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

Abstract

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.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

“…In addition, the applicability of this method can be extended to other in vitro / in situ / ex vivo permeability assays. Although several studies have reported the simultaneous determination of permeability of marker drugs by HPLC (25)(26)(27)(28)(29), to our knowledge, this is the first report of an HPLC-UV method for the determination of the P-gp substrate VIN concomitantly with the corresponding inhibitor VER, and the four passive permeability markers used in this study. Caco-2 permeability assay -suitability demonstration The formation of a functional single monolayer on polycarbonate inserts is an important feature regarding the mimicking of in vivo conditions in the Caco-2 permeability assay.…”

Permeability of atenolol and propranolol in the presence of dimethyl sulfoxide in rat single‐pass intestinal perfusion assay with liquid chromatography/UV detection

“…The rat in situ intestinal perfusion is a commonly used technique for evaluating the permeability of drugs and new chemical entities (Kumar et al, 2005;Chawla et al, 2006). Human perfusion studies have shown an excellent correlation with rat in situ intestinal permeability for a variety of compounds (Amidon et al, 1995).…”

Development and validation of RP-HPLC-UV method for simultaneous determination of buparvaquone, atenolol, propranolol, quinidine and verapamil: A tool for the standardization of rat in situ intestinal permeability studies

“…The rat in situ intestinal perfusion is a commonly used technique for the assessment of permeability of drugs and new chemical entities [5,6] and the functional role of P-gp on the total intestinal transport [7,8]. In this technique the term in situ refers to the methodology in which the animal's blood supply is kept intact, thus the rate of absorption determined by such method would be more consistent with the in vivo situation when compared to other in vitro techniques.…”