An Isocratic Separation of B Vitamins Using Waters XBridge Biphenyl RP Column with MaxPeak Premier Technology

Significance of the topic

Accurate and reproducible quantification of B vitamins is critical across pharmaceutical, food and clinical laboratories because these water‑soluble, polar analytes are present in complex matrices at low concentrations and are labile to environmental factors. Robust chromatographic methods that maintain retention and peak shape under highly aqueous mobile phases are therefore essential to ensure reliable measurement, regulatory compliance, and product quality control.

Objectives and study overview

This application note evaluated the performance of a Waters XBridge Biphenyl reversed‑phase column incorporating MaxPeak Premier Technology for the isocratic separation of four B vitamins (nicotinic acid, nicotinamide, pyridoxal 5'‑phosphate, and thiamine) under 100% aqueous mobile phase conditions. The study compared biphenyl selectivity and column hardware effects against several benchmark stationary phases and stainless‑steel hardware to assess separation, pore‑dewetting susceptibility, peak shape, and response for phosphorylated vitamers.

Methodology and instrumentation

Experimental design and samples:

• Two sample mixes: (A) uracil (10 µg/mL) in 100% acetonitrile; (B) uracil (10 µg/mL), nicotinic acid (32 µg/mL), nicotinamide (80 µg/mL), thiamine (50 µg/mL), pyridoxal 5'‑phosphate (P5P, 100 µg/mL) in 10 mM ammonium acetate pH 4.6 (aq).
• Equilibration protocol: initial pore wetting in 100% acetonitrile (30 min), injections with progressive equilibration to 100% aqueous 10 mM ammonium acetate pH 4.6 (including a stop‑flow test of 10 min followed by 1.2 min re‑equilibration prior to final injections) to probe pore dewetting.

Chromatographic conditions and instrumentation:

• System: ACQUITY UPLC H‑Class (Quaternary Solvent Manager with 100 µL mixer, Sample Manager‑FTN 15 µL needle, ACQUITY Column Manager + Auxiliary Column Manager) with ACQUITY UPLC PDA detector (500 nL flow cell). Data acquired with Empower CDS.
• Column format: 2.1 × 50 mm biphenyl stationary phases evaluated (Waters XBridge Biphenyl with BEH‑based particle and endcapping; a competitor biphenyl; and other columns: ACQUITY Premier HSS T3, XBridge Premier BEH C18, XBridge Premier BEH Phenyl).
• Operating parameters: column temp 30 °C, flow 0.2 mL/min, injection 1.0 µL, detection UV 250 nm. Mobile phases: A = 10 mM ammonium acetate pH 4.6 (aq), B = acetonitrile, C = water.

Key results and discussion

Column selectivity and retention:

• Only the Waters XBridge Biphenyl and a vendor R biphenyl column achieved baseline separation of the four B vitamins under 100% aqueous conditions; C18, HSS T3 and phenyl columns did not provide the same complete separation in this isocratic aqueous mobile phase.
• The competitor biphenyl produced longer retention for thiamine but poor peak symmetry (USP tailing ~2.55). The Waters XBridge Biphenyl gave shorter retention and improved peak symmetry (USP tailing ~1.67), attributed to reduced residual silanol activity from BEH particles and proprietary endcapping chemistry, which reduces ionic interactions with cationic thiamine at pH 4.6.

Pore dewetting assessment:

• After a 10‑min stop‑flow, the Waters XBridge Biphenyl column showed no evidence of pore dewetting when returned to 100% aqueous mobile phase; only a slight increase in thiamine retention time was observed. This supports the column's compatibility with highly aqueous isocratic conditions and stable performance following flow interruption.

Impact of MaxPeak Premier Technology on phosphorylated vitamers:

• P5P (pyridoxal 5'‑phosphate), a phosphorylated vitamin B6 vitamer, exhibited significant adsorption to conventional stainless‑steel column hardware, manifesting as lower peak height/area and a conditioning‑dependent increase in response across successive injections.
• Columns equipped with MaxPeak Premier Technology (inert surface treatment) delivered approximately 36% greater peak height and 15% greater peak area for P5P compared with identical biphenyl columns using stainless‑steel hardware. MaxPeak hardware also reduced injection‑to‑injection variability, eliminating the progressive conditioning effect observed with stainless steel.

Benefits and practical applications

• Enables robust isocratic separations of multiple B vitamins using 100% aqueous mobile phases, avoiding the retention loss that affects many hydrophobic C18 phases.
• Biphenyl selectivity enhances separation of π‑electron–containing vitamins and can improve peak symmetry for ionic/polar species when combined with low‑silanol BEH particles and endcapping.
• MaxPeak Premier inert hardware mitigates adsorption of phosphorylated vitamers and other polar analytes prone to metal surface interactions, improving sensitivity, accuracy and method reproducibility—important for QC and quantitative assays of dietary supplements and formulations.

Future trends and potential applications

• Broader adoption of low‑adsorption hardware (MaxPeak‑type surfaces) and BEH‑based biphenyl chemistries for polar analytes in LC and LC–MS workflows, particularly where phosphorylated or highly polar species are analyzed.
• Application of biphenyl phases to wider panels of water‑soluble vitamins, metabolites and nucleoside/nucleotide analyses where π–π interactions and controlled silanol activity improve selectivity.
• Method transfer to mass spectrometric detection to exploit improved chromatographic peak shape and heightened sensitivity for trace analysis in complex matrices.
• Continued optimization of column hardware and stationary phase design to further minimize surface adsorption, accelerate equilibration in high aqueous backpressures, and support robust high‑throughput quality control methods.

Conclusion

The Waters XBridge Biphenyl RP Column combined with MaxPeak Premier Technology provides a robust solution for isocratic separation of selected B vitamins under 100% aqueous mobile phase conditions. The BEH‑based biphenyl chemistry delivers favorable selectivity and improved peak symmetry while resisting pore dewetting after stop‑flow. MaxPeak Premier inert hardware substantially reduces adsorption of phosphorylated vitamers (notably P5P), increasing signal response and reproducibility versus stainless‑steel hardware. Together these features support reliable, sensitive, and transferable methods for B vitamin analysis in pharmaceutical, food, and supplement testing.

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