Rapid Quantification of Digitoxin and Its Metabolites Using Differential Ion Mobility Spectrometry-Tandem Mass Spectrometry

This study focuses on the quantitative analysis of the cardiac glycoside drug digitoxin and its three main metabolites digitoxigenin-bisdigitoxose, digitoxigenin-monodigitoxose, and digitoxigenin using electrospray ionization-differential ion mobility spectrometry-tandem mass spectrometry (ESI-DMS-MS/MS). Despite large molecular weight differences, gas-phase separation of the four compounds in the DMS drift cell was not possible, even by utilizing additional volatile chemical modifiers. Baseline separation was achieved after adduct formation with alkali metal ions, however, and efficiency was shown to improve with increasing size of the alkali ion, reaching optimum conditions for the largest cesium ion. Subsequently, an assay was developed for quantification of digitoxin and its metabolites from human serum samples and its analytical performance assessed in a series of proof-of-concept experiments. The method was applied to spiked human serum pools with concentration levels between 2 and 80 ng/mL. After a short reversed-phase chromatographic step for desalting the sample, rapid DMS separation of the analytes was carried out, resulting in a total run time of less than 1.5 min. The instrumental method showed good repeatability; the calculated coefficients of variation ranged from 2% to 13%.