DETERMINATION OF TALLIMUSTINE IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A sensitive and reproducible HPLC method for the determination of tall imustine (I) in human plasma has been developed and validated. Compoun d I was extracted from plasma by solid-phase extraction using a C-18 c artridge from which the test compound was eluted with a methanol-formi c acid mixture. The methanol solution was evaporated to dryness and th e residue dissolved in a 0.2 M formic acid in methanol-water (1:1, v/v ) mixture, then injected onto the HPLC column. The chromatographic sep aration was performed isocratically by a reversed-phase column filled with ODS, using a 50 mM KH2PO4-acetonitrile mixture as the mobile phas e. The flow-rate was 1 ml/min. The eluate was monitored at 314 nm. No peak interfering with that of I was observed when blank human plasma w as assayed. Linearity was established in the concentration range 0.5-8 5.5 nanograms of I per millilitre of plasma. Four calibration curves i n plasma, prepared and run on four different days, showed correlation coefficients higher than 0.99 and good reproducibility of the slope (C .V.=4.5%). The intra-day precision, evaluated at three concentrations (in the low, mid and high range of the standard curve) and expressed a s C.V. ranged from 0.9 to 14.4%. The inter-day precision evaluated at the same concentrations was better than 10.2%. The inter-day accuracy evaluated in the same samples and expressed as the ratio of found/adde d amount of I, ranged from 86.2 to 108.5%. The limit of quantitation w as 0.5 ng/ml plasma. The HPLC method described here was successfully e mployed for the determination of I in some plasma samples obtained dur ing a phase I clinical trial with the test compound.