Liquid chromatography refers to the chromatographic technique in which the mobile phase is liquid. It is the oldest one in chromatography. However, by improving the particle size and column pressure of the filler, a gas chromatographic column is introduced on the basis of classical liquid chromatography. Plate theory, the technology uses high-pressure infusion pump, high-efficiency stationary phase and high-sensitivity detector, which realizes fast analysis, high separation efficiency and automatic operation. This chromatographic technique is called high performance liquid chromatography (HPLC). . The composition of traditional Chinese medicine is very complicated. The conventional methods such as thin-layer chromatography cannot meet the needs of modern Chinese medicine because of its precision, accuracy, sensitivity and reproducibility. High performance liquid chromatography is the most important analytical method for traditional Chinese medicine research because of its stability, reliability and high efficiency. At present, high performance liquid chromatography has been widely used in the determination of active ingredients of various traditional Chinese medicines such as alkaloids, saponins, flavonoids, quinones and coumarins. In recent years, there have been many studies on the monitoring of traditional Chinese medicines by high performance liquid chromatography. Because of the advantages of high performance liquid chromatography and classical liquid chromatography and gas chromatography, they have reached or exceeded them in terms of efficiency, selectivity and analysis. The deficiencies of high performance liquid chromatography have been improved to make this technology more sophisticated.
1 Recent developments in high performance liquid chromatography
The application of high performance liquid chromatography in drug analysis mainly considers the pretreatment of the sample and the selection of the analytical column and detector. In the pretreatment of the sample, the solid phase (micro) extraction that has arisen recently has made many low-content components refined and purified, which is suitable for the determination of high performance liquid chromatography. Sun Xinguo has used the countercurrent extraction to determine the content of ligustrazine. Effect. Some of the traditional Chinese medicines have weak UV absorption or no characteristic UV absorption. They are directly determined by high performance liquid chromatography. Their sensitivity and resolution are not satisfactory. These components can be accurately determined by pre-column or post-column derivatization. come out. For the polar and fat-soluble substances, it is not easy to retain on the YWGCl8 column. The sodium dodecyl sulfate is used as the ion-pairing reagent to reduce the polarity and prolong the retention time on the column, so that better separation is achieved. The two powerful analytical techniques of liquid chromatography and mass spectrometry are linked together online. After 30 years of development, it has become a relatively mature analytical tool, but it has problems since its formation: from liquid chromatography When entering mass spectrometry, the changes of mobile phase, the composition of solvent, and the problem of high temperature and high pressure ionization restrict the development of this combined technology. The atmospheric pressure ionization interface has the dual functions of removing solvent and ionization, and its introduction makes the technology Various fields have been widely used. Electrospray ion source is a soft ionization technique. It generally generates only two molecular ion peaks (M+H)+ and (MH)-, selectively monitors the negative molecular ion peak of (mz) 190, and has high sensitivity. Accuracy and specificity meet the needs of low-concentration drug research. The three-dimensional high performance liquid chromatography method studied by Zhang Li et al. can simultaneously determine two indicators of puerarin and ferulic acid. It is proved by experiments that if the appropriate column temperature and other chromatographic conditions are selected, ethanol is used as the mobile phase of reversed-phase high performance liquid chromatography to analyze the active ingredients in traditional Chinese medicines and proprietary Chinese medicines, which is safe and accurate. A substance with similar structure is difficult to detect by ordinary detectors. High performance liquid chromatography-electrochemical method can effectively determine the content of baicalin and baicalein which are only one group in the berberine powder. The sample is collected after separation by chromatography, and the fluorescence intensity is measured by fluorescence spectrophotometer. The influencing factors are numerous and the measurement is complicated. The improved high performance liquid chromatography-fluorescence method can be derivatized and collected without chemical separation. Miscellaneous, can be directly injected after concentration. Good results were also obtained by measuring the content of hypericin in Hypericum perforatum L. by this method. High performance liquid chromatography-differential refractive index measurement of astragaloside IV in Huangqijing oral liquid has also achieved satisfactory results. For the ultraviolet only absorption, the sensitivity is low when UV detection, the baseline is easy to drift, and the refractive index detection is easily interfered by external conditions. The evaporative scattering detector can overcome the above deficiencies, and the response value is only related to the sample quality, and the signal is corresponding to the quality. In proportion, different compounds, the same quality, the signal is basically the same. The evaporative light scattering detection method is based on the fact that the degree of scattering of light by a non-volatile sample molecule is proportional to its mass, regardless of the nature of the group it contains. By selecting the appropriate detector parameters, the mobile phase and solvent are completely vaporized without generating a signal, and the individual components of the sample are present as non-volatile particles that scatter light to be detected. Therefore, the evaporative light scattering detector can be used for simultaneous separation and analysis of multiple components containing different groups. Compared with ultraviolet and fluorescence methods, evaporative light scattering detection has approximately the same response factor for different substances.
Therefore, there is no low concentration, high response or high concentration, low response, which is conducive to accurate determination of mixtures in different proportions. Determination of terpene lactone content, red ginseng and ginseng in Ginkgo biloba leaves by high performance liquid chromatography-evaporative light scattering test Ginseng in fine capsule
Both the content of saponin Rg1 and Re and the content of gambogic acid in garcinia have obtained good results.
2 Research trends in high performance liquid chromatography
2.1 Reduce analysis time and improve separation efficiency. The use of advanced detection instruments and methods to adjust or change the mobile phase and stationary phase, gradient elution, column switching technology is expected to solve this problem. Gradient-eluting high-performance liquid chromatography, which can analyze samples with a wide range of polarities, has great advantages over isocratic elution, but it is somewhat inconvenient for traditional Chinese medicine samples with more complex components and wider polar ranges. Multi-column high performance liquid chromatography is also known as multidimensional high performance liquid chromatography. In addition to the advantages of changing the concentration of the mobile phase with gradient elution, the type of stationary phase, the degree of bonding, the particle size, the length of the column, the diameter of the column, and the type and concentration of the mobile phase can be changed.
2.2 Further development towards automation, intelligence and joint technology. The combination of liquid chromatography and mass spectrometry has become the first choice for the determination of drugs and metabolites in low-concentration biopharmaceuticals. The LC-MS-MS method is used to determine the HIV-1 protease in plasma, which is accurate and efficient, and the endogenous group in plasma. Parts and other drugs do not interfere with the assay, saving material and saving time. Has been applied to the analysis of drugs in body fluids, plasma, serum. The analysis of cholic acid in Chinese herbal compound injection "Qing Kai Ling" was carried out by liquid chromatography-mass spectrometry and mass spectrometry. High performance liquid chromatography-nuclear magnetic resonance (NMR) has a very good role in drug analysis. The newly proposed intelligent multi-column HPLC system utilizes the modular separation performance of the switching technique to switch the sample into blocks of different properties and elute with a suitable mobile phase. Intelligent control is used throughout the process.
3 Application prospect of high performance liquid chromatography in traditional Chinese medicine analysis
The general trend of traditional Chinese medicine research is full-component analysis. Through the study of different compatibility and decoction time from single-flavor medicine to compound, we can discover the changes of chemical composition in traditional Chinese medicine and find the organic connection between Chinese medicine mechanism. There are many kinds of traditional Chinese medicines, and the properties of various ingredients are spread over all polar segments and acids and bases. The easiest way to achieve multi-component analysis is to use a mild mobile phase on a long enough column to elute for a sufficient amount of time. But this is contrary to the simplicity and speed of modern analytical requirements. A large number of applied studies have shown that high-performance capillary electrophoresis has great advantages in the analysis of traditional Chinese medicine components, especially in the analysis of high-polarity chemical components, and shows a high ability in the analysis of a large number of compound preparations. Since the capillary tube is almost free of bed contamination that often occurs in high performance liquid chromatography, and the used capillary column is rinsed in a short time, the second sample can be analyzed, fast and efficient, and resolution. Very high. The emerging capillary electrochromatography is a new type of electro-separation microcolumn liquid chromatography technology that combines the advantages of high performance liquid chromatography and capillary electrophoresis. It is to move various filler particles of high performance liquid chromatography into the capillary to sample and The interaction between the stationary phases acts as a separation mechanism, with electroosmotic flow as the driving principle of the mobile phase. Recently, some advanced detection instruments have been successfully used in high performance liquid chromatography, which makes the application of high performance liquid chromatography more extensive, and makes full use of the advantages of high efficiency, fastness, selectivity and sensitivity to establish a more systematic component analysis. method. Combined with mass spectrometry, gradient elution, column switching technology, advanced detection technology, and molecular biology, modern molecular pharmacology, it will play a significant role in the analysis of traditional Chinese medicine.
1 Recent developments in high performance liquid chromatography
The application of high performance liquid chromatography in drug analysis mainly considers the pretreatment of the sample and the selection of the analytical column and detector. In the pretreatment of the sample, the solid phase (micro) extraction that has arisen recently has made many low-content components refined and purified, which is suitable for the determination of high performance liquid chromatography. Sun Xinguo has used the countercurrent extraction to determine the content of ligustrazine. Effect. Some of the traditional Chinese medicines have weak UV absorption or no characteristic UV absorption. They are directly determined by high performance liquid chromatography. Their sensitivity and resolution are not satisfactory. These components can be accurately determined by pre-column or post-column derivatization. come out. For the polar and fat-soluble substances, it is not easy to retain on the YWGCl8 column. The sodium dodecyl sulfate is used as the ion-pairing reagent to reduce the polarity and prolong the retention time on the column, so that better separation is achieved. The two powerful analytical techniques of liquid chromatography and mass spectrometry are linked together online. After 30 years of development, it has become a relatively mature analytical tool, but it has problems since its formation: from liquid chromatography When entering mass spectrometry, the changes of mobile phase, the composition of solvent, and the problem of high temperature and high pressure ionization restrict the development of this combined technology. The atmospheric pressure ionization interface has the dual functions of removing solvent and ionization, and its introduction makes the technology Various fields have been widely used. Electrospray ion source is a soft ionization technique. It generally generates only two molecular ion peaks (M+H)+ and (MH)-, selectively monitors the negative molecular ion peak of (mz) 190, and has high sensitivity. Accuracy and specificity meet the needs of low-concentration drug research. The three-dimensional high performance liquid chromatography method studied by Zhang Li et al. can simultaneously determine two indicators of puerarin and ferulic acid. It is proved by experiments that if the appropriate column temperature and other chromatographic conditions are selected, ethanol is used as the mobile phase of reversed-phase high performance liquid chromatography to analyze the active ingredients in traditional Chinese medicines and proprietary Chinese medicines, which is safe and accurate. A substance with similar structure is difficult to detect by ordinary detectors. High performance liquid chromatography-electrochemical method can effectively determine the content of baicalin and baicalein which are only one group in the berberine powder. The sample is collected after separation by chromatography, and the fluorescence intensity is measured by fluorescence spectrophotometer. The influencing factors are numerous and the measurement is complicated. The improved high performance liquid chromatography-fluorescence method can be derivatized and collected without chemical separation. Miscellaneous, can be directly injected after concentration. Good results were also obtained by measuring the content of hypericin in Hypericum perforatum L. by this method. High performance liquid chromatography-differential refractive index measurement of astragaloside IV in Huangqijing oral liquid has also achieved satisfactory results. For the ultraviolet only absorption, the sensitivity is low when UV detection, the baseline is easy to drift, and the refractive index detection is easily interfered by external conditions. The evaporative scattering detector can overcome the above deficiencies, and the response value is only related to the sample quality, and the signal is corresponding to the quality. In proportion, different compounds, the same quality, the signal is basically the same. The evaporative light scattering detection method is based on the fact that the degree of scattering of light by a non-volatile sample molecule is proportional to its mass, regardless of the nature of the group it contains. By selecting the appropriate detector parameters, the mobile phase and solvent are completely vaporized without generating a signal, and the individual components of the sample are present as non-volatile particles that scatter light to be detected. Therefore, the evaporative light scattering detector can be used for simultaneous separation and analysis of multiple components containing different groups. Compared with ultraviolet and fluorescence methods, evaporative light scattering detection has approximately the same response factor for different substances.
Therefore, there is no low concentration, high response or high concentration, low response, which is conducive to accurate determination of mixtures in different proportions. Determination of terpene lactone content, red ginseng and ginseng in Ginkgo biloba leaves by high performance liquid chromatography-evaporative light scattering test Ginseng in fine capsule
Both the content of saponin Rg1 and Re and the content of gambogic acid in garcinia have obtained good results.
2 Research trends in high performance liquid chromatography
2.1 Reduce analysis time and improve separation efficiency. The use of advanced detection instruments and methods to adjust or change the mobile phase and stationary phase, gradient elution, column switching technology is expected to solve this problem. Gradient-eluting high-performance liquid chromatography, which can analyze samples with a wide range of polarities, has great advantages over isocratic elution, but it is somewhat inconvenient for traditional Chinese medicine samples with more complex components and wider polar ranges. Multi-column high performance liquid chromatography is also known as multidimensional high performance liquid chromatography. In addition to the advantages of changing the concentration of the mobile phase with gradient elution, the type of stationary phase, the degree of bonding, the particle size, the length of the column, the diameter of the column, and the type and concentration of the mobile phase can be changed.
2.2 Further development towards automation, intelligence and joint technology. The combination of liquid chromatography and mass spectrometry has become the first choice for the determination of drugs and metabolites in low-concentration biopharmaceuticals. The LC-MS-MS method is used to determine the HIV-1 protease in plasma, which is accurate and efficient, and the endogenous group in plasma. Parts and other drugs do not interfere with the assay, saving material and saving time. Has been applied to the analysis of drugs in body fluids, plasma, serum. The analysis of cholic acid in Chinese herbal compound injection "Qing Kai Ling" was carried out by liquid chromatography-mass spectrometry and mass spectrometry. High performance liquid chromatography-nuclear magnetic resonance (NMR) has a very good role in drug analysis. The newly proposed intelligent multi-column HPLC system utilizes the modular separation performance of the switching technique to switch the sample into blocks of different properties and elute with a suitable mobile phase. Intelligent control is used throughout the process.
3 Application prospect of high performance liquid chromatography in traditional Chinese medicine analysis
The general trend of traditional Chinese medicine research is full-component analysis. Through the study of different compatibility and decoction time from single-flavor medicine to compound, we can discover the changes of chemical composition in traditional Chinese medicine and find the organic connection between Chinese medicine mechanism. There are many kinds of traditional Chinese medicines, and the properties of various ingredients are spread over all polar segments and acids and bases. The easiest way to achieve multi-component analysis is to use a mild mobile phase on a long enough column to elute for a sufficient amount of time. But this is contrary to the simplicity and speed of modern analytical requirements. A large number of applied studies have shown that high-performance capillary electrophoresis has great advantages in the analysis of traditional Chinese medicine components, especially in the analysis of high-polarity chemical components, and shows a high ability in the analysis of a large number of compound preparations. Since the capillary tube is almost free of bed contamination that often occurs in high performance liquid chromatography, and the used capillary column is rinsed in a short time, the second sample can be analyzed, fast and efficient, and resolution. Very high. The emerging capillary electrochromatography is a new type of electro-separation microcolumn liquid chromatography technology that combines the advantages of high performance liquid chromatography and capillary electrophoresis. It is to move various filler particles of high performance liquid chromatography into the capillary to sample and The interaction between the stationary phases acts as a separation mechanism, with electroosmotic flow as the driving principle of the mobile phase. Recently, some advanced detection instruments have been successfully used in high performance liquid chromatography, which makes the application of high performance liquid chromatography more extensive, and makes full use of the advantages of high efficiency, fastness, selectivity and sensitivity to establish a more systematic component analysis. method. Combined with mass spectrometry, gradient elution, column switching technology, advanced detection technology, and molecular biology, modern molecular pharmacology, it will play a significant role in the analysis of traditional Chinese medicine.
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