Acquity ultra performance lc

The LC-MS/MS (Waters ACQUITY™ Ultra Performance LC coupled to Waters ACQUITY™ TOQ, Milford, MA, USA) used for identification and quantification of the analytes was equipped with a binary pump, vacuum degasser, thermostated autosampler, thermostated column manager. A Kinetex C18 LC column (50 × 2.1 mm, 2.6 μm, Phenomenex, Torrance, CA, USA) was used for chromatographic separation. MassLynx version 4.1 software (Waters, Milford, MA, USA) was used for instrument control, data acquisition and processing of results.
The LC conditions were as follow: mobile phase flow rate: 0.2 ml/min; column temperature: 40 °C, elution program: 0–1 min, 95 − 80% A; 1–4 min, 80 − 60% A; 4–8 min, 60–95% A; 8–12 min, 95% A; mobile phase A contains: water with 5 mM ammonium acetate, 0.01% formic acid and 0.01% trichloroacetic acid; mobile phase B contains: methanol with 0.1% formic acid, oven temperature: 4 °C; injection volume: 5 µl. The MS/MS conditions were optimized as follows: capillary voltage of 3.0 kV; source temperature of 150 °C; desolvation temperature of 400 °C; cone gas at 100 L/h; desolvation gas at 300 L/h. Both positive and negative electrospray ionization were used along with multiple reaction monitoring (MRM) [10 (link), 26 (link)].

ChemiDocTMMP imaging system with Image lab 5.2 analysis software (Bio-RAD, USA); SPR technique on Biacore T200 biosensor system (GE Healthcare, Madison, USA) with data acquisition software (Biacore T200 Evaluation Software Version 3.0); AcquityUltra Performance LC (Waters, Milford, MA, USA) with Applied Biosystems Triple Quad 5500 (ESI–MS/MS; Foster, CA, USA) in electrospray negative-ion multiple reaction modes; Ultrasonic cleaner (Ningbo Scientz Biotechnology Co., LTD., China); Milli-Q purification system (Millipore, Bedford, USA); Index Cutter-I slitter (Grand Island); IsoFlow dispenser (Imagene Technology, USA); The laser flashlight and the portable fluorescence strip reader (365 nm) were all purchased from Jiening Biological Technology (Beijing, China).

Total amino acid content in tea leaves was determined using the ninhydrin method described by Chen et al. (2017) [25 (link)]. Absorbance was recorded at 570 nm using a spectrophotometer.
Free amino acid contents were determined following the method described previously [26 (link)]. Finely powdered tea leaves (1.0 g) were extracted with 10 mL of 10% formic acid in methanol. After centrifugation (12,000 rpm, 10 min), the resulting supernatants were filtered and 5 μL of each sample was submitted to ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). Waters (Milford, MA, USA) Acquity Ultra Performance LC equipped with an AB 4000 triple quadrupole mass spectrometer was used to detect free amino acids.