Method development for digoxin determination in biological samples using fluorescence quenching

Abstract

Digoxin (DG) is a cardiac glycoside steroid that is commonly used to cure congestive heart failure and arrhythmias. Several studies support that the effective range of plasma concentration is between 0.5 and 2.0 ng/mL, being it toxic at higher plasma concentration (>2.0 ng/mL). It is also well stablished that the effective plasma drug concentration is close to the toxic, and large individual differences in the effects of the drug have been observed. Therefore, simple, and sensitive procedure is required for strict monitoring of DG in blood level to minimize the risk of toxicity. The main objective of this study was the design of new derivatization strategies that allow DG determination by molecular fluorescence. In this research, a standard solution of DG (Sigma Chemical Co.) was taken up in ethanol (1mg mL-1). Bovine seroalbumine (BSA) was acquired of Lab. Alimentos-UNSL, San Luis, Argentina. All experiments were carried out in a 1 cm quartz cell, using a Shimadzu RF-5301PC spectrofluorimeter, equipped with a Xenon discharge lamp. Several factors that affect the derivatization reaction were studied i.g., type and concentration of the fluorophore (BSA, Rhodamine B, eosyne, quinoleine), pH, ionic strength, organic solvents. Also, the effect of different surfactants was evaluated as improvement fluorescence signal (SDS, Bile salt, HTAB), etc. The spectral behavior observed after derivatization were evaluated to establish the optimal experimental conditions (excitation and emission wavelength: 280 nm and 340 nm (BSA) and 555 nm and 575 nm (RhB), slits: 5/5). The attenuation in the fluorescent signal (quenching effect) with increase DG concentration on BSA and RhB was observed. The studied strategies will be incorporated to schemes of high-performance liquid chromatography associated with fluorescent detection to automate the methodology and allow the determination of DG in biological samples.