Abstract
A fully automated high performance liquid chromatography with column-switching was developed for the simultaneous determination of KR60436, a new reversible proton pump inhibitor, and its active metabolite O-Demethyl-KR60436 from rat plasma samples. Plasma sample (50$\mu$l) was directly introduced onto a Capcell Pak MF Ph-1 column ($10{\times}4$ mm I.D.) where primary separation was occurred to remove proteins and concentrate target Substances Using acetonitrile-Potassium Phosphate (PH 7, 0.1 M) (2 : 8, v/v). The drug molecules eluted from MF Ph-1 column were focused in an intermediate column ($10{\times}2$ I.D.) by the valve switching step. The substances enriched in intermediate column were eluted and separated on a Vydac 218MR53 column ($250{\times}3.2$ I.D.) using acetonitrilepotassium phosphate (pH 7, 0.02 M) (47:53, v/v) at a flow rate of 0.5 ml/min when the valve status was switched back to A position. The method showed excellent sensitivity (detection limit of 2 ng/ml) with small volume of samples ($50{\mu}$l), good precision and accuracy, and speed (total analysis time 24 min) without any loss in chromatographic efficiency. The response was linear ($r^2{\geq}0.797$) over the concentration range of 5-500 ng/ml.
