Inter-species variability of haplamine metabolism and identification of its phase I metabolites from liver microsomes.

Haplamine, a pyranoquinoline alkaloid, was isolated from the genus Haplophyllum. The inter-species variability of haplamine metabolism was determined by reversed phase high performance liquid chromatography (HPLC) with UV detection. Microsomes from the liver of rats, mice, rabbits, guinea-pigs and humans were incubated with haplamine. After incubation, samples were extracted with a mixture of ethyl acetate and isopropyl alcohol (90:10; v/v). Haplamine and its metabolites were separated by HPLC using Nucleosil C 18 Nautilus (5 μm) connected with a precolumn of the same type. The HPLC mobile phase consisted of water (A) and a mixture ofmethanol and acetonitrile (85:15; v/v) (B) used in a gradient mode (17 to 27% B for 10 min, 27 to 90% B for 37 min, 90 to 17 % B for 3 min, and finally 17 % B for 3 min) at 1 mL/min. Quantitative and qualitative results showed significant inter-species differences in haplamine metabolism. Qualitative similarities were found between guinea-pigs, rabbits, and humans. The metabolites were isolated by HPLC and identified by GC/MS after silylation. The phase I metabolites identified in human liver microsomes were trams/cis-3,4-dihydroxy-9-O-desmethylhaplamine, trams/cis-3,4-dihydroxyhaplamine and 9-O-desmethylhaplamine.