| Chinese medicine preparation, taking Chinese herbal medicine as raw material, is processed in accordance with the provisions of the prescription and preparation method under the guidance of Chinese medicine theory, and used in clinic directly. The components in the Chinese medicine preparation are complex and changeable, so specific, sensitive analysis methods should be developed for their analysis. Liquid chromatography tandem with mass spectrometry(LC-MS/MS) possesses good chromatographic separation, good specificity, high sensitivity and the ability of structure identification, which makes it especially suitable for the analysis of complicated system such as Chinese medicine. Notoginseng Radix et Rhizoma and Salvia Miltiorrhiza Radix et Rhizoma are two famous traditional Chinese medicines(TCM) and both have the function of promoting blood circulation and removing blood stasis. They are applied together frequently in clinic because of the synergistic function. Fufang Xueshuantong capsule, composed of Notoginseng Radix et Rhizoma, Salvia Miltiorrhiza Radix et Rhizoma, Astragali Radix and Scrophulariae Radix, has the efficacy of blood-activating, stasis-resolving, supplementing qi and nourishing yin and can be used for the retinal vein occlusion and angina pectoris with the blood stasis and qi and Yin deficiency syndrome. Guanxindanshen dropping pill, consisted of Notoginseng Radix et Rhizoma, Salvia Miltiorrhiza Radix et Rhizoma and Dalbergiae Odoriferae Lignum, has the efficacy of regulating qi to alleviate pain, promote blood circulation and remove blood stasis, and is usually used against chest pain, coronary heart disease angina pectoris. In the present study, the two Chinese medicine preparations are taken as the object to explore the application of LC-MS/MS technology in the simultaneous quantitative analysis of components in the complex systems of Chinese medicine. Part one Simultaneous determination of 8 chemicals in Fufang Xueshuantong capsules by LC-MS/MS Objective: To establish a periodic polarity-switching liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for the simultaneous determination of eight components(notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1, tanshinone II A, salvianolic acid B, astragaloside IV, harpagide and harpagoside) in an important traditional Chinese medicinal capsule, Fufang Xueshuantong. Methods: 1 Mass spectrometry conditions: The detection was performed by multiple reaction monitoring(MRM) with a periodic polarity-switching electrospray ionization(ESI) source across five switching periods between positive and negative modes. The periodic polarity-switching method was as follows: 0-3.00 min and 6.23-8.06 min in positive mode for the determination of harpagide and tanshinone Ⅱ A, 3.00-6.23 min in negative mode for the determination of ginsenoside Rg1, harpagoside, astragaloside IV, ginsenoside Rb1, notoginsenoside R1 and salvianolic acid B; 3.00-3.03 min and 6.20-6.23 min are the polarity switching time. 2 Chromatographic condition: LC analysis was carried out on a Kinetex 2.6μ XB-C18 column by gradient elution. A mobile phase containing 0.1% formic acid was found to provide the maximum response for the eight analytes. The mobile phase consists of phases A(acetonitrile-methanol, 1:1) and B(aqueous 0.1% formic acid and 1 m M ammonium acetate) with a linear gradient elution at a flow rate of 300 μL/min. The gradient program was as follows: 0.0-0.5 min, 30-95% A; 0.5-7.0 min, 95% A; 7.0-8.0 min, 95-30% A. The injection volume was 10 μL. 3 Extraction conditions: extraction solvents(25%, 50%, 75% and 100% methanol) and extraction times(20, 40, 60 and 80 min) were selected and evaluated for the extraction of Fufang Xueshuangtong capsules. By comparing the peak areas of the analytes in the chromatogram for each factor, the optimal extraction condition was found with 75% methanol under ultrasonication for 60 min. 4 To comprehensively valid the established method, including the limit of quantification, limit of detection, linearity, range, accuracy, precision, repeatability and stability of the solution. 5 The developed method was successfully employed to analyze three batches of Fufang Xueshuantong capsule samples. Results: Periodic polarity-switching were used to obtain maximal responses and to simultaneously detect eight constituents in one analysis run lasting 8 min. The method was reliable, accurate and specific. The peak area and concentration of 8 components has good linear relationship in the range of determination. All of the analytes showed good linearity(r: 0.9947-0.9998) in the tested ranges. The average recoveries were in the range of 95.4-103.9%. The RSD of the intra- and inter-day variations were in the range of 0.8-3.7%. The RSD of repeatability of the instrument were in the range of 1.3-3.6%. The RSD of stability were in the range of 1.1-4.8%. The determination result showed that the contents of 8 analytes had differences between batches, especially for tanshinone IIA and ginsenoside Rb1. The RSD values of two compounds were 33.45% and 20.51%. The content variance of 4 components(harpagide, ginsenoside Rg1, harpagoside and astragaloside IV) were not significant(RSD<10%). The RSD of notoginsenoside R1 and salvianolic acid B were 10.96% and 13.57%. Conclusion: The new established periodic polarity-switching LC-MS/MS method was proven to be highly sensitive and effective in evaluating the quality of Fufang Xueshuantong capsule. Part two Simultaneous determination of 6 components in Guangxindanshen droping pills by LC-MS/MS Objective: To develop a periodic polarity-switching LC–MS/MS method for the determination of six chemical compositions in Guangxindanshen droping pills, including notoginsenoside R1, ginsenoside Rb1, ginsenoside Rg1, tanshinone IIA, salvianolic acid B and trans-nerolidol and it is applied for the quality control of Guangxindanshen droping pills.Methods: 1 MS conditions: The detection was performed by multiple reaction monitoring(MRM) with a periodic polarity-switching electrospray ionization(ESI) source across three periods of switching between negative and positive modes. According to the elution order of the six analytes under the optimized chromatographic conditions, the polarity was switched across three periods in one experiment. Period 2(3.50-3.53min) was designed to switch polarity with 0.03 min. The dominant ions of notoginsenoside R1, ginsenoside Rg1, salvianolic acid B ginsenoside Rb1 had better sensitivities and reproducibilities in full-scan mass spectra in the negative mode(0-3.50min), and the positive mode was appropriate for trans-nerolidol and tanshinone IIA(3.53-6.03min). 2 Chromatography conditions:LC analysis was carried out on a Synergi 4u fusion-RP(50mm×3.0 mm, 4μm)by gradient elution. The final gradient mobile phase was composed of phases A(acetonitrile) and B(aqueous 0.1% formic acid and 1 m M ammonium acetate). Gradient elution for 7 minutes: 0-0.5 min, 35%-95%A; 0.5-6.0 min, 95%A; 6.0-7.0 min, 95%-35%A. 3 Extraction conditions: To optimize the ultrasonic extraction conditions, extraction solvents(25%, 50%, 75% and 100% methanol) and extraction times(20, 40, 60 and 80 min) were selected and evaluated. The optimal extraction condition was found to be 75% methanol under ultrasonication for 60 min. 4 To comprehensively valid the established method, including the limit of quantification, limit of detection, linearity range, accuracy, precision, repeatability and stability of the solution. 5 Using the established method to determine 6 kinds of chemical components in 3 batches of Guanindanshen dropping pill. Results: Periodic polarity-switching was used to acquire maximal responses and to simultaneously detect six constituents in one analysis run lasting 8 min. The method was reliable, accurate and specific. The peak areas and concentrations of 6 kinds of constituents have good linearity(r: 0.9904-0.9993) in the tested ranges. The average recoveries were between 94.5%-103.7%. The RSD of the intra- and inter-day variations were in the range of 1.6%-3.7% and 1.7%-3.5%, respectively. The RSD of repeatability of the instrument were in the range of 1.3%-3.4%. The RSD of stability were in the range of 1.6%-3.8%. The developed method was successfully applied to analyze the contents of 3 batches of Guangxindanshen drop pills samples. The determination result showed that the contents of 6 analytes had differences between batches, especially for salvianolic acid B and tanshinone IIA. The RSD values of two compounds were 50.51% and 21.30%. The RSD of trans-nerolidol, notoginsenoside R1, ginsenoside Rg1 and ginsenoside Rb1 were 13.48%, 8.84%, 8.75% and 1.75%, respectively. Conclusion: The method was simple, rapid, sensitive and accurate, and also suitable for the simultaneous determination of the Guangxindanshen droping pills samples. |
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