Validation and clinical evaluation of a UHPLC method with fluorescence detector for plasma quantification of doxorubicin and doxorubicinol in haematological patients.

A rapid and simple UHPLC-fluorescence detection method for the quantification of doxorubicin and its main metabolite, doxorubicinol, in human plasma has been developed. The method was also validated for its application in therapeutic drug monitoring, a clinical approach used in the optimization of oncologic treatments. Following a single protein precipitation step, chromatographic separation was achieved using a C18 column (50mm×2.10mm, particle size 1.7μm) at 50°C with a mobile phase consisting of water (containing 0.4% triethylamine and 0.4% orthophosphoric acid)/acetonitrile (77:23, v/v). Flow rate was 0.50mL/min and fluorescence detection with an excitation wavelength of 470nm and an emission wavelength of 548nm was used. The method met the specifications of linearity, selectivity, sensitivity, accuracy, precision and stability of the FDA and EMA guidelines for the validation of bioanalytical methods. Linearity for the drug (8-3000ng/mL) and the metabolite (3-150ng/mL) was observed (R(2)>0.992) and the maximum intra-day and inter-day precision coefficients of variation were less than 14% for both. The lower limits of quantification were 8 and 3ng/mL for doxorubicin and doxorubicinol, respectively. The method was successfully applied to the quantify plasma concentrations of doxorubicin and doxorubicinol in 33 patients diagnosed with haematological malignancies in which broad ranges for drug (8.3-2766.0ng/mL) and metabolite (4.8-104.9ng/mL) levels were measured adequately.