HPLC amino acid analysis was performed on an Agilent 1100 HPLC binary system (Agilent, Santa Clara, United States) equipped with an 1100 Fluorescence detector (FLD) and a Gemini C18 column (2 × 250 mm, 5 μm, Phenomenex, Torrance, United States). Borate buffer (0.4 M H3BO3) was used, and the mobile phases consisted of Solvent A (10 mM Na2HPO4 and 10 mM Na2B2O7, pH 8.2) and Solvent B (mixture of 45:45:10 acetonitrile/methanol/water). An aliquot of 1 μL derivatized sample (ad described above) was injected into the HPLC column equilibrated with Solvent A. The elution was carried out at a flow rate of 0.5 ml/min with the following program: from 0 to 0.5 min in 2% Solvent B, from 0.5 to 20 min gradient step to reach 57% Solvent B, from 20 to 20.1 min gradient step 57–100% solvent B, 20.1 to 23.5 min 100% solvent B, 23.5 to 23.6 min from 100 to 2% solvent B, and at 25 min ended.
The fluorescence detector (FLD) was set to Ex = 340 nm Em = 450 nm for the OPA derivatives. Quantifications of methionine were performed based on a five point calibration line between 5 and 500 μM. Data analysis was performed by using the Chemstation software to quantify methionine. The concentrations of methionine were obtained by measuring the FLD peak areas.
The fluorescence detector (FLD) was set to Ex = 340 nm Em = 450 nm for the OPA derivatives. Quantifications of methionine were performed based on a five point calibration line between 5 and 500 μM. Data analysis was performed by using the Chemstation software to quantify methionine. The concentrations of methionine were obtained by measuring the FLD peak areas.