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

• Type: Article in International Scientific Journal
• Date: 2022

Authors

Silva, LAP (Author) Other; Campagnolo, S (Author) Other; Fernandes, SR (Author) Other; Marques, SS (Author) Other; Luisa Barreiros (Author) FFUP; Sampaio Maia, B (Author) Other; Marcela A Segundo (Author) FFUP

Journal

Title: Analytical and Bioanalytical Chemistry

Vol. 415

Pages: 683-694

ISSN: 1618-2642

Publisher: Springer Nature

Indexing

ISI Web of Knowledge - 1 Citation

Scopus - 1 Citation

Other information

Authenticus ID: P-00X-KSY

DOI: 10.1007/s00216-022-04458-w

Abstract (EN): 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. Multianalyte methods allowing the establishment of a uremic metabolite profile are still missing. Hence, the aim of this work was to develop a rapid and sensitive method based on high-performance liquid chromatography with fluorescence detection for the simultaneous quantification 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 buffer containing an ion-pairing reagent, at a flow rate of 2 mL min(-1). Standards were prepared in plasma and quantification was performed using the background subtraction approach. LOQ values were <= 0.2 mu g mL(-1) for all analytes except for pCS (LOQ of 2 mu g 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 mu L), 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 significantly higher levels of 3-IAA, pC, 3-INDS, and pCS that can be further investigated as biomarkers of disease progression.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 12