VII. ESTIMATlOxM 305 



"(luenching" of the fluorescence of riboflavin by various materials in the 

 extract.'' ' The cHsadvantagc of the method is that the time and work are 

 doubled antl errors in the recovery sample are recorded in the unknown 

 also. As indicated above, the recovery from the Florisil column may be 

 critically related to the amount of riboflavin in the sample. Many workers 

 compromise (see Stiller') and effect considerably saving in time by reading 

 the sample, adding a known amount of riboflavin, and taking another read- 

 ing. After dilution is accounted for, the increment in fluorometer reading 

 is used as a measure of the fluorescence of riboflavin in that extract and the 

 content in the sample is calculated from it. 



Essentially all workers have come to use one or the other of the internal 

 standards mentioned above, since the fluorometer readmg is influenced not 

 only by the amount of riboflavin but by the other materials in the extract. 

 The increment in reading caused by a constant amount of riboflavin will 

 generally fall in about the same range, however, and samples which show 

 widely divergent values should be view^ed with suspicion. 



(5) Blank Readings. Since most biological materials do not yield extracts 

 which contain only riboflavin as the fluorescent material, blank determina- 

 tions for each sample are ordinarily required and simple reagent blanks do 

 not suffice. Reagents must be sufficiently purified to yield low fluorescent 

 extracts, of course. The procedure which is common to most methods^ 

 is the reduction of riboflavin to the leuco form \Wth sodium hydrosulfite, 

 assuming that only riboflavin is reduced and that no fluorescent materials 

 are produced in the reduction. Others recommend strong alkali^'*- -^ or 

 acid-^ to suppress the riboflavin fluorescence, although these have not been 

 widely used as yet. Another, and probably more specific, method is the 

 destruction of riboflavin by irradiation with artificial light or sunlight. 

 After an extensive comparative study of seven methods for the determina- 

 tion of riboflavin in urine, Morell and Slatter-^ find serious fault with the 

 hydrosulfite blank. They conclude: "Methods using the hydrosulfite blanks, 

 even those involving adsorption on Florisil, give high answers because they 

 include 'apparent riboflavin,' i.e., compounds other than riboflavin which 

 fluoresce in the same spectral region and the fluorescence of which is de- 

 stroyed by hydrosulfite. 



"Urine contains not only 'apparent riboflavin' but also precursors of 

 'apparent riboflavin' which are changed to 'apparent riboflavin' by Na2S204- 

 SnClo reduction, K]\In04 oxidation, or during adsorption on Florisil." The 

 authors feel that their method utilizing a blank obtained by sunhght irradia- 



" H. Kahler and E. P. Davis, Proc. Soc. Exptl. Biol. Med. 44, 604 (1940). 

 " B. A. McLaren, S. Cover, and P. B. Pearson, Arch. Biochem. 4, 1 (1944). 

 ^^ A. Gourevitch, Bull. soc. chim. biol. 30, 711 (1948). 

 " D. B. Morell and E. C. Slater, Biochem. J. 40, 652 (1946). 



