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

Significance of the Topic

Vitamins are essential nutrients widely added to food and supplements, requiring accurate quantification for quality control and safety.
Their diverse polarity, spanning water-soluble (WSV) and fat-soluble (FSV) classes, poses analytical challenges in reversed-phase chromatography.
A robust, high-throughput method capable of simultaneous analysis enhances laboratory efficiency and reduces sample preparation complexity.

Objectives and Study Overview

The study aimed to develop and validate a workflow for simultaneous quantitative determination of eight water-soluble vitamins and eleven fat-soluble vitamins in tablets and energy drinks.
This approach leverages the Thermo Scientific Vanquish Flex Duo UHPLC system configured for Dual LC, employing two independent flow paths, injection ports, columns, and detectors.
The method uses Thermo Scientific Acclaim PolarAdvantage II columns optimized for a wide polarity range and diode array detection at multiple wavelengths.

Instrumentation Used

• Vanquish Flex Duo system for Dual LC: Dual Pump F, Dual Split Sampler FT, two Column Compartments H, and two Diode Array Detectors HL
• Acclaim RSLC PolarAdvantage II columns (150×2.1 mm for WSV; 250×2.1 mm for FSV; 2.2 μm, 120 Å)
• Chromeleon 7.2.8 Chromatography Data System
• Auxiliary equipment: pH meter, ultrasonic bath, magnetic stirrer, syringe filters, brown glass vials

Methodology

The workflow executes two independent LC methods in parallel:

• WSV separation: gradient elution from aqueous phosphate buffer to acetonitrile/phosphate, flow 0.4 mL/min, 25°C, detection at 210, 245, 270, and 280 nm.
• FSV separation: gradient from water to methanol, flow 0.4 mL/min, 25°C, detection at 265, 280, 290, and 325 nm.

Sample preparation varied by matrix and vitamin class:

• Tablet WSV: DMSO extraction, acidified water sonication, filtration and dilution.
• Tablet FSV: ethanol–hexane extraction, sonication, phase separation, filtration.
• Energy drink WSV: degassing by sonication, dilution, filtration.

Main Results and Discussion

• Chromatographic performance achieved baseline resolution (Rs >2.0) for most vitamin pairs, with retention time RSD <0.05% for WSV and <0.15% for FSV.
• Peak area precision was <0.3% for WSV (except ascorbic acid at 2.7% due to instability) and <0.6% for FSV.
• Calibration curves exhibited excellent linearity (R² ≥0.9929) over 0.1–100 μg/mL ranges; LODs and LOQs spanned 0.005–0.195 μg/mL and 0.016–0.649 μg/mL, respectively.
• Recovery rates ranged from 81% to 121% for most vitamins; lower values for unstable analytes (ascorbic acid, folic acid, riboflavin, retinol) were attributed to degradation.
• Tablet analysis revealed higher vitamin content than label claims, demonstrating manufacturer overages; retinol was below detection while retinol acetate was detected though unreported.
• Energy drink quantification matched label values for detectable WSV; cyanocobalamin remained below detection limits.

Benefits and Practical Applications

The dual LC setup enables simultaneous, independent operation of two LC methods without complex valve switching, doubling throughput.
It eliminates mobile phase incompatibility and simplifies data system programming.
This workflow supports routine quality control of vitamins in diverse matrices such as beverages and dietary supplements.

Future Trends and Applications

Integration of mass spectrometry detection could further enhance sensitivity, particularly for vitamins below UV detection limits.
Automation of sample preparation and online extraction may reduce manual handling and improve reproducibility.
Extension of the Dual LC concept to other analyte combinations, such as pesticides and pharmaceuticals, could broaden analytical capabilities.

Conclusion

The Vanquish Flex Duo Dual LC workflow provides a reliable, high-throughput solution for simultaneous analysis of water- and fat-soluble vitamins.
It offers excellent precision, linearity, and sufficient sensitivity for most vitamins in tablets and beverages.
The methodology simplifies instrument setup and improves laboratory efficiency, making it well suited for routine QC and research applications.

Reference

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., editors. Handbook of Pharmaceutical Analysis by HPLC. Elsevier; 2005. p. 123–143.