Fast and reliable method for the analysis of methylmalonic acid from human plasma

Importance of the Topic

The accurate quantification of methylmalonic acid (MMA) in human plasma is critical for the diagnosis and monitoring of vitamin B12 (cobalamin) deficiency. Conventional gas chromatography–mass spectrometry methods require lengthy derivatization and extended run times. Interference from endogenous succinic acid (SA), which shares the same molecular weight and similar fragmentation, further complicates selective detection.

Objectives and Study Overview

This study describes a fast, reliable, high-throughput LC-MS/MS workflow for MMA analysis. Key goals include:

• Efficient extraction of MMA from plasma using mixed-mode ion-exchange solid phase extraction (SPE) on SOLAµ WAX microplates.
• Chromatographic resolution of MMA and SA in under 3.5 minutes without derivatization.
• Accurate quantification across a dynamic range (15–1200 ng/mL) with deuterated MMA (MMA-d3) as an internal standard.

Methodology and Instrumentation

Sample Preparation:

• Spike plasma or phosphate-buffered saline (PBS) surrogate matrix with MMA-d3 internal standard.
• Pre-treat samples with ammonium acetate buffer (pH 4) and load onto SOLAµ WAX SPE plates.
• Condition (methanol), equilibrate (15 mM ammonium acetate), load, wash (buffer and methanol), and elute (5% ammonia in water) in a micro-elution format.
• Evaporate under nitrogen or inject directly; reconstitute in starting mobile phase if needed.

Liquid Chromatography-Mass Spectrometry:

• LC system: Thermo Scientific Dionex UltiMate 3000 RSLC with Acclaim Surfactant Plus column (3 µm, 150 × 2.1 mm) and 5 µm guard.
• Gradient: 15 mM ammonium acetate (pH 4) versus acetonitrile; run time 5.5 minutes, flow rate 0.45 mL/min, column at 30 °C.
• MS detection: Thermo Scientific TSQ Vantage triple quadrupole with heated electrospray ionization (HESI) in negative polarity; transitions for MMA (117→73 m/z) and SA (117→99 m/z); scan time 0.02 s.
• Data processing: Thermo Scientific LCQUAN quantitative software.

Main Results and Discussion

Chromatographic Resolution:

• Baseline separation achieved: SA at 1.71 min, MMA at 2.37 min.

Linearity and Calibration:

• MMA standards in PBS produced a linear curve from 15 to 1200 ng/mL (R² = 0.9953) using 1/x² weighting.

Accuracy and Precision:

• PBS QC samples (25, 200, 1000 ng/mL) showed biases within ±11% and RSD <10%.
• Plasma QC adjusted for endogenous MMA (~12.2 ng/mL) delivered biases <10% and RSD <5%.

Recovery and Matrix Effects:

• Recovery from PBS ranged 91–96% with matrix suppression −4% to −10%.
• Plasma recovery ranged 81–105% with matrix suppression −10% to −22%.

Benefits and Practical Applications

• High-throughput capable of processing 96 samples within 1–2 hours without derivatization.
• Low elution volumes enhance sensitivity and reduce sample failures.
• Use of surrogate matrix (PBS) simplifies calibration when blank plasma is scarce.
• Robust reproducibility and minimal matrix interferences support clinical and QA/QC laboratories.

Future Trends and Possibilities

• Automation of SPE and LC integration for fully unattended workflows.
• Extension of mixed-mode SPE methods to other organic acids and biomarkers.
• Development of faster chromatography phases and smaller particle sizes to shorten run times further.
• Integration with high-resolution mass spectrometry and advanced data analytics for broader metabolomic profiling.

Conclusion

The combined use of SOLAµ WAX mixed-mode SPE and fast LC-MS/MS on an Acclaim Surfactant Plus column provides a reliable, high-throughput method for quantifying MMA in human plasma. Excellent recovery, low matrix effects, and clear separation from succinic acid ensure accurate, precise results suited for clinical diagnostics and routine laboratory analysis.