The Analysis of Ro 24–4736 in Human Plasma by Multidimensional Reversed Phase Microbore HPLC/UV

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Abstract
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][l, 4]diazepin-2-y-l]-2-propynyl] = phenanthrydin-6(5H)-one, in human plasma was developed employing multidimensional reversed phase microbore HPLC. First dimension chromatography with a (3 cm × 2 mm i.d.) base deactivated C8 stationary phase column followed by second dimension chromatography on a diisopropyl octyl silane bonded phase HPLC column (15 cm × 1.0 mm i.d.) was used. Detection was by UV (239 nm, ∊ ≈ 61, 000). Differences in the stationary bonding chemistry and pore size provide remarkable selectivity, yielding an assay method limited mainly by the inherent detector sensitivity. Differences in analyte retention caused by differences in the stationary phases allowed use of a weak mobile phase for the first dimension, focusing the analyte on the head of 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 limit of quantitation to 0.05 ng/ml. Plasma concentration versus time profiles of Ro 24–4736 following oral doses in man were obtained using this method. The use of multidimensional chromatography with two reversed phase columns of different selectivity may provide a general technique for exploiting the advantages of microbore chromatography.