Total riboflavin

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. 

All of the B2 vitamers are unstable at alkaline pH the coenzymes are also degraded below pH 5.0 (80 - 82). As a result, analysis of the endogenous coenzymes needs to be carried out at pH 5.0-7.0, while total riboflavin (as riboflavin) is best determined at acidic pH. Riboflavin is soluble in water and short-chain alcohols, but insoluble in ether, chloroform, and acetone. The coenzymes are water soluble. 

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. 

A recent interlaboratory comparison of HPLC and microbiological methods for total riboflavin revealed significant variability between the 13 participating laboratories (42). The extraction and hydrolysis of the riboflavin coenzymes were cited as the most likely sources of this variability. A later intercomparison (70) of riboflavin methods showed lower variability between laboratories, although coefficients of variability (CV) of 12-40% were still reported. 

The HPLC methods published from 1992 to 1997 for total riboflavin and the individual vitamers are summarized in Tables 11-13. The simultaneous quantitation of riboflavin with one or more other vitamins is covered in Sec. XI of this chapter. 

Table 11 HPLC Methods for Quantitating Total Riboflavin in Foods (C18 Columns UV Absorbance Detection)... 

Total riboflavin Bread Acid hydrolysis with 0.1 N Analytical Bon-... 

Total riboflavin Casein Enzymatic hydrolysis with Precolumn ODS... 

As shown in Table 7.1, the total riboflavin concentration in plasma is very much lower than in most tissues. About 50% of plasma riboflavin is free riboflavin, which is the main transport form, with 44% as FAD and the remainder as riboflavin phosphate. The vitamin is largely protein bound in plasma free riboflavin binds to both albumin and a- and /3-globulins, and both riboflavin and the coenzymes also bind to immunoglobulins. The products of photolysis of riboflavin bind to albumin with considerably higher affinity than riboflavin itself this albumin binding may represent a mechanism to prevent tissue... 

A study conducted by the same authors (Scliaus el al., 1955) on 100 samples of the thoracic descending aorta obtained from subjects ranging in age between 1 month and 83 years showed a definite decrease (Fig. 3) in the total riboflavin concentration of this tissue with age (r = —0.39 t = 4.20). 

Vitamin B2 Food contains three B2 vitamers, riboflavin and its two coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, which are the predominant vitamers in foods and are usually bound to proteins. Their analysis usually takes place after extraction with dilute mineral acids with or without enzymatic hydrolysis of the coenzymes (which is necessary to convert all forms to riboflavin and to quantify them as total riboflavin). The extracts may be purified using SPE with Cig cartridges. All the operations performed prior to analysis need to be done under subdued lighting to avoid decomposition of riboflavin upon exposure to light. RP chromatography with Cig columns is used along with fluorescence detection (excitation, 440 nm emission, 520 nm). 

Several difficulties arise when analyzing riboflavin in food. Total riboflavin exists in food in several forms, from which the most representative are... 

Ollilainen, V., Mattila, P., Varo, P., Koivistoinen, P., and Huttunen, J., 1990. The HPLC determination of total riboflavin in foods. Journal of Micronutrient Analysis. 8 199-207. 

For the uptake of riboflavin by brain cells, it is most important to maintain homeostasis. The concentration of riboflavin in the brain remains constant, both at times of riboflavin deficiency and after massive intravenous applications. In rat brains, more than 90% of total riboflavin is found as FAD and FMN (Ball 2004b). 

Free riboflavin appears to be present to a significant extent in the retina of certain animals and fishes, in milk, and in the urine. The findings of Bessey, Lowry, and Love indicate that riboflavin, like the other B vitamins, occurs mostly in bound forms, FAD being the major constituent. The FAD content varies from 90% of the total riboflavin content of the muscle to 70% in the kidney. The remainder occurs mainly as FMN, with free riboflavin being present in only trace amounts. 

Vails, F., Sancho, M.T., Fernandez Muino, M.A., and Checa, M.A., Determination of total riboflavin in cooked sausages, J. Agric. Food Chem., 47, 1067-1070, 1999. 

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