4.4. Determination of Non-Volatile Compounds

Tea infusion preparation: Each dried yellow tea sample was ground using a mixer mill (FW100, Taisite Instrument Co., LTD, Tianjing, China), then sifted through a 450 μM sieve. The tea powder (0.15 g) was extracted with 25 mL of 50% aqueous ethanol at 70 °C for 30 min, and the mixture was centrifuged at 12,000 rpm and 4 °C for 15 min (3K15, Sigma-Aldrich, Milwaukee, WI, USA). The supernatants were collected for non-volatile compound analysis.

Determination of catechins and alkaloids: Catechins and alkaloids (caffeine, theobromine and theophylline) were determined by HPLC with an SPD ultraviolet detector (LC-20A, Shimadzu, Tokyo, Japan) according to a previous study [34]. The HPLC conditions were as follows: Agilent TC–C₁₈ column (4.6 × 250 mm, 5 μm, Agilent Technologies, Inc., Santa Clara, California, USA), column temperature 35 °C, mobile phase A = 1‰ formic acid +999‰ water (v/v) and mobile phase B = 1‰ formic acid + 999‰ acetonitrile (v/v), the linear gradient elution was as follows: Phase B increased from 20% to 40% during the early 45 min, and then maintained at 20% for 10 min for re-equilibrium. Injection volume was 10 μL, flow rate was 1.0 mL/min and the detection was at 360 nm.

Determination of flavones and flavonol glycosides. Flavones and flavonol glycosides were determined by UHPLC-DAD-MS/MS (Waters Corporation, Milford, MA, USA) according to the modified method from a previous study [35]. The chromatographic conditions were as follows: Waters CORTECS T3 column (2.1 × 100 mm, 1.6 μm, Waters Corporation, Milford, MA, USA), column temperature 25 °C, mobile phase A = 0.1% formic acid + 99.9% water (v/v) and mobile phase B = 100% acetonitrile (v/v). Gradient change of mobile phase B: 0–1 min, 0.2%; 1–2 min, 0.2%–10.8%; 2–5 min, 10.8%–15.7%; 5–9 min, 15.7%; 9–11 min, 15.7–16.0%; 11–12 min, 16.0%–16.5%; 12–18 min, 16.5%–18.3%; 18–20 min, 18.3%–60.0%; 20–20.01 min, 60.0%–0.2%; 20.01–21 min, 0.2%. Injection volume was 4 μL and flow rate was 0.15 mL/min. Mass spectrometry (MS) was carried out by electrospray ionization (ESI) technique in a negative ion mode. The ion source conditions were as follows: capillary voltage 3 kV, cone voltage 30 V, extractor 3.0 V, RF lens 0.2 V, ion source temperature 150 °C, desolvation gas nitrogen at a flow rate of 400 L/h and temperature 350 °C. Full scans ranging from 200 to 1000 amu were recorded. The wavelengths from 190 to 400 nm were detected by photodiode array detector (DAD), among which 360 nm was used for quantitative determination of flavones and flavonol glycosides. The flavonol glycosides without references were quantified by standard curve conversion of their corresponding mono-glycosides.

Determination of amino acids. The determination of amino acids was performed on HPLC with a fluorescence detector based on the pre-column derivatization method with O-phthalaldehyde (OPA) and fluolenylmeghyl chloroformate (FMOC), which was modified accord to previous study [36]. The chromatographic conditions were as follows: Zorbax Eclipse-AAA column (4.6 × 150 mm, 3.5 μm, Agilent Technologies, Inc., Santa Clara, CA, USA), column temperature 40 °C, mobile phase A = Na₂HPO₄ aqueous solution (40 mM, pH 7.8) and mobile phase B = 45% acetonitrile + 45% methanol + 10% water (v/v), injection volume 10 μL. Linear gradient change of mobile phase B: 0–18 min, 5%–60%; 18–23 min, 60%–100%; 23–30 min, 5%. The flow rate was 1.5 mL/min and the detection at 340 nm (excitation wavelength) and 450 nm (emission wavelength).

Determination of soluble sugars. The content of soluble sugars was determined by anthraquinone colorimetric method based on a previous study [37], with some modifications. The standard curve of glucose solution was used for quantitative analysis of soluble sugars in tea infusion. Tea infusion (100 μL) was mixed with 400 μL of water and 4 mL of anthrone sulfuric acid solution, and the mixture was placed in a boiling water bath for 7 min. After cooling at room temperature for 10 min, the absorption value of the mixture was determined by ultraviolet spectrophotometer at 620 nm. The soluble sugars were quantified as mg glucose equivalent/g dry weight.