246 PYRIDOXINE AND RELATED COMPOUNDS 



color which can be measured. This method did not gain wide acceptance due 

 to its lack of specificity. 



5. DiAZOTIZED SULFANILIC AciD TeST 



Swaminathan'" used the color reaction between pyridoxine and diazo- 

 tized sulfanilic acid as the basis for its estimation. This reaction is not spe- 

 cific for pyridoxine; complete removal of interfering substances prior to 

 the coupling reaction is essential. The procedure employed to free extracts 

 of interfering substances is long and difficult to perform. Protein and 

 protein derivatives resulting from a pepsin digestion are removed by phos- 

 photungstic acid, and purines, pyrimidines, and imadizole bases are re- 

 moved by a silver nitrate and barium hydroxide treatment. The pyridoxine 

 is then adsorbed on Clarite from a filtrate adjusted to pH 1 to 2, eluted 

 with hot barium hydroxide, adjusted to pH 6, and then coupled with dia- 

 zotized sulfanilic acid. This method has been applied to many foods and 

 to urine samples with excellent results. ^'^^ ^* 



Bina et al}^ improved the chemical procedure with diazotized sulfanilic 

 acid. They adsorbed pyridoxine on Superfiltrol at pH 3 and then used alka- 

 line ethyl alcohol as the selective eluant; this not only aided and simplified 

 the purification process but also served to stabilize the color which, in the 

 Swaminathan procedure, started to fade after 5 minutes. 



Ormsby et al}^ observed that the three members of the vitamin Be com- 

 plex gave different colors when treated with diazotized sulfanilic acid. Pyri- 

 doxine gives an orange color, pyridoxamine an orange to pink color, and 

 pyridoxal a bright yellow color. Although the stability of the colors was 

 limited, absorption spectra could be obtained. It is evident, therefore, that 

 foodstuffs containing more than one member of the Be group can produce 

 a color with diazotized sulfanilic acid which may be difficult to match with 

 standards prepared from pyridoxine. This suggests why individual colori- 

 metric tests are not very accurate when applied to natural materials. 



6. Diazotized p-Aminoacetophenone Test 



Brown et alP introduced this new diazo reaction for pyridoxine; it had 

 first been used for the determination of thiamine.^'* Although the color pro- 

 duced is not as stable as that developed Avith diazotized sulfanilic acid, its 

 sensitivity and specificity are much better. This increased sensitivity is 



12 M. Swaminathan, Nature 145, 780 (1940). 



i» M. Swaminathan, Indian J . Med. Research 28, 427 (1940). 



" M. Swaminathan, Indian J . Med. Research 29, 561 (1941). 



" A. F. Bina, J. M. Thomas, and E. B. Brown, J. Biol. Chem. 148, 111 (1943). 



16 A. A. Ormsby, A.. P'isher, and F. Schlenk, Arch. Biochem. 12, 7!) (1917). 



17 E. B. Brown, A. F. Bina, and J. M. Thomas, /. Biol. Chew. 158, 455 (1945). 



18 H. J. Prebluda and E. V. McCollum, J. Biol. Chem. 127, 495 (1939). 



