REPLICATING WATER AND FAT-SOLUBLE VITAMINS ANALYSES ON A MODERN HPLC SYSTEM

Importance of the Topic

Vitamin supplement usage has risen, making accurate verification of label claims critical for consumer safety and regulatory compliance. High-performance liquid chromatography (HPLC) is the standard for quantifying both water- and fat-soluble vitamins, yet their divergent polarities require distinct chromatographic modes. Adapting a single modern HPLC platform to handle both analyses enhances laboratory efficiency and supports method transfer in regulated settings.

Study Objectives and Overview

This work evaluates the transfer of established HPLC methods from a legacy Waters Alliance 2695 system to a modern Alliance iS HPLC system. It covers: HILIC gradient separation for water-soluble vitamins (B1, B2, B6, B12, folic acid) and normal phase separation for fat-soluble tocopherols (α, β, γ, δ). The goal is to demonstrate equivalent performance and quantitative agreement across both platforms.

Methodology and Instrumentation

Sample Preparation and Chromatography:

• Water-Soluble Vitamins: Multivitamin tablets extracted with water, centrifuged, diluted with acetonitrile, and analyzed under a 30-min HILIC gradient.
• Fat-Soluble Vitamins: Tablet powders and Vitamin E softgels extracted with hexane, diluted, and injected into a normal phase system using a hexanes-ethyl acetate mobile phase.

Both methods used identical mobile phases, columns, and sample handling on each HPLC instrument to isolate the impact of system architecture on chromatographic performance.

Used Instrumentation

• Waters Alliance 2695 HPLC System with TUV detector (UV at 265 nm for HILIC; 295 nm for normal phase).
• Waters Alliance iS HPLC System configured for both HILIC and normal phase modes.
• XBridge BEH HILIC column (4.6×250 mm, 3.5 μm) and normal phase column kit (4.6×150 mm, 5 μm).
• Mobile phases: ammonium acetate buffer/acetonitrile gradient for HILIC; 95% hexanes/5% ethyl acetate with acetic acid for normal phase.

Key Results and Discussion

Both systems achieved system suitability criteria with <2% RSD for peak areas and resolution values exceeding USP thresholds. Retention times shifted slightly due to instrument dead volume differences but remained consistent and reproducible. Quantitative determinations of vitamins in supplement samples agreed within ±9% of label claims, and calibration curves exhibited R2 of 0.9999 on both platforms.

Benefits and Practical Applications

• Seamless migration of HPLC methods ensures continuity in quality control labs during equipment upgrades.
• A single, configurable HPLC platform can handle diverse analyses, reducing capital and training requirements.
• Reliable, robust quantitation supports regulatory compliance and product safety in supplement manufacturing.

Future Trends and Potential Applications

Integration of mass spectrometric detection could expand sensitivity and selectivity for trace analytes. Automation of sample preparation and data processing, combined with green solvent strategies, will further streamline workflows. Advanced software and AI-driven analytics may enable predictive maintenance and real-time quality monitoring in high-throughput environments.

Conclusion

The study confirms that both hydrophilic interaction and normal phase vitamin assays can be migrated from legacy to modern HPLC systems without compromising performance. Comparable precision, accuracy, and chromatographic integrity demonstrate the flexibility of current HPLC platforms to meet diverse analytical needs in nutritional quality control.

References

• Eric S. Grumbach and Kenneth J. Fountain. Comprehensive Guide to HILIC Hydrophilic Interaction Chromatography. Waters Corporation, 2010.
• Kim Tran and Peter Handcock. Analysis of Water-Soluble Vitamins and Caffeine in Beverage and Multivitamin Products by Arc HPLC System With PDA Detection. Waters Application Note, 720007357EN, 2021.
• Waters Corporation Technology Brief. Normal-Phase Separation of Tocopherols with the ACQUITY H-Class System featuring Auto Blend Technology. 720003690EN, 2010.