Quantification of total homocysteine in human plasma or serum by LC-HRAM-MS for clinical research

Importance of the topic

Accurate quantification of total homocysteine in plasma or serum is critical for clinical research into cardiovascular, neurodegenerative and metabolic disorders. Homocysteine, predominantly present as protein‐bound or disulfide forms, requires efficient sample preparation to free all forms for reliable measurement. High-resolution mass spectrometry offers selectivity and sensitivity to support large‐scale studies and nutritional metabolomics applications.

Goals and overview of the study

This study describes the development and evaluation of an LC-HRAM-MS workflow to quantify total homocysteine using a Thermo Scientific Q Exactive Plus hybrid quadrupole‐Orbitrap mass spectrometer. Key objectives included:

• Implementing an offline reduction and protein precipitation protocol with the ClinMass® sample preparation kit
• Establishing chromatographic separation on a Vanquish Duo UHPLC system with a 1.0 minute runtime
• Optimizing PRM acquisition parameters for high mass accuracy and reproducibility
• Validating method performance: linearity, sensitivity, accuracy, precision and carryover

Methodology and instrumentation

Sample preparation involved reduction of homocystine and mixed disulfides to free homocysteine, followed by protein precipitation. A 50 µL plasma or serum aliquot was combined with reduction and internal standard solutions, incubated, precipitated and centrifuged. The supernatant was directly injected.

• Liquid chromatography: Vanquish Duo UHPLC, isocratic elution at 0.7 mL/min, 25 °C, 5 µL injection, 1.0 min runtime
• Mass spectrometry: Q Exactive Plus, HESI-II source in positive mode, PRM acquisition, resolution 17 500 at m/z 200, isolation window 4.0, AGC target 1e6, max IT 100 ms, stepped collision energies 20/30/40

Main results and discussion

The method demonstrated:

• Excellent linearity across 0.837–6.85 µg/mL and reliable quantitation down to an LLOQ of 0.419 ng/mL
• Accuracy within ±15% (±20% at the lowest calibrator) and no detectable carryover
• High precision: intra‐assay CV ≤2.6% and inter‐assay CV ≤2.5%
• Bias of 4.1–5.3% for internal kit controls and 4.1% for NIST SRM 1950 external control

Benefits and practical applications

This streamlined workflow leverages a robust sample prep kit and fast chromatography to deliver high-throughput analysis suitable for clinical research laboratories. The high selectivity of Orbitrap‐based PRM techniques minimizes interferences, enabling accurate homocysteine monitoring in large‐scale nutritional and disease‐related studies.

Future trends and potential applications

Advances may include:

• Integration of online SPE for further automation and throughput gains
• Expansion to multiplex panels covering additional methionine‐cycle metabolites
• Coupling with data‐driven metabolomics and AI tools for deeper phenotyping
• Further miniaturization and high‐resolution instrumentation for point‐of‐care or field applications

Conclusion

The presented LC-HRAM-MS method provides a rapid, precise and accurate approach for total homocysteine quantification in plasma or serum. It meets clinical research needs by ensuring high sensitivity, linearity and reproducibility using readily available sample preparation kits and state‐of‐the‐art Orbitrap technology.