VII. ESTIMATION 249 



this colorimetric proceduie, obscTved that pyridoxamiiic and pyiidoxal were 

 about 31 % and 15 % as reactive, respect i\'ely, as pyridoxine on an eiiuimolar 

 basis. They state, therefore, that tiiis method can be used with precision 

 only wiien pyridoxine constitutes a niajoi' poition of the vitamin I^e content 

 of tiie material tested. W'Iumi applied to metabolically active tissues, like 

 yeast, wliicii ha\e laij2;e amounts of pyi'idoxa! and pyridoxamine, this 

 method can give only approximate \alu(\s. rn(h)ubte(lly, similar conclusions 

 may l)e drawn with respect to other chemical assa}' proce(hnes. 



Canbiick and Lindholm-^ have substituted 2,6-dibroino(iuinonechlori- 

 mide for the chloro compound and have reported good results. 



9. Cyanogen Bromide Test 



Sweeney and Hall-^^ have recently developed a chemical assay procedure 

 which is based on the Konig reaction, often used in the chemical assay for 

 niacin. The pja'idine ring is opened by cyanogen bromide and the resulting 

 product coupled with an aromatic amine to give a colored complex. Al- 

 though pyridoxine is a pyridine derivative, it reacts very little, if at all, 

 with the Konig reagent, for it has -an a-methjd group on the pyridine nu- 

 cleus. However, this methyl group can be removed by first converting it 

 to the carboxyl group and then heating the resulting a-carboxylic acid at 

 120 to 150°. The loss of CO2 results in the formation of a (compound which 

 then reacts with the Konig reagent. 



In this method, pj^-idoxine is remo\'ed from niacin and other interfering 

 substances by adsorption on zeolite at pH 7.6 and subsequent elution with 

 hot normal ammonium hydroxide. The eluate is evaporated to dryness 

 and oxidized, while being heated, with sulfuric acid and selenium. The 

 oxidized mixture is neutralized, treated with cyanogen bromide, and then 

 coupled with sulfanilic acid. The resulting color is read at 480 m/x. Excel- 

 lent recovery experiments (90 to 103%) have been reported. 



Pyridoxal gives the same test as pyridoxine, but pyridoxamine gives onh' 

 a faint color with this procedure. However, pyridoxamine can be converted 

 to pyridoxine with nitrous acid in an additional step. In this way, the 

 procedure affords a means not only for determining the total vitamin Be 

 complex but also for differentiating pyridoxamine from the other two mem- 

 bers of the group. 



C. BIOLOGICAL METHODS 



HENRY SHERM.'VN 



The vitamin Be activity of natural materials is measured most reliably by 

 animal assay. No preliminary hydrolytic, extractive, and concentrating 

 procedures are required to liberate the many bound forms of the \itamin 



''■' T. Callback and M. Lindholm, Finiska Kemistsamfundets Medd. 54, 134 (1945). 

 "» J. P. Sweeney and W. L. Hall, J. Assoc. Offic. Agr. Chemisls 35, 479 (1952). 



