DPP-4 the same Chinese name and are often used in confusion. Hence, it is vital to develop a reliable method of differentiating the three species. However, up to now, studies of RSG have focused on the determination of individual or a few components. Previously, we have developed capillary electrophoresis fingerprinting of RSG. Although CE has the advantages of short analysis time and lack of organic solvent, HPLC is a more commonly used separationtechnique at present and is easily coupled to other techniques, e.g. MS. In the present study, HPLC diode array detection MS/MS was used to identify the chemical constituents and to develop a fingerprint of RSG. Eighteen batches of authenticated RSG were collected from different regions of China for analysis. Nine peaks found in the chromatogram of RSG were all identified. With the fingerprint, RSG and its two confusable JNK signaling pathway species could be easily distinguished. Materials and methods Chemicals and materials HPLC grade acetonitrile and methanol were purchased from RCI Labscan LTD. Throughout the study Milli Q deionised water was used.
Trans resveratrol and taxifolin were purchased from S1P receptors Sigma Chemical Co. Astilbin, 5 O caffeoylshikimic acid and engeletin were purified from RSG by preparative HPLC in our laboratory and their structures were identified by UV, IR, MS and NMR. The detailed isolation procedures and spectra data are provided as Electronic Supplementary Material. Samples of RSG were collected from different areas of China, and were authenticated by Prof. Zhong Zhen Zhao. Three batches of RSG commercial concentrated extract from Taiwan were the products of Kaiser Pharmaceutical Co, Ltd, Sheng Chang Pharmaceutical Co, Ltd. and Sun Ten Pharmaceutical Co, Ltd. Samples of RSC01 and Rhizoma Heterosmilacis were supplied by the National Institute for the Control of Pharmaceutical and Biological Products. The other RSC sample was collected from Ninghua county, Fujian province, China in October 2009. HPLC analysis HPLC analyses were performed on a Waters 600 HPLC system together with a photodiode MG-341 array detector. An Agilent Zorbax SB C18 column was used. The mobile phase consisted of acetonitrile and 0.1% acetic acid aqueous solution using a linear gradient program of 16 21% in 0 15 min, 21 40% in 15 40 min. The flow rate was 1 ml/min and the injection volume was 10 ll.
UV spectra were recorded between 200 and 400 nm and HPLC chromatograms were plotted at 291 nm. All chromatography was carried out at room temperature. HPLC MS/MS analysis An Agilent 1100 system, consisting of a vacuum degasser and a quaternary pump was used. An AB MDS Sciex API 2000 triple quadrupole mass spectrometer equipped with a Turbo IonSpray electrospray ionization source was used for mass damage analysis and detection. Data acquisition was performed with Analyst 1.1 software. An Agilent Zorbax SB C18 column was used. The flow rate was 1 ml/min and about 35% of eluent was introduced into the ESI source after split. The mass spectrometer was operated in the negative ion mode. Ultrapure nitrogen was used as nebulizer, curtain, and collision activated dissociation gas at 20, 10 and 1, respectively. The optimized Turbo IonSpray voltage and temperature were set at 4500 V and 400C, respectively. Preparation of sample Dried RSG was finely homogeniz