RESUMO
A specific, sensitive and fully automated coupled-column LC method for the determination of the anthracycline cytostatic epirubicin and four metabolites in the biological materials human plasma, liver homogenate and liver tumour homogenate has been developed. System-integrated sample processing was achieved using a new restricted access silica precolumn packing. This porous Alkyl-Diol Silica (ADS) was specially designed for the direct and repetitive injection of proteinaceous samples. It consists of a hydrophilic and electroneutral external particle surface (glyceryl-residues) and a hydrophobic reversed-phase internal surface (butyryl-, octanoyl- or octadecyl-residues). These bimodal chromatographic properties allow retention of low molecular analytes by classical RP-chromatography exclusively at the lipophilic pore surface. Macromolecular constituents of the sample matrix (e.g. proteins) are size-excluded by 5 nm pores and quantitatively eliminated in the interstitial void volume. On-line analysis was performed by coupling a C4-Alkyl-Diol precolumn (20 x 4 mm i.d., particle size 25 microns) and LiChrospher RP Select B analytical column (250 x 4 mm i.d., particle size 5 microns) via an electrically driven six-port valve. Separation of the parent compound and its metabolites was achieved with a mobile phase consisting of water (0.1% triethylamine, v/v, pH 2.0 adjusted with trichloroacetic acid)-acetonitrile (70:30, v/v) at a flow rate of 1 ml min-1. The analytes were detected using their natural fluorescence (excitation 445 nm, emission 560 nm). The method described is used for the determination of pharmacokinetics of epirubicin and its metabolites in order to evaluate and optimize treatment regimen of liver cancer chemoembolization therapy.
