BULLARD and COLLINS: IMPROVED METHOD TO ANALYZE TRIMETHYLAMINE 



(K), if present (e.g., for the cold method use 

 Equation (4)): 



(A + 0.007)(250 ml)( lOOHK) .,, . 

 = milligrams 



(0.082X75 g fleshH 1,000 ^g/mg) TMA-N/100 g flesh. (5) 



RESULTS AND DISCUSSION 



In the following sections, the influence of the 

 presence of FA (or not), the type and concentration 

 of base used, and temperature of extraction were 

 determined separately on each component in the 

 reaction and extraction mixture; i.e., 1) NHg and 

 HMTA, 2) DMA and TMMD, and 3) TMA. The four 

 methods were directly compared for precision by 

 replicate analyses of a composite sample of minced 

 fish flesh. Finally, accuracy (recovery of TMA and 

 the interference of DMA) was determined for the 

 four methods by direct addition of known quan- 

 tities of TMA and DMA. 



Reaction of Ammonia 



Sprung (1940) reported that FA reacts with NH3 

 to form HMTA. Under vacuum distillation condi- 

 tions at 30° C, the addition of FA to solutions of 

 NH4CI and NaaCOs rendered the NH3 nonvolatile 

 but in the absence of FA, NH3 was volatile (Benoit 

 and Norris 1942). Dyer (1945) claimed that the 

 addition of 1 or 2 ml of 49c FA did not affect the 

 recovery of TMA, and was sufficient to eliminate 

 the interference of NH3 up to 10-20 times over the 

 usual concentration of trimethylamine nitrogen. 

 Dyer, however, did not directly study the influ- 

 ence of FA on the development of the picric acid 

 color in the presence of NH3 . Researchers studying 

 the TMA method have followed the prior proce- 

 dures and used FA to tie up NH3 in the TMA test. 



Standard solutions of NH4CI in 5% TCA were 

 prepared and used in the three TMA methods. To 

 simulate various stages of spoilage the concentra- 

 tion ranged from 6.6 to 66 /xg N/ml; i.e., 2.2-22.0 

 mg N/100 g flesh. The absorbancies from the 

 NH^^Cl solutions with and without FA (Table 1, 

 columns 1-6) were low and of questionable signifi- 

 cance except if 45% KOH was used. The absorban- 

 cies seemed to increase with concentration of 

 NH4CI and were about the same whether FA were 

 present or not if 50% K2CO3 and 25% KOH were 

 used. Even at the higher concentrations, the 

 absorbancies were low and would contribute little 

 to the TMA value. When 45% KOH was used, the 

 absorbancies were much higher than the absor- 

 bancies with the other bases and were not affected 

 by FA except at the higher concentrations. Accord- 

 ingly, FA and NH3 did not react under these 

 conditions and NH3 could contribute to the TMA 

 value. To determine if FA would react with NH3 at 

 elevated temperatures, the same standard solu- 

 tions in 5% TCA were treated as before but were 

 preheated in the presence of FA at 60° C for 30 

 min, cooled to room temperature, and 45% KOH 

 added and extracted in the usual way. The absor- 

 bancies of the preheated samples were reduced 

 significantly — see columns 5 and 7 in Table 1. The 

 low absorbancies indicated that FA reacted with 

 NH4CI when heated and there was no interference 

 through 26.4 (xg N/ml sample. The color develop- 

 ment in the samples containing 33.0 /xg N/ml and 

 more could be caused by: 1) residual NHg through 

 an incomplete reaction with FA, 2) NH3 through a 

 reverse reaction of the product which is assumed 

 to be HMTA, by the law of mass action or chemical 

 equilibrium, and 3) a partial extraction of HMTA 

 by toluene and subsequent reaction with picric 

 acid. 



Table 1. — Ammonium chloride or hexamethylenetetramine (HMTA): the absorbancies of picrates in 

 the trimethylamine test as affected by the addition of standard solutions of ammonium chloride or 

 hexamethylenetetramine. Samples (columns 1-10) were extracted at room temperature for 15 min 

 (except 9 and 10 which were extracted at -15° C for 60 s by hand), with formaldehyde ( +) and without 

 formaldehyde (0). 



'After addition of FA. samples were heated at 60' C for 30 min to allow FA and NH3 to react. Samples were then cooled, base 

 added, and extracted at room temperature. 



467 



