ANEURINE (thiamine) 



reagent, and the latter by oxidation. Cocarboxylase gave no colour 

 and could be estimated separately after hydrolysis with phosphatase. 

 B. Alexander and J. E. Levi ^^ found that uric acid and vitamin C 

 interfered with the estimation of aneurine by means of the Prebluda- 

 McCollum reagent ; they removed uric acid by precipitation with 

 zinc at pa 7-4 and vitamin C by precipitation with lead acetate at 

 pU 8-2. 



Thiochrome Method 



The other reaction on which is based a chemical test for aneurine 

 was also discovered by R. A. Peters.^^ He observed that when 

 aneurine was oxidised with potassium permanganate or manganese 

 dioxide at a pK not exceeding 6, a blue fluorescent substance was 

 produced. G. Barger et al.^* prepared this fluorescent substance by 

 oxidation of aneurine with potassium ferricyanide solution and 

 obtained it in the pure state as pale yellow crystals having the formula, 

 C12H14N4OS . 2HCI. It showed an intense blue fluorescence in 

 neutral or alkaline solution and had all the other properties of thio- 

 chrome described by R. Kuhn et al.^^ Its constitution was established 

 and its synthesis worked out by Bergel et al.'^^ 



The conversion of aneurine into thiochrome was studied by B. C. P. 

 Jansen/' who established the optimal conditions for the oxidation 

 with potassium ferricyanide ; he extracted the thiochrome with 

 isobutanol and measured the fluorescence of the extract in a fluori- 

 meter calibrated against standard solutions of quinine. The method 

 has been extensively employed for the estimation of vitamin B^ in 

 foodstuffs and urine, and indeed may be said to be the most important 

 method of assay. It was officially adopted in the Seventh Addendum 

 (1945) to the British Pharmacopoeia 1932. 



According to D. J. Hennessy,^^ it is more sensitive than the colori- 

 metric method and capable of estimating lower potencies. This was 

 confirmed by Brown et al.}^ who also obtained good agreement between 

 the two methods. They stated, however, that neither method was 

 satisfactory with very low potencies, only the rat growth or pigeon 

 weight maintenance methods giving reliable results in such instances. 

 Hennessy et al^^ found little difference between the results obtained by 

 means of the thiochrome test, the rat growth test and the fermentation 

 test when pharmaceutical preparations were assayed by these methods. 



J. C. Moyer and D. K. Tressler ^i obtained good agreement between 

 the thiochrome method and the sulphite-cleavage modification of the 

 fermentation method. 



The method has been modified by individual workers to meet 

 their particular requirements and there is, therefore, a bewildering 



38 



