Determination of Digoxin in Human Serum Using Stable Isotope Dilution Liquid Chromatography/Electrospray Ionization-Tandem Mass Spectrometry

Abstract

Digitalis glycosides, represented by digoxin (Dg) and digitoxin, have been commonly used for the treatment of congestive heart failure and other cardiac diseases. In addition to the narrow therapeutic range of the serum digitalis glycosides, their bioavailability is easily affected by kidney or liver failure and the dosing of other medicines. Therefore, it is important to accurately measure the digitalis glycosides in sera from digitalized patients.Dg has been more commonly used than digitoxin and its therapeutic range in serum is 0.8-2.0 ng/ml. 1) Immunoassay is routinely used for therapeutic drug monitoring (TDM) of Dg. However, the commercially available antibodies show a high cross-reactivity with Dg metabolites, such as digoxigenin bisdigitoxoside (Bis-Dggenin), digoxigenin monodigitoxoside (Mono-Dggenin) or digoxigenin (Dggenin) (Fig. 1), which were formed by the successive cleavage of the sugar moiety, as well as with endogenous digitalis-like immunoreactive substances (DLIS).2) These cause a false-elevation or -lowering of the serum Dg concentration due to the inappropriate prescriptions of Dg dosages by physician. To overcome this problem, Ikeda et al. designed a new hapten of Dg and developed a radioimmunoassay (RIA) for Dg in human serum, which can clearly discriminate Dg from the Dg metabolites (Bis-Dggenin, Mono-Dggenin and Dggenin), but not DLIS.3) Liquid chromatography/mass spectrometry (LC/MS) also seems to be suitable for this purpose because of its high sensitivity, specificity and without interfering from DLIS. Recently, the determination of digitoxin or Dg using LC/MS has been reported, [4][5][6][7][8] but its application to real samples obtained from digitalized patients followed by comparison with the data obtained from immunoassay has only been done in our report for determining digitoxin. 4)In this study, we developed a determination method for Dg in human serum using stable isotope dilution LC/electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) and applied this method to real samples. The data obtained in this study were compared to those obtained by using RIA with the anti-serum clearly discriminating Dg from its metabolites.3) Kakuma-machi, Kanazawa 920-1192, Japan: and b Faculty of Pharmaceutical Sciences, Hokuriku University; Ho-3, Kanagawa-machi, Kanazawa 920-1181, Japan. Received April 14, 2007 accepted June 12, 2007; published online June 18, 2007 A method for the determination of digoxin in human serum using a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) technique is reported. Digoxin and the internal standard, [21,21,22-2 H 3 ]digoxin, were extracted from 250 m ml of human serum using a solid phase extraction cartridge (Oasis HLB) and analyzed by LC/ESI-MS/MS in the selected reaction monitoring mode. The intra-and interassay reproducibility and accuracy were satisfactory within the quantification range of 0.20-3.20 ng/ml. The concentrations of digoxin in the serum samples obtained from digitalized patients (n‫)91؍‬ were ...

RETRACTED: Development and validation of an LC–MS method with electrospray ionization for quantitation of digoxin in human plasma and urine: Application to a pharmacokinetic study

“…Liquid-liquid extraction [9][10][11] or solid-phase extraction (SPE) [13][14][15] has been described for the determination of digoxin. We have used liquid-liquid extraction with chloroform-isopropanol (95:5, v/v), the mean extraction efficacies from plasma for digoxin (0.35, 1.96 and 7.80 ng/mL) and IS were found to be (81.7 ± 3.5)%, (85.6 ± 6.9)%, (83.0 ± 3.8)% and (87.2 ± 4.4)%, respectively.…”

“…However, the previously published methods require either large plasma volumes or a tandem mass spectrometer. Some methods utilizing solid-phase column extraction were expensive and time-consuming [13][14][15]. In addition, only one recent LC-MS method to determine digoxin in human urine has been published [16].…”

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P‐glycoprotein (P‐gp) inhibition screen

“…[8] There have also been several papers describing LC-MS/MS methods for digoxin analysis in biological fluids. [9][10][11][12] However, at a total cycle time of more than 4 min per injection, these methods are not suitable for a high-throughput ADME screening environment whereby thousands of samples are generated and analyzed every day. [13,19] To address this challenge and support a high-throughput in vitro P-gp inhibition screen, we developed an ultrafast bioanalytical method for digoxin with a total cycle time of 9 s per injection, using a RapidFire™ 200 system (BIOCIUS Life Sciences, Woburn, MA, USA) with tandem mass spectrometric detection (RF-MS/MS).…”

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

“…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).…”