HPLC methods for the determination of bound and free doxorubicin, and of bound and free galactosamine, in methacrylamide polymer-drug conjugates.

Quantitative acid hydrolysis followed by HPLC separation has been established as an analytical procedure for the determination of polymer-bound doxorubicin (an anti-cancer drug) and D-galactosamine (a liver-targetting 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 required pre-treatment with sodium borohydride and pre-column derivatization with o-phthalalaldehyde and beta-mercaptoethanol. Independent determination of free doxorubicin and glactosamine in untreated polymer samples was also achieved with the same HPLC method up to detection limits of 0.01% and 0.02% respectively. The methods were validated for linearity, 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 free doxorubicin or galactosamine.