CHEMICAL ESTIMATION 



array of methods to choose from. The objects of these various modi- 

 fications are, of course, to eliminate interference from other substances 

 present in the sample being assayed. Some substances prevent the 

 formation of thiochrome, leading to low results, whilst others enhance 

 the fluorescence and produce high results. 



Perhaps the most important modification is that introduced by 

 D. J. Hennessy and L. R. Cerecedo,^^ j^ which the aneurine is ad- 

 sorbed on a zeolite, Decalso, and eluted with hot potassium chloride 

 solution. This method, which is analogous to Melnick and Field's 

 modified procedure, has been adopted by the majority of workers. 

 Perlzweig et al.^^ used Superfiltrol for removing aneurine from urine, 

 and a mixture of pyridine, ethanol and hydrochloric acid for elution. 



Even when adsorbents of this type are used, interfering substances 

 may remain. H. L. Mason and R. D. Williams 2* experienced trouble 

 in assaying urines owing to the presence of fluorescent derivatives of 

 nicotinic acid. Satisfactory results were obtained only when the 

 amount of aneurine in the sample exceeded 100 /xg. When the ex- 

 cretion was low or when 300 to 500 mg. of nicotinic acid were ingested 

 per day, the non-thiochrome material accounted for most of the 

 fluorescence. They suggested that the difflculty could be overcome 

 by repeating the fluorimetric assay after destroying the aneurine in 

 the luine by heating with sodium sulphite at pH 5 for fifteen minutes 

 and then subtracting the intensity of the fluorescence from that given 

 by the untreated solution. 



V. A. Najjar and K. C. Ketron,^^ however, showed that the fluores- 

 cent metabolite of nicotinic acid, " Fg " (see page 254), which was 

 responsible for this phenomenon, was attacked by sodium sulphite, 

 so that Mason and Williams' procedure did not give a true blank. 

 They recommended Hennessy and Cerecedo's method with the rather 

 unsatisfactory expedient of assuming that 21 % of the fluorescence 

 was due to " Fg ". Y. L. Wang and L. J. Harris ^^ destroyed inter- 

 fering fluorescent substances in extracts prepared from foodstuffs by 

 oxidation with hydrogen peroxide before extraction with isobutanol. 



Another group of substances that may cause interference in the 

 fluorimetric assay of urines are salicylates ; these may be removed ^^ 

 by acidification and extraction with isobutanol before oxidation. 



J. G. Organ and F. Wokes ^^ experienced a reverse effect in esti- 

 mating the aneurine content of cereal extracts. These appeared to 

 contain substances that " quenched " the fluorescence, and inter- 

 ference from this source was only satisfactorily overcome by adding 

 known amounts of aneurine equal to at least four times that originally 

 present and then subtracting the amoimt added from the results. 



P. Ellinger and M. Holden ^^ foimd that certain inorganic salts 

 also had a marked quenching effect on the fluorescence of thiochrome 



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