Studies On Component Analysis, Metabolite Identification And Protein Binding Rate Of Related Components In Langdu And Its Preparation

Langdu is the roots of Stellera chamaejasme L., Euphorbia ebracteolata Hayata and Euphorbia fisheriana. It is widely distributed in the northeast, north and southwest of China. Langdu is highly toxic and is used medicinally for the treatment of inflammation, edema, skin disease, tuberculosis and tumor.Until now, studies on Langdu are mainly focused on the extraction and separation of certain chemical components, but there are few reports about the qualitative and quantitative analysis of the chemical compositions as well as metabolites in vivo in Langdu and its preparation.In the present study, we developed microwave-assisted extraction combined with UPLC-Q-TOF-MS/MS method for component identification of Stellera chamaejasme L. and summarized the mass spectrum fragmentations. The method could be used for the study of chemical composition and quality control of Stellera chamaejasme L. A sensitive UPLC-Q-TOF-MS/MS method for identification of scopoletin metabolites in rat urine and bile was established that could provide an important reference for application of scopoletin in clinic. A novel hollow fiber centrifugal ultrafiltration technique associated with HPLC was built for the determination of plasma protein binding of three coumarins. This method could provide effective means for the study of protein binding rate in therapeutic drug monitoring. At last, a preparation procedure for fast extraction and preconcentration of six trace components in “Jieheling” Tablet was developed by using a combination of microwave-assisted extraction(MAE) and ionic liquid-based dispersive liquid-liquid microextraction(IL-based DLLME) coupled with HPLC. The proposed method could provide a new means for quality control of “Jieheling” Tablet. Part 1 Identification of Components in Stellera chamaejasme L. by Microwave-assisted Extraction and UPLC-Q-TOF-MS/MSObjective: To establish microwave-assisted extraction and UPLC-Q-TOF-MS/MS for component identification of Stellera chamaejasme L. and summarize the mass spectrum fragmentations.Methods: The mass spectrum fragmentations of the standards were investigated in the positive and negative ion mode, respectively. The sample was analyzed and data was conducted by "Master View" in “Peak View 2.0”. The compounds in Stellera chamaejasme L. were identified by retention time, mass spectrum fragmentation and reference literatures. Chromatographic separation was performed on a Diamonsil C18 column(100 mm × 2.1 mm, 2.6 μm) at 25 °C. The mobile phase was composed of acetonitrile-0.1% formic acid and eluted at a flow rate of 0.3 mL/min by gradient elution. The injection volume was 2 μL.Results: A total of 37 compounds(10 coumarins, 25 flavones and 2 others) were identified. The proposed fragmentation pathways were explained and summarized.Conclusions: The proposed method was simple and rapid, which could be used for the study of chemical composition and quality control of Stellera chamaejasme L. It also provided an effective means for the component identification of Chinese medicinal herbs. Part 2 Identification of Scopoletin Metabolites in Rat Urine and Bile by UPLC-Q-TOF-MS/MSObjective: To establish UPLC-Q-TOF-MS/MS for identification of scopoletin metabolites in rat urine and bile and explore the main metabolism pathways of scopoletin.Methods: The samples were investigated in the positive ion mode. Chromatographic separation was performed on a Diamonsil C18 column(100 mm × 2.1 mm, 2.6 μm) at 25 °C. The mobile phase was composed of acetonitrile(0.1% formic acid)-0.1% formic acid and eluted at a flow rate of 0.3 mL/min by gradient elution. The injection volume was 5 μL. After administration of scopoletin, the urine and bile were collected from 0~72 h and 0~24 h, respectively. The blank and biological samples were prepared in parallel. The scopoletin metabolites were identified by Metabolite Pilot 1.5.Results: A total of 7 metabolites were identified(7 in urine and 3 in bile). The main metabolic pathways were demethylation, hydroxylation, hydrolization, glucuronidation and sulfatation.Conclusions: The proposed method was specific and sensitive, which could be used for research of metabolic process of scopoletin. It could provide an effective means for the discovery of new active compounds. Part 3 Pretreatment of Plasma Samples by a Novel Hollow Fiber Centrifugal Ultrafiltration Technique Coupled with HPLC for the Determination of Plasma Protein Binding of Three CoumarinsObjective: To establish a novel sample pretreatment method based on hollow fiber centrifugal ultrafiltration(HFCF-UF) to determine plasma protein binding of three coumarins(bergenin, daphnetin and scopoletin) by using HPLC.Methods: The hollow fiber was cut into 15 cm segments, sonicated in methanol and ultrapure water for 5 min respectively and dried at room temperature before use. The hollow fiber was bent to form a U-shape and placed into the glass tube to compose the HFCF-UF device. 900 μL of blank plasma was transferred into a 1.5 mL centrifuge tube and then spiked with 100 μL of standard mixture solution. After vortexing for 30 s, the sample tube was equilibrated in a water bath at 37 °C for 30 min. Then 400 μL of the prepared plasma sample was directly subjected to HFCF-UF at 10000 rpm for 10 min for analysis of unbound concentrations of the three analytes. While for the total concentration determination, another 400 μL of the above plasma sample was spiked with 150 μL of acetone. The mixtures were vortexed for 30 s and then subjected to HFCF-UF. The protein binding ratio was calculated according to the unbound and total concentrations. Chromatographic separation was performed on a Diamonsil C18 column(150 mm × 4.6 mm, 5 μm) at room temperature. The mobile phase was composed of methanol: 1% HAc buffer(pH 3.0)(35: 65, v/v), and eluted at a flow rate of 1.0 mL/min. The injection volume for all experiments was 20 μL. The wavelength was set at 327 nm.Results: The peaks of the three coumarins were well separated and no interference peak from endogenous compounds was shown at the retention time of the analytes. Excellent linearities were obtained in the validated concentration ranges. The correlation coefficients for every calibration curves were higher than 0.9996. The relative standard deviations(RSDs) for intraand inter-day precisions were lower than 3.3% and 3.2%, respectively. The relative recoveries ranged from 97.6% to 101.0%. The absolute recoveries were within 97.5~100.9%. The stabilities of the QC samples were investigated by maintaining the samples at room temperature for 24 h before analysis,-80 °C for 14 days,-80°C for 24 h and thawing at room temperature for three cycles. The RSDs were lower than 4.3%, 4.4% and 4.4%, respectively at three different concentrations.Conclusions: A novel sample preparation method to determine plasma protein binding based on HFCF-UF was developed. The unbound and total concentrations were determined in parallel by the same device. Acetone as a protein binding releasing agent was selected to release the drugs from protein-bound fraction in the plasma sample to allow the determination of the total concentration. Comparing with traditional method, the preparation procedure presented here was simple as it just involved common centrifugation with no special requirement and the filtrate could be injected directly for HPLC analysis without further treatment. Therefore, the recovery and reproducibility were superior to other sample preparation methods. The sensitivity of HPLC analysis was higher because the sample was not diluted too much. More importantly, the method could directly measure drug concentrations under near physiological conditions without significantly disturbing the binding equilibrium than the mostly used “classical” methods. The proposed method provided a reliable alternative for simultaneously determination of unbound and total drug concentrations as well as plasma protein binding in TDM. Part 4 Microwave-assisted Extraction and Ionic Liquid-based Dispersive Liquid-liquid Microextraction Followed by HPLC for Determination of Six Trace Components in “Jieheling” TabletObjective: To establish a simple, novel and efficient sample preparation procedure for fast extraction and preconcentration of six trace components, daphnetin, scopoletin, umbelliferone, daphnoretin, isopimpinellin and ebracteolata compound B in “Jieheling” Tablet by using a combination of microwave-assisted extraction(MAE) and ionic liquid-based dispersive liquid-liquid microextraction(IL-based DLLME) coupled with HPLC.Methods: The sample was prepared by microwave-assisted extraction and ionic liquid-based dispersive liquid-liquid microextraction(MAE-IL-based DLLME). Separations were accomplished on a Kromasil C18 column(150 mm × 4.6 mm, 5 μm) at room temperature. The mobile phase for the validated method was consisted of methanol(solvent A) and THF: 1% HAc buffer = 1: 40, pH 3.0(solvent B) using a gradient elution. The chromatogram was monitored at 325 nm. The mobile phase flow rate was 1.0 mL/min. The injection volume for all experiments was 20 μL.Results: A perfect separation was achieved and there was no interference at the retention times of the analytes. Excellent linearities were obtained in the validated concentration ranges. The correlation coefficients for every calibration curve were higher than 0.9993. The RSDs for measurements of intra- and inter-day precisions were lower than 4.9% and 4.9%, respectively. Measurements of recoveries of the analytes were in the range of 96.0~103.8% with RSDs lower then 4.0%. The enrichment factors were found to be 43, 47, 43, 41, 42 and 45 for daphnetin, scopoletin, umbelliferone, daphnoretin, isopimpinellin and ebracteolata compound B, respectively.Conclusions: The simplicity, facility, low solvent consumption, high sensitivity, short analysis time and high enrichment factors were clear advantages of the suggested approach. The proposed method provided an effective means for quality control of “Jieheling” Tablet and could be further applied for quality control in traditional Chinese medicines and their preparations.