Ultrafiltration and subsequent high performance liquid chromatography for in vivo determinations of the protein binding of etoposide

Etoposide is extensively (approximately 94%) bound to plasma proteins and the free non-protein-bound levels have been shown to correlate more closely to toxicity than total drug concentrations. A rapid and easily performed method, compared to the time consuming equilibrium dialysis, to obtain the free fraction is needed. The aim of this study was to evaluate ultrafiltration and subsequent high performance liquid chromatography (HPLC) for the determination of protein binding of etoposide. Spiked plasma from healthy, drug-free volunteers was used to compare ultrafiltration, using Amicon® Centrifree filters, with equilibrium dialysis at 37 °C. The variability (CV) of the ultrafiltration method was 6.1 and 13.5% (n = 6) at 37 °C and room temperature (RT), respectively. The relative size of the free fraction obtained by ultrafiltration at 37 °C and RT was 1.22 (P = 0.0005) and 0.37 (P = 0.0001), respectively, compared with equilibrium dialysis at 37 °C. The chromatographic separation of metabolites from the mother compound when free etoposide is analyzed is crucial. It is shown that a hydroxyacid metabolite of etoposide is quite dominant in a protein-free plasma fraction. The free concentrations were determined throughout a dose interval of 24 h in a patient receiving etoposide 100 mg/m2 daily. Ultrafiltration and subsequent HPLC is considered convenient and suitable for in vivo pharmacokinetic investigations.