| In this paper, we introduced pharmacological effects of medicines containing saponins from six aspects, and also commented on current extraction and separation of saponins’active ingredients, and then summarized the advantages and disadvantages of different extraction methods. Moreover, we reviewed the present analysis research situation of active ingredients in various kinds of saponins, by comparing the current different determining methods of saponins, we pointed out the limitation of them definitely.In view of the present determining level of active components in medicines containing saponins drugs, and on the basis of their chemical structures, we successfully combined high performance liquid chromatogarphy(HPLC) analysis and mass spectrum(MS) detection technology, depending on the high performance and resolution of HPLC. and high sensitivity and selectivity of MS, we established several new methods of HPLC-MS/MS with electrospray ionization interface (ESI) for the determination of saponins. The methods were proved to be sensitive and selective, and could be used for the quality control of Chinese traditional medicines containing saponins in the future.In this research, we investigated all of the chromatographic separation and mass spectrum detection conditions of three kinds of Chinese herbal medicine containing saponin compounds, such as stationary phase, mobile phase, column temperature and flow rate of the mobile phase for HPLC. and ionization mode, spray needle voltage, drying temperature, drying gas pressure, capillary voltage and collision energy for MS. And under the optimal conditions, we finally established the new separation and analysis methods of corresponding saponin compounds, which were presented in detail as follows:(1) Saikosaponins. Objective:To establish a new HPLC-ESI-MS/MS method for simultaneous determination of saikosaponin a and saikosaponin d in Radix Bupleuri. Methods: The analysis was performed on an Ultimate(?) XB-C8column (150mm×2.1mm,3μm). Mobile phase was water (containing0.1%formic acid) and acetonitrile with gradient elution; the flow rate was0.2mL-min-1; column temperature was35℃. Mass spectrometric conditions were as follows: Ionization of analytes was carried out using negative-mode electrospray ionization (ESI) in the selected reaction monitoring (SRM) mode. Needle potential:-4500V; drying gas (N2) temperature:350℃; drying gas (N2) pressure:22psi; collision gas (Ar) pressure:1.8mT. Results:In the best experimental conditions, we obtained baseline separation of saikosaponin a and saikosaponin d in Radix Bupleuri. By monitoring the quasi-molecular ion peaks and fragment ion peaks in the negative mode, we obtained the structural information of analytes. Here. the quasi-molecular ions of two compounds were proved to be [M-H]-m/z=779. and the fragment ions were [M-H-Glc]-m/z=617. In the best conditions, we observed the good linearity over the range from0.05to5.0mg·L-1for saikosaponin a and from0.05to10.0mg·L-1for saikosaponin d. The regression equations and correlation coefficients of saikosaponin a and saikosaponin d were A=1.31×105c3.9×104, r=0.9994; A=1.34×105c-1.9×104. r=0.9992. respectively. The detection limits of them were15ng·mL-1(a) and12ng·mL-1(d), and the average recoveries were99.3%(a) and99.8%(d), respectively. Conclusion:The method was simple, selective and sensitive, and it could be used for the quality control of medicinal materials and preparations containing saikosaponins.(2) Timosaponins. Objective:To establish a new HPLC-ESI-MS/MS method for simultaneous determination of timosaponin A Ⅲ and timosaponin B Ⅱ in Anemarrhena asphodeloides. Methods:The analysis was performed on an Ultimate(?) XB-C8column (150mm×2.1mm,3μm). Mobile phase was water (containing0.1%formic acid) and acetonitrile with gradient elution; flow rate was0.2mL·min-1; column temperature was35℃. Mass spectrometric conditions were as follows:Ionization of analytes was carried out using negative-mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Needle potential:-4500V; drying gas (N2) temperature:350℃; drying gas (N2) pressure:25psi; collision gas (Ar) pressure:1.8mT. Results:In the best experimental conditions, we obtained baseline separation of timosaponin Alll and timosaponin B II in Anemarrhena asphodeloides. By monitoring the quasi-molecular ion peaks and fragment ion peaks in the negative mode, we obtained the structural information of analytes. Here, the quasi-molecular ions were proved to be [M-H]-m/z=739(AⅢ) and [M-H]-m/z=919(BⅡ). and the fragment ions were [M-H-Glc]-m/z=577(AⅢ) and [M-H-Glc]-m/z=757(BⅡ). In the best conditions, we observed the good linearity over the range from0.01to10.0mg·L-1for timosaponin AⅢ, the regression equation and correlation coefficient were A=2.01×107c+2.0×106. r=0.9996, the detection limit was6ng·mL-1and the average recovery was99.2%; similarly, good linearity over the range from0.01to10.0mg·L-1for timosaponin B Ⅱ, the regression equation and correlation coefficient were A=6.03×106c+3.4×105, r=0.9993, respectively, the detection limit was2ng·mL-1and the average recovery was98.9%. Conclusion:The method was simple, selective and sensitive, and it could be used for the quality control of medicinal materials and preparations containing timosaponins.(3) Astragalosides. Objective:To establish a new HPLC-ESI-MS/MS method for simultaneous determination of astragaloside Ⅰ, astragaloside Ⅱ, and astragaloside IV in Astragalus membranaceus. Methods:The analysis was performed on an Ultimate(?) XB-C8column (150mm×2.1mm.3μm). Mobile phase was water (containing0.5%formic acid) and acetonitrile with gradient elution; flow rate was0.2mL·min-1; column temperature was35℃. Mass spectrometric conditions were as follows:Ionization of analytes was carried out using negative-mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) and selected ion monitoring (SIM) mode. Needle potential:-5000V; drying gas (N2) temperature:320℃; drying gas (N2) pressure:25psi; collision gas (Ar) pressure:1.8mT. Results:In the best experimental conditions, we obtained baseline separation of astragaloside Ⅰ, astragaloside Ⅱ, and astragaloside Ⅳ in Astragalus membranaceus. By monitoring the quasi-molecular ion peaks and fragment ion peaks, we obtained the structural information of analytes. Here, the quasi-molecular ions were proved to be [M+HCOO]-m/z=913(Ⅰ),[M+HCOO]-m/z=871(Ⅱ) and [M+HCOO]’ m/z=829(Ⅳ). and the fragment ions were [M-H]-m/z=867(Ⅰ),[M-H]-m/z=825(Ⅱ) and [M-H]-m/z=783(Ⅳ). In the best experimental conditions, we observed the good linearity over the range from0.01to30.0mg-L-1for astragaloside Ⅰ, the regression equation and correlation coefficient were A=1.04×105c+3.1×104, r=0.9969, the detection limit was3ng·mL-1and the average recovery was99.2%. There were both good linearity over the range from0.01to10.0mg·L-1for astragaloside Ⅱ and astragaloside IV, the regression equations and correlation coefficients were A=1.71×105c-2.1×104. r=0.9996and A=3.81×105c-1.9×103; r=0.9999, respectively. The detection limits of them were4ng·mL-1(Ⅱ) and3ng·mL-1(Ⅳ), and the average recoveries were100.7%(Ⅱ) and100.2%(Ⅳ). respectively. Conclusion:The method was simple, selective and sensitive, and it could be used for the quality control of medicinal materials and preparations containing astragalosides. |
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