Analysis of doxorubicin, 4'-epidoxorubicin, and their metabolites by liquid chromatography

An analytical method based on isocratic HPLC separation and fluorescence detection was developed to allow for sensitive and specific analysis of anthracyclines and their metabolites in plasma and urine. The method is particularly advantageous when comparing the metabolism and/or pharmacokinetics of analogues, such as doxorubicin [(8S, 10S)-10-[(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)- oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-meth- oxy-5,12-naphthacenedione] (1) and 4'-epidoxorubicin (2), since both drugs and their metabolites can be analyzed under identical conditions. The analytical properties of 1, 2, and eight metabolites were studied in plasma, serum, buffer solution, and urine. The detection limit in plasma was 4 X 10(-8) M for the glucuronides, 7 X 10(-9) M for the glycosides, and 1 X 10(-9) M for the aglycones. In plasma, 1, 2, doxorubicinol (3), 4'-epidoxorubicinol (4), doxorubicinone (5), and doxorubicinol aglycone (6) showed a linear concentration-response relationship from their detection limit up to 5 X 10(-6) M. A linear calibration graph for plasma samples was also obtained for 7-deoxydoxorubicinone (7) and 7-deoxydoxorubicinol (8); however, these compounds had a significantly lower upper limit (5 X 10(-7) M). Urine samples were acidified to pH 2.5 and analyzed by HPLC without further purification. A linear calibration curve was obtained in the clinically relevant range. The detection limit in urine was approximately 5 X 10(-8) M. Plasma and urine of two patients who had received 4'-epidoxorubicin by iv bolus injection were analyzed.