VII. ESTIMATION 363 



appears obvious. If only free riboflavin is present (pharmaceuticals), no 

 special extraction should be necessary. The method of Scott et al.^ utilizes 

 acid-acetone as the extraction medium. Acid methanol has also l)een used.^ 



Many workers find an acid extraction alone sufficient,^-'" yet Watts 

 et a/." believe that some of the apparent losses in meats on storage may 

 represent simply insufficient extraction, and the use of acid extraction fol- 

 lowed by enzymatic hydrolysis is common practice. 



(2) Purijication. The procedure in almost universal use is a brief per- 

 manganate oxidation introduced originally l)y Koschara'^ to remove inter- 

 fering pigments. Excess permanganate is removed by H2O2 , usually within 

 2 minutes. However, Klatzien et alP recently reported the production of 

 a non-specific l^lue fluorescence by this oxidation of certain materials. 

 Leviton''* found that in the presence of appreciable amounts of ferrous iron, 

 riboflavin was readily destroj^ed bj^ H2O2 oxidation. Hence, even the use 

 of this common procedure is subject to certain limitations. 



Several methods employ the adsorption of riboflavin on fuller's earth, ^^ 

 such as Florisil used by Conner and Straub,^^ followed by elution with pyri- 

 dine-acetic acid solution. Hoffer et al}'' indicated some possible limitations 

 to this procedure. Often the values for the same material decrease as the 

 size of the sample is increased. It was thought that this represented losses 

 (incomplete adsorption or elution) on the column or possibly adsorption 

 of riboflavin on residues. These authors also reported that the native ribo- 

 flavin in wheat flour extracts is less readily adsorbed than pure riboflavin. 

 In general, the methods which utilize the Florisil adsorption also utilize 

 permanganate oxidation either before or after the adsorption and elution. 

 Whereas permanganate oxidation after the elution is much more effective 

 in decreasing the pigmentation, the non-specific pigment said to be pro- 

 duced by permanganate oxidation in some products'* maj^ be separated 

 from riboflavin by adsorption on Florisil. 



The original procedure of Hodson and Norris'^ and the more recent modi- 

 fication by Rubin et al}^ includes a reduction of riboflavin and other pig- 



8M. L. Scott, F. W. Hill, L. C. Norris, and G. F. Heuser, /. Biol. Chem. 165, 65 



(1946). 

 » A. P. Jansen, Rec. Irav. chim. 69, 1275 (1950). 

 '« D. B. Morell, Australian. J. Exptl. Biol. Med. Sc. 25, 251 (1947). 

 " B. M. Watts, D. H. Peng, and N. C. Esselbaugh, J. Biol. Chem. 172, 707 (1948). 

 »' W. Koschara, Z. physiol. Chem. 232, 101 (1935). 



'' C. Klatzien, F. W. Norris, and F. Wokes, /. Pharm. and Pharmacol. 1, 915 (1949). 

 " A. Leviton, /. Am. Chem. Soc. 68, 835 (1946). 

 15 J. W. Ferrebee, /. Clin. Invest. 19, 251 (1940). 



'« R. T. Conner and G. J. Straub, Ind. Eng. Chem. Anal. Ed. 13, 385 (1941). 

 " A. Hoffer, A. W. Alcock, and W. F. Geddes, Cereal Cham. 21, 515 (1944). 

 " A. Z. Hodson and L. C. Norris, /. Biol. Chem. 131, 621 (1939). 

 " S. H. Rubin, E. De Ritter, R. H. Schuman, and J. C. Bauernfeind,/nrf. Eng. Chem. 



Anal. Ed. 17, 136.(1945). 



