In the drug discovery and development process P-gp inhibition of test compounds are assessed by performing bi-directional permeability studies with digoxin individually or in combination with marketed drugs. Quantification of Digoxin by LC-MS/MS was established with direct injection of the experimental supernatant (HBSS buffer : Acetonitrile, 1:1 v/v) on to the analytical column and detecting with the mass spectrometer operated in negative ion mode with formic acid adduct of the digoxin and internal standard. This method describes the development of a sensitive, accurate, and reproducible LC�MS/MS method for the analysis for digoxin by using digitoxin as IS. Chromatography consists of 0.1 % formic acid in channel A and acetonitrile in channel B with gradient program using XBridge C18 column (2.1 x 50 mm, 3.5 �m). Multiple reaction monitoring (MRM) mode was used with the transition of m/z 825.5 to 45.0 for the digoxin and m/z 810.5 to 45.0 for the IS. The calibration curves were linear in the range of 0.01 to 10 �M in experimental buffer samples with the correlation coefficients (r) obtained from plotting the peak area ratios against the nominal concentration were greater than 0.99. The applicability and reliability of the analytical method was evaluated by successful demonstration of efflux ratio (permeability B to A over permeability A to B) greater than 10 for digoxin in Caco-2 cells.

Development of a sensitive LC�MS/MS method for digoxin and its application to the in vitro P-gp inhibition assay

248th National Meeting (2014)

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