Volatile Compound Analysis in Fish Fillets

The volatile compound content was determined in fresh and refrigerated fillets by headspace solid-phase microextraction coupled with gas chromatography and mass spectrometry (HS-SPME-GC-MS). To perform the analysis, 1 g of sample was weighed into a 10 mL screw-capped vial, and 0.5 mL of a 4 mg/L aqueous solution of 4-methyl-2-pentanol (97%, Sigma-Aldrich®, Merck KGaA, Darmstadt, Germany) was added as internal standard. Subsequently, 0.5 mL of an aqueous antioxidant solution with 4% of EDTA and 0.4% of propyl gallate (both from Sigma-Aldrich®, Merck KGaA, Darmstadt, Germany), 2 mL of double deionized water, and three glass balls were added. The vial was immediately closed and kept in ice until all sample set was prepared. Then, the mixture was homogenized using an ultrasound bath at 4°C for 10 min. Samples were kept in ice at the dark until the HS-SPME-GC-MS determination was carried out. The instrument consisted of an Agilent 6890N Network GC system with an Agilent 5975C Inert MSD quadrupole mass spectrometer (both from Agilent Technologies Santa Clara, CA, USA) and a PAL autosampler (CTC Analytics, Zwingen, Switzerland) configured to perform SPME. After 10 min of sample conditioning at the extraction temperature (45°C), the fiber of divinylbenzene/carboxen/polydimethylsiloxane (2 cm length, 50/30 thickness) from Supelco® (Merck KGaA, Darmstadt, Germany) was exposed to the head space for 30 min and desorbed in the injector at 260°C for 10 min. To perform the separation of the different volatile compounds, a Supelcowax-10 capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) from Supelco® (Merck KGaA, Darmstadt, Germany) was used. The oven temperature program began at 40°C (held 10 min, during fiber desorption time), 3°C/min up to 150°C, and 15°C/min up to 250°C (held for 5 min). Helium was used as gas carrier with a constant flow of 1 mL/min. The temperatures of the ion source and the transfer line were 230 and 280°C, respectively, and the ionization energy was 70 eV. Data were acquired in full scan mode in selected representative samples for the identification of compounds, which was carried out by comparison of their mass spectra and retention times with those of standard compounds or with those available in mass spectrum library Wiley 6 and in the literature. Then, the quantitative assessment of all samples was carried by selected ion mode, considering m/z 44, 45, 55, 56, 57, 58, 70, 81, and 98, which were representative for the compounds of interest. Data were then analyzed by an Agilent MSD ChemStation. Relative amounts of volatile compounds were calculated by the internal standard method, expressing the results as μg of 4-methyl-2-pentanol equivalents/kg of sample.