Vitamin normal-phase separations

FIGURE 5-52. Examples of normal phase separations, (a) Corn-oil tocopherols. Sample 10 /xL of corn oil in 100 /xL of mobile phase. Column Nova Pak Silica (4 jam), 3.9 mm ID x 150 mm. Mobile phase 0.3% isopropyl alcohol in isooctane. Flow rate 1.0 mL/min. Detection fluorescence 290 nm excitation and 335 nm emission. (b) Separation of vitamin E from vitamin A. Mobile phase 0.5% isopropyl alcohol in isooctane. Other conditions are the same as those in a with the exception that retinol was detected with 365 nm excitation and 510 nm emission, (c) Structures of compounds. 

Normal-phase HPLC, using either silica or polar-bonded stationary phases, separates, isocrati-cally, vitamin D2 or D3 from their respective previtamins and inactive isomers (207). Vitamin D (D2 + D3), 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 can be separated from one another and from other hydroxylated metabolites (215), but vitamins D2 and D3 cannot be resolved from one another. The inability to resolve vitamins D2 and D3 means that one vitamer cannot be used as an internal standard for the other. 

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. 

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. 

Normal-phase HPLC on silica columns are also used extensively in D3 analysis of vitamin products with nonpolar mobile phase containing polar modifiers. Krol et al. (66) separated D3 from pre-D3 and from a mixture of other vitamins using an adsorptive silica support introduced in 1972 (Vydac , supplied at that time by Applied Science Laboratories, Inc. State College, Penn.). The hand-packed column was used in conjunction with a mobile phase of pen-tane tetrahydrofuran (97.5 2.5). Sterule (32) used aluminum oxide as column support with chloroform as the mobile phase. Separation of D3 and its isomers and from vitamin A acetate was achieved. 

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. 

Both normal-phase and reversed-phase HPLC have been applied in vitamin E analysis. Reversed-phase HPLC is unable to completely separate all tocopherols and toco-trienols. Because (1- and y-vitamers have very similar structures, their separation cannot be obtained with reversed-phase HPLC. It is, however, applicable when only tocopherols or a-tocopheryl esters are analyzed (Gimeno et al., 2000 Iwase, 2000). There are reversed-phase methods to analyze tocopherols together with other lipid constituents from biological and food samples such as carotenoids (Epler et al., 1993 Salo-Vaananen et al., 2000), ubiquinols and ubiquinones (Podda et al., 1996) or sterols (Warner and Mounts, 1990). 

Determination of four tocopherols and four tocotrienols in vegetable oils and fats by the official American Oil Chemists Society method is based on separation by normal-phase HPLC and fluorescence detection (AOCS, 1990). Oil samples are dissolved in hexane, whereas margarines and other fats containing vitamer esters need a cold saponification step to liberate the vitamers. The American Association of Cereal Chemists has a method to analyze vitamin E in various foods. This method (AACC, 1997) is applicable to a vitamin E range of 1 x 10" - 100%, and it includes hot saponification and separation by reversed-phase HPLC. Results are calculated as a-tocopherol acetate. The Royal Society of Chemistry has approved a method to analyze vitamin E in animal feedstuffs by normal-phase HPLC after the vitamers have been liberated by hot saponification (Analytical Methods Committee, 1990). 

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. 

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. 

Vitamin K Vitamin K exhibits an important anti-hemorrhagic activity, which has increased the interest in developing analytical methods to determine its content in foods. The sample preparation includes various steps such as enzymatic hydrolysis and cleanup using SPE cartridges. Normal-phase LC and RP-LC have been used to separate vitamin K in conjunction with UV detection at 270 nm fluorescence detection can be used only after phylloquinone has been converted to the corresponding hydroquinone after electrochemical or chemical reduction. 

Recent developments have been directed toward the simultaneous determination of multiple vitamins in foods. Separation of vitamins A, E, D2, and D3 in lacteal matrices has been performed using RP-LC with methanohwater as the mobile phase and UV detection at 265 nm (or at the wavelength of maximum absorption of each individual form), after exhaustive saponification. Normal-phase LC has also been used to analyze multiple fat-soluble vitamins in seed oils after extraction or even direct injection of the sample prior to analysis. UV detection has also been used. Sometimes a combination of two detection systems such as UV-visible detection and fluorescence have been used to, for example, determine, respectively, the carotenoids and fat-soluble vitamins in foods. 

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. 

The first category comprise carotenoids, lipids and steroids, the second includes boswellic and fatty acids and vitamins and the last comprises flavones, alkaloids, cannabinoids, berberines and anthraquinones, to give just a few examples. Another method of classification is according to the separation mode, such as normal-phase, reversed-phase, ion-exchange, size-exclusion and affinity-based separations, likewise as in HPLC. In addition, it is worth mentioning that the instrumentation design incorporates pressurized CEC (PEC) and a microchip platform [7]. 

Biesalski and Weiser separated a -trans from l3-cis, W-cis, and 9-cis isomers of retinyl esters (retinyl palmitate, stearate, oleate, palmitoleate, and linole-ate) by isocratic adsorption HPLC (126). Bridges et al. used step gradients to separate geometric isomers of retinyl esters, retinal, retinal oximes, and retinal (both vitamin Ai and vitamin A2 forms), also by adsorption ( normal-phase ) HPLC (139,140). 

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... 

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... 

D-binding immunoaffinity solid phase is used in a batch mode to concentrate vitamin D analogs prior to reverse-phase LC separation and mass spectrometric quantification [16]. Alternatively, an immunoaffinity column can be used in the main separation step of analysis, as has been reported for analysis of transferrin isoforms [17], An important consideration in the use of immunoaffinity columns in the clinical laboratory is the increased cost of the method compared to more common solid phases and the fact that the life time of an immunoaffinity column is shorter than a standard reversed- or normal-phase column. 

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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