HPLC Assay of Water-Soluble Vitamins, Fat-Soluble Vitamins, and a Preservative in Dry Syrup Multivitamin Formulation

Significance of the topic

Reliable quantification of both water-soluble and fat-soluble vitamins is essential in pharmaceutical formulations to ensure accurate dosing and patient safety, particularly for infant supplements prepared as dry syrups.

Objectives and overview of the study

This work presents an HPLC assay for simultaneous determination of water-soluble vitamins (B1, B2, B3, B5, B6, B9, B12, C), fat-soluble vitamins (A, E), and the preservative sodium benzoate in a dry syrup multivitamin formulation. It extends previous methods for bottled waters to a powdered syrup matrix, evaluating extraction techniques and chromatographic settings on the Acclaim PA2 column.

Methodology and instrumentation

Water-soluble vitamins are extracted with water and pH adjustment (KOH), while fat-soluble vitamins are extracted using ethyl acetate or DMSO. Separation is achieved on a Dionex UltiMate 3000 HPLC system equipped with an Acclaim PA2 column (3 µm, 4.6×150 mm) under a gradient of methanesulfonic acid, acetonitrile, and ammonium phosphate at 35 °C. A dual-injection approach within one run injects the water-soluble fraction at time zero and the fat-soluble fraction after a solvent gradient change. An accelerated UHPLC variant uses a 2.2 µm Acclaim PA2 column (2.1×100 mm), reducing analysis time from 28 min to 11 min and cutting solvent use by over 80%.

Main results and discussion

Calibration curves for each vitamin exhibited linearity with correlation coefficients above 0.99. Analysis of the dry syrup and placebo samples yielded water-soluble vitamin recoveries of 100–134% and vitamin E recoveries of 93–99%, reflecting accurate over-fortification assessment. Ethyl acetate extraction matched DMSO performance while better preserving column integrity. Reproducibility was confirmed by RSD values below 3% for most analytes, and peak purity analysis ensured specificity. The UHPLC approach maintained separation quality with significant time and waste reduction.

Benefits and practical applications

• A single-column, dual-injection workflow enables comprehensive vitamin profiling in one chromatogram, streamlining pharmaceutical quality control.
• The Acclaim PA2 column’s compatibility with both fully aqueous and organic eluents accommodates diverse vitamin chemistries under one gradient.
• UHPLC implementation offers rapid analysis, lower solvent consumption, reduced waste, and improved laboratory efficiency.

Future trends and applications

• Coupling with mass spectrometric detection could enhance sensitivity and confirmatory analysis, particularly for low-level vitamins like B12 and folic acid.
• Automated sample preparation and on-line extraction may increase throughput in high-volume testing laboratories.
• Extension of the method to additional micronutrients or excipients could broaden its role in nutritional and pharmaceutical product assessment.

Conclusion

The developed HPLC method using the Acclaim PA2 column on the Dionex UltiMate 3000 platform delivers accurate, reproducible, and efficient analysis of water- and fat-soluble vitamins plus preservative in dry syrup formulations. Its flexibility and UHPLC adaptation make it a valuable tool for routine quality control and research.

References

• Dionex Corporation. Determination of Water- and Fat-Soluble Vitamins in Functional Waters by HPLC with UV-PDA Detection. Application Note 216. 2009.