Aim: To isolate and identify a glucuronide metabolite of SFZ-47 [3H-1,2-dihydro-2-(4-methyl-phenylamino)methyl-1-pyrrolizinone], which is difficult to synthesize because it undergoes hydrolysis and intramolecular acyl migration at physiological pH, in rabbit urine.
Methods: Two rabbits were ig 200 mg doses of SFZ-47. Urine was collected for 24 h, adjusted to pH 4.0 with acetic acid and lyophilized. The residues were reconstituted in 25 mL methanol and centrifuged at 5,000 r.min-1 for 10 min. The supernatant was filtered (0.45 micron) and then isolated with semi-preparative reversed phase HPLC. The eluent collected from individual peaks was evaporated by rotary evaporation and freeze-drying. Compounds were then identified with electrospray ion trap mass spectrometry and 1HNMR spectroscopy.
Results: The 1HNMR and ESI-MSn results indicate that the metabolite is the 1-O-acyl beta-D-glucuronide conjugate of 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino) benzoic acid.
Conclusion: This method was shown to be rapid and simple and gave excellent resolution from endogenous constituents in urine, and it is suitable for preparation of the glucuronide metabolites of SFZ-47 and its analogues.