ENHANCING LC-MS/MS ANALYSIS OF B-GROUP VITAMINS WITH HYBRID SURFACE TECHNOLOGY

Importance of the topic

Phosphate-containing B vitamins such as flavin mononucleotide (FMN) and pyridoxal 5’-phosphate (PLP) are increasingly used in dietary supplements and beverages due to their roles as coenzymated forms that enhance bioavailability. However, analysis of these compounds by LC-MS/MS is challenged by peak tailing, reduced sensitivity, and carry-over arising from interactions between phosphate groups and metal surfaces in conventional HPLC systems. Hybrid surface technology (MaxPeak HPS) offers a solution by passivating metal surfaces and reducing analyte–metal interactions, thereby improving analytical performance.

Objectives and overview of the study

The study aimed to evaluate the impact of Waters MaxPeak HPS hybrid surface technology on the simultaneous LC-MS/MS analysis of 18 B-group vitamers. Two UPLC-MS/MS setups were compared: an ACQUITY Premier System with HPS-coated metal components (HPS setup) and a conventional ACQUITY H Plus System with stainless-steel surfaces (SOP setup). The performance was assessed in terms of peak shape, signal intensity, sensitivity, and carry-over for both phosphate- and non-phosphate–containing B vitamins in energy drink and dietary supplement matrices.

Methodology

A 9-minute gradient method employed an ACQUITY BEH C18 column (1.7 µm, 2.1 × 100 mm) at 40 °C with mobile phases: A) 20 mM ammonium formate in water (pH 5.0) and B) methanol. The gradient progressed from 1% B to 90% B and returned to initial conditions. Flow rate was 0.35 mL/min and injection volume 2 µL. MS detection used a Xevo TQ-S micro in electrospray positive mode with a capillary voltage of 1.4 kV, cone voltage of 70 V, source temperature of 150 °C, and desolvation temperature of 350 °C.

Instrumentation used

• UPLC system: Waters ACQUITY Premier System (HPS setup) and Waters ACQUITY H Plus System (SOP setup)
• Column: ACQUITY Premier BEH C18, 1.7 µm, 2.1 × 100 mm (HPS) or standard BEH C18 (SOP)
• Mass spectrometer: Xevo TQ-S micro
• Vials: LCMS Certified Amber Glass Max Recovery Vials

Main results and discussion

Comparison of the two setups demonstrated that the HPS configuration delivered substantially larger peak areas and sharper peak shapes for both phosphate and non-phosphate B vitamins. Sensitivity improvements ranged from 2- to 7-fold for critical analytes such as FMN and PLP. Chromatograms showed sustained performance on both fresh and heavily used systems, indicating robust HPS surface stability. Carry-over tests revealed no detectable residual peaks with the HPS setup, while the SOP setup exhibited up to 0.1% carry-over for several vitamins.

Benefits and practical application

• Enhanced peak area and reduced tailing for phosphate-containing and other B vitamins
• Increased analytical sensitivity and method robustness
• Elimination of carry-over, improving accuracy in low-level quantitation
• Extended system lifetimes by minimizing surface-induced adsorption

Future trends and potential applications

Adoption of hybrid surface technologies is expected to expand into broader metabolomics workflows, especially for phosphorylated metabolites, organic acids, and oligonucleotides. High-throughput QC laboratories may leverage HPS-coated systems for routine vitamin and supplement testing. Further innovations may integrate HPS surfaces into microfluidic and nanoscale platforms for enhanced trace-level analysis.

Conclusion

The incorporation of MaxPeak HPS on UPLC-MS/MS metal components significantly improves the analysis of B-group vitamins by delivering higher sensitivity, better peak shape, and elimination of carry-over compared with conventional stainless-steel surfaces. This advance supports reliable, accurate quantitation in complex supplement and beverage matrices.

References

• Wakamatsu A, Morimoto K, Shimizu M, Kudoh S. Severe Peak Tailing of Phosphate Compounds Caused by Interaction with Stainless Steel Used for Liquid Chromatography and Electrospray Mass Spectrometry. J Sep Sci. 2005;28:1823–1830.
• Asakawa Y, Tokida N, Ozawa C, Ishiba M, Tagaya O, Asakawa N. Suppression Effects of Carbonate on the Interaction between Stainless Steel and Phosphate Groups of Phosphate Compounds in HPLC and ESI-MS. J Chromatogr A. 2008;1198–1199:80–86.
• Lauber M, Walter TH, DeLano M, et al. Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces. Waters White Paper. 2020;720006930EN.
• Birdsall RE, Kellett J, Ippoliti S, et al. Increasing Chromatographic Performance of Acidic Peptides in RPLC-MS-based assays with ACQUITY Premier featuring MaxPeak HPS Technology. Waters Application Notes. 2020;720007003EN.
• Boissel C, Walter TH. Improved Peak Shape and Wide Selectivity Range with ACQUITY Premier Columns. Waters Application Notes. 2020;720007014EN.
• Smith KM, Rainville P. Utilization of MaxPeak High Performance Surfaces for Improved Separation and Recovery of TCA Cycle Analytes. Waters Application Notes. 2020;720006721EN.
• Brennan K, Lame ML, Donegan M, Rainville PD. Improved Oligonucleotide SPE-LC-MS Analysis Using MaxPeak High Performance Technology. Waters Application Notes. 2020;720007019EN.