Cell Biochemistry and Biophysics

, Volume 70, Issue 1, pp 677–686 | Cite as

Simultaneous Determination of Calycosin-7-O-β-d-Glucoside, Ononin, Calycosin, Formononetin, Astragaloside IV, and Astragaloside II in Rat Plasma After Oral Administration of Radix Astragali Extraction for Their Pharmacokinetic Studies by Ultra-Pressure Liquid Chromatography with Tandem Mass Spectrometry

  • Xiao-hua Liu
  • Jian-bang Zhao
  • Long Guo
  • Ying-lai Yang
  • Fang Hu
  • Rui-juan Zhu
  • Shi-lan Feng
Original Paper


A sensitive and reliable ultra-pressure liquid chromatography with tandem mass spectrometry (UPLC–MS) was developed and validated for simultaneous quantification of six main bioactive components, i.e., calycosin-7-O-β-d-glucoside, ononin, calycosin, formononetin, astragaloside IV, and astragaloside II in rat plasma after oral administration of the 95 % ethanol extraction from Radix Astragali. Plasma samples were extracted with Waters OasisTM HLB 1 cc (30 mg) Extraction Cartridges (SPE) separated on an UPLC™ BEH C18 column and detected by MS with electro spray ionization interface in positive selective ion monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r 2 > 0.99. The method had the lower limit quantification of 1.30, 0.73, 1.17, 2.33, 0.63, and 0.83 ng/mL for ononin, calycosin, calycosin-7-O-β-d-glucoside, formononetin, astragaloside IV, and astragaloside II, respectively, with precision less than 10 %. The RSD of intra- and inter-day variations ranged from 1.66 to 6.46 and 3.39 to 6.58 %. This developed method was applied subsequently to pharmacokinetic studies of the six compounds in rats successfully. The proposed method was for the first time to compare the pharmacokinetic difference between calycosin-7-O-β-d-glucoside and calycosin in rat plasma, so as between ononin and formononetin, and studied to the astragaloside II pharmacokinetics in rat plasma.


UPLC–MS Calycosin-7-O-β-d-glucoside Ononin Calycosin Formononetin Astragaloside IV Astragaloside II Radix Astragali extraction Pharmacokinetic 



The authors greatly appreciate financial support from the great research projects (lzujbky-2013-198) and (No. z2012117) of the Ministry of Education of China, (No. 2011BAI05B00) of the Ministry of Science and Technology of China. The authors would like to thank Professor Rong Wang, for providing instrumental facilities of LC–MS from Department of Pharmacy, Lanzhou General Hospital of PLA.


  1. 1.
    Zhang, Y. (2011). The research status of the main chemical composition of astragalus. Chinese Journal of Clinical Rational Drug Use, 4, 151–152.Google Scholar
  2. 2.
    Luo, Y., Qin, Z., Hong, Z., Zhang, X., Ding, D., Fu, J. H., et al. (2004). Astragaloside IV protects against ischemic brain injury in a murine model of transient focal ischemia. Neuroscience Letters, 363, 218–223.PubMedCrossRefGoogle Scholar
  3. 3.
    Wang, Y. X., Jiang, C. L., Lu, C. L., Song, L. X., You, Z. D., Shao, X. Y., et al. (2000). Distinct domains of IFNa mediate immune and analgesic effects respectively. Journal of Neuroimmunology, 108, 64–67.PubMedCrossRefGoogle Scholar
  4. 4.
    e Nalbantsoy, A., Nesil, T., Yılmaz-Dilsiz, Ö., Aksu, G., Khan, S., & Bedir, E. (2012). Evaluation of the immunomodulatory properties in mice and in vitro anti-inflammatory activity of cycloartane type saponins from Astragalus species. Journal of Ethnopharmacology, 139, 574–581.CrossRefGoogle Scholar
  5. 5.
    Upton, R., & Petrone, C. (1999). American Herbal Pharmacopoeia and Therapeutic Compendium, Astragalus Root, Astragalus membranaceus & Astragalus membranaceus var. mongholicus, Analytical, Quality Control, and Therapeutic Monograph. Santa Cruz: American Herbal Pharmacopoeia.Google Scholar
  6. 6.
    Ying, Hong, Hui, Sarah S C, Chan, Benjiamin T Y, & Jia-yu, Hou. (2002). Study of the inhibitory effect of Huangqi Injection on experimental cardiac hypertrophy in rats. Chinese Traditional Patent Medicine, 24, 525–529.Google Scholar
  7. 7.
    Gui, S. Y., Wei, W., Wang, H., Li, Wu, Sun, W. Y., Chen, Wb, et al. (2006). Effects and mechanisms of crude astragalosides fraction on liver fibrosis in rats. Journal of Ethnopharmacology, 103, 154–159.PubMedCrossRefGoogle Scholar
  8. 8.
    Song, Q. H., Kobayashi, T., Xiu, L. M., Hong, T., & Cyong, J. C. (2000). Effects of Astragali root and Hedysari root on the murine B and T cell differentiation. Journal of Ethnopharmacology, 73, 111–119.PubMedCrossRefGoogle Scholar
  9. 9.
    Lee, Y. S., Han, Ok, Park, C. W., Shin, S. W., Yang, C. H., Jeon, T. W., et al. (2003). Immunomodulatory effects of aqueous-extracted Astragali radix in methotrexate-treated mouse spleen cells. Journal of Ethnopharmacology, 84, 193–198.PubMedCrossRefGoogle Scholar
  10. 10.
    Yan, Q., Jiang, Z., Yang, S., Deng, W., & Han, L. (2005). A novel homodimeric lectin from Astragalus mongholicus with antifungal activity. Archives of Biochemistry and Biophysics, 442, 72–81.PubMedCrossRefGoogle Scholar
  11. 11.
    Ou Yang, G., Wang, D. Y., Li, X. M., et al. (2002). Influence of Huangqi Injection on Bone Density of Patients with Primary Osteoporosis. Tianjin traditional Chinese medicine, 19, 18–20.Google Scholar
  12. 12.
    Chen, G. H., & Huang, W. F. (2008). Progress in pharmacological effects of compositions of Astragalus membranaceus. Chinese Journal of New Drugs, 17, 1482–1485.Google Scholar
  13. 13.
    Qi, L. W., Cao, J., Li, P., Yu, Q. T., Wen, X. D., Wang, Y. X., et al. (2008). Qualitative and quantitative analysis of Radix Astragali products by fast high-performance liquid chromatography-diode array detection coupled with time-of-flight mass spectrometry through dynamic adjustment of fragmentor voltage. Journal of Chromatography A, 1203, 27–35.PubMedCrossRefGoogle Scholar
  14. 14.
    Huang, X., Liu, Y., Song, F., Liu, Z., & Liu, S. (2009). Studies on principal components and antioxidant activity of different Radix Astragali samples using high-performance liquid chromatography/electrospray ionization multiple-stage tandem mass spectrometry. Talanta, 78, 1090–1101.PubMedCrossRefGoogle Scholar
  15. 15.
    Zhang, Y. Z., Xu, F., Dong, J., Liang, J., Hashi, Y., Shang, M. Y., et al. (2012). Profiling and identification of the metabolites of calycosin in rat hepatic 9000 × g supernatant incubation system and the metabolites of calycosin-7-O-β-d-glucoside in rat urine by HPLC–DAD–ESI-IT-TOF-MSn technique. Journal of Pharmaceutical and Biomedical Analysis, 70, 425–439.PubMedCrossRefGoogle Scholar
  16. 16.
    Xiao, H. B., Krucker, M., Albert, K., & Liang, X. M. (1032). Determination and identification of isoflavonoids in Radix Astragali by matrix solid-phase dispersion extraction and high-performance liquid chromatography with photodiode array and mass spectrometric detection. Journal of Chromatography A, 2004, 117–124.Google Scholar
  17. 17.
    Gu, Y., Wang, G., & Fawcett, J. P. (2004). Determination of Astragaloside IV in rat plasma by liquid chromatography electrospray ionization mass spectrometry. Journal of Chromatography B, 801, 285–288.CrossRefGoogle Scholar
  18. 18.
    Singh, S. P., Yadav, D. K., Wahajuddin, Rawat, P., Maury, R., & Jain, G. K. (2010). HPLC coupled with tandem mass spectrometry. Journal of Chromatography B, 878, 391–397.CrossRefGoogle Scholar
  19. 19.
    Zhang, X., Sun, Y. G., Cheng, M. C., Wang, Y. Q., Xiao, H. B., & Liang, X. M. (2007). Simultaneous quantification of three isoflavonoid glycosides in rabbit plasma after oral administration of Astragalus mongholicus extract by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Analytica Chimica Acta, 602, 252–258.PubMedCrossRefGoogle Scholar
  20. 20.
    Tao, L., Wang, Y., Wang, Y., Liang, R., Zhang, D., Zhang, H., et al. (2013). Development of an SPE HPLC MS method for simultaneous determination and pharmacokinetic study of bioactive constituents of Yu Ping Feng San in rat plasma after oral administration. Journal of Ethnopharmacology, 145, 784–792.CrossRefGoogle Scholar
  21. 21.
    Wen, X. D., Qi, L. W., Li, P., Bao, K. D., Yan, X. W., Yi, L., et al. (2008). Simultaneous determination of calycosin-7-O-β-d-glucoside, ononin, astragaloside IV, astragaloside I and ferulic acid in rat plasma after oral administration of Danggui Buxue Tang extract for their pharmacokinetic studies by liquid chromatography–mass spectrometry. Journal of Chromatography B, 865, 99–105.CrossRefGoogle Scholar
  22. 22.
    Yan, L. X., & Guo, D. A. (2005). Quantitation of astragaloside IV in rat plasma by liquid chromatography–tandem mass spectrometry. Journal of Chromatography B, 824, 244–248.CrossRefGoogle Scholar
  23. 23.
    Zhang, W., Zhang, C., Liu, R., Li, H., Zhang, J., Mao, C., et al. (2005). Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry. Journal of Chromatography B, 822, 170–177.CrossRefGoogle Scholar
  24. 24.
    Zhang, W. D., Zhang, C., Liu, R. H., Li, H. L., Zhang, J. T., Mao, C., et al. (2006). Preclinical pharmacokinetics and tissue distribution of a natural cardioprotective agent astragaloside IV in rats and dogs. Life Sciences, 79, 808–815.PubMedCrossRefGoogle Scholar
  25. 25.
    Wu, H., Guo, J., Chen, S., Liu, X., Zhou, Y., Zhang, X., & Xu, X. (2013). Recent developments in qualitative and quantitative analysis of phytochemical constituents and their metabolites using liquid chromatography–mass spectrometry. Pharmaceutical and Biomedical Analysis, 72, 267–291.Google Scholar
  26. 26.
    Liang, J., Liu, X. H., Ren, Y., & Feng, S. L. (2013). A simultaneous determination of five compounds in Radix Astragali by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors. Chinese Journal of Pharmaceutical Analysis, 33, 210–213.Google Scholar
  27. 27.
    Qi, L. W., Yu, Q. T., Li, P., Li, S. L., Wang, Y. X., Sheng, L. H., et al. (2006). Quality evaluation of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors. Journal of Chromatography A, 1134, 162–169.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Xiao-hua Liu
    • 1
    • 2
  • Jian-bang Zhao
    • 3
  • Long Guo
    • 1
  • Ying-lai Yang
    • 1
  • Fang Hu
    • 1
  • Rui-juan Zhu
    • 1
  • Shi-lan Feng
    • 1
  1. 1.School of PharmacyLanzhou UniversityLanzhouPeople’s Republic of China
  2. 2.College of Chemistry and Chemical EngineeringLanzhou UniversityLanzhouPeople’s Republic of China
  3. 3.Food and Drug Control of GansuLanzhouPeople’s Republic of China

Personalised recommendations