Validated HPLC Method for Concurrent Determination of Antipyrine, Carbamazepine, Furosemide and Phenytoin and its Application in Assessment of Drug Permeability through Caco-2 Cell Monolayers

The present work explains the development and validation of a simple, rapid and sensitive liquid chromatographic method for the simultaneous determination of antipyrine (ANT), carbamazepine (CBZ), furosemide (FSD) and phenytoin (PHTN). Chromatographic analysis was carried out by a reversed phase technique on a C18 column, using water pH 3.0 and 50:50 mixtures of methanol and acetonitrile (58:42 v/v) as the mobile phase, at a flow-rate of 1.0 ml/min and a column temperature of 40°C. Detection was carried out at 205 nm for CBZ and PHTN and at 230 nm for ANT and FSD. The proposed method was evaluated for validation parameters including linearity, range, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and specificity. Elution of drugs ANT, FSD, PHTN, and CBZ was observed at 4.1, 5.1, 12.3 and 13.5 min, respectively. The method was found to be linear (R(2) ≥ 0.999) in the concentration range of 5-100 μM, with an acceptable accuracy and relative standard deviation. Results of intra- and inter-day validation (n=3) showed the method to be efficient for routine determination of these permeability markers in Caco-2 cell monolayer permeability studies. The method was successfully utilized for determination of standard compounds in Caco-2 permeability experiments.