Chromatographic analysis of salicylic compounds in different species of the genus Salix

The separation of nine phenol glycosides--salicin, salicortin, 2'-acetylsalicortin, populin, tremulacin, salidroside, triandrin, picein and helicin--by normal phase (NP), reversed phase (RP) HPLC techniques and a coupling of NP and RP monolithic silica columns was studied. Among the above nine compounds only five--salicin, populin, tremulacin, salidroside and triandrin--were resolved in an NP system with a mobile phase comprising hexane/isopropanol/methanol (87:12:1, v/v/v). Optimized separation was performed with two coupled monolithic silica columns of different polarity (bare silica and RP-18). The method was applied to verify the presence of salicylic compounds and other phenolic derivatives in the bark of six species from the genus Salix, namely S. purpurea, S. daphnoides clone 1095, S. alba clone 1100, S. triandra, S. viminalis, and S. herbacea. Gradient elution with a mobile phase composed of acetonitrile and water containing 0.05% of trifluoroacetic acid, with increasing acetonitrile concentration from 3% to 48%, was chosen as optimal. For the selective detection of the salicylic compounds, an evaporative light scattering detector was employed along with a UV detector. The differences in the composition of phenols in the different plant materials were confirmed. Additionally, it must be emphasized that for the first time the presence of 2'-acetylsalicortin was revealed in S. alba clone 1100. Furthermore, an SPE-HPLC method was developed for the rapid analysis of the salicin content, analyzed as free and total fraction, in willow barks. The determined concentrations of total salicin varied from 25.4 mg/g in S. alba clone 1100 to 96.47 mg/g in S. daphnoides clone 1095.