Amino acids were quantified by HPLC after an acidic hydrolysis according to Dhillon, Kumar, and Gujar [66 (link)] and using an AccQ-Tag reagent kit from Waters (Milford, MA, USA) for derivatization of amino acids.
Each RAs sample (ca. 30 mg) was hydrolyzed with 4 mL of HCl and 15 μL of phenol at 110 °C for 24 h, and then entrapped in N2 atmosphere. The hydrolyzate was filtered through syringe filters (0.45 μm), and then dried with N2. Next, 10 μL of the sample was mixed with 70 μL of borate buffer (pH 8.2–9.0), then 20 μL of 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate acetonitrile solution (3 mg/mL) were added to the mixture (AccQ-Tag reagent kit, Waters, Milford, USA). Analogous procedures were used in the case of standards.
The amino acid profile (AAs) was identified on a Dionex Ultimate 3000 HPLC instrument (Thermo Scientific, Germering, Germany). Separation was provided on a reverse-phase Nova-Pak C18 column (4 μm, 150 × 3.9 mm) (Waters, Milford, MA, USA) at 37 °C. Elution was run in a two-component gradient at 1 mL/min; eluent A: acetic-phosphate buffer, eluent B: acetonitrile-water (60:40). The detector (VWD-3400RS) was set at 240 nm wavelength. The AAs peaks were computed from AAs standards (Sigma-Aldrich, Poznan, Poland) run at five concentrations. Individual AA values were expressed as mg/100 g of fresh weight of RAs.
Each RAs sample (ca. 30 mg) was hydrolyzed with 4 mL of HCl and 15 μL of phenol at 110 °C for 24 h, and then entrapped in N2 atmosphere. The hydrolyzate was filtered through syringe filters (0.45 μm), and then dried with N2. Next, 10 μL of the sample was mixed with 70 μL of borate buffer (pH 8.2–9.0), then 20 μL of 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate acetonitrile solution (3 mg/mL) were added to the mixture (AccQ-Tag reagent kit, Waters, Milford, USA). Analogous procedures were used in the case of standards.
The amino acid profile (AAs) was identified on a Dionex Ultimate 3000 HPLC instrument (Thermo Scientific, Germering, Germany). Separation was provided on a reverse-phase Nova-Pak C18 column (4 μm, 150 × 3.9 mm) (Waters, Milford, MA, USA) at 37 °C. Elution was run in a two-component gradient at 1 mL/min; eluent A: acetic-phosphate buffer, eluent B: acetonitrile-water (60:40). The detector (VWD-3400RS) was set at 240 nm wavelength. The AAs peaks were computed from AAs standards (Sigma-Aldrich, Poznan, Poland) run at five concentrations. Individual AA values were expressed as mg/100 g of fresh weight of RAs.