Quantitative analysis of the lipophilic doxorubicin analogue annamycin in plasma and tissue samples by reversed-phase chromatography

A rapid and sensitive HPLC method was developed to detect and quantitate the lipophilic doxorubicin analogue annamycin (Ann) and its metabolites in biological samples. Reversed-phase chromatography coupled with fluorescence detection was used. The emission and excitation wavelengths of the fluorescent detector were set at 550 and 472 nm, respectively. The optimal mobile phase was acetonitrile:methanol:water at a 115:95:90 ratio (v/v/v). The lower limit of detection was 0.35 ng of Ann (7 ng/mL). The retention times of 4-demethoxyadriamycinone (Anone) and Ann were 2.6 and 4.3 min, respectively, and their resolution was 1.3. The precision of the determination of Ann and Anone were 0.5 +/- 0.3 and 0.8 +/- 0.7%, respectively. Ann dissolved in methanol was stable under the determination conditions. With chloroform, 60-90% of Ann was extracted from biological samples. Apart from Anone, two Ann metabolites (retention times 3.4 and 6.0 min) were detected in plasma and tissues from C57BL/6 mice bearing subcutaneous B16 tumors 6 h after intravenous administration of a 5-mg/kg suspension of Ann. No peaks were detected in blank tissues and plasma.