Determination of the N-methyl-D-aspartate receptor NR2B subunit blocker Ro63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated f or the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Hum an plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl-o-aspart ate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 m l) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75 x 4 mm) due to the high ethanol content, the injection solution was diluted, o n-line, using an additional pump and a T-piece. 1% ammonium acetate-ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-lin e dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without an y buffer in the mobile phase) and transferred to the analytical column [con sisting of two coupled columns (125 + 250x4 mm) packed with Superspher 60 R P-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 7 5 mm precolumn and the on-line dilution procedure allowed about one third s horter run times (21 min) and, therefore, a higher sample throughput. The l imit of quantification was 1 ng/ml using 0.4 mi plasma. The method was appl ied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers. (C) 2000 Elsevier Science B.V. All rights reserved.