Analysis of Vitamin E

Significance of the Topic

The analysis of vitamin E is critical for food quality, nutritional assessment and product stability. Tocopherols act as antioxidants and their accurate quantification in food, supplements and biological samples ensures compliance with regulations and supports research in health and nutrition.

Study Objectives and Overview

This study presents a reversed-phase HPLC method for simultaneous separation of four tocopherol isomers (α, β, γ, δ) at a standard concentration of 25 mg/L each. The goal is to establish a straightforward, reproducible procedure suitable for routine determination of fat-soluble vitamins.

Used Instrumentation

• Shim-pack GIST NH2 column (250 mm x 4.6 mm I.D., 5 µm particle size)
• HPLC system equipped with UV detector set at 290 nm

Methodology and Instrumentation

A binary mobile phase consisting of n-hexane (A) and ethyl acetate (B) in a 70/30 v/v ratio was used. The flow rate was set to 1.0 mL/min and the column temperature maintained at 30 °C. Sample injections of 10 µL were performed under isocratic conditions, enabling efficient separation of tocopherol isomers based on polarity differences.

Main Results and Discussion

The method achieved clear baseline separation of all four tocopherol isomers with sharp, symmetrical peaks. Retention behavior reflected the increasing polarity from α- to δ-tocopherol, and the chosen mobile phase composition balanced resolution and analysis time. This protocol demonstrated good reproducibility and reliability for quantifying tocopherols at 25 mg/L.

Benefits and Practical Applications

• Simple isocratic operation without gradient programming
• Short analysis time suitable for high-throughput laboratories
• Robust separation ensuring accurate quantification in food and supplement matrices

Future Trends and Potential Applications

Anticipated developments include coupling with mass spectrometry for enhanced sensitivity and selectivity, method miniaturization using UHPLC formats, and automation for real-time monitoring in industrial processes.

Conclusion

The described HPLC method offers a reliable, reproducible and straightforward approach for separating and quantifying α-, β-, γ- and δ-tocopherols. Its simplicity and robustness make it ideal for routine quality control in food, supplement and research laboratories.

References

• Shimadzu Corporation. Application Note ERAS-1000-0225. First Edition, December 2021.