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

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Abstract

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 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 significantly higher levels of 3-IAA, pC, 3-INDS, and pCS that can be further investigated as biomarkers of disease progression.

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Funding

This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e a Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the projects UIDB/50006/2020 and UIDP/50006/2020, and also from the European Regional Development Fund through COMPETE 2020 — Programa Operacional Competitividade e Internacionalização (POCI) together with FCT/MCTES in the framework of the project PTDC/MEC-MCI/29777/2017. S. R. Fernandes thanks FCT and ESF (European Social Fund) through Norte 2020 (Programa Operacional Regional Norte) for her PhD grant (SFRH/BD/130948/2017 and COVID/BD/152406/2022). L. Barreiros acknowledges funding from FCT through program DL 57/2016–Norma transitória.

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Authors and Affiliations

  1. LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal

    Luís A. P. Silva, Stefano Campagnolo, Sara R. Fernandes, Sara S. Marques, Luisa Barreiros & Marcela A. Segundo

  2. Escola Superior de Saúde, Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida 400, 4200-072, Porto, Portugal

    Sara R. Fernandes & Luisa Barreiros

  3. i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 280, 4200-135, Porto, Portugal

    Benedita Sampaio-Maia

  4. INEB-Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 280, 4200-135, Porto, Portugal

    Benedita Sampaio-Maia

  5. Faculdade de Medicina Dentária, Universidade do Porto, Rua Dr. Manuel Pereira da Silva, 4200-393, Porto, Portugal

    Benedita Sampaio-Maia

Authors
  1. Luís A. P. Silva
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  2. Stefano Campagnolo
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  3. Sara R. Fernandes
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  4. Sara S. Marques
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  5. Luisa Barreiros
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  6. Benedita Sampaio-Maia
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  7. Marcela A. Segundo
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Contributions

LAPS: methodology, investigation, validation, writing — original draft. SC: investigation, validation, formal analysis, writing — review and editing. SRF: methodology, investigation, writing — review and editing. SSM: validation, formal analysis, writing — review and editing. LB: conceptualization, methodology, formal analysis, supervision, writing — original draft, writing — review and editing. BS-M: conceptualization, supervision, resources, funding acquisition, writing — review and editing. MAS: conceptualization, supervision, resources, project administration, funding acquisition, writing — review and editing.

Corresponding author

Correspondence to Luisa Barreiros.

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The study protocol was approved by the Ethics Committee at Centro Hospitalar de São João (ref. CES 87–15) and performed in agreement with the 1964 Helsinki declaration and subsequent revisions. Written informed consent was obtained from all participants before sample collection.

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The authors declare no competing interests.

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Luís A. P. Silva and Stefano Campagnolo contributed equally to this work.

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Silva, L.A.P., Campagnolo, S., Fernandes, S.R. et al. Rapid and sustainable HPLC method for the determination of uremic toxins in human plasma samples. Anal Bioanal Chem 415, 683–694 (2023). https://doi.org/10.1007/s00216-022-04458-w

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