Quantification of total homocysteine in human plasma or serum by liquid chromatography tandem mass spectrometry for clinical research

Significance of the Topic

A precise measurement of total homocysteine in human plasma and serum is a critical tool in clinical research and diagnostic studies. Elevated homocysteine levels are associated with cardiovascular and neurodegenerative disorders, making reliable quantification essential for risk assessment, intervention monitoring, and epidemiological investigations.

Study Objectives and Overview

This study aimed to develop and validate a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for quantifying total homocysteine in human plasma or serum. The method converts all homocysteine forms (protein‐bound, dimerized, and mixed disulfides) into the free thiol and employs a Thermo Scientific Transcend II TLX-1 system coupled to a TSQ Endura triple quadrupole mass spectrometer for rapid and accurate analysis.

Methodology and Instrumentation

Sample Preparation:

• Fifty microliters of plasma or serum mixed with 50 µL of reducing solution and 50 µL of d8-homocystine internal standard.
• Incubation at room temperature for 5 minutes to convert all homocysteine forms to free homocysteine and homocysteine-d4.
• Protein precipitation with 200 µL of cold precipitant, vortex mixing, 5 minutes at 4 °C, and centrifugation at 10 000 × g.

Chromatography and Mass Spectrometry:

• Isocratic elution on the Transcend II TLX-1 system with a 70-second runtime.
• Detection by positive-mode heated electrospray ionization on a TSQ Endura triple quadrupole.
• Selected reaction monitoring (SRM) transitions for homocysteine (m/z 136.2→90.3 and 136.2→56.2) and d4-homocysteine (m/z 140.3→94.2 and 140.3→60.3).
• Data acquisition and processing in Thermo Scientific TraceFinder 3.3.

Results and Discussion

The method demonstrated a linear response over 0.117–50.7 µmol/L (R2 = 0.991), extending well beyond the calibration range by dilution of the lowest calibrator. The lower limit of quantification was 0.117 µmol/L. Analytical accuracy, assessed with NIST SRM 1955 at three concentration levels, showed biases between 3.7% and 5.2%. Precision studies yielded maximum intra-assay and inter-assay coefficients of variation of 2.0% and 3.3%, respectively. Representative chromatograms confirmed clear peak separation and signal stability at the lowest calibrator level.

Benefits and Practical Applications

This method offers:

• Simple and rapid sample preparation with minimal handling steps.
• Comprehensive measurement of all homocysteine forms via a single reduction step.
• High throughput with a total runtime of just over one minute.
• Strong analytical performance meeting clinical research requirements for sensitivity, accuracy, and precision.

Future Trends and Opportunities

Advancements may include automation of sample preparation, integration of multiplexed assays to measure related biomarkers simultaneously, and further miniaturization of LC-MS/MS systems. Expanded applications could cover routine screening programs, population studies, and personalized medicine approaches targeting homocysteine-linked pathologies.

Conclusion

The validated LC-MS/MS assay provides a robust, high-performance tool for total homocysteine quantification in human plasma and serum. It supports clinical research demands and offers a platform adaptable to evolving analytical and diagnostic needs.

Reference

Claudio De Nardi et al. Technical Note 64917, Thermo Fisher Scientific, 2017
RECIPE MS2000 ClinMass® Complete Kit Homocysteine in Plasma/Serum
NIST Standard Reference Material® 1955