DEVELOPMENT OF AN LC-MS/MS-BASED PANEL FOR PROTEIN-BOUND UREMIC TOXINS

Project no. 31853 · Status: Kapanmış (Yayın bekleniyor) · 2024

Summary

Objective: The accumulation of protein-bound uremic toxins (PBUTs) in chronic kidney disease (CKD) accelerates kidney function loss and contributes to complications such as cardiovascular diseases and oxidative stress. This study aims to develop a sensitive and reliable LC-MS/MS method for the quantification of PBUTs (p-cresol sulfate [PCS], indoxyl sulfate [IxS], indole-3-acetic acid [IAA], and CMPF), establish reference intervals for healthy individuals, and compare PBUT levels in hemodialysis patients.Materials and Methods: Serum samples from 32 hemodialysis patients and 120 healthy individuals were analyzed for total and free PBUTs using LC-MS/MS. The method development included the optimization of mass spectrometric parameters, pH, and mobile phase conditions. Various solvent systems were tested to enhance extraction efficiency, and the optimal system was selected. Method accuracy, precision, recovery, matrix effects, and quantification limits were validated according to FDA and EMA guidelines.Results: The optimized LC-MS/MS method demonstrated high sensitivity and accuracy for all analytes. The ACN/acetone (1:1) mixture was identified as the most effective solvent system for extraction. Reference intervals for healthy individuals were calculated as follows: PCS, 30.72-11,410.49 ng/mL; IxS, 110.83-2,160.75 ng/mL; IAA, 126.25-1,814.82 ng/mL; and CMPF, 108.48-1,157.18 ng/mL. Strong positive correlations between total and free PBUT levels were observed in hemodialysis patients (R values: 0.70-0.88). PBUT levels were significantly higher in hemodialysis patients compared to healthy individuals (p<0.001).Conclusion: The developed LC-MS/MS method provides a sensitive and reliable quantification of PBUTs in serum. The reference intervals established for healthy individuals can be utilized in clinical diagnosis and monitoring. This study offers valuable insights into the potential role of PBUTs as biomarkers in the pathophysiology of CKD.Keywords: LC-MS/MS, Uremic toxins, Extraction, Serum, p-Cresol Sulfate, Indoxyl Sulfate, Methodology

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