Development and validation of a RP-HPLC method for simultaneous determination of cimetidine, metoprolol tartrate and phenol red for intestinal perfusion studies.

A new reversed phase high-performance liquid chromatography (RP-HPLC) method, with a short analysis time and easy to apply, was developed for the simultaneous detection of cimetidine (CIM), metoprolol tartrate (MT) and phenol red (PR) for use in intestinal perfusion studies. The analysis was performed with phosphate buffer (pH 5.0, 12.5 mM)-acetonitrile mixture as mobile phase and C column (Inertsil ODS-3; 5 µm, 4.6 × 250 mm) as stationary phase. Gradient analysis conditions were used and the acetonitrile ratio in the mobile phase varied from 10 to 50% in 10 min. Total run time for analysis was 10 min and the injection volume was 20 µL. Detection of compounds was performed at 207 nm. Under optimum HPLC conditions, retention times were 4.03 min for CIM, 6.99 min for MT and 8.49 min for PR. The method was validated according to ICH Q2 (R1) guideline for specificity, linearity, sensitivity, precision, accuracy, stability and robustness. Developed method was linear and determination coefficients of the calibration curves were 0.9993, 0.9991 and 1.0 for CIM, MT and PR, respectively. The limits of quantification were 6.20, 2.78 and 0.45 μg/mL for CIM, MT and PR, respectively. The precision and accuracy values of the developed analytical method met the ICH Q2 (R1) limits. The applicability of the method was demonstrated by preliminary in-situ intestinal perfusion studies. In conclusion, samples obtained from in-situ intestinal perfusion studies performed to examine the absorption/permeability of CIM, MT, and PR can be analyzed with the developed HPLC method.