**2. Materials and methods**

### **2.1 HPLC sample preparation and analysis**

Golden Wulong (regular Oolong tea) and Organic GABA Body and Mind tea (GABA-enriched Oolong tea) both originated from Taiwan and were purchased from www.teas.com.au. Standard cups of each oolong tea were prepared and analysed to quantify both the differences in active constituent content between the teas and also the consistency of GABA, theanine, caffeine and EGCG extraction between separate brews of the same tea. Teas were prepared in accordance with manufacturer's instructions for consumption, by the addition of 5 g tea to 200 mL of 90°C deionised Milli-Q water (dH2O) and infused for 10 min.

Samples (20 mg) of the authentic standard compounds, GABA (RBI; Natick, MA, U.S.A.), theanine (Tocris; Ballwin, MO, U.S.A.), caffeine (Sigma, St. Louis, MO, U.S.A.) and EGCG (Sigma, St. Louis, MO, U.S.A.), were weighed accurately and added to a 100 mL volumetric flask before being dissolved in 12.5% acetonitrile, yielding final stock solutions of 200 μg/mL. Standard solutions in the range of 5.0–200 μg/mL were made by diluting the stock solution with 12.5% acetonitrile directly before each run. The standard solutions were filtered through 0.45 μm, 13 mm diameter HPLC nylon syringe filters (Grace Davison Discovery Science; Deerfield, IL, U.S.A.). Analysis was via reverse-phase HPLC using a Varian ProStar 210 solvent delivery system coupled to a Varian autosampler model 410 with cooling tray set at 4°C, Degassit degasser (Varian Inc.; Walnut Creek, CA, U.S.A.). Data were collected and analysed using Varian Star Chromatography Workstation, Interactive Graphic, System Control and Method Builder version 6.30 (Varian Inc.).

### **2.2 GABA and theanine measurements**

GABA and theanine were determined using pre-column derivatisation of standards and tea samples at the same time before each run for detection of amino acids. Immediately prior to derivatisation, 50 μL tea samples were mixed with 50 μL 0.2 M borate buffer (pH 8.5). To derivatise the amino acids, 100 μL 15.5 mM 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) was added and allowed to react for 1 min before addition of 60 μL cleavage reagent (0.5 M hydroxylamine hydrochloride: 1.7 M sodium hydroxide: water) to remove excess FMOC-Cl which can interfere with amino acid separation. The reaction was stopped after three minutes with 100 μL quenching

reagent (1:4 glacial acetic acid: acetonitrile). Each sample was then diluted 1:3 in water and filtered through a 0.45 μm HPLC nylon syringe filter (13 mm diameter). All samples were stored at 4 °C and analysed within 24 hours.

For amino acid detection, a Shimadzu RF-10AXL spectrofluorometric detector (excitation 263 nm; emission 313 nm) with a Varian Microsorb-MV 100 C18, 5 μm, 250 × 4.6 mm column maintained at room temperature were used.
