Water-soluble vitamins sample preparation

A large number of methods have been developed for analysis of water-soluble vitamins simultaneously in pharmaceutical products (like multivitamin tablet supplements). In fact, for these products no particular sample preparations are required and the high concentrations simplify the detection, enabling the use of UV [636]. The use of MS is also reported [637]. As well, Moreno and Salvado [638] reports also the use of a unique SPE cartridge (C18) for separating fat-soluble and water-soluble vitamins, which are, then analyzed using different chromatographic systems. 

Jegle [41] described the separation of water soluble vitamins by CZE, and gave an example of analysis from a commercial vitamin preparation (Figure 7). Lambert, et al. compared the analysis of B12 and analogues by HPLC and CZE. The CZE method was tested on multi-vitamin preparations [42]. Ma, et al., used CZE and laser induced fluorescence for the fast microassay of vitamin A in serum samples [43]. 

In general, the methods available for water-soluble vitamins are less successful for real samples than those described for the fat-soluble vitamins. The problems are, in general, related to sample preparation prior to LC analysis since naturally occurring vitamins are often bound to other food constituents such as carbohydrates or proteins. 

Electrochemical detection in LC provides a sensitive assay method for certain vitamins, such as AA, folates, and flavins. AA may be easily detected with femtomolar sensitivity. Sample preparation and matrix interference problems limit the routine applicability of electrochemistry in the analysis of water-soluble vitamins currently to AA. 

Chromatographic methods, especially LC, have offered increased selectivity and sensitivity in vitamin assays. This is reflected in the many methodological publications on the topic over the past decades. Analyses for water-soluble vitamins in physiological samples are now performed routinely using these methods. The problems with these methods are related to sample preparation and to the sensitivity of the detection method that is used. Only a few chromatographic methods enable simultaneous assay of several water-soluble vitamins in physiological samples, and so separate assays are needed. Less complex samples (e.g., pharmaceuticals or fortified foods) are easier to analyze in this respect. 

Chromatographic analysis of the vitamin Bg complex, including sample preparation and pre-TLC extraction have been reviewed (14). Separation of pyridoxine from other water-soluble vitamins in pharmaceutical preparations may be facilitated by impregnating silica gel plates with zinc acetate to provide a self-indicating system after separation (7). Impregnation of plates with hexadecyltrimethyl-ammonium bromide has been used to improve the TLC analysis of vitamin in foods (9). Overpressure layer chromatography was found to provide better separation and resolution of B( from other compounds than HPTLC (11). 

Reversed-phase HPLC systems are often used for the simultaneous assay of thiamine and other water-soluble vitamins in multivitamin preparations. No special treatment of the sample is required before chromatography and UV detection at 270 nm or 254 nm is usually employed. 

Nuttall and Bush (102) described a TLC chromatographic method for the analysis of multivitamin preparations. After extraction of fat-soluble vitamins, water-soluble vitamins and water-soluble materials were separated in three TLC systems. Biotin was resolved with acetone-acetic acid-benzene-methanol (1 1 14 4) as solvent and visualized by spraying o-toluidine-potassium iodide. Standards can be included if quantitative results are required. However, the reproducibility of the technique has not been tested. Groningsson and Jansson (105) worked out a TLC method for the determination of biotin in the presence of other water-soluble vitamins. After dissolution of the lyophilized preparation and addition of the internal standard (2-imidazolidone), the sample was applied on a silicagel plate and eluted with chloroform-methanol-formic acid (70 40 2). Biotin was visualized by spraying with p-DACA and determined in situ by reflectance measurements. The sensitivity of the method could be increased by spraying with paraffin after the coloring procedure. LFnder these conditions the detection limit was 10 ng. 

The use of a mass spectrometer as chromatographic detector offers a great advantage in vitamin analysis the possibility of simplifying the extraction procedure. The selectivity of the LC—MS technique reduces problems due to intrusive peaks from matrix components, while its sensitivity (ng or pg injected for real samples) allows the direct injection of an extract, eliminating the concentration step and the exposure to heat (most water-soluble vitamins posses low thermal stability). Sample preparation time is reduced as well as the duration of exposition to air and light (most vitamins and carotenoids are susceptible to these factors). 

The extraction and cleanup steps for riboflavin are similar to those proposed for thiamin. Special care should be taken to protect samples and standards from UV hght and alkaline conditions. Strict control in the oxidation process is also necessary to avoid riboflavin decomposition. Although riboflavin is classified as a water-soluble vitamin, some problems can arise when dissolving the vitamin in water, and this must be taken into consideration when preparing the standard solutions. 

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... 

Liquid-liquid extraction is generally reserved for more complex samples because it offers poorer precision than other techniques. It is most commonly used for the preparation of biological samples in which less precise methods can be tolerated. Occasionally, however, an extraction is necessary for the determination of a water-insoluble compound in a water-soluble matrix, such as the analysis of fat-soluble vitamins in tablets or menthol in pharmaceutical lozenges. In these cases, the water-soluble matrix must be treated with water to gain access to the analytes, but the solvent cannot be made sufficiently nonpolar to dissolve the analytes by adding a water-miscible solvent. 

Overview. Polychlorinated Biphenyls. Polycyclic Aromatic Hydrocarbons Determination. Polymers Natural Rubber Synthetic Polyurethanes. Quality Assurance Quality Control Instrument Calibration Interlaboratory Studies Reference Materials Production of Reference Materials Method Validation Accreditation Clinical Applications Water Applications. Sample Handling Comminution of Samples Sample Preservation Automated Sample Preparation Robotics. Sampling Theory Practice. Solvents. Supercritical Fluid Chromatography Overview Applications. Vitamins Overview Fat-Soluble. 

See also Carbohydrates Overview. Extraction Solid-Phase Extraction Supercritical Fluid Extraction. Food and Nutritional Analysis Sample Preparation Antioxidants and Preservatives Mycotoxins Oils and Fats. Lip-Ids Overview. Peptides. Proteins Overview. Toxins Mycotoxins Neurotoxins. Vitamins Overview Fat-Soluble Water-Soluble. 

The hydrophilic vitamins are a disparate group of compounds with few conunon properties apart from water solubility. This single fact, however, makes TLC an obvious choice for their separation, particularly in pharmaceutical preparations and food products. At physiological concentrations, TLC may equally well be employed, following extraction of the vitamins fi om tissues or body fluids, but this must be under acid conditions as most of these compounds are unstable at high pH. Some are also light-sensitive. Special means of detection may also be required unless samples have been greatly concentrated, since tissue levels of most hydrophilic vitamins are low or very low. 

The work covers both the fat-soluble (Chapters 1—4) and the water-soluble (Chapters 5-13) vitamins, with emphasis on state-of-the-art chromatography, sample preparation, and final measurement. Following present analytical evolution, sections on recent techniques such as capillary electrophoresis and mass spectrometry have been added or expanded. Information on metabolism and biochemical function has also been revised to incorporate current knowledge. 

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