DETERMINATION OF TUROSTERIDE, A NEW INHIBITOR OF 5-ALPHA-REDUCTASE, IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ULTRAVIOLET DETECTION

A sensitive and specific HPLC method for the determination of turoster ide in human plasma was developed and validated. Turosteride was extra cted from plasma with diethyl ether. Further purifications of the frac tion extracted were performed sequentially by solid-phase extraction u sing a CN cartridge and by liquid-liquid partition between n-hexane an d acetonitrile. Finally the acetonitrile solution containing the test compound was evaporated to dryness and the residue dissolved in the mo bile phase, then injected onto the HPLC system. The chromatographic se paration was performed isocratically by a reversed-phase column filled with ODS using a water-acetonitrile-methanol mixture. The eluate was monitored at 210 nm. No peak interfering with that of turosteride was observed when blank human plasma was assayed. Linearity was obtained i n the turosteride concentration range of 5-1000 ng/ml plasma. Six cali bration curves in plasma prepared and run on six different days showed correlation coefficients higher than 0.99 and good reproducibility of the slope (C.V.=4.3%). The intra-day precision, evaluated at three co ncentrations (in the low, mid and high range of the standard curve) an d expressed as C.V., ranged from 0.81 to 13.25%. The inter-day precisi on evaluated at the same concentrations was better than 10.7%. The int er-day accuracy evaluated in the same samples and expressed as the rat io of found/added amount of turosteride ranged from 97.66 to 98.38%. T he limit of quantitation was 5 ng/ml plasma. The HPLC method described was successfully employed for the determination of turosteride in pla sma samples obtained during a phase I clinical trial with the test com pound.