Riboflavin assay methods

Three recent reviews specifically cover HPLC methods for quantitating riboflavin in foods. In addition to HPLC methods, Nielsen (81) summarized paper chromatography, TLC, and open-column chromatography procedures for quantitating total riboflavin and the individual vitamers in foods, pharmaceuticals, and biological samples. Russell (44) included a brief discussion of the standard methods, along with HPLC and flow injection analyses published between 1990 and 1994 for total riboflavin and the individual vitamers in foods. Ball (45) reviewed HPLC methods for quantitation of riboflavin, as well as chemical and microbiological riboflavin assays for foods. 

The use of solid-phase extraction cartridges is now well established in the analysis of clinical specimens. However, although this method provides efficient purification of the sample, it may lead to a loss of protein-bound vitamins. Direct injection of plasma samples into liquid chromatography (LC) columns is possible in some applications. Dilute filtered or centrifuged urine can be injected in certain LC applications, as is the case in urinary riboflavin assay. 

Shah J., Riboflavin, in Methods of Vitamin Assay, 4th ed. Augustin, J., et al., Eds., John Wiley Sons, New York, 1984. 

Snell and Strong (363, 364) developed the growth response of Lactobacillus casei to riboflavin as an assay method, and it was soon used, for example by Fraser, Topping, and Isbell (102) to assay riboflavin in the urine and tissues of normal and riboflavin-deficient dogs. The microbiological riboflavin assay has since been widely used e.g., 394). 

Riboflavin can be assayed by chemical, en2ymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of intense yeUow-green fluorescence with a maximum at 565 nm in neutral aqueous solutions. The fluorometric deterrninations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the irradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

The microbial assay is based on the growth of l ctobacillus casei in the natural (72) or modified form. The lactic acid formed is titrated or, preferably, the turbidity measured photometrically. In a more sensitive assay, l euconostoc mesenteroides is employed as the assay organism (73). It is 50 times more sensitive than T. casei for assaying riboflavin and its analogues (0.1 ng/mL vs 20 ng/mL for T. casei). A very useful method for measuring total riboflavin in body fluids and tissues is based on the riboflavin requirement of the proto2oan cHate Tetrahjmenapyriformis which is sensitive and specific for riboflavin. 

Although riboflavin can be assayed more readily by chemical or microbiological methods than by animal methods, the latter are preferred for nutritional studies and as the basis of other techniques. Such assays depend upon a growth response the rat or chick is the preferred experimental animal. This method is particularly useful for assaying riboflavin derivatives, since the substituents frequently reduce or eliminate the biological activity. 

The recognition of their structure permits the determination of vitamins by the tools of analytical chemistry, but while such methods are widely used in industrial production, the minute quantities in body fluids and tissues limit the purely chemical approach to a few members of this group present in relatively high concentration, e.g., vitamin C (K5). Microchemical methods are in use for the determination of thiamine, riboflavin, and some of the fat-soluble vitamins, based on the most sensitive colorimetric and, in particular, fluorometric techniques. Vitamin D, on the other hand, is determined by animal assay. 

Kawasaki (68) briefly reviewed HPLC methods for determining total thiamine alone and in combination with riboflavin. Russell (44) provided a more detailed summary of HPLC methods, published between 1990 and 1994, for thiamine alone and in conjunction with other vitamins. Ball (45) reviewed selected HPLC analyses for thiamine in various foods, as well as other chemical and microbiological assays. 

Photolysis of riboflavin leads to the formation of lumiflavin in alkaline solution and lumichrome in acidic or neutral solution (see Figure 7.2). Because lumiflavin is chloroform extractable, photolysis in alkaline solution, followed by chloroform extraction and fluorimetric determination, is the basis of commonly used chemical methods of assaying riboflavin. The photolysis proceeds by way of intermediate formation of cytotoxic riboflavin radicals, and the addition of riboflavin and exposure to light has been suggested as a means of inactivating vimses and bacteria in blood products (Goodrich, 2000). 

Flavins are lost from the body as intael riboflavin, rather than as a breakdown product of riboflavin. Hence, vitamin status may be assessed by measuring the level of urinary riboflavin. Generally, the loss of 30 ig of riboflavin/g creatinine or less per day indicates a deficiency. This metht>d of assessment is not preferred because it is influenced by a number of factors unrelated to vitamin status. Another problem with this method is its great sensitivity to a short-term deficiency thus, it does not necessarily reflect the true concentrations of FAD and FMN in tissues. The most reliable way to assess riboflavin status is by a functional test. The test involves the assay of glutathione reductase, using red blood cells as the source of... 

Riboflavin status is assessed by (1) determination of urine riboflavin excretion, (2) a functional assay using the activation coefficient of stimulation of the enzyme glutathione reductase by FAD, or (3) direct measurement of riboflavin or its metabolites in plasma or erythrocytes. The advantages and disadvantages of functional or direct methods have been discussed in the section on thiamine. 

Rapid freeze epr can be used as a direct assay of superoxide dismutase, as described in the earlier work by Ballou et al., (1969) who studied the effect of superoxide dismutase on the decay of the signal of O2. Superoxide was trapped by rapid freezing during the reaction with oxygen of anaerobically reduced tetraacetyl riboflavin. The O2 available for reaction with superoxide dismutase was ca. 10 M, and under these experimental conditions, it was possible to estimate that the turnover rate number of superoxide dismutase was at least 3 X 10 min . As a consequence of the difficulty inherent in the method, rapid freeze epr has not resulted in routine assay of superoxide dismutase. It has been used in a different approach for mechanistic studies (Fielden et al., 1974). In this case O2 was generated by pulse radiolysis, and the valence state of the enzyme estimated by epr. 

The nitroblue tetrazolium assay (111) is another indirect method that is used especially for detecting SOD activity on gel electrophoresis. Superoxide radicals are generated by xanthine/xanthine oxidase or by the photoreduction of flavins (typically riboflavin), which oxidize H2O to O2. The gel on which SOD samples have been loaded is then stained with nitroblue tetrazolium chloride. This reagent is reduced by superoxide to the blue-colored formazan. SOD competes with nitroblue tetrazolium and produces colorless zones on the blue gels. This method, which is highly speciflc toward superoxide dismutase, is limited by its low reliability with respect to quantitative determinations. 

Several B vitamins, including folic acid, niacin, pyridoxine, and pantothenic acid, are routinely determined using microbiological assays, details of which can be found in the AOAC Official Methods of Analysis. Standard methods for thiamine determination using fluorimetric detection are also detailed in the AOAC methods in addition, LC techniques are now being used routinely for thiamine and other B vitamins, e.g., riboflavin. 

Coverage includes B vitamins and folate in the context of a historical background, disease, cardiovascular effects and the importance of vitamins in biochemistry as illustrated by a single vitamin. Thereafter there are chapters on the chemistry and biochemistry of thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folate and cobalamin. Methodical aspects include characterization and assays of B vitamins and folate in foods of all kinds, dietary supplements, biological fluids and tissues. The techniques cover solid-phase extraction, spectrofluorimetry, mass spectrometry, HPLC, enzymatic assay, biosensor and chemiluminescence. In terms of fimction and effects or... 

A nonchromatographic method for the sensitive determination of riboflavin in human plasma and urine by enzyme-linked ligand-sorbent assay (ELLSA) was recently described (35). Using standard microtiter plate formate, a conjugate of flavin with bovine serum albumin is immobilized on a plastic surface. The RF... 

Validity. The HPLC results for the riboflavin content in foods have been found to agree well with those obtained by the Association of Official Analytical Chemists (AOAC) microbiological assay and fluorimetric assay. However, lower values for the HPLC method compared to the AOAC method have also been reported in some foods. This might indicate the presence of interfering material in the AOAC fluorimetric assay and the higher selectivity of the HPLC method. 

S Harbron, HJ Eggelte, BR Rabin. Amplified colorimetric assay of alkaline phosphatase using riboflavin 4 -phosphate a simple method for measuring riboflavin and riboflavin 5 -phosphate. Anal Biochem 198 47-51, 1991. 

Although the growth of rats and chicks may occasionally be used to assay riboflavin in mixed diets, the biologic method of assay has been generally superseded by microbiological and chemical methods. 

Reyes, E. and Subryan, L., An improved method of simultaneous HPLC assay of riboflavin and thiamin in selected cereal products, J. Food Comp. Anal, 2, 41 7, 1989. 

Although the measurement of the fluorescence of riboflavine solutions at known pH values has been widely used for assay and forms the basis of the U.S.P. method, riboflavine is best determined in simple formulations by extraction and measurement of the extinction at 444 m. ... 

Capsules of Vitamins, J5.P.C., may be assayed for riboflavine either by the microbiological method given below for dried yeast using 5 capsules or by the method of Brealey and Elvidge. ... 

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