**2.4 Gradient elution**

All chromatographic experiments were performed using gradient elution. For GABA and theanine, optimised mobile phase A consisted of 2 M ammonium phosphate buffer (pH 6.7), methanol and water (0.75:15:84.25). Mobile Phase B was acetonitrile and water (90:10). For caffeine and EGCG, the composition of the optimised mobile phase A was water/acetonitrile/formic acid (94.7/4.3/1) and mobile phase B was water/ acetonitrile/formic acid (49.5/49.5/1). Each mobile phase was filtered through a 0.45 μm Teflon membrane filter under vacuum prior to use. The flow rate was 1.0 mL/min.

The gradient elution profile for GABA and theanine was 0 min 12% B, 5 min 30% B, 20 min 34% B and 22–27 min 99% B. The gradient elution profile for caffeine and EGCG was 0 min 1% B, 10 min 35% B, 13.3 min 90% B, 19.5 min 90% B, 21 min 1% B and 26 min 1% B. The injection volume was 5 μL and the detection wavelength was set at 254 nm. GABA, theanine, caffeine and EGCG were identified by comparison of the retention time of peaks produced by the tea sample against that of the respective GABA, theanine, caffeine and EGCG standards.
