RIBOFLAVINE 



W. S. Jones and W. Cluustiani.en ^^ Ubcd a similar method, in which, 

 however, the reduction and re-oxidation steps were omitted ; they 

 employed a fluorophotometer for measuring the fluorescence. 



G. E. Shaw ^^ used another modification of the fluorimetric method. 

 The alkaUne riboflavine solution was irradiated and extracted with 

 chloroform, and the fluorescence of the chloroform extract, which 

 contained the lumiflavine, was then compared with that of a standard 

 solution. A similar method had previously been used, though not 

 very successfully, by Warburg and Christian. A. R. Kemmerer ^^ 

 found that the fluorimetric method of Hodson and Norris and the 

 microbiological method of Snell and Strong gave more reliable results 

 than a colorimetric method in which a methanolic solution containing 

 acetic acid was treated with potassium permanganate and then with 

 hydrogen peroxide. 



A. E. Schumacher and G. F. Heuser ^3 adopted a modification of 

 the Hodson and Norris method, in which the fluorescence was measured 

 after reduction with sodium dithionite solution and again after re- 

 oxidation by air ; the increase in the intensity of the fluorescence 

 was proportional to the riboflavine concentration. The results were 

 in good agreement with those obtained by biological assay, using 

 chicks or rats. Other workers ^*» ^^' ^^ also reported good agreement 

 between the two types of assay, although the fluorimetric method 

 gave unsatisfactory results with skeletal muscle, a blue fluorescent 

 substance being obtained in the digest together with the riboflavine. 



A somewhat different modification was used by Rubin et al.^"^ In 

 this method, the material was extracted in a Waring blendor, digested 

 with clarase at pH 4-5, treated with potassium permanganate solution 

 at the same pK and then twice reduced with sodium dithionite at 

 pH 4-5, instead of at pB. 7-0 to 7-5, and re-oxidised. Good agreement 

 with the microbiological method of assay was obtained. 



The fluorimetric method was applied to urine by V. A. Najjar,^^ 

 whose method has been adopted, with minor modifications, by other 

 workers. The urine was acidified with acetic acid and saturated with 

 sodium sulphate. The riboflavine was then extracted with pyridine- 

 Dutanol, and interfering substances were destroyed by oxidation with 

 potassium permanganate. After decomposing the excess of the latter 

 by treatment with hydrogen peroxide, the fluorescence was measured, 

 and the riboflavine content calculated from a standard curve. Urines 

 low in riboflavine were first treated with lead sulphide to adsorb the 

 riboflavine, which was subsequently eluted with a mixture of water, 

 pyridine and acetic acid. 



E. C. Barton-Wright and R. G. Booth ^^ adopted Najjar's method 

 for the assay of cereals, but found Super-filtrol to be more satisfactory 

 than lead sulphide for adsorption of the vitamin. M. Swaminathan *® 



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