Development and Validation of a RP-HPLC Method for Simultaneous Determination of Metformin Hydrochloride, Phenol Red and Metoprolol Tartrate for Intestinal Perfusion Studies

A novel, fast, easy liquid chromatographic method was developed for simultaneous determination of metformin hydrochloride, phenol red, metoprolol tartrate for intestinal perfusion studies. The analysis was performed on a C-18 column with a mobile phase composed of phosphate buffer (pH 3.0, 100 mM)-acetonitrile (30: 70, v/v). Method was validated for selectivity, sensitivity, linearity, precision, accuracy, stability. All calibration curves were linear (r > 0.9986). Quantification limit was 2.5 mu g/mL for metformin, 0.38 mu g/mL for phenol red, 0.77 mu g/mL for metoprolol. Detection limit was 0.85 mu g/mL for metformin, 0.12 mu g/mL for phenol red, and 0.25 mu g/mL for metoprolol. Precision and accuracy values of the method fulfilled the required limits. The mean permeability values of metformin across jejunum, ileum and colon were 5.59 +/- 3.09, 11.5 +/- 1.26, 7.86 +/- 2.65 x 10(-5) cm/s, respectively; the corresponding values for metoprolol were 5.77 +/- 3.37, 4.33 +/- 0.48 and 10.5 +/- 3.15x10-5 cm/s. These results demonstrate that permeability of metformin across jejunum and colon can be classified as low, whereas its permeability is high across ileum.