Automated Preparation and LC-MS/MS Analysis of Methylmalonic Acid in Serum Using the Andrew+ Pipetting Robot and Xevo TQ-S micro

Importance of the Topic

Accurate measurement of methylmalonic acid (MMA) in serum is critical for assessing vitamin B-12 status, as MMA accumulates when B-12 is deficient, even if serum B-12 appears normal or elevated. High-throughput, reliable methods are essential for clinical research, diagnostics, and quality control in biochemical and pharmaceutical laboratories.

Objectives and Study Overview

This work aimed to develop and validate an automated sample preparation protocol for MMA in human serum using the Andrew+ pipetting robot combined with LC-MS/MS analysis on a Xevo TQ-S micro system. The goal was to reproduce and enhance an existing manual workflow to improve throughput, precision, and reproducibility while reducing analyst hands-on time.

Methodology and Instrumentation

A streamlined extraction protocol employed Waters’ Ostro 96-well protein precipitation and phospholipid removal plate. Key steps, including reagent addition, mixing, and sample transfer, were encoded in cloud-native OneLab software, with user prompts for an off-deck evaporation stage. Optimized pipetting parameters replaced manual vortexing to ensure consistent mixing and high recovery.

Instrumentation Used

• Andrew+ Pipetting Robot with OneLab control software
• Ostro 96-well Plate for protein precipitation and phospholipid removal
• ACQUITY UPLC I-Class System
• ACQUITY UPLC CSH C18, 1.7 μm column
• Xevo TQ-S micro Tandem Quadrupole Mass Spectrometer

Main Results and Discussion

Comparison of automated versus manual preparation demonstrated:

• Precision at two QC levels: ≤3.5% RSD (automation) vs. ≤5.2% RSD (manual)
• Recovery differences under 4% between automated and manual methods

The optimized liquid handling parameters and in-plate mixing delivered clean extracts and consistent analyte quantification.

Benefits and Practical Applications

• Significant reduction in hands-on analyst time
• Enhanced method transferability across laboratories
• Minimized human error and improved data quality
• High-throughput capacity with 96-well processing
• Comparable or superior performance to manual protocols

Future Trends and Potential Applications

Further integration of robotics and cloud-based control is expected to expand automated workflows for other small-molecule biomarkers. Advances in multiplexed LC-MS/MS methods, combined with automated sample handling, will support large-scale clinical studies, personalized medicine, and real-time diagnostics.

Conclusion

The Andrew+-based sample preparation for MMA analysis offers a robust, precise, and high-throughput alternative to manual workflows. Coupled with UPLC-MS/MS detection on the Xevo TQ-S micro, this platform delivers reliable quantification suitable for clinical research and routine diagnostics.

References

1. Vashi P, Edwin P, Popiel B, Lammersfeld C, Gupta D. Methylmalonic Acid and Homocysteine as Indicators of Vitamin B-12 Deficiency in Cancer. PLoS ONE. 2016;11(1):e0147843.
2. Foley D, Van Hulle M, Brown HA, Calton LJ. Analysis of Methylmalonic Acid in Serum Using the Xevo TQ-S micro for Clinical Research. Waters Application Note 720006806. 2020.