Vitamins, HPLC analysis

Gel permeation ehromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard referenee material (SRM) samples of a fortified eoeonut oil (SRM 1563) and a eod liver oil (SRM 1588). The on-line GPC/normal-phase proeedure eliminated the long and laborious extraetion proeedure of isolating vitamins from the oil matrix. In faet, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC eolumns used for the vitamin determinations were a 10 p.m polystyrene/divinylbenzene gel eolumn and a semipreparative aminoeyano eolumn, with hexane, methylene ehloride and methyl tert-butyl ether being employed as solvent. 

High-pressure pumps operating at up to 6000 psi are required to force solvent through a tightly packed HPLC column, and electronic detectors are used to monitor the appearance of material eluting from the column. Alternatively, the column can be interfaced to a mass spectrometer to determine the mass spectrum of every substance as it elutes. Figure 12.18 shows the results of HPLC analysis of a mixture of 10 fat-soluble vitamins on 5 jam silica spheres with acetonitrile as solvent. 

Figure 12.18 Results of an HPLC analysis of a mixture often fat-soluble vitamins.

The effect of vitamin E supplementation on a-tocopherol and /(-carotene concentrations in tissues from pasture- and grain-fed cattle was also elucidated with HPLC analysis. The investigation was motivated by the fact that a-tocopherol influences beneficially meat colour and stability [53], and the presence of /(-carotene can modify the amount of a-tocopherol in tissues [54], Samples were extracted with hexane and the concentration of /(-carotene was assessed by HPLC. Some data are listed in Table 2.20. It was concluded from the data that... 

Burns [27] described a fully automated approach for HPLC analysis of vitamin tablets. A sample valve provides the injection interface in this apphcation. Tablets direct from the production plant are dispensed into the sample cups on a Technicon Sohd Prep sampler, they are dissolved, and the fat-soluble vitamins are extracted. The solution is concentrated... 

Recently we published data that even in countries with excellent food sources and availability, insufficient vitamin A supply will occur (Schulz et ah, 2007). The aim of this trial was to analyze vitamin A and p-carotene status and investigate the contribution of nutrition to vitamin A and p-carotene supply in mother-infant pairs of multiparous births or births within short birth rates. Twenty-nine volimteers aged between 21 and 36 years were evaluated for 48 hours after delivery. In order to establish overall supply, retinol and p-carotene were determined in maternal plasma, cord blood, and colostrum via HPLC analysis. A food frequency protocol was obtained from all participants. Regardless of the high-to-moderate socioeconomic background, 27.6% of participants showed plasma retinol levels below 1.4 pmol/liter, which can be taken as borderline deficiency. In addition, 46.4% showed retinol intake <66% of RDA and 50.0% did not consume liver at all, although liver contributes as a main source for preformed retinol. Despite a high total carotenoid intake of 6.9 3.9mg/day, 20.7% of mothers showed plasma levels <0.5 pmol/liter p-carotene. 

A list of selected applications of HPLC for vitamin A analysis is reported in Table 19.10. 

Selected applications of HPLC for vitamin D analysis are listed in Table 19.13. 

HPLC analysis of polycyclic aromatic hydrocarbons (PAH) in drinking water is one of the current and classical applications of fluorescence. In this case, the detector contains a fluorescence flow cell placed after the chromatographic column. This mode of detection is specifically adapted to obtain threshold measurements imposed by legislation. The same process allows the measurement of aflatoxins (Fig. 12.11) and many other organic compounds (such as adrenaline, quinine, steroids and vitamins). 

R Andreoli, M Careri, P Manini, G Mori, M Musci. HPLC Analysis of fat-soluble vitamins on standard and narrow bore columns with UV, electrochemical and particle beam MS detection. Chro-matographia 44 605-612, 1997. 

The discussion of the physicochemical properties of each vitamin will highlight those properties having a significant impact on HPLC analysis. Recent methodological reviews will be listed for each vitamin. 

In foods, the simultaneous HPLC analysis of several vitamins is feasible only under special conditions, for example, determination of fortified, free vitamins rather than endogenous ones, which are often bound to other food components, or determination of vitamins in relatively simple foodstuffs, such as fruit juice. The HPLC separation per se of multiple vitamins is not difficult it is their simultaneous, nondegradative extraction from foods that is problematic. Developing a single set of extraction conditions that satisfies the diverse physicochemical properties and stability requirements of several vitamins is a challenge. 

If fat-soluble vitamins (A, D and E) are added to a product, the first step in the analysis will often be hydrolysis. In fortified products this converts any esters, for example, vitamin A palmitate or vitamin E acetate, to their free alcohols. The product is then extracted with a hydrophobic solvent, such as hexane or diethyl ethyl or a mixture of these solvents, prior to HPLC analysis often using a reverse-phase (Ci8) column. 

In recent years HPLC procedures have evolved as the methods of choice for the analysis of vitamin Do in bulk drug (32,33,53-61), pharmaceutical preparations (52-73), fortified milk (74-82), animal feed supplements (83-89), margarine (79), infant formulae (79), cod liver oil (90-93), and chicken egg (96). It may be pointed out that these reports represent the latest in the state-of-the-art in the HPLC analyses of vitamins Dg and D3. In spite of the intensive effort by a number of investigators and a few collaborative studies, there is no consensus on widely acceptable HPLC methods for vitamin D analysis in the bulk drug, pharmaceutical preparations, and fortified milk. 

In this procedure, the HPLC analysis is carried out on a DuPont Zorbax ODS column using methanol, acetonitrile and water 10 2 1 at the mobile phase. This system resolves vitamins D3 from D2 and from degradation products and other fat soluble vitamins. Didecyl and dinonyl phthalate were used as the internal standards. The latter was included for use in the event that extraneous peaks interfered with the didecyl phthalate peak. Figure 11 shows a chromatogram of an extract of a multivitamin formulation with the two added internal standards. 

Fuchs, J. et al., HPLC analysis of vitamin E isoforms in human epidermis correlation with minimal erythema dose and free radical scavenging activity, Free Radio. Biol. Med., 34, 330, 2003. 

FIGURE 1-8. Journal references to the HPLC analysis of antibiotics and vitamins, 1969-1988. (Adapted from reference 14 with permission.)... 

Sundl, I. Murkovic, M. Bandoniene, D. Winklhofer-Roob, B.M. 2007. Vitamin E content of foods comparison of results obtained from food composition tables and HPLC analysis. Clin. Nutr. 26 145-153. 

Papadoyannis, I.N. Tsioni, G.K. Samanidou, V.F. Simultaneous determination of nine water and fat soluble vitamins after SPE separation and RP-HPLC analysis in pharmaceutical preparations and biological fluids. J. Liq. Chromatogr. 1997, 20 (19), 3203-3231. 

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). 

Salo-Vaananen, P, Ollilainen, V., Mattila, P., Lehikoinen, K., Salmela-Molsa, E., andPiironen, V. 2000. Simultaneous HPLC Analysis of Fat-Soluble Vitamins in Selected Animal Products after Small-Scale Extraction. Food Chem. 71 535-543. 

The structure of vitamin B12, called cyanocobalamin, is shown in Figure 8.13 and is a water-soluble vitamin. It has a molecular weight of 1355 and has a complex chemical structure. The molecule is hydrophilic and is not easily extractable into organic solvents. Thus, SPE is an excellent way to isolate it from aqueous solutions followed by HPLC analysis. It is isolated by reversed-, phase sorption onto a C-18 sorbent. 

Vitamin Kj (Fig. 9.10) is primarily known for its role in the synthesis of coagulation proteins, and recent work has shown that it has a much wider role for other proteins in the body thus, methods for its analysis in food are important (Booth et al., 1994). Concentrations of vitamin K, in foods have not been frequently reported thus dietary intake is not well understood. This recent method by Booth and co-workers (1994) addresses the need for methods for vitamin K, and uses a silica-gel clean-up followed by HPLC analysis. 

The sample preparation and FIPLC analysis are more elaborate for formulations with multiple APIs (e.g., over-the-counter (OTC) products) or with natural products. Examples of HPLC analysis of two OTC multi-vitamin products are shown in Figure 6.5, with a summary of method performance for both water-soluble and fat-soluble vitamins17 listed in Table 6.5. Other examples of HPLC analysis of extracts of natural products (white and red ginseng)18 are... 

Figure 7.7. HPLC analysis of water-soluble vitamins in a beverage powder sample (Tang ) and a baby formula (Similac ) using ion-pair reversed-phase chromatography and UV detection. Reprinted with permission from reference 17.

Experiment 27. Ion pair chromatography of vitamins Experiment 28. Techniques in HPLC analysis of analgesics Experiment 29. Analysis of amino acids as their DNP derivatives Experiment 30. Analysis of paraben preservatives by HPLC with photodiode array detection... 

Many vitamins are extremely labile and considerable attention has been devoted to improving their stability during extraction and HPLC analysis. As a result of their instability, the major difficulty in the analysis of vitamins by HPLC is to resolve them from the complex molecules found in their environment without causing their degradation. No attempt will be made to cover the entire area of extraction and sample preparation and the reader is referred to a review (Parrish, 1980) however, specific examples are cited in the text where appropriate. References are presented along with specific apphcations in Section 11.8.4. 

HPLC analysis of the D vitamins is particularly difficult because they are only present at very low concentrations. Many methods of extraction have been used, but the most common is to saponify the sample and extract it into an organic phase (usually hexane or dimethyl-sulphoxide). A mobile phase of diethyl ether has been suggested to be preferable to hexane since this solvent allows an excellent separation... 

Experimental Results For control experiments, 20% cellulose slurry was made in a 2L flask by combining it with 200 mL of TM2 medium. Following addition of 13 ml (1300 BFPU) SPEZYME CP (Genencor) and 20 mg spectinomycin and 1 mg vitamin B12, samples were taken from the vessel at varying reaction times, centrifuged, and the supernatants frozen to terminate enzyme action. The supernatants were subjected to HPLC analysis. This... 

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