Fluorometric Determination of Vitamin Constituents in Human Plasma Using Ultra Performance Liquid Chromatography

Edward C Bell

doi:10.4172/2157-7064.1000143

Abstract

Purpose: A rapid, selective and sensitive ultra performance liquid chromatography method has been developed for the detection and quantification of several vitamins in human plasma. Methods: Alpha tocopherol, gamma tocopherol, and retinol are assayed using fluorescence detection. Excitation/emission wavelengths are 295 nm/330 nm and 325 nm/470 nm for the analysis of both tocopherols and retinol, respectively. Retinol acetate is employed as the internal standard. The reversed phase method incorporates gradient elution with a mobile phase consisting of methanol and acetonitrile. Separation of vitamin compounds is achieved using a bridged ethyl-hybrid C18 column. Results: The retention times for retinol, retinol acetate, gamma tocopherol and alpha tocopherol are 1.6 minutes, 1.8 minutes, 3.9 minutes and 4.3 minutes, respectively. The limits of quantification for retinol, gamma tocopherol and alpha tocopherol, were 0.02 μg/ml, 0.02 μg/ml, and 0.1 μg/ml, respectively. Conclusion: The assay method is suitable for the analysis of these vitamins in human plasma following the ingestion of foods fortified with these fat soluble vitamins.

Highlights

Vitamins commonly found in foodstuffs and nutritional supplements within the human diet are frequently monitored for a variety of applications
We have developed a rapid and sensitive fluorometric assay for the simultaneous detection and quantification of alpha (α)-tocopherol, gamma (γ)-tocopherol, and retinol using ultra performance liquid chromatography
In similar fashion to post-extraction recovery calculations, concentration levels of 0.05, 0.5 and 5 μg/mL for gamma tocopherol and retinol, and concentration levels of 0.5 and 5 μg/mL for alpha tocopherol were used for intra-day and inter-day calculations (Table 1)

Summary

Introduction

Vitamins commonly found in foodstuffs and nutritional supplements within the human diet are frequently monitored for a variety of applications. An additional advantage of fluorescence detection methods is its ability to achieve appreciable quantitative analysis even in the instance of less than optimal column resolution [48]

Results

Discussion

Conclusion