FISHERY BULLETIN: VOL. 78, NO. 2 



To determine if HMTA would react with picric 

 acid, purified HMTA in dry toluene was added to 

 the picric acid reagent. A strong color developed 

 which indicated that HMTA reacted with picric 

 acid. To determine if HMTA would be extracted 

 under the standard conditions of the TMA test 

 using 45% KOH, standard solutions of HMTA 

 were prepared and used in the same manner as 

 NH^Cl solutions. The absorbancies for the HMTA 

 samples (Table 1, column 8) were similar to the 

 absorbancies of the samples represented by col- 

 umns 5 and 7 of Table 1. Further, the absorbancies 

 for HMTA samples were not as would be expect- 

 ed from the literature and indicated that either 

 HMTA was extracted by toluene and reacted with 

 picric acid or a partial reverse reaction occurred 

 and NH3 was extracted and reacted with picric 

 acid. Whether the picrate color was caused by NH3 

 or by HMTA, FA did not eliminate the interfer- 

 ence of NHj when 45% KOH was used. Formalde- 

 hyde gave some protection, however, if the reac- 

 tion mixture was heated at 60° C for 30 min prior 

 to the addition of 45% KOH. 



Since NH.j or HMTA was extracted by toluene 

 at room temperature and reacted with picric acid, 

 NH3 was extracted at a low temperature to see if 

 the interference could be reduced. The same stan- 

 dard solutions of NH4CI were treated as before but 

 were extracted by the cold method. The data given 

 in columns 9 and 10 of Table 1 showed that the 

 absorbancies were at the same general level as the 

 preheated samples and the presence of FA had 

 little effect on absorbance. We conclude that 45% 

 KOH was less effective in releasing NHg at -15° C 

 than at room temperature (Table 1, compare col- 

 umns 5 with 9, 6 with 10) or NH3 was less 

 extractable by toluene at -15° C. Although other 

 researchers have used FA to eliminate the inter- 

 ference of NH3, our data showed that FA does not 

 tie up NH3 under the usual conditions in the 

 analysis for TMA. On practical grounds, the 



Table 2. — Dimethylamine hydrochloride: the absorbancies of 

 picrates in the trimethylamine test as affected by the three bases 

 used and temperature of extraction. Samples were extracted for 

 60 s with vigorous hand shaking using 4 ml 15.9 fxg DMA-N/ml, 

 with formaldehyde ( +) and without formaldehyde (0). 



contribution of NK, to the TMA value would be 

 quite low because the bases (except 45% KOH) 

 released only a small amount of NH3 or NH3 was 

 only slightly extracted by toluene. Even in ad- 

 vanced spoilage such as 22 mg NH.j-N/100 g flesh 

 (66 ixg N/ml), the contribution of NH3 to the TMA 

 value would be equivalent to 0.45 mg TMA-N/100 

 g flesh if extracted at room temperature with 45% 

 KOH but only 0.10 mg TMA-N/100 g flesh if 

 determined by the cold method. 



Reaction of DMA 



Several researchers have found that DMA and 

 TMA are not completely extracted by toluene 

 under conditions used in the TMA test unless 

 replicate extractions are made (Castell et al. 

 1974). Further, different bases resulted in differ- 

 ent extractabilities of DMA. Accordingly, we de- 

 termined the absorbancies of the picrate color 

 using a standard solution of DMA (15.9 /xg DMA- 

 N/ml; i.e., 5.3 mg DMA-N/100 g flesh) under 

 various conditions of the TMA test; temperature, 

 base (KjCOg and KOH), replicate extraction, and 

 presence or absence of FA. 



A standard solution of DMAHCl in 5% TCA 

 was extracted with and without FA by the usual 

 methods but the temperature of extraction was 

 varied (-15° C to +30° C) and the tubes were 

 vigorously shaken by hand for 60 s. The extraction 

 of DMA was strongly influenced by the base and 

 by temperature (Table 2). In the absence of FA, the 

 differences in reactivity of the bases in releasing 

 DMA from the hydrochloride salt are shown in 

 columns 2, 4, and 6 (Table 2). At -15° C, 45% KOH 

 released about half of the DMA present, 25% KOH 

 released l/20th, and K2CO3 was unreactive and 

 did not release DMA. The bases were more reac- 

 tive at higher temperatures than at lower temper- 

 atures but had the same order of reactivity. The 

 date also showed that, if released from the salt, 

 DMA was extracted by toluene even at low tem- 

 peratures. When FA was present however, a 

 different order of release was evident (Table 2, 

 columns 1, 3, and 5) and showed that FA reacted 

 with DMA to give a product having different 

 reactivity with the bases. The order of release (or 

 extractability) for the product was different than 

 for DMA, i.e., high absorbance with K2CO3, in- 

 termediate with 45% KOH, and low with 25% 

 KOH. Considerable amounts of the product were 

 extracted in the presence of FA at all tempera- 

 tures in the carbonate system and would result in 



468 



