Normal-phase chromatography vitamin

Reversed-phase chromatography also separates, isocratically, vitamin D2 or D3 from their respective previtamins and inactive isomers (207), but, unlike normal-phase chromatography, it can separate vitamin D2 from D3 using nonendcapped stationary phases (198). The 25-hydroxylated metabolites of vitamins D2 and D3 can be separated from one another using a Vydac 201 TP column (37). The separation of vitamin D2 from vitamin D3, and 25-hydroxyvitamin D2 from 25-hydroxy vitamin D3, allows the D2 form of the vitamin or its metabolite to be used as an internal standard for quantifying the corresponding D3 form. 

A reverse-phase HPLC assay, as part of the Association of Official Analytical Chemists report on analysis of fat-soluble vitamins, was described by DeVries et. al. (65). Analysis were made with a Merck LiChrosorb RP-18 column (Manufacturing Chemists, Inc., Cincinnati, OH) and a acetonitrile propionitrile water (79 15 6) mobile phase. Although adequate chromatography was realized, the authors were concerned that problems arose concerning influence of temperature, dissolution of sample and purification of solvents in the mobile phase. For these reasons they recommended normal-phase chromatography. Separation of vitamins D2 and Dj with their systems was not discussed. 

Diack, M. Saska M. 1994. Separation of vitamin E and y-oryzanols from rice bran by normal-phase chromatography. J. Am. Oil Chem. Soc. 71 1211-1217. 

Normal-Phase Chromatography Example. Vitamin E, an antioxidant, is a complex made up of tocopherols and tocotrienols (Figure 8-62), which are sometimes used to stabilize formulations. Tocopherols are a series of related benzopyranols with a C16 saturated side chain. Tocotrienols contain three double bonds on the C16 side chain [32]. 

Normal-phase sorbents such as silica and Florisil are used to isolate low to moderate polarity species from nonaqueous solutions. Examples of applications include lipid classification, plant pigment separations, and separations of fat-soluble vitamins from lipid extracts, as well as the clean-up of organic solvent concentrates obtained from a previous SPE method or liquid-liquid extraction. Alumina is used to remove polar species from nonaqueous solutions. Examples include vitamins in feeds and food and antibiotics and other additives from feed. Normal-phase chromatography has been used for a number of years, and most applications for normal-phase column chromatography may be easily transferred over to normal-phase SPE. 

Solvents commonly used in normal phase chromatography are aliphatic hydrocarbons, such as hexane and heptane, halogenated hydrocarbons (e.g., chloroform and dichloromethane), and oxygenated solvents such as diethyl ether, ethyl acetate, and butyl acetate. More polar mobile phase additives such as isopropanol, acetone, and methanol are frequently used see Table 2). The technique is particularly suited to analytes that are very hydrophobic, e.g., fat-soluble vitamins such as tocopherols (6J and other hydrocarbon-rich metabolites that exhibit poor solubility in the water-miscible solvents employed in other separation modes. In addition, since the geometry of the polar adsorbent surface is fixed, the technique is useful for the separation of positional isomers the proximity of functional groups to the adsorbent surface, and hence the strength of interaction, may well differ between isomers. 

Extensive development of a two-stage chromatographic system for the analysis of the K vitamins has been reported (Lefevre et al., 1979). This method, which involves initial normal phase chromatography followed by reversed phase chromatography, was necessary because only small quantities of vitamin K are present in the extracts of milk... 

Normal Phase. As rationalized in III.A.2, the strength of normal-phase chromatography for vitamin E lies in its ability to separate all tocopherols and tocotrienols, including the positional isomers, particularly in connection with the analysis of vegetable oils and foods. Silica continues to be the most popular column material for this purpose, but polar bonded phases have increasingly gained a foothold (Table 3). Diol phases in particular can be readily substituted for silica and can be eluted with similar binary mobile phases containing a hydrocarbon as a base solvent and an alcohol, an aliphatic ether, or a cyclic ether as... 

Normal-phase/reversed-phase chromatography is the ideal combination for semipreparative and quantitative separations in two-dimensional HPLC. Vitamins D2 and D3 coelute during the semipreparative stage, allowing a narrow retention window to be collected for analysis using internal standardization. By this means, Johnsson et al. (201) obtained a vitamin D3 detection limit of 0.1 yug/kg for milk and milk products. 

JKG Kramer, L Blais, RC Fouchard, RA Melnyk, KMR Kallury. A rapid method for the determination of vitamin E forms in tissues and diet by high-performance liquid chromatography using a normal-phase diol column. Lipids 32 323-330, 1997. 

T-S Shin, JS Godber. Improved high-performance liquid chromatography of vitamin E vitamers on normal-phase columns. J Amer Oil Chem Soc 70 1289-1291, 1993. 

Gel permeation chromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard reference material (SRM) samples of a fortified coconut oil (SRM 1563) and a cod liver oil (SRM 1588). The on-line GPC/normal-phase procedure eliminated the long and laborious extraction procedure of isolating vitamins from the oil matrix. In fact, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC columns used for the vitamin determinations were a 10 im polystyrene/divinylbenzene gel column and a semipreparative aminocyano column, with hexane, methylene chloride and methyl te/t-butyl ether being employed as solvent. 

Figure 10.9 Chromatograms of fortified coconut oil obtained by using (a) normal-phase HPLC and (b) GPC/normal-phase HPLC. Peak identification is as follows 1 (a,b), DL-a-toco-pheryl acetate, 2 (b), 2,6-di-terf-butyl-4-methylphenol 2 (a) and 3 (b), retinyl acetate 3 (a) and 4 (b), tocol 4 (a) and 5 (b), ergocalciferol. Reprinted from Analytical Chemistry, 60, J. M. Brown-Thomas et al., Determination of fat-soluble vitamins in oil matrices by multidimensional high-performance liquid chromatography , pp. 1929-1933, copyright 1988, with permission from the American Chemical Society.

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., Blais, L., Fouchard, R.C., Melnyk, R.A., and Kallury, K.M.R. 1997. A Rapid Method for the Determination of Vitamin E Forms in Tissues and Diet by High-Performance Liquid Chromatography Using a Normal-Phase Diol Column. Lipids. 32 323-330. 

HPLC fulfills all of these criteria and is now used extensively in the analysis of samples from a variety of sources including mammalian tissue, plant tissue and food extracts. The overall efficiency of HPLC and the variety of chromatographic modes allows the majority of analyses to be performed by either reversed phase, reversed phase ion-pair, normal phase or ion-exchange HPLC. Other chromatographic modes such as size exclusion and affinity have found limited application in the chromatography of the vitamins. 

Alternatively, where samples are extracted into organic solvents, normal phase HPLC can also be used. This option is particularly valuable for the resolution of optical isomers of the A vitamins and, for example, 13-cw-retinol can be resolved using this system (Egberg et al., 1977). Retinoyl species may be converted to retinol prior to chromatography but the subsequent profile is difficult to interpret because of the number of possible intermediates that may also be generated. A computer-assisted analysis of vitamin A extracts from two species of tobacco leaf using non-aqueous reversed phase is shown in Fig. 11.8.2. 

HPLC is now accepted as the method of choice for the analysis of the E vitamins (the tocopherols) from plant tissue extracts. However, difficulties in the extraction of the E vitamins do not always leave the samples in a convenient chromatographic buffer and consequently other methods of analysis have also been used (Desai, 1984). Vitamin E activity resides in a number of chemical species which are generally classified as tocopherols and tocotiienols. The most common forms are a-, /8-, y- and 5-tocopherols and separation of each can be performed using either normal phase or reversed phase chromatography. 

Liquid chromatography On silica or polar bonded phases (nitro, cyano), the separation of vitamin D metabolites occurs according to the number and position of hydroxyl groups in the molecule. Binary mobile phases are usually based on hexane-2-propanol mixtures. Improved resolution of normally co-eluting D2 and D3 hydroxylated metabolites is afforded by ternary solvent systems containing dichloromethane as a third component. However, the D2 and D3 parent compounds remain unresolved in any normal-phase system. 

See also Bioassays Overview Microbial Tests Bioautography. Blood and Plasma. Fluorescence Clinical and Drug Applications Food Applications. Food and Nutritional Analysis Dairy Products. Gas Chromatography Mass Spectrometry. Immunoassays, Techniques Enzyme Immunoassays. Liquid Chromatography Normal Phase Reversed Phase Food Applications. Microbiological Techniques. Radiochemical Methods Food and Environmental Applications. Vitamins Fat-Soluble. 

Lindemans (1992) reviewed the use of TLC and paper chromatography for the analysis of cobalamins. His review contains extensive information on extraction procedures bioautographic techniques for the analysis of cobalamins from the plasma of human subjects tabular data on values for cobalamins on reversed- and normal-phase TLC and tabular data on various mobile phases used for the TLC analysis of vitamin B derivatives on cellulose. 

Reversed-Phase. Reversed-phase chromatography continues to form the backbone of most assays of tocopherols and, rarely, tocotrienols in biological materials. Its popular status in the vitamin E area has been rationalized in ni.A.2. When the methods for the simultaneous determination of tocopherols and retinoids/carotenoids are also taken into account, reversed-phase systems outnumber their normal-phase counterparts by a factor 2. A survey of reversed-phase systems for the separation and quantitation of tocopherols, tocopheryl esters, tocotrienols, and a-tocopherolquinone is presented in Table 2. Methods specifically... 

The choice of fhe LC mode for the analysis of water-soluble vitamins depends on the extraction procedure employed and the vitamin form to be quantified (Figure 18.1). The most popular LC modes are normal-phase (NP), reversed-phase (RP), ion-pair RP, ion-suppression RP, and ion-exchange chromatography. 

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... 

Similar to adsorption chromatography, also on normal bonded phase packing materials, cis- and traws-vitamin Ki(20) can be separated. 

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