Robust, reliable assay to quantify total homocysteine in human plasma or serum by liquid chromatographytandem mass spectrometry for use in clinical research

Importance of the Topic

Total homocysteine is a critical biomarker reflecting the efficiency of the methionine cycle and redox balance in humans. Elevated homocysteine levels have been associated with cardiovascular disease, cognitive impairment, and other chronic conditions. Reliable quantification in plasma or serum supports nutritional metabolomics, clinical research, and patient management.

Objectives and Study Overview

This study aimed to develop and validate a sensitive, accurate, and high-throughput LC-MS/MS assay for total homocysteine quantification in human plasma employing a Thermo Scientific Transcend II UHPLC system coupled to a TSQ Quantis triple-stage quadrupole mass spectrometer.

Methodology

• Sample preparation: 50 µL plasma reduced with DTT and spiked with d8-homocystine internal standard, followed by protein precipitation with methanol and centrifugation.
• Chromatography: Acclaim PolarAdvantage II column (100 × 3.0 mm, 3 µm) at 30 °C; mobile phases water and methanol (0.1% formic acid); gradient elution over 3.5 min; injection volume 5 µL.
• Mass spectrometry: Heated electrospray ionization in positive mode; SRM detection of homocysteine and d4-homocysteine; optimized source parameters (350 °C, 4000 V) and collision gas at 1.5 mTorr.

Used Instrumentation

• Thermo Scientific Transcend II UHPLC system
• Thermo Scientific TSQ Quantis triple-stage quadrupole mass spectrometer
• Acclaim PolarAdvantage II analytical column
• TraceFinder software version 4.1 for data acquisition and processing

Main Results and Discussion

• Linearity: Calibration range 0.794–6.86 µmol/L with correlation coefficients > 0.999.
• Sensitivity and Carryover: No significant carryover; low limits of quantification.
• Accuracy: Bias of –0.3% at two QC levels.
• Precision: Intra-assay CV ≤ 3.3%; inter-assay CV ≤ 2.7%.

Benefits and Practical Applications

• Simple and robust sample preparation requiring only 50 µL plasma.
• Rapid analysis with 3.5-minute runtime supports high throughput.
• High accuracy and reproducibility suitable for clinical research laboratories.
• Applicable to nutritional studies, disease biomarker research, and patient monitoring.

Future Trends and Applications

• Integration with broader metabolomic panels to explore multi-pathway interactions.
• Automation of sample processing and data analysis to increase throughput.
• Expanding the assay to other biological matrices and related metabolites.
• Potential development of point-of-care testing platforms for homocysteine monitoring.

Conclusion

The validated LC-MS/MS method delivers robust, reliable, and rapid quantification of total homocysteine in human plasma, meeting stringent clinical research requirements for sensitivity, accuracy, and precision.

References

• RECIPE Chemicals + Instruments GmbH calibrators and controls
• Thermo Fisher Scientific Transcend II, TSQ Quantis, TraceFinder 4.1