4.4. Analyses of Free Amino Acids in Tea Samples

l-Theanine, l-glutamine, and l-glutamic acid were detected using an ultra-performance liquid chromatography/quadrupole time-of-flight (UPLC−QTOF-MS) system (Waters, Milford, MA, USA) as described previously [25]. Metabolites were separated on a HSS T3 column (1.8 μm, 100 mm × 2.1 mm; Waters, Milford, MA, USA) at 40 °C, with two elution binary gradients at a flow rate of 0.25 mL/min. The mobile phase consisted of water as solvent A containing 0.1% formic acid and acetonitrile as solvent B containing 0.1% formic acid with a gradient program as follows: 0−5 min, 100% A; 5−5.1 min, 100% A to 10% A; 5.1−10 min, 10% A and 90% B; 10−10.1 min, 10% A to 100% A; 10.1−13 min, 100% A. The injection sample volume was 5 μL. The eluted solutions were detected using the following instrument settings in positive ESI mode: capillary voltage was set to at 2 kV with the sampling cone voltage set at 50 V. The source and desolvation temperatures were set at 100 and 300 °C, respectively. The cone and desolvation gas flow rates were set at 50 and 600 L/h, respectively. Data were analyzed by using Mass LynxTM software. The characteristic ions for l-theanine, l-glutamine, and l-glutamic acid are m/z 175.1083, 147.0770, and 148.0610, respectively.

The method of other free amino acids analyses was referenced from a previous study [73]. The total amount of 100 mg fresh tea samples was extracted with 0.5 mL precooled methanol through ultrasonic extraction for 10 min. For phase separation, 0.7 mL chloroform and 0.2 mL water were added into the centrifuge tubes, and the mixture was centrifuged at 5000× g for 10 min. The upper layer was dried using a centrifugal concentrator under vacuum (45 °C, 1400 r/min) and then was dissolved with 5% sulfosalicylic acid (1 mL), standing for 1 h. The solution was centrifuged at 5000× g for 10 min and the upper layer was filtered through a 0.22 µm nylon filter membrane. The content of amino acids was determined using an automatic amino acid analyzer (Sykam S-430D, Eresing, Germany). The mobile phase was lithium citrate pH 2.9, pH 4.2, and pH 8.0 with the flowrate of 0.45 mL/min, and the flowrate of the derivatizating reagent was 0.25 mL/min. The instrument parameters were set as follows: automatic injector (5 °C), column temperature (38 °C), post column reaction equipment (130 °C), UV-Vis detection wavelengths (570 nm and 440 nm), and injection volume (50 μL).