Analysis of Methylmalonic Acid in Serum Using the Xevo TQ-S micro for Clinical Research

Importance of the Topic

The quantification of methylmalonic acid (MMA) in serum is critical for assessing vitamin B12 status in clinical research. Elevated MMA levels reflect impaired conversion of methylmalonyl-CoA to succinyl-CoA, indicating functional B12 deficiency. A robust, high-throughput method enables reliable biomarker measurements at low physiological concentrations and supports large-scale studies of B12-related metabolic disorders.

Objectives and Study Overview

This study aimed to develop and validate a rapid, selective, and sensitive LC-MS/MS workflow for quantifying MMA in human serum. Key goals included achieving baseline chromatographic separation from isobaric/isomeric interferences, minimizing sample volume, and demonstrating agreement with an independent reference method.

Methodology

Sample Preparation:

• Aliquot 100 µL serum, add 25 µL 13C4-MMA internal standard and 400 µL 1% formic acid in acetonitrile.
• Process samples on an Ostro phospholipid removal plate under vacuum.
• Evaporate eluate under nitrogen at 50 °C, reconstitute in 60 µL 1% formic acid in water.

Chromatography:

• System: ACQUITY UPLC I-Class with fixed loop and column heater at 40 °C.
• Column: ACQUITY UPLC CSH C18, 2.1×100 mm, 1.7 µm, with inline filter.
• Mobile phases: A) water + 0.2% formic acid; B) acetonitrile + 0.2% formic acid.
• Gradient: 1%–95% B over 1.5 min, total run 3.3 min, flow 0.45 mL/min.
• Injection volume: 10 µL.

Mass Spectrometry:

• Instrument: Xevo TQ-S micro, ESI negative mode.
• Acquisition: MRM, transitions 117.1>73.1 (quantifier), 117.1>55.1 (qualifier) for MMA; 121.1>76.1 for internal standard.
• Source temp 150 °C, desolvation 600 °C, capillary 0.50 kV.

Used Instrumentation

• Waters ACQUITY UPLC I-Class System with Fixed Loop and Column Heater
• ACQUITY UPLC 1.7 µm CSH C18 Column
• Ostro Phospholipid Removal Plate
• Waters Xevo TQ-S micro Tandem Quadrupole Mass Spectrometer
• MassLynx v4.2 and TargetLynx Application Manager

Main Results and Discussion

Chromatographic Separation and Specificity:

• Baseline separation of MMA and succinic acid achieved, with no significant interference from endogenous compounds (albumin, bilirubin, lipids) or structural isomers.
• System carry-over <20% of lowest calibrator.

Sensitivity and Linearity:

• Limit of quantification at 21 nmol/L with S/N >10 and precision <20% RSD.
• Calibration linear from 6.8 to 1,524 nmol/L, r2 >0.995.

Precision and Accuracy:

• Total precision across QC levels: 4.4%–6.3% RSD; repeatability 3.7%–5.5% RSD.
• Dilution integrity (1:5) maintained accuracy (103%) at 4,234 nmol/L.

Matrix Effects and Stability:

• Matrix factor normalized by internal standard: 0.89–1.04, RSD 4.2%.
• Extracted samples stable on autosampler at 8 °C for 67 h (±2% bias).

Method Comparison:

• Deming regression vs. independent LC-MS/MS (n=94): y=1.05x–11.77, no significant bias (p>0.19).
• Bland-Altman mean bias –0.8%, 95% limits –22.9% to 21.3%.

Benefits and Practical Application

This workflow combines minimal sample volume (100 µL), rapid 3.3 min runtime, and simplified phospholipid removal for high throughput. The method’s sensitivity and selectivity support reliable MMA biomarker quantification in clinical research, epidemiology, and nutritional studies.

Future Trends and Potential Uses

Advancements may include automation of sample cleanup, integration into multiplex panels for metabolic profiling, and application to other organic acids. Miniaturized UPLC-MS systems and data-driven quality control will enhance large-scale screening and personalized nutrition research.

Conclusion

A robust LC-MS/MS method on the Xevo TQ-S micro was established for precise MMA measurement in serum. The approach delivers high sensitivity, selectivity, and throughput, with excellent agreement to an independent reference method, making it well suited for clinical research into vitamin B12 status.