A rapid and sensitive LC/MS/MS assay for quantitative determination of digoxin in rat plasma

Abstract

Digoxin is a cardiac glycoside that is widely used for the treatment of congestive heart failure. To evaluate pharmacokinetics of digoxin in rats, a sensitive LC/MS/MS assay was developed and validated for the determination of digoxin concentration in rat plasma. For detection, a Sciex API3000 LC/MS/MS with atmospheric pressure ionization (API) mass spectrometry turbo ion spray inlet in the positive ion-multiple reaction monitoring mode was used to monitor precursor→product ions of m/z 798.6→651.6 for digoxin and m/z 577.6→433.3 for oleandrin, the internal standard (IS). The standard curve was linear (r²≥0.999) over the digoxin concentration range of 0.1–100 ng/ml in plasma for digoxin. The mean predicted concentrations of the quality control samples deviated by <5.8% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8.6% relative standard deviation. At the lower limit of quantitation (LLQ) of 0.1 ng/ml, the mean deviation of predicted concentrations from the nominal value was within 3.7%. The extraction recoveries of digoxin and internal standard were 82.7±3.9 and 105.9±2.3%, respectively. The present method was successfully applied to characterization of pharmacokinetic profiles of digoxin in rats after oral administration.

Introduction

Digoxin (Fig. 1) is a cardiac glycoside that is widely used for the treatment of congestive heart failure. Digoxin has a very narrow therapeutic index and thus, digoxin therapy requires strict monitoring of blood levels to minimize toxicity [1], [2]. Digoxin has been shown to be a substrate of P-glycoprotein (P-gp) both in vitro [3] and in vivo [4]. Drug–drug interactions of digoxin with P-gp modulators have been well documented. For example, P-gp inhibitors, such as verapamil [5] and quinidine [6], have been shown to increase blood concentrations of digoxin. On the other hand, rifampin, a P-gp inducer, increased the intestinal P-gp expression and led to a significant decrease in oral exposure of digoxin [7]. Therefore, identification of drug candidates that are neither substrates nor inhibitors of P-gp would reduce the likelihood of a clinical drug–drug interaction. Implementation of small animal models (e.g. rats) to study digoxin interactions has been difficult mainly because of lack of a sensitive and easy to use bioanalytical methodology for digoxin quantitation.

There are numerous methods reported for digoxin quantitation, including radioimmunoassay (RIA), high performance liquid chromatography (HPLC) assay with RIA or fluorescence detection and LC/MS or LC/MS/MS assay. RIA is currently the most commonly used method for digoxin quantitation in biological matrices [8], [9]. While this method is sensitive and used frequently in clinical and non-clinical studies, it has been reported that RIA is not specific and cross-reacts with digoxin metabolites and endogenous digoxin-like substances [10], [11], [12]. HPLC methods are capable of avoiding interference, but are generally not sensitive enough to quantitate digoxin at lower levels (e.g. <1 ng/ml). For digoxin interaction studies, [³H]digoxin is often preferred even though significant effort has to be devoted to digoxin peak resolution and radioactive safety precautions. There are several reports describing LC/MS or LC/MS/MS methods for the identification and quantitation of digoxin, digoxin metabolites and digoxin-like substances in biological matrices [12], [13]. For digoxin quantitation, these methods are generally sensitive but require large sample volume (0.5–4 ml plasma), which is not feasible in small animal models (e.g. rats).

To evaluate digoxin pharmacokinetics in rats, a sensitive and robust LC/MS/MS method was developed and fully validated. The method was successfully applied to digoxin pharmacokinetic interaction studies in rats and can be easily extended to other animal species. The current method offers a number of advantages over existing methods, such as shorter analysis time, smaller sample volume (200 μl blood), amenable to serial sampling studies and devoid of extensive sample cleanup.