In Vitro Permeability of Poorly Aqueous Soluble Compounds Using Different Solubilizers in the PAMPA Assay with Liquid Chromatography/Mass Spectrometry Detection

PURPOSE: This study compares the use of UV-VIS detection with liquid chromatography/mass spectrometry (LC/MS) detection for the PAMPA (Parallel Artificial Membrane Permeability Assay) permeability determination of compounds in the drug discovery stage. LC/MS detection offers a selective and sensitive method for the determination of the PAMPA permeability for compounds that do not contain a UV chromophore or possess a low UV extinction coefficient. To enhance the reliability of our permeability measurements for compounds with low aqueous solubility, we demonstrated the use of LC/MS detection as a means for facilitating the study of solubilizing agents to enhance aqueous solubility that normally would interfere with UV-VIS detection. In doing so, the PAMPA assay can be expanded to study the in vitro permeability of poorly water soluble compounds and evaluate the effects of solubilizers' on the membrane permeability of different compounds. This might be useful in selecting solubilizers for poorly water soluble compounds to be used for further in vivo studies. METHODS: A diverse set of 20 drugs using UV-VIS detection were compared with data using LC/MS detection. A PAMPA screening method was designed which used solubilizers (Brij 35, Cremophor EL, ethanol, and Tween 80) for compounds with low aqueous solubility. The stability of the artificial membrane was determined using various solubilizer concentrations (0.1-5% w/v) to ensure the phospholipid membrane was not disrupted. Two compounds, amiodarone and miconazole, with low aqueous solubility yielding an undetected response in the PAMPA assay using UV-VIS detection were subjected to the different solubilizing agents and their PAMPA permeability was measured using LC/MS detection. RESULTS: Most of the compounds showed similar PAMPA permeability using the two detection systems. However, for compounds lacking a UV chromophore or with a low UV extinction coefficient, LC/MS was the detection method of choice for determination of PAMPA permeability values. LC/MS also gave reliable quantification data for compounds containing impurities, as well as compounds that were not stable during the assay. Although many solubilizers were found to interfere with UV-VIS detection, the LC/MS approach was applicable to determine the permeability values of compounds with normally low aqueous solubility. CONCLUSIONS: LC/MS detection offered greater sensitivity and selectivity as compared with UV-VIS detection for the PAMPA assay. With this added versatility in detection, PAMPA can be used in both discovery and pre-formulation applications, which has not been described before.