52 THE BIOCHEMISTRY OF B VITAMINS 



Another means of eliminating the effect of interfering substances is to 

 reduce with stannous chloride and sodium hydrosulfite all the fluorescent 

 pigments including riboflavin, and then reoxidize by contact with air; 

 this procedure brings back riboflavin to its original fluorescent form, but 

 leaves in reduced form some of the substances which would otherwise 

 interfere. 



Another procedure involves the use of dilute permanganate under con- 

 trolled conditions to oxidize interfering pigments without affecting the 

 riboflavin. The excess permanganate is removed with hydrogen peroxide. 



Another procedure, regarded as necessary only when the materials 

 tested are of relatively low potency or highly pigmented, involves adsorb- 

 ing the riboflavin selectively upon a column of "Florosil" and eluting it 

 with 20 per cent pyridine in 2 per cent acetic acid. This, of course, leaves 

 behind many fluorescing substances, but introduces an extra step into 

 the procedure; hence it is likely to introduce errors as well as expend 

 time. 



Scott and co-workers 43 have recently published a complete procedure 

 which has been applied with excellent results to many types of materials, 

 including milk and milk products, 44 dried leguminous seeds, 45 miscel- 

 laneous cereal products, fermentation residues, leaf meals, meat and fish 

 scrap, and yeasts. 43 In this method, only two of the expedients discussed 

 above are used: permanganate oxidation and hydrosulfite reduction at 

 the end to obtain a blank reading. Details of procedure are to be found 

 in the original article. 43 It is said to require less time than the micro- 

 biological method. 45 



A fuller discussion of the various alternate procedures involved in the 

 fluorometric method will be found elsewhere. 46 



Microbiological Method. This method, which is essentially that of 

 Snell and Strong, 47 has been widely used and in spite of the natural handi- 

 cap inherent in the use of an unfamiliar type of technique, it has been 

 adopted by the U. S. Pharmacopoeia. 48 



The method is based upon the fact that the growth of Lactobacillus 

 casei and its ability to produce lactic acid requires riboflavin in the 

 medium. The riboflavin can be in the free form, but its combined forms, 

 e.g., flavoprotein, flavin adenine dinucleotide, and presumably riboflavin 

 phosphate, are equally effective under the experimental conditions pre- 

 scribed for the assay. 49 



Theoretically, for this or any other comparable assay, one should 

 have a basal medium perfect in every other respect except for the lack 

 of the one item to be assayed for. Actually, it is only necessary in this 

 case that the basal medium be such that under laboratory conditions other 



