Ab 4000 triple quadrupole mass spectrometer

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.

The concentration of ghrelin in serum was determined according to the kit instructions (Jiancheng Bioengineering Institution, Nanjing, China). The determination of serum amino acid concentration was completed by Suzhou panomic Biomedical Technology Co., Ltd. (Suzhou, China). Briefly, 200 μL serum were transferred into a 2 ml EP tube, and then fully mixed with 400 μL 10% formic acid methanol-H₂O (1:1. v/v) solution, followed by centrifugation at 12,000 rpm at 4 °C for 5 min. Next, after mixing 10 μL centrifuged supernatant with 490 μL 10% formic acid methanol-H₂O (1:1. v/v) solution, we took 100 μL diluted samples and added 100 μL internal standard (13 C-labelled Phe, 100 ppb) into them. Finally, the mixed solution were filtered with the 0.22μM-pore-size membrane for subsequent detection by the liquid chromatography-tandem mass spectrometric (LC-MS) method. The LC-MS instrument consisted of a Waters ACQUITY UPLC system (Waters, Milford, Massachusetts, USA), equipped with an analytical C18 column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA, USA) and an AB 4000 triple quadrupole mass spectrometer.