Accelerated method development for the separation of water-soluble vitamins by RP-HPLC with UV detection using an empirical approach to predict separation

Significance of the Topic

Analysis of water-soluble vitamins is essential for ensuring nutritional quality and safety of foods, beverages, and supplements. Their diverse structures and high polarity make reversed-phase separations challenging. A fast, UV-based method accessible to routine laboratories offers cost-effective quality control without requiring mass spectrometry.

Study Objectives and Overview

This work aimed to develop a single reversed-phase HPLC-UV method capable of baseline separating all known water-soluble vitamins within a short runtime. A software-driven empirical approach using ChromSword was applied to predict optimal gradient profiles and retention times, followed by method transfer to a UHPLC system for validation.

Methodology

An empirical workflow involved:

• Screening four RP stationary phases and various aqueous buffers with methanol or acetonitrile.
• Performing a few initial isocratic experiments at selected organic percentages.
• Entering experimental retention times into ChromSword Offline software to model retention behavior and propose gradient conditions.
• Dividing vitamins into polar and non-polar subsets for initial modelling, then combining predicted and experimental data to optimize a single method.

Used Instrumentation

• UHPLC: Thermo Scientific Vanquish Horizon system (pump, autosampler, column compartment, diode array detector with LightPipe flow cell).
• Columns tested: Hypersil GOLD aQ C18; Acclaim Vanquish Polar Advantage II; Acclaim Polar Advantage; Accucore aQ C18.
• Software: ChromSword Auto Developer and Offline modules; Chromeleon 7.2.8 for data acquisition and processing.
• Detection: UV diode array at multiple wavelengths (210–280 nm).

Main Results and Discussion

The final workflow produced an 11-minute gradient on Hypersil GOLD aQ C18 using 50 mM ammonium formate pH 3.8 with methanol. Key outcomes included:

• Baseline separation (resolution >2.0) of eleven water-soluble vitamins in a single run.
• Retention time prediction errors below 0.10 min for six vitamins, and below 0.82 min for all.
• Retention time RSD <0.16%, peak area RSD <1% (with two exceptions at ~3%).
• Calibration linearity (R² >0.996) over relevant concentration ranges.
• Limits of detection between 0.03 µg/mL and 76 µg/mL; limits of quantitation from 0.10 µg/mL to 253 µg/mL.

Advantages and Practical Applications

This all-in-one RP-HPLC-UV method accelerates routine analysis of water-soluble vitamins in quality control, research, and industrial settings. The empirical modelling approach reduces experimental workload, ensures transferability, and delivers predictable retention times.

Future Trends and Applications

Empirical software-driven method development is likely to expand to other complex analyte classes. Integration with high-throughput UHPLC platforms and advanced detection technologies may further shorten analysis time and enhance robustness.

Conclusion

A predictive, empirical strategy using ChromSword enabled the first single reversed-phase HPLC-UV method resolving all known water-soluble vitamins in under 11 minutes. The approach combined rapid method development with high retention time accuracy and robust performance suitable for routine laboratories.

References

1. Kamangar F, Emadi A. Vitamin and Mineral Supplements: Do We Really Need Them? Int J Prev Med. 2012;3(3):221–226.
2. Walash MI, Sheribah ZA, Salim MM. Kinetic Spectrophotometric Determination of Biotin in Pharmaceutical Preparations. Int J Biomed Sci. 2008;4(3):238–243.