Water-soluble vitamins methods

Description of Method. The water-soluble vitamins Bi (thiamine hydrochloride), B2 (riboflavin), B3 (niacinamide), and Be (pyridoxine hydrochloride) may be determined by CZE using a pH 9 sodium tetraborate/sodlum dIhydrogen phosphate buffer or by MEKC using the same buffer with the addition of sodium dodecyl-sulfate. Detection Is by UV absorption at 200 nm. An Internal standard of o-ethoxybenzamide Is used to standardize the method. 

Ascorbic acid commonly known as vitamin C, is one of the most important water soluble vitamins. Ascorbic acid is involved in many biological processes and it is an essential compound in the human diet [1]. The determination of ascorbic acid has gained increase significance in pharmaceutic, clinical, and food applications. So far, different methods have been developed for determination of ascorbic acid [2, 3]. 

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. 

Reversed micelles can be used to concentrate water-soluble materials in the water pool of SCCO2. The extraction of water-soluble vitamins into reversed micelles has been examined. The efficiency of extraction was strongly affected by the extraction temperature and the concentration of reversed micelles, and the selectivity depended on the size of micelles. Water-soluble vitamins could be efficiently and rapidly extracted. The selective extraction of a model mixture of vitamins from pharmaceutical preparations was also demonstrated. Moreover, the usefulness of the proposed method for the determination of vitamins in various commercial tablets was also demonstrated. Using this method, the surfactant remains mixed with the extracted com-... 

This chapter does not constitute a comprehensive review of all recently published HPLC methods for the analysis of water-soluble vitamins. It is a summary of selected methods and is intended to serve as a tool for the analyst in search of a method for quantitating one or more of the water-soluble vitamins in foods. The selected methods must ... 

The water-soluble vitamins are a highly diverse group of compounds with differing physicochemical properties. A single vitamin generally consists of several vitamers, or chemical species, each of which exhibits the same biological activity in vivo. Individual vitamers with the same biological functions often exhibit vastly different physicochemical properties. This necessitates unique extraction and separation procedures for each vitamin. As a result, each vitamin is considered individually in this chapter. A section on methods that determine multiple vitamins simultaneously is also included. 

Vitamin C continues to be the water-soluble vitamin upon which the bulk of the method development attention is focused. The HPLC methods published from 1992 to 1997 for total vitamin C... 

JW DeVries. Water-soluble vitamins. In DM Sullivan, DE Carpenter, eds. Methods of Analysis for Nutritional Labelling. Arlington, VA AOAC International, 1993, pp 131-135. 

PCH Hollman, JH Slangen, PJ Wagstaffe, U Faure, DAT Southgate, PM Finglas. Intercomparison of methods for the determination of vitamins in foods. Part 2. Water-soluble vitamins. Analyst 118 481-488, 1993. 

A better method is to first add an equal volume of dimethylsulfoxide (DMSO) or dimethylformamide (DMF) to the aqueous sample. This breaks both biological and encapsulation membranes and pulls polar and nonpolar material into solution. The second step is to dilute the sample with 10 volumes of water. At this point, nonpolars can be removed by solvent extraction or with a Cig SFE. Charged molecules can be recovered with pH-controlled extraction or with ion pairing reagents. The DMSO or DMF stays with the water layer. Customers have told me they can achieve almost complete recovery of both fat-soluble and water-soluble vitamins from polymer-encapsulated mixtures. Vitamins are encapsulated to protect potency from air-oxidation. Water-soluble vitamins have nonpolar encapsulation fat-soluble vitamins have polar encapsulation. Either vitamin can be extracted by themselves, but they are difficult to extract under the same condition unless DMSO or DMF are used to break both capsules. 

Several methods have been reported for the separation of water soluble vitamins including vitamin Bg. Some of these systems are described as follows ... 

Modern Analytical Methodologies in Fat and Water Soluble Vitamins. Edited by Won O. Song, Gary R. Beecher and Ronald R. Eitenmiller Modern Analytical Methods in Arts and Archaeology. Edited by Enrico Ciliberto and Guiseppe Spoto... 

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]. 

GH Rolando, SM Laritza. Methods for the simultaneous analysis of water-soluble vitamins. Rev Mex Cienc Farm 30 9-14, 1999. 

Cheese contains a high concentration of essential nutrients relative to its energy content. Its precise nutrient content is influenced by the type of milk used (species, stage of lactation, whole fat, lowfat, skim), method of manufacture, and to a lesser extent the degree of ripening. As outlined in detail elsewhere in this review, water-insoluble nutrients of milk (casein, colloidal minerals, fat, and fat-soluble vitamins) are retained in the cheese curd whereas the water-soluble constituents (whey proteins, lactose, water-soluble vitamins, and minerals) partition into the whey. However, loss of water-soluble B vitamins in whey may be compensated to a certain extent by microbial synthesis during ripening (Renner, 1987). 

When acceptable frying practices are applied and the oil is discarded regularly, the oil remaining in the fried food retains its nutritive value and a significant portion of the liposoluble vitamins. The fried product retains water-soluble vitamins, minerals, and other nutrients to a greater extent than in other cooking methods. 

Vitamin Bg exists as six separate forms in the pyridine group of water-soluble vitamins. The common forms are pyridoxal and pyri-doxamine together with their corresponding phosphate esters and pyridoxine forms. These compounds function as cofactors in a wide variety of enzyme reactions, but most notably in the transamination reaction of amino acid biosynthetic pathways. Extraction of this group of vitamins can be performed by the same methods as those described for the B2 vitamins (Section 11.8.3.3). 

Vitamins are the foodstuff components most often quantified using fluorimetric means. There are several official fluorimetric methods for the determination of three water-soluble vitamins vitamin Bi (thiamine) (AOAC 942.23 and 957.17), B2 (rib-oflavine) (AOAC 970.65 and 981.15), and C (ascorbic acid) (AOAC 984.26). Thiamine is determined by oxidation to fluorescent thiochrome with alkaline hexacyanoferrate(III) or an alternative oxidant (Figure 1). The method is quite simple, reproducible, and selective and provides good recoveries. Many LC methods for thiamine determination in foods have... 

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. 

At present, much effort is being devoted to simultaneous separation and detection of water-soluble vitamins. Undoubtedly, multiple water-vitamin analysis using LC as a separation method is effective with RP ion pair chromatography with acidified methanol or acetonitrile water as the mobile phase. Detection is performed using combined systems such as UV absorbance and fluorescence systems, depending on the vitamins to be determined. Analysis in real food... 

The method of choice for the determination of most vitamins is HPLC due to its high separation capability, its mild analytical conditions, and the possibility to use various specifically adapted detection methods, e.g., LTV, fluorescence, or MS detection. All fat-soluble vitamins and most water-soluble vitamins have chromophores suitable for UV detection. Separation of vitamers and stereoisomers can be achieved. If a higher sensitivity is required HPLC with fluorescence detection can be used, either directly (e.g., vitamins A and E) or after derivatization (e.g., thiamine). A further improvement in sensitivity and specificity has been achieved by introducing HPLC with mass spectrometric detection in vitamin analysis. Due to the structural information retrievable, e.g., molecular mass, fragmentation pattern, this is the method of choice for analysis of samples with complex mixtures or low vitamin concentrations. Examples for the use of HPLC-MS in vitamin analysis include the determination of 25-hydroxy-D3 and pantothenic acid. However, one drawback of mass spectrometry is the need for an isotopically labeled reference compound for reliable quantification. Due to the structural complexity of many vitamins, these reference compounds are often expensive and difficult to synthesize. An interesting unique application is the determination of vitamin B12 by HPLC-IPC-MS, which is possible due to its cobalt content. 

The earliest methods for vitamin analysis were based on animal bioassays. Later, when it was recognized that vitamins are essential for certain microorganisms, microbiological tests were evolved. The chemical methods for determination of water-soluble vitamins were quite common in the past, before the development of more specific and selective analytical methods. 

The physicochemical properties of the water-soluble vitamins are extensively utilized in chemical methods. A method for quantitative vitamin C (ascorbic acid, AA) measurement in food and physiological samples is based on a reaction of the keto groups in dehydroascorbic acid (DHA) with o-phenylenediamine (OPD) to give a fluorescent quinoxaline. This method involves the oxidation of AA to DHA, followed by the measurement of total AA in the sample. The reductive capabilities of AA can especially be utilized for direct electrochemical (amperometric or coulometric) measurement when coupled with HPLC separation. 

Three methods of detection (UV absorbance, fluorescence, and electrochemical activity) are applicable for the analysis of water-soluble vitamins. Currently, UV absorption detection is used in many LC applications for water-soluble vitamins. A simultaneous assay of several vitamins with several wavelengths along with information of peak purity can be accomplished with a diode array absorbance detector. The detection limit of a UV detector is in the order of 1-10 ng (10-100 pmol), which is poorer than that of fluorescence and electrochemical detectors but often sufficient for analysis for many vitamins in foods and physiological samples (Table 4). The lack of selectivity of UV detection may cause problems with interfering and co-eluting contaminants especially in biological samples, thus necessitating sample purification prior to LC. 

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. 

Immunoassays, specific protein-binding assays, and radioisotope tests are sometimes used for the determination of water-soluble vitamins. These are the only feasible and practical methods for the quantification of certain vitamins in physiological samples. The principle of competitive protein binding (CPB) using labeled radioactive or fluorescent tracer is still routinely applied to quantification of serum vitamin... 

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. 

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