Improved methodology for subnanogram quantitation of doxorubicin and its 13-hydroxy metabolite in biological fluids by liquid chromatography

Abstract

Sensitive and specific methodology for quantifying doxorubicin (DOX), a potent antineoplastic drug, and its 13-hydroxy metabilite, doxorubicinol (DOX-OL), in plasma and urine has been developed and validated. The plasma method uses solid-phase extraction for analyte isolation and a narrow-bore (2.0 mm i.d.) column for liquid chromatographic separation with optimized fluorescence detection. The dynamic ranges for both drug and meatbolite in plasma are linear from 0.2 to 100 ng ml −1. Drug and metabolite are quantified in unextracted, diluted urine over a 16 to 400 ng ml −1 range. Epirubicin, an epimeric analogue of doxorubicin, is used as an internal standard. Mean extraction efficiencies for drug, metabolite and internal standard from plasma are 88, 86 and 90%, respectively. The instrumental detection limit (signal-to-noise ratio = 3) for doxorubicin or metabolite was 18 pg on column, while the lower limit of quantitation (LLOQ) was 0.3 and 0.6 ng ml −1, respectively for DOX and DOX-OL. The typical intra-dry accuracy and imprecision in plasma was < 8% bias and < relative standard deviation (R.S.D.) for DOX at or above 0.3 ng ml −1, and < 15% and < 10% for DOX-OL at or above 0.6 ng ml −1. For the urine method, the average intra-day imprecision was < 7% R.S.D. and the average bias was < 5% for both drug and metabolite over the entire dynamic range. Determinations of these two components in patient samples has verified the robustness and utility of the method.