VII. ESTIMATION 245 



plied to the deteimiimtioii of pyridoxine. The test depends upon the reac- 

 tivity of the a-melhyl <i;roups. The vitamin must first he comerted into the 

 methyl ether by diazomethane and then into a (luaternary pyridine com- 

 pound hy methyl iodide or dimethylsulfate. The resulting salt, upon heat 

 treatment with a solution of sodium in ethyl alcohol and then with chloro- 

 form, produces a violet color, which has al)sorption maxima at 599 and 555 

 m/i- l^y this color test, 0.1 mg. of pyridoxine can be detected. It has not 

 l)een used extensively in food analysis because of the difficulty encountered 

 in the preparation of the phenolic ether in (luantitative yields. 



2. Phthaleins^ 



Stiller and his associates oxidized pyridoxine to the 4 , 5-dicarbo.xylic acid 

 and then fused the resulting acid with resorcinol to obtain the correspond- 

 ing phthalein, which had a greenish-j^ellow fluorescence. However, this 

 method is not readily adaptable to assay procedure because of its lack of 

 specificity. It can be used, however, to measure pyridoxine in pure solutions. 



The most widely used color reactions depend upon the phenolic nature 

 of the vitamin Be group. One series of tests invoh'es the active participa- 

 tion of the phenolic hydroxyl group. Another series of tests depends upon 

 the coupling reaction which is common to most phenolic compounds. Each 

 of the three acti\e forms of the vitamin Be group has a phenolic group in 

 the 3 position and an unsubstituted position para to this phenolic group. 

 These two factors favor the condensation of the vitamin with chromogenic 

 reagents to form dyes which can be measured colorimetricall}^ ; this funda- 

 mental reaction is the basis of the color tests that have been applied most 

 successfully to the chemical assay of the vitamin Be complex. 



3. Ferric Chloride Method 



When pyridoxine is reacted with a 10% aqueous solution of FeCls, a 

 red-brown color is formed, indicating the presence of the phenolic hydroxyl 

 group.* This reaction has been used to estimate pyridoxine in more highly 

 refined concentrates, by comparing the color developed in the unknown 

 sample with that of standards.^ Pharmaceutical preparations containing 

 only pyridoxine of the vitamin Be group are especially amenable to such a 

 test; other members of the B vitamin group do not interfere.^'* No literature 

 is available on the reactions between pyridoxal or pyridoxamine with FeCls. 



4. Phenol Test with Folin-Denis Reagent^^ 



Pyridoxine, like most phenols, reacts with the Folin-Denis reagent (a 

 mixture of phosphotungstic and phosphomolybdic acids) to form a blue 



9 R. D. Greene, J. Biol. Chem. 130, 513 (1939). 

 »• G. G. Villela, Anais assoc. quim. Brasil 7, 168 (1948). 

 " O. Folin and W. Denis, J. Biol. Chem. 22, 305 (1915). 



