HILIC/MS METHOD FOR THE QUANTIFICATION OF THE POLAR BIOMARKER ITACONIC ACID

Importance of the Topic

Itaconic acid is a small polar metabolite identified as a biomarker for conditions including gestational diabetes mellitus and rheumatoid arthritis. Sensitive quantification in human serum supports early detection and improved patient care.

Objectives and Study Overview

This study aimed to establish a reliable hydrophilic interaction chromatography–tandem mass spectrometry (HILIC-MS/MS) method for quantifying endogenous itaconic acid in human serum, eliminating the need for derivatisation and reducing sample preparation time.

Methodology and Instrumentation

Sample preparation combined protein precipitation with 0.1% trifluoroacetic acid (TFA) and a weak cation exchange (WCX) solid‐phase extraction pass‐through on Waters Oasis WCX µElution plates. Chromatographic separation used a 100 × 2.1 mm, sub-2 µm amide HILIC column with a mobile phase of >80% acetonitrile buffered at pH 9 with 5 mM ammonium acetate. Detection was performed by multiple reaction monitoring (MRM) in negative electrospray ionization on a Waters ACQUITY UPLC H-Class coupled to a Xevo TQXS tandem quadrupole mass spectrometer. Ostro™ PPT plates facilitated protein removal.

Main Results and Discussion

Optimization of protein precipitation and SPE conditions achieved recoveries up to 94% and significantly reduced matrix effects compared to mixed‐mode SPE and precipitation alone. Calibration curves were linear over 2.5–500 ng/mL (r² > 0.997) with mean accuracy between 88.7% and 110.8% and an LLOQ near 1 ng/mL. The validated range covers reported endogenous concentrations (5.2–260 ng/mL).

Benefits and Practical Applications

The developed HILIC-MS/MS assay delivers high sensitivity, rapid workflow without derivatisation, and robust quantification suitable for clinical laboratories and research settings monitoring itaconic acid as a disease biomarker.

Future Trends and Potential Applications

Potential improvements include incorporation of isotopically labelled internal standards, further reduction of LC cycle times, exploration of alternative MRM transitions to minimize interferences, and automation of sample handling. The platform could be extended to other polar metabolites in clinical metabolomics studies.

Conclusion

This feasibility study confirms that HILIC-MS/MS with a sub-2 µm amide column and optimized sample preparation provides reliable, reproducible, and sensitive quantification of itaconic acid in human serum, supporting its application in biomarker research.

References

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