Simultaneous quantification of digoxin, digitoxin, and their metabolites in serum using high performance liquid chromatography‐tandem mass spectrometry

The aim of this work was the development of a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for simultaneous quantification of two cardiac glycosidic drugs in human serum, namely digoxin (DG) and digitoxin (DT), as well as several of their metabolites: digoxin‐bis‐digitoxose and digoxin‐mono‐digitoxose, digitoxin‐bis‐digitoxose and digitoxin‐mono‐digitoxose, digoxigenin, digitoxigenin, dihydrodigoxin and acetyl‐ and methyldigoxin. The assay also allowed for semi‐quantitative analysis of two structurally similar compounds, deslanoside and lanatoside. As internal standards, deuterated analogues were synthesized for some of the target analytes. Highly abundant proteins were initially removed by protein precipitation using zinc sulfate before samples were extracted by supported liquid extraction. Chromatographic separation was achieved on a pentafluorphenyl stationary phase prior to electrospray ionization triple quadrupole mass spectrometry in multiple reaction monitoring mode. The assay allowed quantification of the analytes with lower limits of quantification between 0.04 and 2.0 ng/mL. Linearity was shown over the range 0.16–9.5 ng/mL for DG and its metabolites, and 1.6–95 ng/mL for DT and its metabolites with correlation coefficients R >0.991. The quantification range was determined as 1.1–8.9 ng/mL for DG and its metabolites and 12–90 ng/mL for DT and its metabolites. Within this range, DG and DT were determined with an accuracy of ±2% and precision <7% RSD. Trueness was confirmed by analyzing native samples and comparing the results to values obtained by a certified analysis laboratory. In conclusion, the assay represents a useful reference method for immunoassay‐based digoxin and digitoxin tests, which are easily biased by the presence of metabolites of the target analytes or structurally similar substances.