developed from information obtained from a de- 

 tailed study of how temperature, type and concen- 

 tration of base, and the presence (or not) of FA 

 affect the extraction and subsequent absorbancies 

 of the picrate color of NH3, DMA, and TMA. 



EXPERIMENTAL 



Purification Procedures 



Trimethylamine hydrochloride (TMA  HCl) and 

 dimethylamine hydrochloride (DMA HCl) were 

 crystallized twice from hot 2-propanol and dried 

 under high vacuum overnight. Reagent grade and 

 previously used toluene was purified by shaking 

 and partitioning with concentrated sulfuric acid 

 in a separatory funnel followed by water, sodium 

 hydroxide, and water; filtering through anhydrous 

 sodium sulfate (NA2SO4); and distilling at 110° C. 

 Reagent grade Formalin^ (37% FA) was shaken 

 with magnesium carbonate, filtered, and diluted 

 1:9 with water Hexamethylenetetramine (HMTA) 

 from Pfaltz and Bauer was crystallized twice from 

 hot 2-propanol or from hot, dry toluene and dried 

 overnight under high vacuum. N N N'N'- 

 tetramethylmethanediamine (TMMD) from 

 Pfaltz and Bauer was distilled using a column 

 packed with glass helices. The first 25 ml fraction 

 (72°-81° C) was discarded, the next 25 ml fraction 

 (81° C) was used for analysis, and the final 25 ml 

 distillate was discarded. Reagent grade ammoni- 

 um chloride (NH4CI) was crystallized twice from 

 hot water and dried under high vacuum overnight. 



Extraction Procedure for Fish Flesh 



Procedures cited in the literature for the extrac- 

 tion of fish have used a specific amount of flesh 

 plus water or TCA followed by shaking or blend- 

 ing and filtration. These methods assume a speci- 

 fic moisture content of the fiesh, a total volume, 

 and a complete extraction or uniform dispersion 

 of TMA in the extract, e.g., 100 g flesh at 

 SO^f moisture plus 300 ml TCA solution would 

 give a 1/95 aliquot for a 4 ml sample. To improve 

 accuracy of the method, we used an exhaustive 

 extraction-filtration procedure and dilution to a 

 volume. Details of the procedure are given in the 

 Recommended Procedures section. 



^Reference to trade names does not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



FISHERY BULLETIN: VOL. 78, NO. 2 



Methods of Analyses for TMA 



Unless otherwise indicated, the three common- 

 ly used methods of analyses for TMA were modi- 

 fied slightly to fit our equipment and to reflect 

 recent advances in the methods. The methods of 

 Dyer (1945), Tozawa et al. (1971), and Murray and 

 Gibson (1972) were used as follows: a 4 ml sample 

 of a standard solution in 57f TCA or a 5% TCA 

 extract of fish flesh was added to a 25 x 150 mm 

 screw top test tube, followed by the addition of 10 

 ml toluene, 1 ml of 3.79?^ FA solution, and left to 

 stand 5 min before the addition of 3 ml of base 

 (25% KOH, 45% KOH, or 50% K2CO3). The tube 

 was tightly sealed using a gasket of a double layer 

 of 1 mil polyethylene film under the cap and 

 shaken for 15 min at room temperature on a 

 Burrell wrist action shaker modified by building- 

 up the platform 20.3 cm with Styrofoam. After 

 standing several minutes, about 7 ml of the 

 toluene layer were removed and dried with 0.5 g 

 anhydrous NagSO^. After drying, 5 ml were added 

 to 5 ml of 0.02% picric acid in dry toluene and the 

 absorbance was determined at 415 nm on a 

 Gilford modified Beckman D.U. spectrophotome- 

 ter. A fourth method will be referred to as the "cold 

 method" of extraction and uses 45% KOH (Murray 

 and Gibson 1972) but the extraction is done at -15° 

 C. The details of this method are given in the 

 section on Recommended Procedures. 



Standard Curves 



Since four distinctly different methods were 

 used to analyze for TMA content, complete blank 

 determinations and standard curves were made 

 for each method. The equations for the regression 

 lines (standard curves) of absorbance on concen- 

 tration of TMA (micrograms TMA- N/milliliter) 

 for each of the methods were as follows: 



25% KOH, room temperature Y = O.OllX - 0.007 (1) 



45% KOH, room temperature Y = G.OSTX - 0.001 (2) 



50% K2CO3, room temperature Y = 0.067X - 0.012 (3) 



45% KOH, cold ( - 15° C) Y = 0.082X - 0.007 (4) 



where Y = absorbance and X = micrograms TMA- 

 N/milliliter. 



The trimethylamine values in milligrams TMA- 

 N/100 g flesh were calculated from these equa- 

 tions and from the total volume (250 ml) of 5% 

 TCA extract, weight of extracted fish flesh (75 g), 4 

 ml of sample extract per tube, and a dilution factor 



466 



