Simultaneous determination of naproxen, ketoprofen and phenol red in samples from rat intestinal permeability studies: HPLC method development and validation

A simple reversed-phase high performance liquid chromatographic method with UV detection at 270 nm was developed for simultaneous quantitation of ketoprofen and naproxen sodium along with phenol red as a non-absorbable marker for in situ permeability studies. The mobile phase was a mixture of 20% methanol, 28% of acetonitrile, 52% water and 0.4 ml triethylamine (adjusted to pH 3.2 using orthophosphoric acid). Analysis was run at a flow of 1.5 ml/min with a 20 min run time. The calibration curves were linear for all three compounds (r > 0.999) across the concentration range of 15.6-250 μg/ml with a limit of quantitation of 0.3, 0.25 and 0.2 ng/ml for naproxen, ketoprofen and phenol red, respectively. The coefficient of variation for intra-assay and inter-assay precision was less than or equal to 5.3% and the accuracy was between 95.36 and 101.6%. Using the SPIP technique and the suggested HPLC method for sample analysis, the mean values of 1.17e−4 (±0.28) cm/s and 0.97e−4 (±0.2) cm/s were obtained for naproxen and ketoprofen, respectively.