Abstract

Chronic kidney disease (CKD) is characterized by accumulation of uremic toxins. Many of the uremic toxins are Protein bound uremic solutes (PBUS), associated with progression to end stage renal disease in patients with early stages of CKD which originate due to gut dysbiosis. Monitoring the levels of PBUS plays important role for studying the therapeutic effect of probiotic treatment and adequacy of dialysis. Objective: Here we developed a Chromatography (HPLC) based method for the simultaneous determination of predominant uremic solutes - indole-3-acetic acid (3-IAA), Indoxyl sulfate (3-INDS) and p-cresol sulfate (p-CS) in human serum and application of method for analysis of uremic solutes level in CKD patients. Materials & Methods: HPLC method was developed by using ion-pairing mobile phase with an isocratic flow and Kinetex C18 reverse phase 250mm X 4.6mm, 5 µ analytical column in High performance liquid Chromatography (HPLC), shimadzu, japan. Chromatographic peaks were monitored with fluorescence detector for 20 minutes. The sample preparation involves protein precipitation with methanol and followed by centrifugation to collect clear supernatant, which is injected on the column, and the chromatogram corresponding to all three uremic solutes is obtained in less than 20 min. Results: Method was validated in line with International Council for Harmonization (ICH) M10 bioanalytical method validation guidelines. The limit of quantification for 3-IAA, 3-INDS and p-CS were 0.10 ng/mL, 0.20 ng/mL and 0.50 ng/mL respectively. Method was found linear with regression coefficient (r 2) value greater than 0.99 throughout validation. Conclusion: This HPLC method permits the simultaneous and quick quantification of uremic solutes for daily analysis of large numbers of samples giving an opportunity to monitor their serum levels in CKD patients. This method could be of clinical importance for the follow up of these patients with probiotic treatment, the assessment of cardiovascular risk and the checking of blood purification adequacy during dialysis treatment

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