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An improved GC-Mass method to assess the short chain fatty acid in fish gut fecal compounds

An improved GC-Mass method to assess the short chain fatty acid in fish gut fecal compounds Chiara Ceccotti ¹, Giuseppe Scollo¹, Luca Chiodaroli¹, Genciana Terova¹, Marco Saroglia¹ ¹ Department of Biotechnology and Life Sciences (DBSV), University of Insubria, Varese, Italy

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An improved GC-Mass method to assess the short chain fatty acid in fish gut fecal compounds

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  1. An improved GC-Mass method to assess the short chain fatty acid in fish gut fecal compounds Chiara Ceccotti¹, Giuseppe Scollo¹, Luca Chiodaroli¹, Genciana Terova¹, Marco Saroglia¹ ¹Department of Biotechnology and Life Sciences (DBSV), University of Insubria, Varese, Italy Corresponding author: chiara.ceccotti@uninsubria.it Introduction: Butyric acid, a short chain fatty acid (SCFA), has been reported to protect fish gut structure and having an anti-inflammatory property. It may be either introduced with feed or produced by intestinal bacteria, resulting of great relevance when high percentage fish meal (FM) substitutions with vegetable proteins (VP) is operated (Plöger et al., 2012, Tacon et Metian, 2008). So, monitoring the residual butyrate concentration in the fish gut content, may result of paramount importance when tuning fish feed formulation. The volatility of butyrate allowed to quantify the molecule in fish feces by the headspace method with GC-Mass (Valero et al.,2000).The method consisted in acidification and heating of feces in order to evaporate the butyrate. Many parameters (initial temperature and the characteristics of the ramp) have been challenged, in order to find the best analytical conditions and to avoid errors due to contaminants. • Materials and Methods. • Best temperature identification for extracting butyric acid in the vial headspace (Fig.1). • GC-MS (Thermo Scientific Focus GC/DQII). Protocol applied: headspace method (RT:9,04 of butyric acid) (Fig.2). • Samples for standards calibration: 400 µl of butyric acid and 800 µl of sulphuric acid. Samples heated at 60°C. • Volume injected : 250 µl. • Three butyric acid calibration standards have been measured at 3 different temperatures (25°C,60°C,100°C). Standards concentrations: 1.0; 5.0; 10.0; 50.0;100.0 ppb. (Fig. 3). Fig.1: temperature VS constant pressure of headspace. (Yaws & McGraw-Hill,1999, modif.) Fig.3: butyric acid area has been evaluated in Selected Ion Mass (SIM) using the most abundant mass peak (m/z=88). Fig.2: samples in vials heated at 60°C, quantitative headspace injection in GC-MS and chromatogram of butyric acid, with the retention time of the molecule (RT: 9.04). • Results and Discussion • Temperature ranging 40ºC-100ºC resulted to improve the method sensibility; that is strongly reduced at 25°C. The temperature 60°C has been chosen as the most profitable. • The detection limit of GC-MS with butyric acid sample in methanol resulted 0.05 µM. The minimum detectable butyric acid concentration in the sample resulted 1.0 ppb. Bibliography: Plöger S. et al, Ann. N.Y. Acad.Sci,1258, 52-58,2012 Tacon, A., Metian M., Aquaculture, 146-158, 2008 Valero, E.; Villaseñor, M.J.; Sanz, J.; Martínez Castro, I. Chromatographia, 2000, 52, 5-6. Yaws C.L.; MacGraw-Hill. Chemical Properties Handbook, 1999.

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