Using on-line solid phase extraction for determination of amiloride in human urine by sequential injection technique

This presented paper deals with a new methodology for the direct determination of amiloride in human urine. The methodology described is based on the sequential injection analysis technique (SIA) coupled with solid phase extraction (SPE) microcolumn. SPE microcolumn was used for selective retention of amiloride, while the urine matrix components were eluted with water carrier flow to the waste. Due to the acid-basic and polarity properties of amiloride molecule and principles of ion-exchange chromatography, it was possible to retain amiloride on the ion-exchange sorbent (SPE BAKER WCX—carboxy group). Eluting solution was 0.01 M HNO3 + 0.1 M KCl, flow rate 20 μl s−1. The fluorescence detection of amiloride was performed at λem 385 nm (secondary filter). Recovery was found in the range 96.8–99.4% for 10 times diluted urine, linearity of determination in the range 0.5–100 μg ml−1 (r = 0.998), and 3σ limit of detection (LOD) was 0.05 μg ml−1. The whole procedure comprising raw sample pre-treatment, analyte detection and column reconditioning took 8 min. The proposed SIA–SPE method has been applied for direct determination of amiloride in human urine.