Developing an Analysis Method for Plasma Concentration of Indoxyl Sulfate in Chronic Kidney Disease Patients

Introduction: Indoxyl sulfate, a protein-bound uremic toxin, accelerates the progression of chronic kidney disease (CKD). Few methods are available for the quantification of IS in biological fluids and most of them require extensive preparation and long laboratory run time. The goal of this study was to evaluate the serum levels of IS in CKD patients using salting-out assisted liquid-liquid extraction (SALLE) and fluorescence. Methods: Plasma samples were extracted and deproteinized by the addition of nine part of acetonitrile to one part of serum and centrifuged for 10 min at 8000 rpm. The supernatant was transferred to a microtube and 500μl of different salting out agents i.e. sodium chloride were added. The fluorescence intensity of separated organic phase was determined by a spectroflurimnetry. Results: The affecting parameters on extraction i.e. volume of solvent, pH, the concentration of salt optimized and developed method was validated for quantification of IS with a good precision and accuracy less than 15%. Discussion: IS plays a significant role in the progression of CKD. A sensitive and reproducible SALLE extraction method was developed and validated for the quantitative determination of IS in plasma. It provides similar simplicity to protein precipitation, but cleaner extracts due to a true phase separation. The precision and accuracy are within the limits required for biological analytical assays.

Keywords:

Chronic Kidney Disease Patients , Indoxyl Sulfate , Salting-out assisted liquid-liquid extraction