Identification of doxorubicin and an imine derivative from liquid and solid samples utilizing liquid chromatography

Doxorubicin is an anthracycline glycoside used to treat a variety of cancer conditions from breast cancer to Wilms' tumor. The appearance of doxorubicin resistance has initiated studies of doxorubicin derivatives. The formation of the doxorubicin imine derivative is shown with means of identification by HPLC. Both the parent doxorubicin and imine derivative can be extracted from aqueous samples into 2-pentanol or 2-methyl-2-butanol by utilizing aggressive salting-out techniques. Both forms of doxorubicin are bright red in color. A mixture of methanol/water or straight ethanol can be used to solubilize from solid samples. Determination of molecular properties indicated the imine group substantially increases lipophilicity and decreases aqueous solubitlity. Doxorubicin and its imine derivative are separated using Alltech Altima 18C 5 mu column, UV/Vis detection (200 nm), and a mobile phase of methanol:water (80%/20%) at 1.0 mL/minute. Utilizing non-destructive detection allows the collection of the eluted drug. The resolution achieved was 1.81 minutes, with 784 plates for doxorubicin separation, and 6834 plates for the imine derivative. The lipophilic substituent constant of the imine group is 3.48, indicating increased lipophilicity. The imine derivative is stable for weeks when stored dry at -10 degrees C.