Extraction of indoxyl sulfate to its determination in patients with kidney disease

Abstract

Introduction: Indoxyl sulfate(IS), a protein-bound uremic toxin which is related to different pathological conditions such as chronic kidney disease (CKD) and cardiovascular disease (CVD). Few methods are available for quantification of IS in biological fluids and most of them require long laboratory run time. Salting-out assisted liquid-liquid (SALLE) extraction method is used for distinct solvents from plasma. Aims: The aim of this study was to propose a simple analytical method for IS in plasma samples of CKD patients using SALLE and fluorescence quantification. Methods: Various parameters affecting the fluorescence intensity i.e. salt type and concentration and pH were optimized using SALLE and fluorescence methodology. Then, the optimized parameters were applied to method validation in different samples. Plasma samples were extracted and deproteinized by addition of nine part of acetonitrile to one part of plasma and centrifuged for 10 min at 8000 rpm. The supernatant was transferred to microtube and sodium chloride solution was added. The fluorescence intensity of separated organic phase was determined by a spectroflurimetry. Results: Under the optimum conditions, the method provided a linear range between 2.5 to 40 mg/L with a correlation coefficient (R2) of 0.995 with limit of detection (LOD) and limit of quantification (LOQ) of 0.75 mg/L and 2.5 mg/L, respectively. The accuracy and precision (as relative standard deviation were within ±15% of the nominal concentration and less than 15%, at all concentrations, respectively. Conclusion: IS plays a significant role in progression of CKD. A sensitive and reproducible SALLE extraction method was developed for the determination of IS in plasma, provides clean extracts due to true phase separation.