3.2. HPLC-DAD validation
The HPLC method developed for this study achieved adequate separation among the eight standards, GV and 1–7, in 19 min, Fig. 2A. The linear regression parameters obtained for all eight calibration curves were optimal in the concentration range established for each compound (Table 2). The same could be argued for the sensitivity of the method, with LOD values in the range of 0.008–0.212 μg/ml and LOQ of 0.027–0.707 μg/ml. In terms of linear range, sensitivity, and HPLC run-time, the results are comparable to those achieved by two other validated HPLC methods available in the literature for the quantification of compounds 1–6 (Cicchetti and Chaintreau, 2009 and Sinha et al., 2007). However, this is the first validated HPLC method to assess GV and anisyl alcohol in the presence of aglycones 1–6. A good linearity was achieved for both of these compounds, GV and compound 7, over a wide concentration range, 1–1000 μg/ml, Table 2. Precision (retention time and peak area) was addressed through intra-day and inter-day repetitive analysis (n = 5) at four different concentrations per compound. In general, the results were satisfactory, displaying coefficient of variation (CV) below 2%, as shown in Table 3 for a particular concentration of each standard.