RP-HPLC-UV Method for Estimation of Fluorouracil–Epirubicin–Cyclophosphamide and Their Metabolite Mixtures in Human Plasma (Matrix)

Abstract

A combination of 5-fluorouracil (FU), epirubicin (EP) and cyclophosphamide (CP) is routinely employed in the treatment of breast cancer. The objective of this study was to develop a reverse phase high-performance liquid chromatography (HPLC)-UV method for simultaneous quantitative analysis of the triple-drug and their metabolites in plasma. RP-HPLC system with a C18 column and a diode array detector was employed. The plasma samples were precipitated with acetonitrile and the supernatant was dried under a flow of nitrogen gas. The mobile phase comprised of two combinations, water (pH 4.0) and methanol (98:2 v/v), and water (pH 4.0):methanol:acetonitrile (70:13:17 v/v/v). The retention times for the compounds were determined and the parameters of validation established in plasma indicated the robustness and reliability. The corresponding HPLC peaks were confirmed using electron spray ionization mass spectrometry. FU and metabolites had a recovery of >93%; EP, epirubicinol and CP were >78% from plasma. Stability at 28-30°C in water (pH 4.0) of FU, 5,6-dihydro-5-fluorouracil and EP were higher followed by CP, EPol, fluorodeoxyuridine and fluorouridine (FUR). Storage of the drug-spiked plasma at -80°C assessed for 72 h showed a small but significant (P < 0.05) change in the recovery of FUR and EP. The method was validated in patient's plasma samples (n = 6).