[Show abstract][Hide abstract] ABSTRACT: The chemical profile of four main bioactive constituents including harpagoside (HS), 8-O-(E-p-methoxycinnamoyl)harpagide (HG), E-cinnamic acid (CA), and E-p-methoxycinnamic acid (MCA), in the extract of Scrophularia buergeriana was studied using HPLC-diode array detector-electrospray-MS (HPLC-DAD-ESI-MS). The HPLC-DAD conditions were optimized for the simultaneous analysis of these four compounds. This method was validated in terms of specificity, linearity (r(2) >0.9998), precision (< 2.0% RSD), and recoveries (94.4-115.1%). The LOD of these compounds were ranged from 5.9 to 37.8 ng. In addition, the main compounds in S. buergeriana and S. ningpoensis were quantified using this validated method, which resulted in significant difference in the contents of these compounds between the species.