High performance liquid chromatographic analysis of a new proton pump inhibitor KR60436 and its active metabolite O-demethyl-KR60436 in rat plasma samples using column-switching

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 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (10 × 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 × 2 mm I.D.) by the valve switching step. The substances enriched in intermediate column were eluted and separated on a Vydac 218MR53 column (250 × 3.2 mm I.D.) using acetonitrile-potassium 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 μl), good precision and accuracy, and speed (total analysis time 24 min) without any loss in chromatographic efficiency. The response was linear (r² ≥ 0.999) over the concentration range of 5-500 ng/ml.