Abstract
A high performance liquid chromatographic assay method was established to simultaneously determine lenampicillin (ampicillin prodrug), ampicillin, and an absorption marker compound (antipyrine) for the high‐throughput in vitro assessment of intestinal absorption and metabolism using Caco‐2 cells. These compounds were determined using an octyl (C8) column, mobile phase consisting of 37% methanol and 10 mM NaH2PO4 in water (pH 4.5) at a flow rate of 1.5 mL/min, and a UV‐detector (200 nm). For lenampicillin, the slope and its S.D. of a typical calibration line going through the coordinate origin (peak area ratio of lenampicillin to internal standard versus lenampicillin concentration from 0.5 µM to 0.5 mM) were 2.91×10−3 and 1.83×10−5, respectively. The correlation coefficient (r) of the calibration line in the inter‐assays of lenampicillin was 0.9994 (coefficient of variation=0.04%). For ampicillin, the slope and its S.D. of a typical calibration line going through the coordinate origin (peak area ratio of ampicillin to internal standard versus ampicillin concentration from 0.5 µM to 0.5 mM) were 2.28×10−3 and 1.04×10−5, respectively. The correlation coefficient (r) of the calibration line in the inter‐assays of ampicillin was 0.9999 (coefficient of variation=0.01%). Lenampicillin absorption and metabolism to ampicillin were shown by this assay method.
Acknowledgment
This study was supported in part by a Grant‐in‐Aid for Scientific Research (C) (No. 15590141) from the Japan Society for the Promotion of Science.