Study On Chemical Constituents And The Quality Control Of Traditional Chinese Medicine Injection

This paper mainly investigated the chemical components in Shuangshen injection for the quality control of injections. Chemical components in Shuangshen injection are the main and important points studied. The best extracting method was selected according to the amount of multiple components. The best chromatographic condition was chosen through optimizing the resolution of liquid chromatographic peaks. High performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-ESI-TOF-MS and HPLC-ESI-MSn were used for isolation, identification and structural analysis of the constituents in Radix Salvia miltiorrhizae and Panax ginseng. Basing on the fundamental study of chemical components, the peaks in the chromatograms can be qualified unambiguously. The fingerprints of crude drugs, intermediate and injection can be performed, which can be used for the effect quality control of the injection. The paper can be mainly divided into two sections:Part I The qualification and analysis of the crude drugs, intermediate and Shuangshen injectionThe extracting conditions of Dan-shen and Ren-shen were systematic optimized in the experiments. The crude drugs of Dan-shen was selected basing on the indexes of seven target components including extraction method, extracting time, extracting solvents and extracting times. The Dan-shen samples including both water-soluble phenolic compounds and nonpolar diterpenoid compounds were prepared by sonication in 70% methanol. Ren-shen samples were extracted by the method of Pharmacopeia 2005(I). The two samples were then detected in a gradient HPLC run. In this study, two ion-detecting techniques: High resolution time of flight mass spectrometry and ion trap techniques were used. The liquid chromatograms acquired by DAD, total ion chromatograms (TIC) from TOF-MS and ion trap were compared. Each analytical technique had different advantages and disadvantages in identifying the constituents in Dan-shen and Ren-shen. The various techniques, which were used in turn for the isolation, identification and structural elucidation of these compounds, could provide complementation for the identification of constituents in Radix Salvia miltiorrhizae. Forty components were identified in 30min based on the corresponding positive-ion and negative-ion ESI mass spectra and liquid chromatographic information in Dan-shen. And 42 compounds were fundamental qualified according to the HPLC-DAD and HPLC-TOF/MS in Ren-shen. The salvianolic acids were appeared between 0-15min, and the tanshinones appeared between 15-30min. The fragmentation routs of the detached [M-H]- ions of Protocatechuic acid, protocatechualdehyde, caffeic acid, rosmanric acid and salvianolic acid B were investigated. The fragment ion [M-H-44]- corresponding to the loss of"CO2"indicated the existence of carboxyl group in the structure. The fragment ion [M-H-28]- resulted from the loss of CO corresponding to the carbonyl group in the structure. Rosmarinic acid is the dimer of caffeic acid, and salvianolic acid B is the tetramer of caffeic acid and danshensu according to the structures. Therefore, the neutral loss of 198 and 180 corresponded to the molecule of danshensu (198) and caffeic acid (180). The characteristic components in Panax ginseng was ginsenosides. It was reported that the ginsenosides could be divided into three kinds including protopanaxadiol, protopanaxatriol and oleanane-type ginsenosides. The three ginsenosides showed their characteristic dissociation rules in the ion trap mass spectrometry. Forty components in Dan-shen including 22 salvianolic acids and 18 tanshinones were identified in 30min through systematic study, depending on three analytical techniques. Forty-two ginsenosides in Ginseng were detected using the method in pharmacopeia(2005), in which thirty ginsenosides were identified unambiguously. The chemical components were classified after systematic study in the chemical components in the two drugs, and identified using the TOF/MS and ion trap mass spectrometry. It can provide reliable basis for the fingerprints of crude drugs, intermediate and injections.Part II The study of fingerprints of crude drugs, intermediate and injection of Shuangshen injection.The chromatographic peaks were clearly identified after identification and structural elucidation for crude drugs, intermediate and injection. The qualitative identification and structural elucidation of the chemical constituents in Danshen provided essential scientific data for further pharmacological and clinical study. Both HPLC-DAD- TOF-MS and HPLC-DAD- MSn methods can provide more information about compounds in Danshen than the single method. It is suggested that the fingerprints of Danshen should be acquired by HPLC-UV, HPLC- TOF-MS and HPLC- MSn. Several techniques of HPLC-DAD, HPLC-TOF-MS and HPLC-MSn, which were in turn used for the isolation, identification and structural elucidation of these compounds, could provide complementation for identification of the constituents in Danshen. The fingerprints of crude drugs, intermediate and injections can provide demonstrating study for the fingerprint study and quality control of herbal medicines.