Simultaneous Determination of Procaspase Activating Compound 1 and Permeability Markers in Intestinal Perfusion Samples and Application to a Rat Intestinal Absorption Study

A sensitive and simple HPLC method for simultaneous determination of PAC-1 (first procaspase-activating compound), phenol red, and permeability markers (carbamazepine and furosemide) in perfusion samples was developed and validated to assess intestinal absorption of PAC-1 using single-pass intestinal perfusion technique (SPIP) in rats. The chromatographic separation was carried out on a Kromasil C₁₈ column (150 mm × 4.6 mm, 5 μm) with acetonitrile–methanol–30 mmol L^?1 phosphate buffer (pH 3.0, 25:10:65, v/v/v) as mobile phase at a flow rate of 1.0 mL min^?1, and the wavelength of the UV detector was set at 281 nm. The calibration curves were linear in the ranges of 2.40–48.0 μg mL^?1 for PAC-1; 3.60–72.0 μg mL^?1 for carbamazepine; 3.20–64.0 μg mL^?1 for furosemide, and 4.80–96.0 μg mL^?1 for phenol red (r > 0.999). Both the intra- and inter-day precisions (RSD%) of all analytes were less than 6.8 % at three concentration levels, while accuracy ranged from 95.4 to 104.5 %. Data obtained in all method validation studies indicated that the method was suitable for the intended purpose. The effective permeability values (P _eff) considering water flux with the help of non-permeable marker phenol red was calculated to be 0.42 × 10^?4, 0.62 × 10^?4, 0.32 × 10^?4 cm s^?1 for PAC-1; 0.72 × 10^?4, 0.77 × 10^?4, 0.52 × 10^?4 cm s^?1 for carbamazepine; 0.20 × 10^?4, 0.16 × 10^?4, 0.12 × 10^?4 cm s^?1 for furosemide in duodenum, jejunum and ileum, respectively. The P _eff value can be increased by co-perfusion with verapamil, indicating that absorption of PAC-1 is efficiently transported by P-glycoprotein (P-gp) in the gut wall.