Analysis of Vitamins by Liquid Chromatography

Department of Chemistry, Faculty of Mathematical, Physical and Natural Sciences, University of Rome La Sapienza, Italy 

Before performing a LC analysis, it is advisable to adopt some preventive measures to restrain losses due to vitamin instability. The most important factors that lead to inactivation are light, air, temperature, pH, trace metals, and ionic strength. Since the majority of the vitamins are photosensitive, the use of low actinic amber glassware and subdued light is recommended for the whole duration of the analysis. Another precaution that cannot be disregarded is the addition of a proper antioxidant to the solvents employed both for the preparation of standard solutions and extraction procedures. 

LIQUID CHROMATOGRAPHIC DETERMINATION OF WATER-SOLUBLE VITAMINS 

Vitamin Bi [1] exists in nature both in free (thiamin) and esterified form (thiamin monophosphate, diphosphate, and triphosphate), while thiamin hydrochloride is used as a supplement [4]. To evaluate the total content of vitamin Bi in a food, extraction usually consists of an acid hydrolysis (0.1 M HCl in a water bath at 100°C or in an autoclave at 121°C) followed by an enzymatic digestion (diastases possessing a phosphatase activity) [1,2,5,6]. The acid treatment frees protein-boimd forms and converts starch into soluble sugars. The enzymatic treatment may require several hours (on average 3 hr) of incubation for complete dephosphorylation of the thiamin esters. 

Owing to its low molar absorptivity, the use of UV detection is mainly indicated for the analysis of fortified foods containing high concentrations of thiamin [1,7]. Low content of endogenous vitamin and high quantities of interfering substances in an extract require 

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JN Thompson. Trace analysis of vitamins by liquid chromatography. In JF Lawrence, ed. Trace Analysis. Vol 2. New York Academic Press, 1982, pp 1-67. 

Sanchez-Perez, A., Delgado-Zamarreno, M.M., Bustamante-Rangel, M., and Hernandez-Men-dez, J. 2000. Automated analysis of vitamin E isomers in vegetable oils by continuous membrane extraction and liquid chromatography-electrochemical detection. J. Chromatogr. A 881 229-241. 

DB Dennison, JR Kirk. Quantitative analysis of vitamin A in cereal products by high-speed liquid chromatography. J Food Sci 42 1376-1379, 1977. 

Chavez-Servin, J.L. Castellote, A.I. Lopez-Sabater, M.C. 2006. Simultaneous analysis of vitamins A and E in infant milk-based formulae by normal-phase high-performance liquid chromatography-diode array detection using a short narrow-bore column. J. Chromatogr. A. 1122 138-143. 

Kramer, J.K.G., Fouchard, R.C., and Kallury, K.M.R. 1999. Determination of Vitamin E Forms in Tissues and Diets by High-Performance Liquid Chromatography Using Normal-Phase Diol Column, in Analysis of Vitamin E Forms. New York, NY Academic Press Ltd., pp. 318-329. 

LG Rushing, WM Cooper, HC Thompson Jr. Simultaneous analysis of vitamins A and E in rodent feed by high-pressure liquid chromatography. J Agric Food Chem 39 296-299, 1991. 

After extraction into hexane, a solid-phase extraction, using silica and nonpolar eluents, or sometimes reversed-phase extraction with polar solvents, is common in many assays. Some research groups used a semipreparative HPLC method (silica column and acetonitrile [62,67,68] or di-isopropylether [89] in hexane) with UV detection for cleanup. Blanco-Gomis et al. (60,61), introducing narrow-bore HPLC or capillary liquid chromatography for small-sample analysis of vitamins, were able to get appropriate results without solid-phase cleanup by reextracting the hexane layer with methanol water 9 1 to remove interfering lipids, as described elsewhere (24,88,108). 

Dimartino G. Convenient analysis of vitamin D in cheese and other food matrixes by liquid chromatography/mass spectrometry. J AOAC Int 2007 90(5) 1340—5. 

Wong, F.F., Analysis of vitamin Be in extractives of food materials by high-performance liquid chromatography, J. Agric. Food Chem., 26, 1444—1446, 1978. 

Taylor, P. Barnes, P. (1981). Analysis of vitamin E in edible oils by high performance liquid chromatography. Chem. Ind, 7 7, 722-726. 

Lamidsen C, Leonard SW, Griffin DA, Liebler DC, McQme TD, Traber MG. Quantitative Analysis by Liquid Chromatography— Tandem Mass Spectrometry of Deuterium-Labeled and Unlabeled Vitamin E in Biological Samples. Anal Biochem 2001 289 89-95. 

Currently, high-performance liquid chromatography (HPLC) methods have been widely used in the analysis of tocopherols and tocotrienols in food and nutrition areas. Each form of tocopherol and tocotrienol can be separated and quantified individually using HPLC with either a UV or fluorescence detector. The interferences are largely reduced after separation by HPLC. Therefore, the sensitivity and specificity of HPLC methods are much higher than those obtained with the colorimetric, polarimetric, and GC methods. Also, sample preparation in the HPLC methods is simpler and more efficiently duplicated than in the older methods. Many HPLC methods for the quantification of tocopherols and tocotrienols in various foods and biological samples have been reported. Method number 992.03 of the AOAC International Official Methods of Analysis provides an HPLC method to determine vitamin E in milk-based infant formula. It could probably be said that HPLC methods have become dominant in the analysis of tocopherols and tocotrienols. Therefore, the analytical protocols for tocopherols and tocotrienols in this unit are focused on HPLC methods. Normal and reversed-phase HPLC methods are discussed in the separation and quantification of tocopherols and tocotrienols (see Basic Protocol). Sample... 

F Zonta, B Stancher. Quantitative analysis of phylloquinone (vitamin K,) in soy bean oils by high-performance liquid chromatography. J Chromat 329 257-263, 1985. 

DA Sampson, LA Eoff, XL Yan, K Lorenz. Analysis of free and glycosylated vitamin B6 in wheat by high-performance liquid chromatography. Cereal Chem 72 217-221, 1995. 

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