High-throughput UHPLC-MS/MS method for 15 protein-bound uremic toxins in human serum.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Nephrology

Background:

• Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a severe complication of chronic kidney disease (CKD).
• CKD-MBD is associated with the accumulation of protein-bound uremic toxins (PBUTs).
• Existing PBUT analysis methods have limitations in analyte coverage and high-throughput quantification.

Purpose of the Study:

• To develop and validate a high-throughput targeted Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method for quantifying multiple PBUTs in human serum.
• To assess the utility of the developed method for differentiating between uremic patients and healthy controls.

Main Methods:

• Development and validation of a targeted UHPLC-MS/MS method for 15 PBUTs, including Nδ-(carboxymethyl)lysine (CML) and pentosidine (PE).
• Utilized 96-well protein/phospholipid removal plates for sample preparation.
• Optimized chromatographic and mass spectrometric conditions for reproducible quantification.

Main Results:

• The validated method demonstrated excellent linearity (R² > 0.99), low limits of quantification (10-20 ng/mL), and acceptable precision (RSD < 15%).
• Analysis of serum samples revealed significantly elevated levels of 10 PBUTs in 40 uremic patients compared to 20 healthy controls.
• Specific PBUTs, such as p-cresyl sulfate (PCS), were found at approximately 400-fold higher concentrations in patients, with five PBUTs undetectable in healthy individuals.

Conclusions:

• A reliable and accurate high-throughput UHPLC-MS/MS method for PBUT quantification has been established.
• The findings underscore the critical role of PBUTs in the pathogenesis of CKD-MBD.
• This method advances PBUT-related metabolomic research and clinical diagnostics for CKD complications.