A Systematic AQbD Approach for Optimization of the Most Influential Experimental Parameters on Analysis of Fish Spoilage-Related Volatile [PERSON]  
1 CQM-Centro Quimica da Madeira, Campus Universitario da Penteada, 9020-105 Funchal, Portugal; [EMAIL_ADDRESS] (J.F. ); [PERSON]L_ADDRESS] (P.S.) [PERSON]  
1 CQM-Centro Quimica da Madeira, Campus Universitario da Penteada, 9020-105 Funchal, Portugal; [EMAIL_ADDRESS] (J.F. ); [PERSON]L_ADDRESS] (P.S.) [PERSON]  
2 CQ-Centro Quimica da Madeira, Campus Universitario da Penteada, 9020-105 Funchal, Portugal; [EMAIL_ADDRESS] (J.F. ); [PERSON]L_ADDRESS] (P.S.) [PERSON]  
1 CQM-Centro Quimica da Madeira, Campus Universitario da Penteada, 9020-105 Funchal, Portugal; [EMAIL_ADDRESS] (J.F. ); [PERSON]L_ADDRESS] (P.S.) 2 CUBAS-Abeel Salazar Institute for the Biomedical Sciences, University of Porto, R. [PERSON], 228, 4050-313 Porto, Portugal; [EMAIL_ADDRESS]  
CIMAR-Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros de Leixoes, Av. General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal  
Faculty of Exact Sciences and Engineering, University of Madeira, Campus Universitario da Penteada, 9020-105 Funchal, Portugal  
Correspondence: [EMAIL_ADDRESS]; Tel. : +351-291-705112; Fax: +351-291-705149 202  
parasites [2; 3]. The most common consequences of these processes are pH and physical alterations as well as the production of slime, off-odors, and off-flavors attributed to volatile compounds, mainly amines and short-chain alcohols. Among the amines, the volatile amines trimethylamine (TMA) and dimethylamine (DMA) can be used as potential markers to monitor the spoilage state of fish, assisting in the determination of the time in which the fish will remain acceptable for consumption, retaining its sensory and eating qualities [4; 5; 6]. The impact of secondary amines on fish products is relevant because of the involvement of amines as probable precursors of carcinogenic compounds. The most common methods to quantify volatile amines in fish include total volatile base-nitrogen determination (TVB-N) and TMA quantification [7]. The TVB-N measures the ammonia (intrinsic and formed during analysis), TMA, and DMA. The increment in these components, in combination or alone, will increase TVB-N amounts [4; 8]. The ammonia present in the immediate postmortem flesh (after death during harvesting or after a few hours in ice storage) has its origin in the inosine monophosphate formed through adenine nucleotide deamination [2; 4]. Additionally, trimethylamine oxide demethylase (TMAOase), a molybdenum oleenzyme present in the periplasm of specific spoilage organisms, catalyzes the cleavage of trimethylamine oxide (TMAO) into DMA and formaldehyde (FA). However, several days (\(\pm\)10 days) after the catch, bacterial reduction (genus _Shewanella_, if fish are from cold water, or _Pseudomonad_ if fish are from warm water) of TMAO to TMA is the dominant process. During storage, the increment in TMA levels is characterized by a fast increase in the first 5-7 days, followed by an apparent stable concentration. DMA formation, besides not being present in all species, is more relevant during frozen storage even though in small amounts [4; 8]. In Europe, chemical analysis of fish samples follows the Regulation (EC) No. 2074/2005, which determines the quantification of TVB-N through steam distillation and establishes acceptable amounts according to fish species [9; 10; 11]. In the case of the determination of TMA concentration, the official recommended method is based on [PERSON]'s method [10]. Other common methodologies to quantify the TVB-N, ammonia, TMA, and DMA include steam distillation and titration of the amines, using muscle samples or extracts from the muscle, as described in Table S1 (Supplementary Material). Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), colorimetry, gas chromatography, flow injection analysis using potentiometric detection, and electronic nose have also been used [8; 12].