Development of a quantification method for digoxin, a typical P-glycoprotein probe in clinical and non-clinical studies, using high performance liquid chromatography–tandem mass spectrometry: The usefulness of negative ionization mode to avoid competitive adduct-ion formation

A sensitive method for digoxin determination using formate-adduct ion based on the effect of ionization enhancement in liquid chromatograph–mass spectrometer

“…One previous report [2] inspired us to investigate ion full scan mass spectra (Q1) of digoxin (500 ng/mL dissolved in 60:40 (v/v) acetonitrile:water) in negative ion mode. Interestingly, in this experiment, the intensity of the [M+HCOO] − ion stood out from the other fragments including [M−H] − that was reported to be responsible for the high sensitivity [2].…”

“…In addition, digoxin is a substrate for permeability-glycoprotein (P-gp). Many other substrate-binding sites are also present on Pgp, and co-administered drugs transported by P-gp may interact with each other, which can influence the final concentrations in the blood and ultimately affect toxicity and therapeutic effectiveness [2]. In this case, digoxin is commonly used as a probe compound to study drug-drug interaction (DDI).…”

“…LC/MS/MS is a highly efficient analytical tool for biological and nonbiological analysis. Some LC/MS/MS methods for quantifying digoxin have been reported, but in this study we developed a novel rapid and sensitive approach using formate-adduct ions [M+HCOO] − , found in Q1 scanning exhibited higher response than any other fragment ions [2,[5][6][7][8][9][10][11][12][13]. Formate-adducts afforded a limit of quantitation (LOQ) of 0.2 ng/mL and an amount on column (AOC) of 0.001 ng, which are both lower than most previously reported methods (Table 1).…”

“…Formate-adducts afforded a limit of quantitation (LOQ) of 0.2 ng/mL and an amount on column (AOC) of 0.001 ng, which are both lower than most previously reported methods (Table 1). Despite advantages in LOQ and AOC, one previous method involved a basic mobile phase consisting of 0.05% ammonium carbonate (pH 9.0) may degrade the silica in some columns [2]. Similarly, a large injection volume of 80 L reported in another method (using a column of 50 mm × 2.1 mm, 5 m) is considerably bigger than http://dx.doi.org/10.1016/j.jchromb.2014.11.023 1570-0232/© 2014 Elsevier B.V. All rights reserved.…”

“…The method developed here has an injection volume of 5 L (using a column of 100 mm × 2.1 mm, 5 m), well within the suggested limit, which allows analysis of trace amounts of sample and improves column performance in the LC/MS/MS procedure. Additionally, unlike previously described methods that use gradient elution [2,[5][6][7][8][9][10][11][12][13], which leads to a higher column pressure and compromises HPLC analysis, our method does not use gradient elution. Other advantages include a short analysis time, a simple mobile phase and sample extraction procedure, and the used of Oasis HLB Elution plates in the final optimized method, which can determine digoxin from only 100 L of biological sample (rat plasma).…”