Validation of Doxepin Quantitative Determination Methods for their Application to In Vitro, Ex Vivo and In Vivo Studies

Doxepin is a tricyclic antidepressant drug that has proven effective anesthetic and analgesic activity in oral mucositis when administered as a topical oral rinse. Aimed to study alternative doxepin formulations for buccal application, we have developed three different quantitative methods to determine doxepin. Each method was specifically developed for its intended use: in vitro release, ex vivo permeation or in vivo studies analyzing plasma. Simple, rapid and easy to perform UV-vis spectrophotometry analysis was chosen for doxepin quantitation in the in vitro release studies with artificial membranes. A reversedphase HPLC method with UV-vis detection using a C18 column was developed for the quantitative determination of doxepin permeated across porcine buccal mucosa. Finally, to determine doxepin plasma concentrations in pigs, a HPLCcoupled with tandem mass spectrometer analytical method with simple sample preparation was developed. The proposed methods were validated according to ICH guidelines with respect to specificity, linearity, accuracy and precision. Therefore, the developed methods were found to be precise, accurate and selective which is suitable for the estimation of doxepin pharmacokinetics and biopharmaceutics parameters.