Fast and Reliable Method for the Analysis of Methylmalonic Acid from Human Plasma

Importance of the Topic

Accurate measurement of methylmalonic acid in human plasma is vital for diagnosing metabolic disorders and monitoring vitamin B12 status. Traditional gas chromatography–mass spectrometry approaches involve lengthy derivatization and extended run times, which limit sample throughput. A faster, reliable workflow enhances clinical decision making and laboratory efficiency.

Objectives and Overview

This study presents a high-throughput LC-MS/MS method for quantifying methylmalonic acid using mixed-mode ion-exchange micro-elution solid phase extraction. The approach aims to separate methylmalonic acid from isobaric succinic acid interference, ensure minimal matrix effects, and achieve accurate, precise results across a broad concentration range.

Methodology

The analytical workflow comprises:

• Sample Pretreatment: Spiking plasma or phosphate buffer saline with deuterated MMA internal standard, followed by acidic dilution.
• SPE Cleanup: Mixed-mode SOLAµ WAX 96-well plate conditioned with methanol and ammonium acetate buffer for micro-scale extraction.
• Chromatography: Acclaim Surfactant Plus column with a 3.5-minute gradient from aqueous ammonium acetate to acetonitrile for baseline separation of MMA and succinic acid.
• Mass Spectrometry: Negative-mode heated electrospray ionization on a triple quadrupole instrument monitoring specific precursor-product ion transitions for MMA, MMA-d3, and succinic acid.
• Data Processing: Quantitation using weighted linear calibration (1/x2) over 15–1200 ng/mL with dedicated software.

Used Instrumentation

• SOLAµ WAX mixed-mode ion-exchange SPE plate
• Thermo Scientific Dionex UltiMate 3000 RSLC system
• Acclaim Surfactant Plus analytical column and guard
• Thermo Scientific TSQ Quantiva triple quadrupole mass spectrometer
• HyperSep 96 vacuum manifold and compatible vacuum pump

Main Results and Discussion

The method demonstrated linear response (R2 > 0.995) from 15 to 1200 ng/mL. Recoveries exceeded 90% in buffer and 80% in plasma, with matrix suppression below 12% in buffer and 22% in plasma. Precision and accuracy met acceptance criteria, with bias within ±11% and RSDs under 10% across low, mid, and high quality control levels. Chromatographic resolution achieved separation of succinic acid (1.7 min) and MMA (2.4 min), eliminating isobaric interference.

Benefits and Practical Applications

• High sample throughput with up to 96 samples processed in under two hours.
• Robust selectivity for MMA over succinic acid without derivatization.
• Reduced solvent consumption and low elution volumes enhance sensitivity.
• Flexible use of surrogate matrices simplifies calibration preparation.

Future Trends and Potential Applications

Ongoing advances in mixed-mode SPE and fast chromatography could further shorten analysis times and improve sensitivity. Integration with fully automated platforms and expansion to other small organic acids offers broader clinical and research utility. Emerging high-resolution mass spectrometers may enable multiplexed assays in a single run.

Conclusion

The described LC-MS/MS method using mixed-mode SPE provides a fast, reliable, and high-throughput solution for methylmalonic acid analysis in human plasma. It combines effective interference removal, excellent analytical performance, and streamlined workflow to support clinical and research laboratories.