A, B, D, E – Simultaneous determination of water- and fat-soluble vitamins

Importance of the Topic

Vitamins are essential micronutrients divided into water-soluble (B-complex, C) and fat-soluble (A, D, E, K) classes with distinct absorption and storage characteristics. Routine analysis typically requires separate chromatographic modes, leading to increased solvent use, instrument time, and operational complexity. A unified analytical approach streamlines workflows, reduces resource consumption, and supports timely quality control in food and pharmaceutical industries.

Objectives and Study Overview

This study aimed to establish a single reversed-phase HPLC method capable of simultaneous quantification of three water-soluble vitamins (B2, B9, B12) and three fat-soluble vitamins (A palmitate, D3, E) in a single run. The approach seeks to maintain analytical performance while lowering solvent consumption and total analysis time.

Methodology and Instrumentation

Sample preparation involved individual stock solutions of each vitamin followed by a combined mixed standard. Water-soluble vitamins were dissolved in deionized water; fat-soluble vitamins were prepared in methanol or provided as ready-to-use ampoules.

Analytical conditions:

• Column: Eurospher II C18, 150×3 mm with precolumn
• Mobile phases: A) water + 0.01% TFA (pH 4); B) methanol
• Gradient: initial 95% A, 5% B transitioning to 100% B over 12 min, held, then re-equilibration
• Flow rate: 0.5 ml/min; temperature: 30 °C; injection volume: 5 µl
• Detection: UV wavelength program switching between 270 nm, 318 nm, 360 nm

Instrumentation Used

• Pump: AZURA P 6.1L
• Autosampler: AZURA AS 6.1L with cooling/heating
• Detector: AZURA DAD 6.1L with LightGuide flow cell
• Column thermostat: AZURA CT 2.1
• Software: ClarityChrom 8.2.3 with PDA extension

Main Results and Discussion

A five-level calibration yielded excellent linearity (R2 > 0.9996) for all vitamins. Limits of quantification ranged from 0.01 µg/ml (B2) to 0.668 µg/ml (A palmitate), with detection limits meeting regulatory requirements. The chromatographic separation achieved baseline resolution of all six analytes within 28 min. The method demonstrated reproducible retention times and consistent peak shapes across replicates.

Benefits and Practical Applications

The unified reversed-phase method eliminates the need for multiple chromatographic systems, cutting solvent usage and instrument costs. It is directly applicable to quality control of fortified foods, dietary supplements, and clinical samples, enhancing laboratory throughput and sustainability.

Future Trends and Potential Applications

Emerging directions include coupling with mass spectrometry for expanded vitamin profiling, miniaturized HPLC formats for rapid field testing, and automated sample preparation to further increase throughput. Integration into high-throughput screening platforms will support large-scale nutritional studies and industrial quality assurance.

Conclusion

The described HPLC method provides a robust, efficient, and sensitive solution for simultaneous quantification of water- and fat-soluble vitamins. By consolidating analyses into a single run, it offers significant advantages in resource conservation, operational efficiency, and versatility for routine analytical laboratories.

References

• Verywell Health. Fat vs. Water-Soluble Vitamins; accessed 2021-09-27.
• Colorado State University Extension. Fat-Soluble Vitamins: A, D, E, and K; Fact Sheet No. 9.315; accessed 2021-09-27.