VII. ESTIMATION 535 



However, the large number of modifications of this basic method which 

 have appeared in the past twelve years attests to the fact that the proce- 

 dure is not so simple as it seems when applied to a variety of biological 

 materials. Difficulties are encountered in hydrolysis in obtaining complete 

 liberation and maximum conversion of nicotinamide to nicotinic acid with- 

 out formation of interfering chromogens and without conversion of tri- 

 gonelline. The choice of reaction temperatures and proper pH, the choice 

 of the proper aromatic amine to yield the maximum stable color, and proper 

 blank corrections are important factors. Many of these difficulties have been 

 discussed and examined in detail in other publications.^' ^ Gyorgy and 

 Rubin^ present detailed instructions for three methods: (1) a procedure 

 using metol, devised by Perlzweig et al}^ and improved by Dann and 

 Handler," which has been used extensively in examining animal tissues; 

 (2) the aniline procedure of Melnick,^- which has been applied extensively 

 to cereals; and (3) a p-aminoacetophenone (or p-aminopropiophenone) pro- 

 cedure developed by a British group.'^- '* Dennis and Rees^^ have recently 

 proposed a method which uses procaine hydrochloride for color develop- 

 ment. The Association of Vitamin Chemists'^ has adopted two carefully 

 checked chemical methods, one of which is essentially the method of Friede- 

 mann and Frazier.^ A new method has recently been proposed for adoption 

 by the Association of Official Agricultural Chemists.'^' '* The method uti- 

 lizes sulfanilic acid or Tobias acid (2-naphthylamine-l -sulfonic acid) for 

 color development and permits both a quantitative and a qualitative dif- 

 ferentiation of nicotinic acid and nicotinamide. 



2. Differential Assay of Nicotinic Acid, Nicotinamide, and Related 



Compounds 



These compounds may be differentially quantitated either by micro- 

 biological (p. 538) or by chemical methods. In addition to the method of 

 Sweeney and Hall, mentioned above, '^' '^ paper chromatography has proved 

 useful in separating and determining small quantities of these sub- 

 stances. ^^'^^ 



8 P. Gyorgy and S. H. Rubin in Vitamin Methods, Vol. I, pp. 223-239. Academic 

 Press, New York, 1950. 



9 T. E. Friedemann and E. I. Frazier, Arch. Biochem. 26, 361 (1950). 



10 W. A. Perlzweig, E. D. Levy, and H. P. Sarett, J. Biol. Chem. 136, 729 (1940). 



" W. J. Dann and P. Handler, /. Biol. Chem. 140, 201 (1941). 



'2D. Melnick, Cereal Chem. 19, 553 (1942). 



'3 E. M. James, F. W. Norris, and F. Wokes, Analyst 72, 327 (1947). 



'< C. Klatzkin, F. W. Norris, and F. Wokes, Anabjst 74, 447 (1949). 



'5 P. O. Dennis and H. G. Rees, Analyst 74, 481 (1949). 



'^ Association of Vitamin Chemists, Methods of Vitamin Assay, 2nd ed., pp. 184-203. 



Interscience Publishers, New York, 1951. 

 " J. P. Sweeney, J. Assoc. Offic. Agr. Chemists 34, 380 (1951). 

 '8 J. P. Sweeney and W. L. Hall, Anal. Chem. 23, 983 (1951). 



