FISHERY BULLETIN: VOL. 78, NO. 2 



amounts of TMA and DMA in the sample. Only 

 the cold method gave TMA values that were 

 nearly independent of the DMA content of all 

 levels of DMA and TMA. If methods other than the 

 cold method are used to analyze for TMA, the 

 history of the fish and sample storage should be 

 known or the DMA content should be determined 

 separately. 



RECOMMENDED PROCEDURES 



Extraction Procedure for Fish Flesh 



Blend a thoroughly mixed composite sample of 

 fish flesh (75 g) with 90 ml of 8.27^ (weight/volume) 

 TCA for 5 min at high speed in a Vertis blender. 

 Pour contents of blender jar into a 150 ml medium 

 porosity sintered glass funnel and filter under 

 vacuum. To prevent foaming and plugging of 

 filter, clamp off the suction line after filtering 

 starts and briefly open when required. Reextract 

 residue with 70 ml of 59^ TCA for 2 min and filter 

 into the same filter flask and rinse with 5% TCA 

 from a wash bottle. Quantitatively transfer the 

 combined filtrates and washings to a 250 ml 

 volumetric flask and dilute to the mark with 59c 

 TCA. The extract (4 ml) is used in the TMA 

 analysis without dilution but, if required, 4 ml of a 

 diluted extract is used rather than smaller vol- 

 umes of extract. 



Cold Method of Analysis for TMA 



Add 4 ml samples of standard solutions of 

 TMAHCl in 5% TCA or 57c TCA extracts, 10 ml 

 toluene, and 1 ml of 3.77f FA to 25 x 150 mm screw 

 top test tubes. Allow to stand for 5 min then place 

 tubes in an ice- water bath in an effort to avoid the 

 possible yellow color caused by the addition of 

 concentrated KOH (Castell et al. 1974). When 

 completely chilled, add 3 ml 45*^ KOH and tightly 

 seal tubes, invert twice, and place in a mixture of 

 salt and precooled saturated brine-ice at -15° C. 

 Use a pump or stirring motor to maintain constant 

 temperature by circulating the brine through the 

 salt, brine-ice mixture. After 2 min, remove the 

 test tubes and shake vigorously by hand for 15 s 

 and replace in the cold bath for 2 min. Repeat this 

 procedure three times for a total of 60 s of vigorous 

 hand shaking. After settling ( almost immediately), 

 transfer about 7 ml of the toluene layer to clean 

 dry 18 x 150 mm test tubes and dry with about 0.5 

 g anhydrous Na2S04 by swirling (Vortex Mixer). 



After drying, remove 5 ml and add to 5 ml of 0.02% 

 picric acid in dry toluene. Determine the absor- 

 bance at 415 nm using 1 cm standard silica cells 

 and a Gilford modified Beckman D.U. spectro- 

 photometer. Determine the blank in the same 

 manner but use 4 ml TCA. Calculate the TMA 

 content in mg TMA-N/100 g from the absorbance 

 and Equations (4) and (5). 



SUMMARY 



Although NH.j, DMA, and other amines contrib- 

 ute to the TMA value, the TMA content of some 

 marine fish, especially gadoid species, is accepted 

 internationally as an index of spoilage. Variations 

 in the conditions of the three methods used to 

 analyze for TMA were studied to determine the 

 best condition to extract TMA and to reduce the 

 extraction of HN3, DMA, and other amines. We 

 found that NHg was not tied up by FA as suggested 

 in the literature but has little affect on the TMA 

 value of fish even in advanced spoilage. The 

 amount of DMA extracted was strongly dependent 

 on the temperature of extraction, the base used, 

 and the presence or absence of FA. Formaldehyde 

 and DMA reacted to form a compound that was 

 rapidly extracted by 507c KaCOy and very slowly 

 by 25% KOH. If DMA and TMMD were extracted, 

 the absorbancies were nearly the same which 

 infers that the compound formed from FA and 

 DMA was TMMD. The amount of TMA extracted 

 was strongly dependent on the temperature of 

 extraction when 25% KOH or 50% K2CO3 was 

 used as the base but nearly independent when 

 45% KOH was used. A cold method of extraction 

 (45% KOH and -15° C) was developed that essen- 

 tially eliminated the contribution of DMA to the 

 TMA value. Trimethylamine was determined in 

 spoiled fish flesh by the cold method and the three 

 other methods. Standard deviations were similar 

 for all four methods. The KgCO^ method gave the 

 highest value and the cold method gave the lowest 

 value. If varying amounts of TMA and DMA were 

 added to Pacific cod flesh and analyzed by the 

 three published TMA methods, the recovery of 

 TMA and interference from DMA was strongly 

 influenced by the relative amounts of TMA and 

 DMA present. Relative to the other methods, the 

 cold method gave TMA values that were indepen- 

 dent of the presence of DMA or the relative 

 amounts of DMA and TMA. We recommend that 

 the cold method be used because it extracts most of 

 the TMA (97%), gives good recovery of added 



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