Increased LC/MS Sensitivity for Methylmalonic Acid Using Mixed-Mode Reversed-Phase/Anion-Exchange Chromatography and MaxPeak Premier Columns

Importance of the Topic

Methylmalonic acid is a key biomarker for vitamin B12 deficiency, impacting clinical diagnostics. Sensitive detection of MMA is critical for assessing vitamin B12 status, particularly in aging and vulnerable populations.

Objectives and Study Overview

The study aimed to enhance LC/MS sensitivity for MMA by comparing a MaxPeak Premier CSH Phenyl-Hexyl column with an inert surface to a conventional stainless-steel version, focusing on analyte retention and signal quality.

Methodology and Instrumentation

• Column types: ACQUITY Premier CSH Phenyl-Hexyl with MaxPeak HPS (1.7 µm, 2.1 × 50 mm) vs stainless-steel CSH Phenyl-Hexyl (130 Å, 1.7 µm, 2.1 × 50 mm).
• LC system: ACQUITY I-Class with Xevo TQ-S micro, electrospray ionization in negative mode, SIR at m/z 117.
• Mobile phase: 0.1% formic acid in water, isocratic flow at 0.555 mL/min for 2.5 min.
• Sample prep: 0.02 mg/mL MMA in 50:50 methanol:water, stored at 4 °C, 2 µL injection.

Key Results and Discussion

• Both columns provided similar retention and peak widths via mixed-mode reversed-phase/anion exchange by CSH particles.
• MaxPeak Premier achieved a 103% increase in peak area and 90% increase in peak height compared to stainless-steel.
• Peak area precision improved, with RSD reduced from 7.51% to 3.36%, reflecting reduced analyte adsorption.

Benefits and Practical Applications

• Enables sensitive, robust quantitation of small polar acids without derivatization or ion-pairing.
• Improves recovery and reproducibility in clinical and metabolomic analyses.
• Minimizes metal-induced adsorption and adduct formation in LC/MS workflows.

Future Trends and Opportunities

• Adoption of inert surface technologies for broader metabolomic targets and complex matrices.
• Integration into high-throughput clinical assays for nutrient deficiency screening.
• Development of multi-modal stationary phases combining HILIC and CSH features for polar analytes.

Conclusion

The inert-surfaced MaxPeak Premier CSH Phenyl-Hexyl column significantly enhances LC/MS sensitivity and precision for methylmalonic acid, offering a robust solution for polar metabolite analysis and clinical diagnostics.

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