Simultaneous determination of metoprolol, propranolol and phenol red in samples from rat in situ intestinal perfusion studies

Single-pass intestinal perfusion technique (SPIP) is the most used classic technique employed in the study of intestinal absorption of compounds in which a non-absorbable marker such as phenol red is used to correct the water flux. A simple and rapid reversed-phase high performance liquid chromatographic method with UV detection at 227 nm was developed for simultaneous quantitation of propranolol and metoprolol along with phenol red for in-situ permeability studies. The mobile phase was a mixture of 55% methanol, 45% of 0.05 M KH2PO4 aqueous solution (adjusted to pH 6) and 0.2 % (v/v) triethylamine. Analysis was run at a flow rate of 1 ml/min with a 9 min run time. The calibration curves were linear for all three compounds (r > 0.999) across the concentration range of 7.5-125 μg/ml with a limit of detection of 4.24, 2.18 and 8.57 ng/ml and limit of quantification of 14, 7.2 and 28.3 ng/ml for metoprolol, propranolol and phenol red respectively. The coefficient of variation for intra-assay and inter-assay precision was less than 8% and the accuracy was between 93.6-107%. Using the SPIP technique and the suggested HPLC method for sample analysis, the mean values of 0.49 e-4 (±0.19) cm/sec and 0.32 e-4 (± 0.09) cm/sec were obtained for propranolol and metoprolol intestinal permeability coefficients respectively.