Analytical and pharmacological validation of the quantification of phenol red in a mouse model: An optimized method to evaluate expectorant drugs.

INTRODUCTION: The evaluation of expectorant activity has been extensively studied in murine models, involving the secretion of phenol red in the trachea or bronchus to estimate the secretory capacity of lower airway mucosa. However, differences in the experimental protocols of several studies evidenced the need of to standardize the quantification of phenol red in the bronchoalveolar fluid (BALF). METHODS: The analytical methodology for the quantification of phenol red in the BALF was optimized by investigation of pH influence, quantity of the alkali agent added and appropriate wavelength for quantification of phenol red by UV-VIS spectroscopy. Different phenol red suspensions (0.05, 0.5, 1.25, 2.5 and 5%) were prepared and administered intraperitoneally in mice at doses 5, 25, 50, 250 or 500 mg/kg. RESULTS: It was shown that phenol red should be used at dose 500 mg/kg and intraperitoneal administration should be performed from a suspension at 1.25% (w/v). Furthermore, the alkalinizing agent of choice would be NaOH (0.1 M). The pharmacological validation of the analytical method showed that ambroxol (30, 60 or 120 mg/kg), guaifenesin (100 mg/kg), NH4Cl (2000 mg/kg) or salbutamol (4 mg/kg) can be used as positive controls. DISCUSSION: The phenol red quantification in the BALF is a rapid and low cost assay for the discovery of new expectorant drugs. Thus, it was proposed a standardization of the analytical and pharmacological methods to ensure the reliability of BALF processing and reproducibility of phenol red quantification for data analysis.