Enzymatic determination of trimethylamine in fish

Resumen del póster presentado a la XXXVIII Reunión Bienal de la Real Sociedad Española de Química, celebrada en el Palacio de Congresos de Granada, del 27 de junio al 30 de junio de 2022.Trimethylamine (TMA) is the substance responsible for the fishy odour and it is produced by the reduction of trimethylamine N-oxide (TMAO) by microorganism. Fresh seafood contains low levels TMA but higher of TMAO and its concentration rapidly increases after death. This is because, its concentration is used as an indicator of the freshness of fish. On the other hand, in humans, TMA is metabolized to TMAO (odourless) catalyzed by flavin-containing monooxygenase (FMO). Trimethylaminuria or "fish odour syndrome" is a human disorder characterized by the difficulty of converting foul-smelling TMA to its odourless N-oxide. In this case, the intake of excess TMA is a serious problem so it is necessary to control the concentration of TMA in food. The most frequent methods of analysis of TMA in foods involve GC, HPLC and EC. These methods are not appropriate for in-situ determination so it is necessary to develop rapid methods for determination of TMA. In this work, a rapid and “in-situ” method for the determination of TMA based on its enzymatic reaction with FMO3 is presented. FMO3: TMA + NADPH + H+ +O2 → TMAO + NADP + H2O. It has been found that the slope of the variation of absorbance at 340 nm due to the disappearance of NADPH (Figure 1a: [NADPH]=3.45.10-4 M, pH= 7.74 , Tª=38 ºC, [FMO3]=500 U.) can be related to the concentration of TMA and used as analytical signal of the proposed method. FMO3 concentration, pH, NADPH concentration and Tª have been optimized. In the optimal conditions ([NADPH] = 8·10-4 M, pH= 9, Tª=38ºC,FMO3=2000 U), the method shows a linear response to TMA between 2·10-5 and 1.5·10-4 M, with a RSD of 9.67% (n=3). An exhaustive interference study (putrescine, cadaverine, histamine, tyramine, monomethylamine and dimethylamine (DMA) were considered) show that only DMA is an interference but if the ratio DMA: TMA is 10:1. The method was applied to the determination of TMA in codfish by the addition standard method with good results.This work has been supported by the Agencia Estatal de Investigación of Spain (PID2019-105408GB) and by the founding to research group of the DGA-FEDER (E25_20R).Peer reviewe