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

Importance of the Topic

Measurement of total plasma homocysteine and methylmalonic acid (MMA) is critical for diagnosing inherited and acquired disorders of methionine and cobalamin metabolism. Elevated homocysteine is associated with cardiovascular risk and inborn errors such as homocystinuria. Increased MMA indicates vitamin B12 deficiency or methylmalonic acidemia. A rapid and robust analytical method enhances clinical decision making and quality control in research and diagnostic laboratories.

Objectives and Overview of the Study

The study aimed to develop and validate a four-minute LC-MS/MS assay for simultaneous separation and quantitation of total homocysteine and MMA in human plasma. Key goals included optimizing sample reduction, achieving baseline chromatographic resolution from isobaric interferents, and demonstrating linearity, accuracy, precision, and repeatability across multiple days and plasma lots.

Methodology and Used Instrumentation

Sample Preparation

• Plasma (100 µL) spiked with isotopic internal standards (DL-homocysteine-d4 and MMA-D3, 5 ng/mL).
• Reduction of disulfide bonds with 20 µL of 0.5 M dithiothreitol (DTT), incubated in darkness at room temperature for 30 minutes.
• Protein precipitation with 300 µL methanol, vortexing and centrifugation at 4,000 rpm for 10 minutes.
• Supernatant transfer to vial insert for LC-MS/MS injection (5 µL).

Used Instrumentation

• Liquid chromatograph coupled to a tandem mass spectrometer.
• Analytical column: Raptor Polar X, 50 × 2.1 mm, 2.7 µm; guard column: Raptor Polar X cartridge.
• Mobile phases: A = 0.5% formic acid in water; B = acetonitrile; flow rate 0.6 mL/min; column temperature 40 °C.
• Gradient elution over 4 minutes, achieving separation of homocysteine monomer and dimer, methionine, cysteine, succinic acid and MMA.

Main Results and Discussion

Optimization of Reduction

• A 30-minute incubation with DTT provided maximum conversion of homocystine to homocysteine with minimal extra processing time.

Linearity and Calibration

• Analytes exhibited linear response over 50–5,000 ng/mL for homocysteine and 25–5,000 ng/mL for MMA (1/x2 weighting, r2 ≥ 0.995).

Accuracy and Precision

• Inter-day accuracy for quality control samples remained within ±15% of nominal values; precision (%RSD) under 15% for all levels.

Repeatability

• Intra- and inter-day repeatability across three plasma lots showed % differences below 15% and %RSD under 12% for both homocysteine and MMA.

Chromatographic Performance

• All target analytes were resolved with good peak shape and retention time stability over 500 injections, with no carryover observed.

Benefits and Practical Applications

• Rapid sample preparation with DTT reduction and protein precipitation simplifies workflow.
• Four-minute run time increases sample throughput in clinical and research settings.
• Simultaneous quantitation of homocysteine and MMA supports differential diagnosis of cobalamin disorders without additional assays.

Future Trends and Applications

• Integration of automated sample preparation to further increase throughput.
• Expansion to multiplex panels including additional metabolic markers for comprehensive nutritional assessment.
• Application of high-resolution mass spectrometry for even greater specificity and sensitivity.
• Adaptation of the method for dried blood spot analysis or microfluidic platforms for point-of-care testing.

Conclusion

A fast, sensitive and reliable LC-MS/MS method was established for the simultaneous analysis of total homocysteine and methylmalonic acid in plasma. The optimized protocol demonstrates strong analytical performance and practical utility for clinical diagnostics and research applications.

References

1. Jiang Y, et al. Simultaneous determination of plasma total homocysteine and methionine by liquid chromatography-tandem mass spectrometry. Clin Chim Acta. 2017;464:93-97.
2. Persichilli S, et al. A Simplified Method for the Determination of Total Homocysteine in Plasma by Electrospray Tandem Mass Spectrometry. J Sep Sci. 2010;33(20):3119-3124.
3. Weaving G, et al. Simultaneous quantitation of homocysteine, cysteine and methionine in plasma and urine by liquid chromatography-tandem mass spectrometry. Ann Clin Biochem. 2006;43:474-480.