Simultaneous determination of N, N-di(n-butyl)doxorubicin-14-valerate and its 8 urinary metabolites by HPLC.

Aim: To develop an HPLC assay for simultaneous determination of AD202 and its metabolites and to examine metabolites of AD202 in urine of rats.

Methods: Reverse-phase HPLC with fluorescence detection and gradient elutionusing a Waters Chromatograph equipped with a 710 B WIFP autosampler, a power Mate SX plus computer, C18 Nova-pak (4 microns) 5 mm x 10 cm radial compression column connected with a guard micro-column, and a Waters 991 photodiode array detector for online observation of UV spectrum of related fraction.

Results: Detection limit was 1-3 ng for AD202 and 1-3 ng for its metabolites per injection. After i.v. AD202 20 mg.kg-1, only 4.9% dose as total anthracycline fluorescence signal was recovered in urine over 72 h. The predominant urinary metabolites were AD285 (desacyl AD202) and AD284 (N-mono-debutyl AD285). Six minor metabolites including aglycones and 13-keto reductive product were identified and 3 as-yet unknown metabolites were seen. Enzymatic and acid-hydrolysis failed to reveal the presence of glucuronides in urine.

Conclusion: The sample analysis techniques developed in this study proved to be very efficient, sensitive, and specific, a total of 11 compounds achieved resolution with detection limit of 2 ng and no interference from matrix. Urine samples can be injected directly into chromatograph without any extraction, making sample analysis simple and time-saving.