ESTIMATION 



freed from benzene, the residue was fused with 2 : 4-dinitro-i-chloro- 

 benzene and the melt dissolved in ether and the solution extracted 

 with water. The intensity of the colour formed on adding one or two 

 drops of potassium hydroxide solution was found to be proportional to 

 the nicotinic acid content. Substantially the same method was 

 used by Vilter et al.^ for the estimation of nicotinic acid and nicotin- 

 amide in urine. Urine contains other pyridine compounds in addition 

 to these two substances, however, and, although trigonelline does not 

 give a colour with dinitrochlorobenzene, nicotinuric acid, the con- 

 jugated compound of nicotinic acid with glycine interferes with the 

 estimation, giving high results. 



Chemical Methods based on the Konig Reaction 



The Vongerichten reaction proved to be rather inconvenient for 

 analytical purposes, and the majority of workers have made use of 

 the Konig reaction,® in which the pyridine compound is warmed with 

 cyanogen bromide solution, and an organic base is then added ; a 

 yellow colour is formed, the intensity of which is proportional to the 

 amount of pyridine compound present. The method was first applied 

 to the estimation of nicotinic acid in foodstuffs by M. Swaminathan,'' 

 who used aniline as the base ; this was also used by H. Kringstad and 

 T. Naess.^ E. Bandier and J. Hald ^ used metol (^-methylamino- 

 phenol sulphate) as the organic base in place of aniline, and found 

 that nicotinamide gave a stronger colour than nicotinic acid, so that 

 it was necessary to hydrolyse the amide to the acid before applying 

 the reaction. E. Bandier ^^ also used the method for the estimation 

 of nicotinic acid in urine but, like Vilter et a/., ^encountered difficulties 

 due to the presence of other pyridine compounds (page 252). These 

 were overcome by hydrolysis with alkali in order to convert all the 

 derivatives of nicotinic acid into the free acid, the values thus obtained 

 being a measure of the total (free and combined) nicotinic acid in the 

 urine. 



Von Euler et al.^'^ used /S-naphthylamine as the base, whilst L. J. 

 Harris and W. D. Raymond ^^ worked out a method of estimating 

 nicotinic acid in urine, using _/)-aminoacetophenone. This method 

 was modified by E. Kodicek,^^ who stressed the importance of adhering 

 rigidly to the detailed conditions laid down, if satisfactory results 

 were to be obtained. Like Bandier, Kodicek used alkaline hydrolysis 

 of biological materials to convert nicotinamide and other derivatives 

 into nicotinic acid ; impurities were then removed by precipitation 

 with alcohol. W. S. Jones ^* used substantially the same method in 

 conjunction with a fiuorophotometer. 



Hydrolysis with alkali, however, did not completely eliminate the 



219 



