Simultaneous determination of water- and fat-soluble vitamins in tablets and energy drinks by using a novel Vanquish Flex Duo system for Dual LC

Importance of the topic

Vitamins are critical micronutrients in foods, beverages, and dietary supplements. They are categorized into water-soluble (WSV) and fat-soluble (FSV) classes based on polarity. Conventional reversed-phase chromatographic methods struggle to analyze both classes simultaneously due to their vastly different retention behaviors. A streamlined, high-throughput approach for co-analysis of WSV and FSV improves laboratory efficiency and supports quality control in food safety and nutritional research.

Objectives and Study Overview

This study demonstrates a workflow using the Thermo Scientific™ Vanquish™ Flex Duo UHPLC system for Dual LC coupled with Acclaim™ PolarAdvantage II columns. The goals were to develop independent, optimized methods for WSV and FSV that run concurrently, validate analytical performance, and apply the procedure to commercial vitamin tablets and an energy drink.

Methodology and Instrumentation

Sample preparation protocols were tailored for tablets and beverages. WSV extraction used DMSO and aqueous acetic acid, followed by sonication and filtration. FSV extraction employed ethanol–hexane partitioning. Calibration standards covered eight concentration levels; thiamine used standard addition due to matrix effects.

Instrumentation

• Vanquish Flex Duo Dual LC system: Dual Pump F, Dual Split Sampler FT, two Column Compartments H, two Diode Array Detectors HL
• Columns: Acclaim RSLC PolarAdvantage II (150×2.1 mm, 2.2 µm) for WSV; Acclaim RSLC PolarAdvantage II (250×2.1 mm, 2.2 µm) for FSV
• Detection: UV wavelengths optimized for each vitamin class; 3D spectral scanning
• Software: Chromeleon™ 7.2.8 CDS

Main Results and Discussion

Both methods achieved baseline separation with resolution (RS) >2.0 for most critical pairs. Retention time repeatability (RSD) was <0.05% for WSV and <0.15% for FSV. Peak area RSD generally fell below 0.5%. Calibration curves were linear (R2≥0.999), with limits of detection and quantification in the low ng/mL range. Recoveries for stable vitamins ranged from 90% to 121%; less stable analytes (ascorbic acid, folic acid, riboflavin) showed lower recoveries but measured concentrations agreed with labeled values.
Quantification in a supplement tablet revealed actual vitamin levels often exceeded label claims, while certain compounds (retinol, cyanocobalamin) were below detection. In the energy drink, measured WSV concentrations matched declared content.

Benefits and Practical Applications of the Method

• Simultaneous, unattended analysis of WSV and FSV in a single instrument
• Elimination of complex valve switching and solvent compatibility issues
• High throughput via parallel operation of two independent flow paths
• Robust quantitation suitable for quality control in food and beverage industries

Future Trends and Potential Applications

Future developments may include coupling Dual LC with mass spectrometry for enhanced sensitivity, expanding the analyte scope to related micronutrients and contaminants, integrating green solvent strategies, and implementing fully automated sample handling to further increase throughput and reproducibility.

Conclusion

The Vanquish Flex Duo Dual LC workflow with Acclaim PolarAdvantage II columns provides a simple, reliable, and high-throughput solution for simultaneous determination of water- and fat-soluble vitamins in complex matrices. The method offers excellent chromatographic performance, quantitative accuracy, and practical applicability for routine analysis in food, beverage, and supplement quality control.

References

1. Thermo Fisher Scientific Technical Note 72488: Determination of water- and fat-soluble vitamins by HPLC.
2. Thermo Fisher Scientific Application Note 252: HPLC Assay of Water-Soluble Vitamins, Fat-Soluble Vitamins, and a Preservative in Dry Syrup Multivitamin Formulation.
3. Choi C.K.; Dong M.W. Sample preparation for HPLC analysis of drug products. In Ahuja S, Dong M.W. (eds) Handbook of Pharmaceutical Analysis by HPLC, Elsevier, 2005.