Determination of Ro 23-7637 in dog plasma by multidimensional ion-exchange-reversed-phase high-performance liquid chromatography with ultraviolet detection

Ro 23-7637 (I) is a new drug under development for the treatment of metabolic diseases. A high-performance liquid chromatographic-ultraviolet detection (HPLC-UV) analytical procedure for its analysis in dog plasma was developed and is reported here. Following C18 solid-phase extraction, the sample is applied to a strong cation-exchange column in the first dimension. The analyte and internal standard, Ro 24-4558 (II), are transferred to a base-deactivated C18 reversed-phase column in the second dimension (orthogonal separation mechanism), with UV detection at 254 nm. The reversed-phase solid-phase extraction provides a gross isolation of the drug, based on hydrophobicity. The first-dimension ion-exchange separation allows neutral species and anions to elute with the column void volume, while separating basic species according to PKA. The second dimension provides a high-resolution separation dependent upon the hydrophobicity of the sample species. The rationale for using orthogonal multidimensional chromatography was that an exhaustive examination of reversed-phase and normal-phase columns invariably resulted in co-elution of I with endogenous plasma components, which limited the sensitivity of the method. We have utilized C18 solid-phase extraction, followed by multidimensional HPLC-UV, to develop an accurate and precise analytical method whose limit of quantitation, 5 ng/ml using 0.5 ml of plasma, is determined by inherent detector sensitivity. Increased sensitivity can be readily achieved by using more sample or by using microbore HPLC on the second dimension.