were monitored by validated HPLC procedures. Geometric mean ratios and 90% confidence intervals (90% CIs) were calculated for Cmax, AUCinfinity, the truncated areas of AUC, AUCinfinity/Cmax, and the truncated areas of AUC/Cmax. RESULTS: The results indicated that the two formulations were bioequivalent in terms of the conventional parameters Cmax and AUC for all three analytes in the sense that the 90% CIs fitted entirely within preset bioequivalence limits of 80-125%. Moreover, the 90% CIs for the truncated areas AUC2.0hr through AUClast and Cmax/AUC1.0hr through Cmax/AUClast of all three analytes also fell entirely within bioequivalence limits of 80-125%. It was concluded that it was unnecessary to have harvested plasma samples beyond 12 hours, in which case additional plasma samples could have been devoted to the more precise estimation of tmax and Cmax. CONCLUSION: Of the three analytes, test and reference individual plasma concentration versus time curves of 8-hydroxyamoxapine were more closely superimposable than those of the other two analytes. Any decision to use metabolite data in bioequivalence studies, however, must be made a priori to avoid introduction of bias arising from selective post hoc manipulation of the raw data; and to facilitate the design of blood sampling schedules based on prior information about the tmax of the selected analyte.

Pharmacokinetics of fluphenazine, a highly lipophilic drug, estimated from a pulse dose of a stable isotopomer in dogs at steady state.

The potential utility of a pulse dose of a deuterium-labeled isotopomer (FLU-D(4)) in elucidating the pharmacokinetics of fluphenazine (FLU) at steady state was investigated in dogs. The single-dose oral pharmacokinetics of FLU in dogs were established. After resting the dogs for 3 weeks, the animals were dosed to steady state with oral FLU administered at 12-h intervals. Following 15 doses, one dose of FLU was replaced by a pulse dose of FLU-D(4), after which dosing with FLU was resumed. FLU and FLU-D(4) plasma concentrations were determined by tandem mass spectrometry. Comparable estimates of apparent oral clearance were calculated from (i) a single dose of FLU, (ii) a pulse dose of FLU-D(4), and (iii) over a dosing interval at steady state. Average steady-state plasma concentrations were reliably predictable from a pulse dose of FLU-D(4).

Bioequivalence: switchability and scaling.

This study demonstrated that the mere fact that two multisource drug formulations are bioequivalent with the same reference formulation does not guarantee that they are bioequivalent with each other. The present unscaled bioequivalence limits (BEL) of 0.80 to 1.25 permitted far greater deviation from unity of the geometric mean ratio (GMR) for multisource formulations with low within-subject variabilities than for drugs with high variabilities. Scaling the BEL drastically reduced the maximum deviation from unity of GMRs for two multisource formulations each bioequivalent with the same reference product while broadening the BEL for highly variable drugs. It was concluded that scaling was consistent with the principle of switchability for toxic drugs with low variability and for safe, highly variable drugs. On the other hand, scaling need not be applied to safe drugs with low variability and should not be applied in the unusual case of a highly variable drug with a narrow therapeutic range.

The roles of depot injection sites and proximal lymph nodes in the presystemic absorption of fluphenazine decanoate and fluphenazine: ex vivo experiments in rats.

PURPOSE: The release and presystemic absorption of fluphenazine and its decanoate ester from intramuscular depots were investigated. METHODS: Rats were sacrificed in groups of three at various times after injection of drug or prodrug in sesame oil. Muscle tissues at the injection sites and various lymph nodes were excised. Blood (plasma) was harvested by cardiac puncture. RESULTS: Following administration of fluphenazine decanoate, the amount of prodrug at the sites of injection declined exponentially (half-life 3.4 days). Highest concentrations of drug and prodrug were found in iliac and hypogastric lymph nodes nearest to injection sites in which both analytes were detectable 28 days post dose. The half-life for the decline of fluphenazine from lymph nodes (4.6 days) was similar to that from plasma (4.3 days). Following administration of fluphenazine base, only 2.8% of the dose remained at the sites of injection after 2 days. Concentrations of drug in iliac and hypogastric lymph nodes were comparable to those in distal lymph nodes. Fluphenazine concentrations in the lymphatic tissues decreased at about the same rate as plasma concentrations. CONCLUSIONS: The rate limiting step appeared to be slow partitioning of the decanoate from oily deposits at the injection site and proximal lymph nodes with subsequent hydrolysis of the ester group.

Studies on the mechanism of absorption of depot neuroleptics: fluphenazine decanoate in sesame oil.

PURPOSE: The purpose of the present study was to investigate the pharmacokinetic characteristics of fluphenazine (FLU) and its decanoate (FLU-D) after intravenous and intramuscular administration to dogs. METHODS: A group of four beagle dogs was used in all intravenous and intramuscular experiments, with washout periods of no less than three months between doses. RESULTS: After intravenous FLU-D, the pharmacokinetics of the prodrug (mean +/- SD) were as follows: Clearance (CL) 42.9 +/- 6.3 L/h; terminal half-life (t1/2) 3.5 +/- 0.8 h; volume of distribution (Vd) 216 +/- 61 L. The fractional availability of FLU was 1.0 +/- 0.2. After intravenous FLU, the volume of distribution of FLU (51 +/- 17.8 L) was some 4 fold less than that of the prodrug. Simulations (Stella II) suggested that the rate limiting step was slow formation of FLU from the prodrug in the tissue compartment. After intramuscular FLU-D in sesame oil, the apparent t1/2 of FLU was 9.7 +/- 2.0 days whereas after intramuscular FLU base in sesame oil, the apparent t1/2 was only 7.7 +/- 3.4 h showing that the absorption of FLU itself from the intramuscular site and proximal lymph nodes is relatively rapid. CONCLUSIONS: The rate limiting step after intramuscular FLU-D appeared to be the slow partitioning of the prodrug out of the sesame oil at the injection site and in proximal lymph nodes.

alpha-Adrenergic activity and cardiovascular effects of besipirdine HCl (HP 749) and metabolite P7480 in vitro and in the conscious rat and dog.

Besipirdine displays potent adrenergic activity in a variety of pharmacological and behavioral tests. Based on this property, we evaluated the effects of besipirdine and its N-despropyl metabolite N-despropyl-besipirdine (P7480) on cardiovascular function in rats and dogs. Besipirdine and P7480 bind alpha-2 adrenoceptors (K(I): 380 and 10 nM, respectively) and facilitate the stimulated release of [3H]norepinephrine from rat cortical slices due to presynaptic autoreceptor blockade. In rat aorta rings and the pithed rat, P7480, but not besipirdine, also behaved as a postsynaptic alpha-1 adrenoceptor agonist. In conscious rats, besipirdine (2-10 mg/kg, p.o.) and P7480 (3-10 mg/kg, p.o.) produced dose-related increases in mean arterial pressure. Inhibition of hepatic cytochrome P-450 enzyme activity blocked the pressor effect of besipirdine, but not of P7480; therefore, P7480 mediated besipirdine's pressor effect. The bradycardia after either agent was unaffected. In conscious dogs, besipirdine (0.1-2 mg/kg, p.o.) also produced dose-related hypertension and bradycardia. The hypertension, but not the bradycardia, were sensitive to prazosin (3 mg/kg, p.o.), but not hexamethonium (10 mg/kg, p.o.). Muscarinic and beta-adrenergic receptor blockade studies in anesthetized dogs demonstrated the bradycardia to be due to withdrawal of cardiac sympathetic tone. These findings suggest that besipirdine's peripheral hypertensive effect is primarily mediated by the pressor metabolite P7480, although facilitated norepinephrine release may contribute. Besipirdine's bradycardic action appears to be centrally mediated, because both compounds lacked direct negative chronotropic activity on spontaneously beating guinea pig atria in vitro.

Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography.

Doxepin is a tricyclic antidepressant marketed as an irrational mixture of cis- and trans-geometric isomers in the ratio of 15:85. A convenient high-performance liquid chromatographic (HPLC) procedure for simultaneous quantitation of geometric isomers of doxepin and N-desmethyldoxepin in plasma and urine is described. The HPLC procedure employed a normal phase system with a silica column and a mobile phase consisting of hexane-methanol-nonylamine (95:5:0.3, v/v/v), a UV detector and nortriptyline as the internal standard. The liquid-liquid extraction solvent was a mixture of n-pentane-isopropanol (95:5, v/v). The limit of quantitation was 1 ng/ml for each isomer. The calibration curves were linear over the ranges 1-200 ng/ml (plasma) and 1-400 ng/ml (urine). In plasma, the accuracy (mean +/- S.D.) (97.53 +/- 1.67%) and precision (3.89 +/- 1.65%) data for trans-doxepin were similar to corresponding values for urine, i.e., 97.10 +/- 2.40 and 3.82 +/- 1.14%. Accuracy and precision data for trans-N-desmethyldoxepin in plasma were 97.57 +/- 2.06 and 4.38 +/- 3.24%, and in urine were 97.64 +/- 3.32 and 5.26 +/- 1.83%, respectively. Stability tests under three different conditions of storage indicated no evidence of degradation. The recovery of doxepin was 61-64% from plasma and 63-68% from urine. The method has been applied to analyses of plasma and urine samples from human volunteers and animals dosed with doxepin.

Sensitive method for the simultaneous measurement of fluphenazine decanoate and fluphenazine in plasma by high-performance liquid chromatography with coulometric detection.

A highly sensitive and specific high-performance liquid chromatographic method with coulometric detection was developed for the simultaneous assay of fluphenazine decanoate and fluphenazine in plasma. The extraction and sample clean-up procedures are simple, rapid to execute, yet yield chromatograms relatively free of any interference from endogenous plasma constituents, such that the extraordinary sensitivity of the coulometric detector can be exploited fully. This is the first analytical procedure for the simultaneous determination of fluphenazine decanoate and fluphenazine. The detection limits for both fluphenazine decanoate and fluphenazine were 0.1 ng/ml plasma and the limits of quantitation were 0.25 ng/ml plasma. Standard curves from 0.25 to 10 ng/ml were linear with coefficients of variation < 10%. The method was applied to measure plasma levels of fluphenazine decanoate and fluphenazine in patients under medication with 25-50 mg biweekly intramuscular (i.m.) injections of fluphenazine decanoate. It was possible to monitor the plasma levels of fluphenazine in all cases. Fluphenazine decanoate was present in measurable concentration in the plasma of 4 out of 5 patients who received biweekly i.m. injections of 50 mg fluphenazine decanoate. In a pilot experiment with a dog, the method was used to follow fluphenazine decanoate and fluphenazine plasma levels up to 13 days, at least, after i.m. single dose (10 mg/kg).

Stereoselective in vivo and in vitro studies on the metabolism of doxepin and N-desmethyldoxepin.

1. Doxepin is marketed as an irrational mixture of geometric isomers such that the more active Z-isomer comprises only 15% of the total doxepin whereas the less active E-isomer makes up the remaining 85%. 2. The ratio of isomers of the doxepin remains approximately Z:E = 15:85 in the plasma of depressed patients whereas plasma levels of the active Z-N-desmethyl metabolite are similar to those of E-N-desmethyldoxepin. 3. After examination of four animal species (dog, rabbit, guinea pig, rat), rat was closest to human in terms of the Z:E ratio of the geometric isomers of N-desmethyldoxepin excreted in the 0-24-h urine. 4. Changes in the urinary Z:E ratio of the metabolite were observed after oral but not after intravenous or intraperitoneal administration of commercial doxepin to rat. 5. There was no evidence of Z/E interconversion after administration of the pure isomers to rat in vivo, or after incubation of rat or human liver homogenates with pure isomers. 6. In vitro data suggested that the distortion of the Z:E ratio of N-desmethyldoxepin was a consequence of faster metabolism of the E-isomer in comparison with Z-N-desmethyldoxepin rather than 'enrichment' of the Z-isomer at the expense of the E-isomer.

Application of electrospray mass spectrometry in the identification of intact glucuronide and suplate conjugates of clozapine in rat.

1. The phase II metabolites in the bile, urine and faeces of rat dosed with clozapine were investigated by means of electrospray mass spectrometry (ESMS) in both positive and negative ion modes. 2. When operated at a cone voltage of 45 V, this soft ionization technique permitted the detection of quasi molecular ions of both sulphate and glucuronide conjugates of hydroxylated phase I metabolites of clozapine. With the cone voltage set at 90 V, however, the ESMS also contained highly diagnostic ions resulting from the loss of 80 Daltons (sulphur trioxide) or 176 Daltons (the glucuronide moiety) from sulphates and O-glucuronides respectively. 3. A sufficient quantity of one metabolite was isolated from rat bile to permit further analysis by 1H-nmr. This metabolite, which was also found in rat urine, was proved to be 7-O-glucuronyl-7-hydroxyclozapine. The analogous sulphate metabolite was also identified in bile by ESMS. 4. Correspondingly glucuronide and sulphate conjugates of a hydroxylated N-desmethyl clozapine were similarly detected in rat bile. There was insufficient material to permit analysis by 1H-NMR, but it appears likely that conjugation was also at the 7-position of N-desmethylclozapine. 5. Finally, the sulphate conjugate of a hydroxy dechlorinated derivative of clozapine was identified by ESMS in both urine and bile. By analogy with a previous report of a similar metabolite in man, the metabolite was tentatively identified as 8-hydroxy-8-deschloroclozapine.