Enhancing the LC-MS/MS Analysis of B-group Vitamins with MaxPeak High Performance Surfaces Technology

Significance of the Topic

B-group vitamins are essential micronutrients involved in numerous metabolic pathways, and accurate quantification in food and supplement matrices is critical for quality control and regulatory compliance. Conventional LC-MS/MS analyses of phosphate-containing vitamers often suffer from interactions with stainless-steel surfaces, causing peak tailing, reduced sensitivity, and carry-over, which compromise data integrity. Waters MaxPeak High Performance Surfaces (HPS) technology addresses these limitations by minimizing analyte–metal interactions.

Objectives and Study Overview

This work evaluates the impact of MaxPeak HPS on LC-MS/MS performance for 18 B-group vitamins. Key objectives include comparing sensitivity, peak shape, calibration linearity, and carry-over between an ACQUITY Premier HPS system and a conventional stainless-steel LC system when analyzing energy drink and dietary supplement samples.

Methodology and Instrumentation

Standards of individual B vitamins and stable isotope–labeled internal standards were prepared in aqueous solutions under low-light conditions. Energy drink (ED) samples were filtered and analyzed both undiluted and at 1:50 dilution; liquid dietary supplement (DS) samples were diluted at multiple ratios. Chromatographic separation employed Waters ACQUITY Premier System with a MaxPeak HPS BEH C18 column (1.7 µm, 2.1 × 100 mm) at 40 °C. Mobile phase A was 20 mM ammonium formate (pH 5.0) and B was methanol at 0.35 mL/min, with a 9 min gradient and 2 µL injection. Detection used a Xevo TQ-S micro triple quadrupole MS in positive ESI mode.

Main Results and Discussion

The HPS setup produced 3–10× greater sensitivity for key vitamins (riboflavin, thiamine, nicotinamide, FMN, PLP, 5-MTHF) and improved calibration linearity (R² ≥ 0.995) compared with stainless-steel surfaces. Peak tailing was markedly reduced, and carry-over for riboflavin, pyridoxal, 5-MTHF, and methylcobalamin was eliminated. Performance benefits persisted even after extensive use. In ED and DS analyses, recovery ranged from 91–107% (ED) and 78–122% (DS), with repeatability ≤7% RSD for most analytes.

Benefits and Practical Applications

The adoption of MaxPeak HPS surfaces in LC-MS/MS workflows delivers:

• Enhanced peak intensity and sensitivity enabling lower limits of quantitation.
• Reduced peak tailing and sharper chromatographic profiles.
• Elimination of carry-over issues for phosphate-containing vitamers.
• Improved calibration linearity, supporting robust quantitative analysis.

Future Trends and Possibilities

Advances in inert surface coatings may extend analytical improvements to other challenging analyte classes (e.g., phospholipids, oligonucleotides). Integration with ultra-high-throughput LC-MS/MS platforms and emerging detection technologies will further streamline quantitative assays in clinical diagnostics, nutritional profiling, and pharmaceutical quality control.

Conclusion

Waters MaxPeak HPS technology significantly enhances LC-MS/MS analysis of B-group vitamins by mitigating deleterious metal-surface interactions. The ACQUITY Premier Solution offers superior sensitivity, peak shape, linearity, and carry-over prevention, establishing a robust platform for routine vitamin quantitation in complex matrices.

Reference

1. Wakamatsu A. et al. J. Sep. Sci. 2005, 28, 1823–1830.
2. Asakawa Y. et al. J. Chromatogr. A 2008, 1198–1199, 80–86.
3. Lauber M. et al. Waters White Paper, 720006930EN, 2020.
4. Birdsall R.E. et al. Waters App. Note 720007003EN, 2020.
5. Boissel C.; Walter T.H. Waters App. Note 720007014EN, 2020.
6. Smith K.M.; Rainville P. Waters App. Note 720006727EN, 2020.
7. Brennan K. et al. Waters App. Note 720007019EN, 2020.
8. AOAC Official Method 2015.14, 21st Ed., AOAC INTERNATIONAL, 2019.