Solid-phase extraction and optimized separation of doxorubicin, epirubicin and their metabolites using reversed-phase high-performance liquid chromatography

Abstract

A reversed-phase isocratic high-performance liquid chromatographic method is described in which a formal structured procedure was applied to predict the mobile phase composition giving optimal baseline resolution of the clinically important anticancer agents doxorubicin and 4′-epidoxorubicin (epirubicin), their principal metabolites, and daunorubicin (internal standard). These formal statistical procedures included the simultaneous techniques of solvent selectivity triangle and factorial design for range-finding preliminary studies, followed by use of the modified simplex, a sequential procedure. These were used to select the parameters of organic modifier, buffer strength and pH necessary for use with a Spherisorb ODS 1 column, to achieve optimal separation of eight anthracycline solutes. Ultraviolet and fluorescence detection was used (λ ex = 254 nm, λ em = 560 nm), and the latter gave a low detection limit for doxorubicin in serum of 1 ng ml −1. The optimal mobile phase composition was determined to be acetonitrile-0.06 M Na 2 HPO 4 containing 0.05% (v/v) triethylamine adjusted to pH 4.6 with 0.03 M citric acid (35:65, v/v). A solid-phase extraction method was developed to enable the selective isolation of anthracyclines by adsorption onto C 8 Bond-Elut cartridges, and is based on extraction of serum spiked with a mixture of the anthracycline solutes. The anthracyclines were eluted using acetonitrile-0.2 M Na 2 HPO 4 containing 0.05% (v/v) triethylamine adjusted to pH 3.6 with 0.1 M citric acid (67.5:32.5, v/v). Reproducible recoveries for doxorubicin (94 ± 8%) and for epirubicin (96 ± 8%) were obtained ( n = 5). In particular, recoveries for the 7-deoxyaglycone metabolite (99%) were higher than other extraction methods cited. The solid-phase extraction method described enables rapid and reproducible determinations of these anthracyclines and their metabolites in biological matrices regardless of their disparate physicochemical properties.