Separation and Quantitation of Total Plasma Homocysteine and Methylmalonic Acid by LC-MS/MS Analysis

Importance of the Topic

Quantifying total plasma homocysteine and methylmalonic acid by LC-MS/MS provides critical insights into B-vitamin related metabolic disorders such as hyperhomocysteinemia and methylmalonic acidemia. Rapid and accurate measurement of these biomarkers supports differential diagnosis, patient monitoring, and quality control in clinical and research laboratories.

Aims and Study Overview

This study developed and validated a streamlined LC-MS/MS method for simultaneous separation and quantitation of homocysteine, methylmalonic acid (MMA), and related metabolites in plasma with a total run time of 4 minutes. The method targets improved sample throughput, robustness, and analytical performance for routine clinical applications.

Methodology and Instrumentation

The sample preparation protocol uses dithiothreitol (DTT) reduction of disulfide-bound homocystine to free homocysteine, followed by protein precipitation with methanol. Chromatographic separation employs a Raptor Polar X 50×2.1 mm, 2.7 µm column and guard cartridge, with a gradient elution using 0.5% formic acid in water (A) and acetonitrile (B) at 0.6 mL/min and 40 °C column temperature. Detection is performed on a tandem mass spectrometer in positive and negative electrospray ionization modes with multiple reaction monitoring (MRM) transitions optimized for each analyte and internal standards (DL-homocysteine-D4 and MMA-D3).

Main Results and Discussion

The method demonstrated robust linearity (r2≥0.995), accuracy within ±15% at low, mid, and high QC levels, and precision (intra- and inter-day %RSD <15%). Optimal DTT reduction time was 30 minutes. Baseline separation of homocysteine from homocystine and MMA from succinic acid was achieved in 4 minutes without carryover over 500 injections. Inter- and intra-day repeatability assessed across multiple plasma lots confirmed method reproducibility.

Benefits and Practical Applications

• Rapid turnaround time (4 minutes run) and simplified sample prep increase laboratory throughput.
• Simultaneous measurement of homocysteine, MMA, methionine, cysteine, succinic acid, and cystathionine supports comprehensive metabolic profiling.
• High analytical performance ensures reliable differential diagnosis of cobalamin and B-vitamin disorders.

Future Trends and Applications

Potential developments include automation of the DTT reduction step, integration with high-throughput workflows, expansion to additional biomarkers of one-carbon metabolism, and adaptation to microsampling techniques for remote or pediatric testing. Advanced data analysis and machine learning could further refine diagnostic algorithms.

Conclusion

A robust LC-MS/MS method for rapid, accurate, and reproducible quantitation of total plasma homocysteine and MMA was established, meeting clinical laboratory demands for high throughput and reliability. The approach enhances metabolic disorder diagnostics and supports expanded clinical research applications.

Instrumentation Used

• Raptor Polar X 50×2.1 mm, 2.7 µm analytical column with guard cartridge
• Mobile phases: 0.5% formic acid in water and acetonitrile
• Tandem mass spectrometer with electrospray ionization and MRM capability
• Microcentrifuge, vortex mixer, and 4000 rpm centrifuge

References

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