HPLC analysis of doxorubicin, epirubicin and fluorescent metabolites in biological fluids

Summary

A specific, sensitive and reliable HPLC method for the determination of doxorubicin (DX), epirubicin (epiDX) and their known fluorescent metabolites [doxorubicinol (DXol), epirubicinol (epiDXol) 7-deoxy-adriamycinone (metabolite C), adriamycinone (metabolite D), 7-deoxy-13-dihydro-adriamycinone (metabolite E), 13-dihydro-adriamycinone (metabolite F), 4'-O-β-d-glucuronyl-4'-epiDX (metabolite G) and 4'-O-β-d-glucuronly 13-dihydro-4'-epiDX (metabolite H)] in biological fluids (human plasma, bile and urine) has been developed and tested. Plasma samples were solid-phase-extracted (C₁₈-bonded silica cartridges). Urine and bile samples were injected directly after the addition of the internal standard and dilution with 0.3 M phosphoric acid. Complete separation of unchanged drugs and metabolites was achieved with a mobile phase consisting of 75.6% 10 mM KH₂PO₄ and 24.4% CH₃CN (the pH of the solution was adjusted to 4.3 with 0.03 M H₃PO₄) at a flow rate of 1.5 ml/min. For the analyses we used a cyanopropyl chromatographic column (25 cmx4.6 mm i.d.; particle size 5 μm) and fluorescence detection with excitation wavelength set at 470 nm and emission at 580 nm. Sensitivity was better than 0.3 ng/ml for all substances analysed. The mean absolute recovery of unchanged drugs and metabolites was between 88.3% (metabolite E) and 98.92% (metabolite G). Intraand interassay precisions (plasma samples) were better than 10.6% and 13.0%.