ULTRAFILTRATION AND SUBSEQUENT HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHYFOR 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 mor e closely to toxicity than total drug concentrations. A rapid and easi ly performed method, compared to the time consuming equilibrium dialys is, to obtain the free fraction is needed. The aim of this study was t o evaluate ultrafiltration and subsequent high performance liquid chro matography (HPLC) for the determination of protein binding of etoposid e, Spiked plasma from healthy, drug-free volunteers was used to compar e ultrafiltration, using Amicon(R) Centrifree filters, with equilibriu m dialysis at 37 degrees C. The variability (CV) of the ultrafiltratio n method was 6.1 and 13.5% (n = 6) at 37 degrees C and room temperatur e (RT), respectively, The relative size of the free fraction obtained by ultrafiltration at 37 degrees C and RT was 1.22 (P = 0.0005) and 0. 37 (P = 0.0001), respectively, compared with equilibrium dialysis at 3 7 degrees C, The chromatographic separation of metabolites from the mo ther compound when free etoposide is analyzed is crucial. It is shown that a hydroxy-acid metabolite of etoposide is quite dominant in a pro tein-free plasma fraction. The free concentrations were determined thr oughout a dose interval of 24 h in a patient receiving etoposide 100 m g/m(2) daily. Ultrafiltration and subsequent HPLC is considered conven ient and suitable for in vivo pharmacokinetic investigations.