The standard series used for construction of the calibration curve for caffeine was prepared in methanol. On the other hand, the calibration curve for GA was constructed by spiking GA standard in the extract itself. This was carried out in order to ensure perfect resolution and investigate possible matrix interference.
Calibration curves were constructed by plotting the integrated peak area versus the corresponding concentrations in the range of 0.20–2.40 μg/band and 0.50–5.00 μg/band for caffeine and GA, respectively. Second order polynomial regression was found superior to linear regression model for both standards with respect to assay range, goodness of fit and standard deviation of residuals. Regression equation parameters are summarised in Table 2. The concentration of caffeine, GA internal standard and EGCG expressed as GAE were calculated from the corresponding regression equations. The assay was validated according to ICH guidelines (ICH, 2005); results are summarised in Table 2. Calibration curves were employed in subsequent experiments to assess the stability of GT preparation.