248 PYRIDOXINE AND RELATED COMPOUNDS 



tion depend for their accuracy upon the complete removal of interfering- 

 substances prior to the formation of the color complex. 



In this procedure, Scudi^^ used a two-phase water-butanol solution to aid 

 in the removal of interfering substances at pH 6.8 to 7.2. However, the 

 neutralized solution was then weakly buffered with veronal prior to coloi 

 development. No provision was made to control interfering compounds 

 like salts and bases. A borate buffer blank was introduced to correct for 

 compounds which would react with the chlorimide reagent, but the results 

 were not very ciuantitative. Pyridoxine forms a complex with boric acid, 

 which does not give the indophenol salt. It was originally thought that the 

 difference in values obtained in veronal and borate buffers would represent 

 vitamin Be-active materials, but with the discovery of pyridoxal and pyri- 

 doxamine the borate buffer blank lost its original purpose. Pyridoxal and 

 pyridoxamine react with the chlorimide reagent to form a blue pigment 

 even in the presence of borate. Therefore, when all three compounds are 

 present in natural materials, the total blue color formed with the chlori- 

 mide reagent is equal to the sum of the members of the vitamin Be group. 

 The difference, in the presence and absence of borate, represents the color 

 contribution of pyridoxine alone, and not the vitamin Be group. 



Bird et al.-^ modified the Scudi colorimetric method so that it was simpler 

 and more rapid. Pyridoxine was adsorbed on Superfiltrol at pH 3.0, washed, 

 and then eluted by adding a butanol solution of 2 , 6-dichloroquinonechlori- 

 mide to the Superfiltrol. Veronal buffer was then added, so that the pH 

 was raised to 7.8 to 8.0 and the characteristic blue color then developed. 

 Elution and color development were thus carried out simultaneously. 



Hochberg et alr^ developed a method which was based on the coupling 

 of pyridoxine and the chlorimide reagent in isopropanol, a one-phase sys- 

 tem. The use of a strong NH4OH-NH4CI buffer of high basicity and salinity 

 eliminated the interference caused by different kinds and amounts of bases 

 and salts in the test solution. The color development reached a maximum 

 in 60 seconds and was three times as sensitive as other tests; similar tests 

 required 20 to 40 minutes for complete color development. The use of an 

 internal standard eliminated the influence of other compounds upon the 

 rate, extent, and stability of color formation. A borate blank made the re- 

 action fairly specific for pyridoxine. When this method was applied to bio- 

 logical materials, the pyridoxine was first adsorbed on Lloyd's reagent, 

 eluted, and then submitted to the coupling reaction.-^ Bottomley-^ has sug- 

 gested that products which are high in fat content be subjected to an ether 

 extraction prior to isolation and coupling. Melnick and coworkers,^ using 



" o; D. Bird, J. M. Vandenbelt, and A. D. Emmett, /. Biol. Chem. 142, 317 (1942). 

 26 M. Hochberg, D. Melnick, and B. L. Oser, J. Biol. Chem. 155, 109 (1944). 

 " M. Hochberg, D. Melnick, and B. L. Oser, ./. Biol. Chew. 155, 119 (1944). 

 28 A. C. Bottomlcy, Biochem. J. 38, V (1944). 



