An Improved Method to Analyze Trimethylamine in Fish and the Interference of Ammonia and Dimethylamine

The trimethylamine content of most marine fish, especially the gadoid species, is internationally accepted as an index ofspoilage. However, ammonia, dimethylamine, and other aminesalso contribute to the trimethylamine value. Variations in the conditions of the three current methods used to analyze for trimethylamine content were studied in detail to determine the best condition to extract trimethylamine and to reduce the extraction of ammonia, dimethylamine, and other amines. Formaldehyde does not inhibit the interference from ammonia but the interference is negligible even in advanced spoilage. Formaldehyde inhibits the interference from dimethylamine to some extent if KOH is used as the base but increases the interference in the K,CO method. The extraction ofdiand trimethylamine are highly dependent upon the base used and the 'temperature of extraction. A new method ofextracting at -150 C using 45% KOH was developed that essentially eliminates interference from ammonia, dimethylamine, and other amines. To directly compare the methods, the trimethylamine content of a sample of spoiled walleye pollock, Theragra chalcogramma, flesh was determined by the three currently used methods and the cold method of extraction. All methods gave similar standard deviations but the K2 CO. method gave higher values than the KOH methods and the cold method gave the lowest value. Various levels of trimethylamine and dimethylamine simulating different qualities of fish and frozen storage times were added to samples of Pacific cod, Gadus macrocephalus, flesh. The cold method consistently extracts more accurate amounts oftrimethylamitle with less interference from dimethylamine than any of the other extraction methods. The trimethylamine (TMA) content of most marine fish, especially the gadoid species, is accepted internationally as an index of spoilage. Dyer's 1959 method of analysis for TMA, except for the concentration of formaldehyde (FA), has been adopted by the Association of Official Analytical Chemists (Horwitz 1975). Trimethylamine is produced by the reduction oftrimethylamine oxide by microorganisms (Poller and Linneweh 1926). Ammonia, dimethylamine (DMA), and other volatile bases are also formed when fish spoil and to some extent interfere with the measurement of TMA. In advanced spoilage, some of the higher aliphatic amines are formed by decarboxylation of amino acids and may cause interference (Dyer 1945). A number of investigators studied the TMA method to improve the accuracy and reduce the effects of ammonia, DMA, and other amines. Dyer (1945) adapted the method ofdetermining amines to fish and used 0.02% picric acid in dry toluene instead of chloroform (Richter 1938; Richter et al. 1941). Dyer and Mounsey (1945) used a trichloro'Northwest and Alaska Fisheries Center Kodiak Investigations-Utilization, National Marine Fisheries Service, NOAA, P.O. Box 1638, Kodiak, AK 99615. Manuscript accepted December 1979. FISHERY BULLETIN: VOL. 78, NO.2, 1980. acetic acid (TCA) extract offresh cod instead of the unstable press juice or weighed samples. Hashimoto and Okaichi (1957) claimed that variation of temperature caused serious errors in the determination ofTMA, and recommended a 30° C extraction with 25% KOH rather than 50% ~C03 and room temperature. Tozawa et al. (1971) confirmed these findings and showed that 25% KOH reduced the interference caused by DMA and claimed the formation of a compound from FA and DMA which was not extracted in the presence of hydroxide ions. Murray and Gibson (1972) found that 45% KOH extracted more TMA and gave more linear and reproducible results than if extracted with 25% KOH or 50% K2 C03· The three current methods of analysis for TMA employ 25% KOH, 45% KOH, or 50% K2C03 to release TMA for extraction into the toluene layer and result in different absorbancies for the picrate color with DMA and TMA. In general, the use of K2 C03results in a higher extraction ofDMA and a lower extraction of TMA than if KOH were used. Ideally, the best method to measure TMA content should result in complete extraction of TMA and zero extraction of ammonia (N~), DMA, and other amines so that these components will not contribute to the TMA value. A new method was