ИСТИНА

Ginseng is a very old Chinese medicine. Panax ginseng has been used in Asia for more than 5000 years as a tonic and a panacea. The pharmacological properties of Ginseng are generally attributed to its triterpene glycosides, called ginsenosides. Up to now more than 600 ginsenosides have been isolated from Panax species. But there are still several challenges in ginseng research. For ginsenoside profiling and quality control purposes HPLC methods combined with UV and ELSD are being used. Sample extraction and preparation techniques for P. ginseng roots should be improved and validated. Dealing with such challenges commonly requires the use of high informative MS detection techniques. The apparatus consisted of a HPLC Dionex Ultimate 3000 (USA), system with a binary analytical pump, an automatic sample injector coupled on-line with an AB Sciex Qtrap 3200 (Canada) mass spectrometer with an electrospray ionization interface. The HPLC separation LC separation was conducted on a C18 column (3 µm, 2.1 × 150 mm, RSLC 120 Acclaim) at a flow rate of 0.4 mL/min. Two solvents were used: (A) 0.5% HCOOH aqueous solution, and (B) MeCN. Good peak separation was achieved during 45 min of the chromatographic run in gradient elution mode. The column effluent was analyzed by ESI-MS in positive ion mode and the mass spectra were acquired and processed using the Analyst software (AB Sciex) and in-house software developed. A rapid single-run analytical approach based on high-performance liquid chromatography coupled with electrospray positive ionisation linear ion trap mass spectrometry (HPLC/ESI-LITMS) suitable to achieve a comprehensive characterization of ginsenosides – main bioactive compounds present in plant materials from Panax species and ginseng based products was developed. The meticulous study of the fragmentation of ginsenosides in the linear ion trap and its application for analysis of these compounds was made in this work. Main ions in the ESI-LITMS spectra were attributed to molecular adducts with sodium and potassium and fragments corresponding to cleavage of the glycosidic bonds. The MS/MS spectra of the [M+Na]+ ions of ginsenosides provide more structural information on the sapogenin and the attached sugar sequences of ginsenosides than similar spectra of [M-H]- ions. One chromatographic peak – one mass-spectrum pairs were used for the fast semi-automated identification of the analytes. Specific sapogenin fragmentation patterns were suggested for several ginsenoside groups. The parameters of the extraction procedure were optimized and the stability of the analytes during ultrasonication at 30 °C was shown. Mass-spectra obtained in EMS mode allowed to distinguish F11 and Rf chosen as biomarkers of P. Quinquefolius and P. ginseng correspondingly. The isomeric ginsenosides Rb3, Rc and Rb2 were identified by injection of the reference compounds. Saponin profiles of several root and marketed products extracts were compared. Quantitative evaluation was conducted using ginsenoside standards. The results of this study indicated that HPLC/ESI-LITMS is easily applicable for quality control purposes of marketed products and allows the rapid and direct identification of ginsenosides in crude plant extracts. Developed HPLC/ESI-LITMS approach allows to avoid sophisticated information depended acquisition and leads to the opportunity of fast screening of ginsenosides.