THE ANALYSIS OF RO 24-4736 IN HUMAN PLASMA BY MULTIDIMENSIONAL REVERSED-PHASE MICROBORE HPLC UV/

A highly sensitive HPLC method for Ro 24-4736 5-[3-[4-(2-chlorophenyl) -9-methyl-6H-thieno[3, 2-f] = [1,2,4] triazolo[4,3-a][1,4]diazepin-2-y -1]-2-propynyl] = phenanthrydin-6(5H)-one, in human plasma was develop ed employing multidimensional reversed phase microbore HPLC. First dim ension chromatography with a (3 cm x 2 mm i.d.) base deactivated C8 st ationary phase column followed by second dimension chromatography on a diisopropyl octyl silane bonded phase HPLC column (15 cm x 1.0 mm i.d .) was used. Detection was by UV (239 nm, epsilon approximate to 61,00 0). Differences in the stationary bonding chemistry and pore size prov ide remarkable selectivity, yielding an assay method limited mainly by the inherent detector sensitivity. Differences in analyte retention c aused by differences in the stationary phases allowed use of a weak mo bile phase for the first dimension, focusing the analyte on the head o f the second column. Use of smaller i.d. second dimension columns (2.0 mm and 1.0 mm) further concentrated the analyte and decreased the lim it of quantitation to 0.05 ng/ml. Plasma concentration versus time pro files of Ro 24-4736 following oral doses in man were obtained using th is method. The use of multidimensional chromatography with two reverse d phase columns of different selectivity may provide a general techniq ue for exploiting the advantages of microbore chromatography.