Simultaneous Determination of Water- and Fat Soluble Vitamins in Tablets and Energy Drinks by Using a Novel UHPLC System

Importance of the Topic

Vitamins play a crucial role in human health and are widely supplemented through tablets and energy drinks. Accurate quantification of both water- and fat-soluble vitamins is essential for quality control in food and pharmaceutical industries. Traditional methods struggle to analyze compounds with vastly different hydrophobicity in a single run, creating a need for more efficient workflows.

Objectives and Overview of the Study

This study aimed to develop and validate a simultaneous analytical method for water-soluble vitamins (WSV) and fat-soluble vitamins (FSV) in tablets and energy drinks. By leveraging a dual-path UHPLC system, two optimized chromatographic methods were run in parallel, enabling comprehensive and rapid vitamin profiling.

Methodology and Instrumentation

A Thermo Scientific Vanquish Flex Duo UHPLC system equipped with a Dual Pump F, Dual Split Sampler FT, and two column compartments was used. Separate Acclaim Vanquish Polar Advantage II columns (150×2.1 mm, 2.2 µm for WSV; 250×2.1 mm, 2.2 µm for FSV) delivered optimal retention across a wide polarity range. Water-soluble vitamins were extracted by sonication in DMSO and acidified water or directly from energy drinks after degassing. Fat-soluble vitamins were extracted with ethanol and hexane. Chromeleon CDS software controlled data acquisition.

Main Results and Discussion

WSV Separation
The PA2 column achieved baseline separation of key polar compounds, including ascorbic acid, thiamine, folic acid, and cyanocobalamin, with resolution values above 2.0. Analysis of an energy drink revealed four WSVs (pyridoxine, nicotinamide, pantothenic acid, cyanocobalamin) in close agreement with label claims, except for traces of cyanocobalamin near the detection limit.

FSV Separation
Fat-soluble vitamins were well resolved (Rs>2) except for minor overlaps between menaquinone and delta-tocopherol (Rs=1.4) and α-tocopherol acetate and γ-tocopherol (Rs=1.0). Tablet analysis showed measured content often exceeding label values. Notably, retinol was undetectable (<0.0002 mg/tablet) despite a 0.6 mg claim, while retinol acetate appeared at 0.9 mg/tablet though not declared.

Benefits and Practical Applications of the Method

• Simultaneous dual-path analysis doubles throughput without additional hardware.
• Robust separation across a broad polarity range simplifies method transfer.
• Streamlined sample preparation fits routine QA/QC workflows in food and pharmaceutical settings.

Future Trends and Opportunities

The dual UHPLC approach may be further enhanced by integrating mass spectrometric detection for increased selectivity, automating sample preparation to reduce hands-on time, and expanding the method to cover comprehensive micronutrient panels. Miniaturized and portable platforms could enable on-site analysis in manufacturing or field studies.

Conclusion

The novel dual-path UHPLC workflow provides a powerful, high-throughput solution for simultaneous quantification of water- and fat-soluble vitamins in complex matrices. It offers accurate, reliable results and has strong potential for routine implementation in quality control laboratories.

References

1. Thermo Fisher Scientific TN 72488: Determination of water- and fat-soluble vitamins by HPLC.
2. Choi C.K., Dong M.W.: Handbook of Pharmaceutical Analysis by HPLC.
3. Thermo Fisher Application Note 72592: Simultaneous determination of water and fat-soluble vitamins in tablets and energy drinks by using a novel Thermo Scientific Vanquish Flex Duo UHPLC system.