High-performance liquid chromatography method development and validation for simultaneous determination of five model compounds, antipyrine, metoprolol, ketoprofen, furosemide and phenol red, as a tool for the standardization of rat in situ intestinal permeability studies using timed wavelength detection

A simple, precise, accurate and rugged reversed-phase high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of five permeability model compounds, viz. antipyrine, metoprolol, ketoprofen, furosemide and phenol red. The method was intended to standardize rat in situ single-pass intestinal perfusion studies to assess the intestinal permeability of drugs in the market as well as new chemical entities. Optimum resolution was achieved by gradient elution on a Symmetry Shield C-18 analytical column with the mobile phase consisting of a mixture of aqueous potassium dihydrogen orthophosphate (pH 5.5; 0.01 m) and methanol at a flow rate of 1.5 mL/min. The retention times of antipyrine, metoprolol, ketoprofen, phenol red and furosemide were about 9, 12, 13, 16 and 17 min, respectively. Data acquisition was carried out using a photo diode array detector in the wavelength range 210-600 nm. Extraction of chromatograms was carried out by timed wavelength. Data obtained in all studies indicated that the method was suitable for the intended purpose. The validated method was found to be linear and precise in the working range. Suitability of storage under various conditions and freeze/thaw impact at cold temperature were established to ensure complete sample recovery without any stability issues. Recovery very close to the spiked amounts indicated that the method was highly accurate and suitable for use on routine basis.