An LC–MS/MS method for the determination of digitoxigenin in skin samples and its application to skin permeation and metabolic stability studies
An LC–MS/MS method was developed for the determination of digitoxigenin in mice skin samples. Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 column. Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI interface operating in a positive ionization mode. Quantification was performed using selective reaction monitoring of the precursor-product ion transitions of m/z 375.5 → 339 for digitoxigenin and m/z 391.5 → 337 for internal standard (IS). The calibration curves were linear over the concentration range of 1.00–500 ng/mL. The intra- and inter-batch precision was no more than 10.6% of the coefficient of variation and the accuracy was within ±8.1% of the actual values. This validated method has been successfully applied to skin permeation and skin metabolic stability studies of digitoxigenin in mice. The steady-state flux and lag time of digitoxigenin permeated across the full-thickness mice skin were 1.86 ± 0.45 μg/cm2/h and 0.46 ± 0.18 h, respectively. The metabolism of digitoxigenin in the skin was not detected in our study.