Replicating Water and Fat-Soluble Vitamins Analyses on a Modern HPLC System

Significance of the topic

Analysis of water- and fat-soluble vitamins in dietary supplements is critical for quality control, regulatory compliance and consumer safety. High-performance liquid chromatography (HPLC) provides the selectivity and sensitivity required to quantify a broad range of vitamin chemistries. The ability to migrate established methods across HPLC platforms ensures continuity of data, facilitates system upgrades and reduces downtime in regulated laboratories.

Objectives and Study Overview

The study aimed to demonstrate method transferability for both water-soluble vitamins under HILIC gradient conditions and fat-soluble vitamins under normal phase conditions between a legacy Alliance 2695 HPLC system and a modern Alliance iS HPLC system. Key goals included verifying chromatographic performance, quantitative agreement with label claims and minimal reconfiguration requirements.

Instrumentation

• HPLC Systems: Alliance 2695 and Alliance iS HPLC equipped with TUV detectors
• Columns: XBridge Amide 4.6×250 mm, 3.5 µm for water-soluble vitamins; XBridge BEH HILIC 4.6×150 mm, 5 µm as alternative; normal phase kit for fat-soluble analysis
• Detectors: UV at 265 nm (2 Hz) for water-soluble; UV at 295 nm (10 Hz) for fat-soluble vitamins
• Mobile phases: ammonium acetate buffer/pH 5.5, acetonitrile, water for HILIC; hexanes/ethyl acetate/acetic acid for normal phase

Methodology and Instrumentation

Sample preparation varied by vitamin class:

• Water-soluble vitamins: tablets shaken in water, centrifuged, diluted with acetonitrile, cleaned and injected under a 30 min HILIC gradient
• Fat-soluble vitamins: multivitamin tablets ground, softgel capsules extracted with hexanes, centrifuged and injected in an isocratic normal-phase run

No major system modifications were required beyond check-valve configuration on the Alliance iS for normal phase operation.

Main Results and Discussion

Both HPLC platforms produced nearly identical standard and sample chromatograms.

• Water-soluble vitamins: retention times and peak shapes for B-complex components showed excellent overlap. Quantitation of B12 and folic acid matched label claims within ±8 % on both systems.
• Fat-soluble vitamins: four tocopherol isomers (α, β, γ, δ) were resolved with consistent system suitability on legacy and modern instruments. Vitamin E content in softgels and multivitamin tablets agreed with labels within ±10 %.

The migration preserved resolution, sensitivity and linearity, confirming method robustness across hardware generations.

Benefits and Practical Applications

• Seamless method transfer reduces revalidation time and cost when upgrading HPLC systems.
• One instrument can handle both polar and non-polar vitamin analyses with simple reconfiguration, maximizing lab productivity.
• Regulatory laboratories gain confidence in data continuity and compliance during instrument transitions.

Future Trends and Potential Uses

Advances in UHPLC, core-shell columns and integrated software will further streamline multivitamin analysis. Coupling HILIC/normal phase separation with mass spectrometry promises enhanced selectivity for trace vitamins in complex matrices. Automation of sample pretreatment and AI-driven data analysis will improve throughput and method optimization. Green solvent systems and miniaturized flow cells may reduce environmental impact and solvent consumption.

Conclusion

The study successfully migrated HILIC and normal phase vitamin methods from an Alliance 2695 to an Alliance iS HPLC system without loss of performance. Quantitative agreement with label claims and preserved chromatographic integrity demonstrate the feasibility of updating legacy systems while maintaining regulatory compliance and analytical reliability.

References

1. Grumbach ES, Fountain KJ. Comprehensive Guide to HILIC Hydrophilic Interaction Chromatography. Waters Corporation; 2010.
2. Tran K, Handcock P. Analysis of Water-Soluble Vitamins and Caffeine in Beverage and Multivitamin Products by Arc HPLC System with PDA Detection. Waters Application Note 720007357EN; 2021.
3. Waters Corporation Technology Brief. Normal-Phase Separation of Tocopherols with the ACQUITY H-Class System Featuring Auto Blend Technology. 720003690EN; 2010.