Determination of unbound etoposide concentration in ultrafiltered plasma by high-performance liquid chromatography with fluorimetric detection.

Etoposide is a highly protein bound drug, and monitoring the concentration of free drug could help individualize dosage in oncological patients. The cost and difficulty of the standard techniques (equilibration dialysis) has hampered the monitoring of free drugs. We describe a simple HPLC method for the measurement of free etoposide concentration in plasma. Sample preparation involves the ultrafiltration of plasma by a Centrifree device for 30 min at 2000 g and extraction with chloroform. The isocratic separation is performed with a mu Bondapak phenyl analytical column. Fluorimetric detection is used (288-328 nm excitation and emission wavelengths). Linearity of the calibration curve is excellent between 0.05 and 1 microgram/ml. Accuracy and precision are reported at the concentrations 0.06 and 0.4 microgram/ml: within-run accuracy is 10% and 6.2%, respectively; between-run accuracy is < or = 1%; within-run coefficients of variation (C.V.) are 10.6 and 5.0%; between-run C.V. are 11.6 and 6.8% respectively. The range of the assay is 0.05 to 1 microgram/ml. The feasibility of the technique has been tested in 7 patients treated with oral etoposide for hepatocarcinoma (mean protein binding 91%). We found no interference from endogenous substances, co-administered drugs (alizapride, furosemide, ranitidine) and other antineoplastic agents (doxorubicine, idarubicine, vinblastine, vinorelbine).