Background Sequential injection chromatography (SIC) is certainly a young, ten years aged, separation technique. validation Background Furosemide (FSM) is usually a loop diuretic. It is an anthranilic acid derivative, which is usually chemically 4-chloro-N-furfuryl-5-sulfamoylanthranilic acid (Physique?1a). FSM functions inhibiting the co-transportation of sodium, potassium and chloride. It further causes the excretion of calcium, magnesium and bicarbonate ions [1,2]. In another context, amiloride (AML) is usually chemically N-amidino-3,5-diamino-6-chloropyrazine-2-carboxamide (Physique?1b). AML, as another potent loop diuretic, acts primarily by blocking sodium and chloride reabsorption in the ascending limb of the loop of Henle. FSM helps to conserve potassium and minimize the risk of alkalosis. It PD173074 is also used in the treatment of oedema associated with hepatic cirrhosis and congestive heart failure [3,4]. Physique 1 Chemical structures of (a) amiloride and (b) furosemide. The association of AML and FSM furnishes a valuable natriuretic VCL agent with a diminished kaliuretic effect and minimizes the risk of alkalosis in the treatment of refractory oedema associated with hepatic PD173074 cirrhosis or congestive heart failure [5]. Due to the benefits of their simultaneous use, AML and FSM PD173074 are being prepared as binary dosage forms. Accordingly, the development of assay methods for those two drugs is usually desirable for the purpose of quality control. In this issue, various analytical techniques were exploited including high performance liquid chromatography [4,6,7], spectrophotometry [8-10], fluorometry [2] and electroanalytical [11]. On the other hand, athletes use diuretics, in general, for flushing previously taken prohibited substances with forced diuresis [12] to achieve acute weight loss. Hence, the World Anti-Doping Company (WADA) prohibits the usage of diuretics [13]. Besides as an condemned practice ethically, the risk towards the sportsmen health must be considered being that they are generally self-administered in an incorrect way; i.e. overdoses, connections with other medications or the usage of medications of illicit origins [14-16] even. Evidently, a delicate and dependable analytical solution to determine diuretics in urine and/or plasma is certainly a prerequisite in sport actions. Toward this final end, WADA establishes the very least detection capacity for testing strategies called the Least Required Performance Limitations (MRPL). That is to make sure that the presence could be reported by all doping control laboratories of prohibited substances uniformly. The limit for every analyte in the course of diuretics is certainly 250?ng/mL [17,18]. The dominant techniques employed for screening diuretics in charge urinalysis are HPLC and GC. However, both methods have the limitations of the high cost of instrumentation and maintenance. Moreover, other difficulties in GC namely are the low volatility of the compounds and the necessity of the additional step of derivatization. HPLC has also the limitation of large consumption of solvent volumes, which is due to the continuous circulation of mobile phase and large instrumentation dimension. Recently, sequential injection chromatography (SIC) was launched to overcome some difficulties in separation techniques [19]. In theory, the procedure of SIC is based on a sequential injection, i.e. a discontinuous-flow approach, of a mobile phase and samples. The separation process is usually carried PD173074 out into a monolith column using programmable miniaturized modules. The association of the three methods of the discontinuous-flow approach, monolith separation column and system miniaturization renders SIC process simple, rapid and reagent-saving [20-22]. On the other side, the major limitation of SIC is the limited pressure of the syringe pump. The maximum is usually 900?psi. This causes back-pressure in separation column and hence limits the use of longer separation column and therefore reduces the parting.