The application of in vitro models for production of metabolites: Isolation and characterisation of hydroxysecobarbital

Secobarbital was incubated in vitro with freshly prepared porcine hepatocyt es using a commercially available CELLine CL 1000 cartridge from INTEGRA Bi osciences. The incubation medium was concentrated and the metabolites purif ied by HPLC. The fractions containing the metabolites were initially identi fied by HPLC-UV analysis. Aliquots from these fractions were derivatised (m ethylation and silylation) and analysed by positive ion Fl and Ci (El+ and Cl+) GC-MS. To confirm the identity of the required analyte, hydroxysecobar bital, structure elucidation was confirmed by GC-MS and NMR. Using these te chniques approximately 300 mug hydroxysecobarbital was isolated in relative ly pure form (yield 6%). The techiques developed in the course of this work have been applied for the production of other compounds of interest.