For total amino acid analysis, psoas major muscles (~1 g) were hydrolyzed with HCl (10 ml, 6 mol/l) at 110°C for 22 h. Hydrolysates were transferred, filtered, and diluted with deionized water to volume. The filters were dried under nitrogen and the residue redissolved in HCl aqueous solution (2 ml, 0.02 mol/l) for further analysis. Amino acid concentrations were determined on an L-8900 amino acid analyzer (Hitachi, Tokyo, Japan). Identification of amino acids was performed by comparison with authentic standards.
For free amnio acid analysis, psoas major muscles (~100 mg) and 1 ml of sulphosalicylic acid (30 g/l) were mixed, homogenized, and centrifuged for 15 min at 14000 rpm at 4°C. The supernatant was vortex-mixed with hexane, centrifuged, and filtered through a 0.22 μm membrane. The filtrate was carried out an ACQUITY ultra-performance liquid chromatography (UPLC, Waters Corp., Milford, MA, United States) coupled with triple-quadrupole mass spectrometer (SCIEX QTRAP 6500, Framingham, MA, United States) in the electron spray ionization (ESI) mode. Two microliters of the samples were injected into LC–MS/MS system using an autosampler kept at 10°C. An ACQUITY UPLC HSS T3 column (2.1 × 100 mm, particle size 1.8 μm, pore size 100 Å) was applied to separate target analysts in meat samples. Mobile phase A was deionized water while mobile phase B was methanol. The 10-min gradient conditions were set as follows: 0–3 min, 2% B; 3–7 min, 2–95% B; 7–8 min, 95% B; 8–8.1 min, 95–2% B; 8.1–10 min, 2% B. The MS parameters was an ion-spray voltage of 4500 V, turbo gun source temperature of 400°C and curtain gas of 300 psi. Free amino acids were quantitated by external standard curves containing varying concentrations of reference standards.
For free amnio acid analysis, psoas major muscles (~100 mg) and 1 ml of sulphosalicylic acid (30 g/l) were mixed, homogenized, and centrifuged for 15 min at 14000 rpm at 4°C. The supernatant was vortex-mixed with hexane, centrifuged, and filtered through a 0.22 μm membrane. The filtrate was carried out an ACQUITY ultra-performance liquid chromatography (UPLC, Waters Corp., Milford, MA, United States) coupled with triple-quadrupole mass spectrometer (SCIEX QTRAP 6500, Framingham, MA, United States) in the electron spray ionization (ESI) mode. Two microliters of the samples were injected into LC–MS/MS system using an autosampler kept at 10°C. An ACQUITY UPLC HSS T3 column (2.1 × 100 mm, particle size 1.8 μm, pore size 100 Å) was applied to separate target analysts in meat samples. Mobile phase A was deionized water while mobile phase B was methanol. The 10-min gradient conditions were set as follows: 0–3 min, 2% B; 3–7 min, 2–95% B; 7–8 min, 95% B; 8–8.1 min, 95–2% B; 8.1–10 min, 2% B. The MS parameters was an ion-spray voltage of 4500 V, turbo gun source temperature of 400°C and curtain gas of 300 psi. Free amino acids were quantitated by external standard curves containing varying concentrations of reference standards.
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