Dried Matrix Spot Analysis of Loxapine Phase II Metabolites in Rat Urine

Loxapine is a dibenzoxazepine antipsychotic. Gender differences in the plasma pharmacokinetic and metabolic profiles in male and female Sprague Dawley rats were observed following oral or intravenous administration of loxapine succinate. Plasma was collected and analyzed, and in order to fully investigate the differences, urine samples were collected from 0-24 hours post-dose, and analyzed for loxapine and its metabolites. Urine volumes were measured, and glacial acetic acid (0.1%) added. The samples were stored at -70ºC until analysis. One set of samples were spotted (15 µL each spot) onto Whatman FTA-DMPK cards (Type A, B, and C), dried, and then stored for 96 hours at ambient temperature. Another set of samples was treated by centrifugation after thawing. Hydrolysis of Phase II metabolites was accomplished by the addition of glucuronidase/sulfatase, followed by incubation for 2 hours at 37ºC. The hydrolyzed samples were also spotted onto DMPK cards or treated by centrifugation. Samples were punched (6mm) from each card, and extracted using methanol containing internal standards. The extracts and centrifuged samples were analyzed by LC-MS-MS for loxapine, amoxapine, loxapine N-oxide, 7-OH loxapine, and 8-OH loxapine using authentic standards and their respective deuterated internal standards. Following both intravenous and oral administration of loxapine to rats, the major metabolite observed in plasma was 7-OH-loxapine, with amoxapine and loxapine N-oxide observed to a lesser extent. No 8-OH loxapine (major human metabolite) was observed in rat plasma samples. Very little (<3%) loxapine or its metabolites were excreted into the urine over 24 hours post dose following either route of administration. The main metabolites recovered from the urine were 7-OH loxapine and loxapine N-oxide, with the amount of 7-OH-loxapine recovered from female rats higher than from the male rats. Treatment with a glusulase enzyme indicated that loxapine, 7-OH loxapine and 8-OH loxapine were excreted as glucuronides, and males appeared to exhibit more glucuronidation than females. DMPK Card B had the highest % recovery of all aglycones, and showed that 47% of 7-OH loxapine was conjugated into phase II metabolites. The results from Card B were comparable to the analysis with centrifuged processed samples, and therefore, dried matrix spot analysis is suitable for analysis of loxapine Phase II metabolites in rat urine.