Vitamin reversed-phase separations

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

Fig. 11.8.1. Reversed phase separation of vitamin A alcohol and esters. Chromatographic conditions stationary phase, Zorbax C-8 (250 x 4.6 mm I.D.) mobile phase, 99.8% water-0.2% perchloric acid (60%), and 99.8% methanol-0.2% perchloric acid flow rate, 1.6 ml/min detection, UV at 254 nm. Peaks 1, vitamin A alcohol 2, impurity 3, vitamin A acetate 4, vitamin A palmitate. Reproduced from Dupont Analytical Systems with permission.

Gundersen and Blomhoff (1999) used online dilution with online SPE to measure vitamin A (retinol) and other active retinoids in animal plasma. The intention of online dilution in this application was on optimizing SPE extraction conditions rather than on peak focusing during analytical separation. An SPE cartridge packed with Bondapak C18 materials (37 to 53 jt/M, 300 A, Waters, Milford, Massachusetts) and a reversed-phase analytical column (250 x 2.1 mm inner diameter, Superlex pkb-100, Supelco, Bellefonte, Pennsylvania) were controlled by a six-port switching valve (Rheodyne, Cotati,... 

Water-soluble vitamins in formulations have been determined by use of ion-pair chromatography. The vitamins include several B vitamins as well as niacin, folic acid, and ascorbic acid (565). Vitamins D and Da were rapidly separated on reverse phase columns (247) as are vitamins A, D, and E in multivitamin tablets (564). Addition of silver ions to the mobile phase has been shown to increase the flexibility inherent in RPC by complexing with the unsaturated bonds and thereby decreasing the retention factor. This effect is also observed with other unsaturated drug molecules including steroids (247). Vitamin A and related compounds have... 

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... 

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. 

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. 

Reversed-phase chromatography can separate phylloquinone from closely related structures, but it cannot separate cis- and tram-phylloquinonc. The postcolumn reduction of vitamin K and... 

Reverse-phase HPLC procedures for vitamin D3 in bulk drug were reported in the early 701s by William et al. (54) using a DuPont Permaphase 0DS column (DuPont, Wilmington, Del.) and 78% methanol in water as the mobile phase. A similar column and mobile phase (DuPont s Zorbax 0DS Column and 95% methanol in water) were reported to give increased resolution of the two forms of vitamin D (62) in multivitamin formulations. Complete separation of vitamins Dg and D3 by reverse-phase chromatography in model multivitamin preparations were reported by Osadca and Araujo (63). In this... 

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. 

M. C. Gennaro, Separation of water-soluble vitamins by reversed-phase ion-interaction-reagent high-performance liquid chromatography Application to multivitamin pharmaceuticals, J. Chromatogr. Sci., 29 410(1991). 

Reversed-phase chromatography is the most popular mode for the separation of low molecular weight (<3000), neutral species that are soluble in water or other polar solvents. It is widely used in the pharmaceutical industry for separation of species such as steroids, vitamins, and /3-blockers. It is also used in other areas for example, in clinical laboratories for analysis of catecholamines, in the chemical industry for analysis of polymer additives, in the environmental arena for analysis of pesticides and herbicides, and in the food and beverage industry for analysis of carbohydrates, sweeteners, and food additives. 

Several reversed-phase HPLC methods have also been reported for the quantitative analysis of tocopherols and tocotrienols (Table 11.6). To be able to separate all eight tocols a pentafluorophenylsilica column (Abidi, 2003) was used. A C30-bonded phase silica column separated the three tocotrienols, a-tocopherol, and a-tocomonoenol in palm oil (Ng et al., 2004). C30-bonded silica columns have also been used to simultaneously analyze tocopherols, other fat-soluble vitamins, and carotenoids (Gentili and Caretti, 2011). 

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). 

The more usual way of preparing reversed phase papers is by impregnation with silicones, rubber, or liquid ion exchangers [15]. These papers, only some of which are available commercially, can be used to separate hydrophobic substances such as amines, fatty acids, sterols, triglycerides, vitamins and pesticides. 

At first reverse phase sorbents were prepared by impregnating the support with long-chain hydrocarbons such as paraffins and silicone oils to give packings of defined composition with the stationary phase held to the support by purely physical forces of attraction [53]. These materials though of use for the analysis of lipophilic substances, fats and waxes, steroids and fat soluble vitamins and dyes were unsatisfactory as the stationary phase can be washed off by the eluant which consequently lowers the capacity and effectiveness of the partition separation. 

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. 

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