HPLC METHODS FOR THE DETERMINATION OF BOUND AND FREE DOXORUBICIN, ANDOF BOUND AND FREE GALACTOSAMINE, IN METHACRYLAMIDE POLYMER-DRUG CONJUGATES

Quantitative acid hydrolysis followed by HPLC separation has been esta blished as an analytical procedure for the determination of polymer-bo und doxorubicin (an anti-cancer drug) and D-galactosamine (a liver-tar getting moiety) in the polymer-drug conjugates FCE 28068 and FCE 28069 . Optimal conditions of hydrolysis were determined in both cases: 1 N HCl, 50 degrees C, 1.5 h for doxorubicin, and 6 N HCl, 60 degrees C, 5 h for galactosamine. Appropriate HPLC quantitation of galactosamine r equired pre-treatment with sodium borohydride and pre-column derivatiz ation with o-phthalalaldehyde and beta-mercaptoethanol. Independent de termination of free doxorubicin and glactosamine in untreated polymer samples was also achieved with the same HPLC method up to detection li mits of 0.01% and 0.02% respectively. The methods were validated for l inearity, precision and repeatability. Validation for accuracy before and after acid hydrolysis was achieved by testing hydrolysis on model compounds and by assessing recovery in polymer solutions spiked with f ree doxorubicin or galactosamine.