Direct Determination of Amiloride in Urine Using Isopotential Fluorimetry

Abstract

A method for the determination of amiloride at concentrations between 15 and 152 ng ml ^{- 1} by means of matrix isopotential synchronous fluorescence spectrometry and derivative techniques is proposed. This method is useful for the determination of compounds in samples with unknown background fluorescence without the need for tedious pre-separation. As amiloride is widely used as a doping substance in sport, the method was successfully applied to the determination of amiloride in urine. To obtain maximum sensitivity and adequate selectivity, factors affecting fluorescence intensity were studied in the amiloride band centered at λ _ex = 362 nm and λ _em = 415 nm. As a result, the determination was performed in an ethanol–water (1 + 1, v/v) medium at pH 6.3, adjusted by using sodium citrate–citric acid (0.1 M) as buffer solution. The concentration of amiloride in urine samples can be calculated by recording its total luminescence spectrum and applying the isopotential trajectory of the urine that cuts the selected band of amiloride. The unknown analytical signal of urine is eliminated in the MISF spectrum obtained, by means of its first derivative. A calibration graph was constructed by measuring first derivative values at λ _ex = 357 nm and λ _em = 392 nm. Analytical parameters of the proposed method were calculated according to the error propagation theory. The sensitivity, repeatability, reproducibility and limit of determination achieved with the proposed method are adequate for the determination of amiloride in urine.