| Recently, the traditional Chinese medicine (TCM) has attracted more and more attention for their therapeutic effects, which has accelerated the modernization process of TCM. Two aspects must be realized during the modernization of TCM. First, TCM is a complicated system, which contains a number of bioactive compounds. Herbal medicinal preparations are combined mixtures of herbal medicines. In order to ensure their maximal therapeutic effect, it is necessary and important to develop a simple and sensitive quality control method for TCM and the herbal medicinal preparations. Second, the bioactive compounds in TCM are the precursor of the new drug. However, the pharmacokinetics of the bioactive compounds is not very clear. So the method of determination of the bioactive compounds in biological sample is urgent to establish. The proposed method can applied to the pharmacokinetic studies. Based on the above situation, this thesis was concentrated on the quality control of the TCM and the determination of bioactive compounds in biological samples. The details are summarized as follows:(1) Based on molecularly imprinted solid-phase extraction, a new molecularly imprinted polymer (MIP) was prepared by using epicatechin as the template molecule. The synthesized MIP was characterized by infrared spectroscopy and scanning electron microscopy and the MIP with 15-38μm particle size was selected as a solid-phase extraction (SPE) sorbent. It showed good selectivity and affinity for epicatechin in contrast to the structurally related compounds. The MIP cartridge enabled the selective extraction and preconcentration of epicatechin from tea samples.(2) A rapid, specific, and reproducible high-performance liquid chromatography (HPLC)-photodiode array detection (DAD)-electrospray ionization mass spectrometry (ESI/MS) method has been developed for determination of geniposide in Gardenia jasminoides and its preparation. For full quality control of herbal medicinal preparations, the multiple bioactive compounds were analyzed. Dehydroandrographolide, andrographolide and geniposide are used as the markers to control the quality of such herbal preparations. A simple and sensitive high-performance liquid chromatographic (HPLC) method coupled with photodiode array detection (DAD) and electrospray mass spectrometry (ESI/MS) was developed to determine the three compounds simultaneously in the extracts of medicinal herbs and herbal preparations. Compared with the previously reported method, the proposed method is more simple and effective.(3) The major active biological constituents in Citrus herbs are flavonoids and alkaloids, especially hesperidin, naringin and synephrine. However, they can not be detected in one run due to their different chromatographic retention behaviors. In this thesis, perfluorinated carboxylic acids, which are appropriate for MS detection due to their volatility, were used as ion pairing agents. The problems of the synephrine separation, such as band tailing and low retention were solved successfully. A new ion pairing chromatographic method was developed to exclude the most polar solute (synephrine) from the void volume and to maintain selectivity among the two other solutes (hesperidin, naringin). The two different kinds of the analytes were well separated in a single run using an isocratic eluent. The method is employed successfully to determine of hesperidin, naringin and synephrine in several Citrus herbs.(4) High cone voltage was used to improve the quantification sensitivity of large molecular weight compounds in HPLC-ESI/MS, with ginsenosides as example. Investigations on the effect of cone voltage showed that within a voltage range of 3-?130 V, for all the ginsenosides tested, i.e., Rb1, Rb2, Rc, Rd, Re, Rf and Rg1, the increase of the applied cone voltage can significantly increase the sensitivity of the method. At the high cone voltage, both the molecular weight and structural information `were obtained from a single mass spectrum. It can also be used for isomer differentiation and structure determination of ginsenosides. Linear relationships between the peak area response and concentration were observed in the range of 50-2×10~5 ng/mL, with the correlation coefficients >0.99. The limits of detection reached down to pg for ginsenosides. The method was successfully applied to the determination of ginsenosides in commercial ginseng samples. It offers a simple and effective method to quality control of the ginseng.(5) Based on the unique capacity of magnetite beads to absorb microwave radiation, a new magnetite nano-beads based microwave-assisted (MBMAE) technique was developed to improve the extraction of bioactive compounds from plant tissues, Scutellaria baicalensis Georgi and its herbal preparations were taken as application examples. In the presence of magnetite beads, 40 s microwave irradiation, was sufficient for carrying out the whole extraction process, Furthermore, it yields a higher extraction efficiency compared with the conventional microwave extraction technique and a lower consumption of organic solvent. The extracts were analyzed by high performance liquid chromatography–electrospray ionization mass spectrometry. The recovery of this method was in the range of 97.6–101.4% with relative standard deviation lower than 2.5 %. The limits of detection were 0.05, 1.0 and 2.0 ng/mL for wogonin, baicalein and baicalin, respectively. The present method has been successfully applied to the extraction determinination of wogonin, baicalein and baicalin in real samples. The results demonstrated the powerful application foreground of the proposed method in analysis of bioactive compounds contained in other Chinese herbal medicine.(6) In order to protect the animal, decrease or replace the animal experiments, the in-vitro assessment of the toxicity of the drug using the cell mode is urgently. Many clinical researches showed that CPT-11 is useful in the treatment of many types of cancer, such as colorectal, ovarian and small cell lung cancers, while there are few reports about the treatment of breast tumors. Human breast cancer cell line MCF-7 was selected as a model to test the potent anticancer activity of CPT-11 in vitro. A a simple, rapid and sensitive method of liquid chromatography-electrospray mass spectrometry has been developed to assay the concentrations of anticancer drug irinotecan in human breast cancer cells. A single step protein precipitation with the -80℃methanol was used for sample preparation. High specificity and little matrix effects was obtained by this method. After exposure to irinotecan at different time interval, the intracellular drug concentration was successfully determined by the proposed method. The pharmacokinetics of irinotecan in human breast cancer cells was investigated.(7) The sample pretreatment is the key step in the biological sample analysis by HPLC. The sample pretreatment contains the protein precipitation and the analyte extraction, thus the operation is relatively laborious and time-consuming. Meantime, the recovery is low. A simple and sensitive micellar liquid chromatography (MLC) method is proposed for the determination of Cryptotanshinone (CT) and tanshinone IIA (TS) in biological samples. No extraction step is required. A C18 column and a micellar mobile phase of 0.15 M SDS and 8% n-butanol were used. CT and TS are the major active compounds contained in traditional Chinese medicine Radix salvia miltiorrhiza (Danshen), with powerful pharmacological activities. Satisfactory recoveries in the biological matrices were achieved for CT and TS. Compared with the conventional reversed-phase chromatography method, the proposed MLC method is more sensitive, time-saving and less organic solvent is required. The proposed method could be used for the pharmacokinetic studies of the CT and TS. |
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