48 THE BIOCHEMISTRY OF B VITAMINS 



omitted and advantage is taken of the fact that the pigment produced 

 by the oxidation of cocarboxylase is not soluble in isobutyl alcohol, 

 whereas thiochrome is. Experimental details of procedure and a partial 

 bibliography will be found in the reference cited. 5 



As ordinarily performed, the thiochrome solution examined (Step 7) 

 corresponds to the order of 1.0 fxg of thiamine. A micromethod using a 

 Spekker Fluorometer has been devised 6 which is accurate to =•= 20 per cent 

 when 0.001 /*g of thiamine is determined, and to about ±3 per cent when 

 the amount of thiamine is 0.05 /*g. 



N S 



/ \ /\ 



H 3 C— C C— NH 2 HC1 HC C— CH 2 — CH 2 OH 



II I II II 



N C N + C— CH 3 



\ y \ / ci- 



CH 2 



Thiamine hydrochloride 



N N S 



/ \ / \ /\ 



H 3 C— C C C C— CH 2 — CH 2 OH 



I II I II 



N C N C— CH 3 



V \ X 



H H 2 



Thiochrome 



The thiochrome procedure has been successfully applied to the analysis 

 of cereals and cereal products, 7 - s to miscellaneous foods and tissues, 9, 10 

 to urine, 11, 12 to blood, 13, 14, 14a and to pharmaceuticals. 15, 16 



Colorimetric Method. Another chemical method of some value involves 

 the formation of a colored pigment (usually red) when thiamine is 

 allowed to react in alkaline medium with a diazotized aromatic amine. 

 The most important amine for this purpose, p-aminoacetophenone, which 

 yields a red dye insoluble in water but soluble in xylene, was first used 

 by Prebluda and McCollum. 17 The development of quantitative methods, 

 suitable for laboratory use, involving this reagent is the work of Melnick 

 and Field. 18 



The Melnick and Field method, including modifications thereof, has 

 not been used as extensively as the thiochrome method mainly because 

 of its lack of sensitivity, though in some laboratories, at least, it has been 

 found more reliable. 15 It requires, in order to be applied, a concentration 

 of 2-3 fig of thiamine per ml, whereas the thiochrome method is applicable 

 to solutions containing as little as 0.05 /*g per ml. For the analysis of 



