Concurrent analysis of fat and water soluble vitamins in biological fluids using reverse phase-high performance liquid chromatography technique

Abstract

Background: Vitamins are essential micronutrient which plays an important role as cofactor of enzymes in energy metabolism. Methods used for vitamin analysis in biological samples are usually based on chemical or microbiological assays that are not very accurate and is time-taking. HPLC method were developed and validated for the simultaneous detection and quantitation of three fat soluble and eight water-soluble vitamins in plasma and urine samples. Methods: Separation was achieved at 40˚C on a reversed-phase C18 column using isocratic mode with methanol and ethanol (75:25) for fat soluble and multi-step gradient mode with methanol and water for water soluble vitamins. Total run time was 20 minutes for fat soluble and 34 minutes for water soluble vitamins. Detection was performed with quaternary diode array detector set at maximum absorption wavelengths for quantification of each vitamin. Spectral comparison was used for peak identification in real plasma samples. Results: The novel analytical method detailed in this paper has proved to be specific, robust, and time-efficient for the simultaneous detection and quantification of fat and water-soluble vitamins in complex biological matrices such as plasma and urine. Conclusion: Method reported can be used in multivitamin analysis in matrixes where low concentrations of vitamins are expected. Recovery percentages ranged from 93% to 100%.

Keywords

• Isocratic mode
• multi-step gradient mode
• quaternary diode array detector
• Run time
• time-efficient method