Rapid and sustainable HPLC method for the determination of uremic toxins in human plasma samples

Protein-bound uremic toxins, mainly indoxyl sulfate (3-INDS), p-cresol sulfate (pCS), and indole-3-acetic acid (3-IAA) but also phenol (Pol) and p-cresol (pC), are progressively accumulated during chronic kidney disease (CKD). Their accurate measurement in biomatrices is demanded for timely diagnosis and adoption of appropriate therapeutic measures. Multiana lyte methods allowing the establishment of a uremic metabolite profle are still missing. Hence, the aim of this work was to develop a rapid and sensitive method based on high-performance liquid chromatography with fuorescence detection for the simultaneous quantifcation of Pol, 3-IAA, pC, 3-INDS, and pCS in human plasma. Separation was attained in 12 min, using a monolithic C18 column and isocratic elution with acetonitrile and phosphate bufer containing an ion-pairing reagent, at a fow rate of 2 mL min−1. Standards were prepared in plasma and quantifcation was performed using the background sub traction approach. LOQ values were≤0.2 µg mL−1 for all analytes except for pCS (LOQ of 2 µg mL−1). The method proved to be accurate (93.5–112%) and precise (CV≤14.3%). The multianalyte application of the method, associated to a reduced sample volume (50 µL), a less toxic internal standard (eugenol) in comparison to the previously applied 2,6-dimethylphenol and 4-ethylphenol, and a green extraction solvent (ethanol), resulted in the AGREE score of 0.62 which is in line with the recent trend of green and sustainable analytical chemistry. The validated method was successfully applied to the analysis of plasma samples from control subjects exhibiting normal levels of uremic toxins and CKD patients presenting signifcantly higher levels of 3-IAA, pC, 3-INDS, and pCS that can be further investigated as biomarkers of disease progression.