Water-soluble vitamins.
Simultaneous Determination of Vitamins.--Klejdus et al. described a simultaneous determination of 10 water- and 10 fat-soluble vitamins in pharmaceutical preparations by liquid chromatography-diode-array detection (LC-DAD). A combined isocratic and linear gradient allowed separation of vitamins in 3 distinct groups: polar, low-polar, and nonpolar. The method was applied to pharmaceutical preparations, fortified powdered drinks, and food samples, for which results were in good agreement with values claimed. Heudi et al. described a separation of 9 water-soluble vitamins by LC-UV. The method was applied for the quantification of vitamins in polyvitaminated premixes used for the fortification of infant nutrition products. The repeatability of the method was evaluated at different concentration levels and coefficients of variation were <6.5%. The concentrations of vitamins found in premixes with the method were comparable to the values declared. A disadvantage of the methods mentioned above is that sample composition has to be known in advance. According to European legislation, for example, foods might be fortified with riboflavin phosphate or thiamin phosphate, vitamers which are not included in the simultaneous separations described. Vitamin B2.--Viñas et al. elaborated an LC analysis of riboflavin vitamers in foods. Vitamin B2 can be found in nature as the free riboflavin, but in most biological materials it occurs predominantly in the form of 2 coenzymes, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD). Several methods usually involve the conversion of these coenzymes into free riboflavin before quantification of total riboflavin. According to the authors, there is growing interest to know flavin composition of foods. The described method separates the individual vitamers isocratically. Accuracy of the method is tested with 2 certified reference materials (CRMs). Vitamin B5.-Methods for the determination of vitamin B5 in foods are limited because of their low sensitivity and poor selectivity. Pakin et al. proposed a post-column derivatization of pantothenic acid as a fluorescent compound and used this principle in a specific and sensitive method for the determination of free and bound pantothenic acid in a large variety of foods. A French laboratory invited European laboratories to participate in a series of collaborative studies for this method, which will be carried out in 2005/2006. A more sophisticated method was described by Mittermayer et al. They developed an LC-mass spectrometry (LC/MS) method for the determination of vitamin B5 in a wide range of fortified food products. Application of the method to various samples showed consistent results with those obtained by microbiology. Vitamin B6.-Method 2004.07, an LC method for the analysis of vitamin B6 in reconstituted infant formula, was published by Mann et al. In contrast with this method, which quantifies vitamin B6 after converting the phosphorylated and free vitamers into pyridoxine, Viñas et al. published an LC method which determines 6 vitamin B6 related compounds, the 3 B6 vitamers, their corresponding phosphorylated esters, and a metabolite. Accuracy was determined using 2 CRMs. Results were within the certified ranges. Vitamin C.-Franke et al. described an extensive study to vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. Vitamin C was determined by measuring ascorbic acid in its reduced state by LC and coulometric detection along with UV absorbance detection at 245 nm. No attempts were made to assess levels of dehydroascorbic acid. Most recent research revealed that cell uptake of dehydroascorbic acid is unlikely to play a major role, which may explain the very low vitamin C activity of orally administered L-dehydroascorbic acid in rats. The food levels found by Franke et al. are variably lower, higher, or equal in comparison to other studies. Iwase described a method for the determination of ascorbic acid in foods using L-methionine for the pre-analysis sample stabilization. Electrochemical detection was used for the quantification. Traditionally, metaphosphoric acid was proven to be a useful dissolving agent for the determination of ascorbic acid. However, it dissolves in water very slowly, it is hygroscopic, and accurate weighing is not easy. Adjustment at pH 2-3 takes a long time. It appeared to be possible to replace metaphosphoric acid by 0.2% phosphoric acid. Methionine played an important role on the stability of ascorbic acid. The method seemed to be applicable to the routine analysis of ascorbic acid in foods. Folic Acid.-Microbiological analysis of total folate in foods is often considered as the golden standard compared to other methods based on, for example, LC. Koontz et al. showed results of total folate concentrations measured by microbiological assay in a variety of foods. Samples were submitted in a routine manner to experienced laboratories that regularly perform folate analysis fee-for-service basis in the United States. Each laboratory reported the use of a microbiological method similar to the AOAC Official Method for the determination of folic acid. Striking was, the use of 3 different pH extraction conditions by 4 laboratories. Only one laboratory reported using a tri-enzyme extraction. Results were evaluated. Results for folic acid fortified foods had considerably lower between-laboratory variation, 9-11%, versus >45% for other foods. Mean total folate ranged from 14 to 279 microg/100 g for a mixed vegetable reference material, from 5 to 70 microg/100 g for strawberries, and from 28 to 81 microg/100 g for wholemeal flour. One should realize a large variation in results, which might be caused by slight modifications in the microbiological analysis of total folate in foods or the analysis in various (unfortified) food matrixes. Furthermore, optimal combination of enzymes and reaction conditions may vary depending on the composition of the food. Padrangi and Laborde showed recently that treatment with alpha-amylase had no significant effect on measured folate in spinach, although addition of protease significantly increased the release of folate. LC/MS applications gain increasing attention because of their specificity. Rychlik used stable isotope dilution assays for the determination of the folate content of broccoli and bread. Compared to data in the literature and food data bases, amounts were significantly lower. Pawlosky et al., however, found comparable values for 5-methyltetrahydrofolic acid and folic acid by HPLC analysis with fluorescent detection and HPLC/MS. Among samples analyzed were CRMs and broccoli. Besides folic acid, other water-soluble vitamins were also determined by LC/MS/MS by Leporati et al. The method was applied to the quantitative analysis of the natural content of vitamins in typical Italian pasta samples, as well as in fortified pasta samples produced for the U.S. market. Biotin.-A paper from Staggs et al. included the assertion that existing biotin data in food composition tables are inaccurate because the majority are based on bioassays with all relevant disadvantages. Data in most cases are overestimated with consequences for recommendations for dietary biotin intake. An HPLC/avidin-binding assay was used to analyze 87 foods to support the hypothesis mentioned.