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Combination of UHPLC/Q-TOF-MS, NMR spectroscopy, and ECD calculation for screening and identification of reactive metabolites of gentiopicroside in humans

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Abstract

The metabolic investigation of natural products is a great challenge because of unpredictable metabolic pathways, little knowledge on metabolic effects, and lack of recommended analytical methodology. Herein, a combined strategy based on ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, and electronic circular dichroism (ECD) calculation was developed and employed for the human metabolism study of gentiopicroside (GPS), a naturally hepato-protective iridoid glycoside. The whole metabolic study consisted of three major procedures. First, an improved UHPLC/Q-TOF-MS method was used to separate and detect a total of 15 GPS metabolites that were obtained from urine samples (0 to 72 h) of 12 healthy male participants after a single 50-mg oral dose of GPS. Second, a developed “MS-NMR-MS” method was applied to accurately identify molecular structures of the observed metabolites. Finally, given that the associated stereochemistry may be a crucial factor of the metabolic activation, the absolute configuration of the reactive metabolites was revealed through chemical calculations. Based on the combined use, a pair of diastereoisomers (G05 and G06) were experimentally addressed as the bioreactive metabolites of GPS, and the stereochemical determination was completed. Whereas several novel metabolic transformations, occurring via oxidation, N-heterocyclization and glucuronidation after deglycosylation, were also observed. The results indicated that GPS has to undergo in vivo metabolism-based activation to generate reactive molecules capable of processing its hepato-protective activity.

Identification of metabolites of gentiopicroside using a combined strategy

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Acknowledgments

The financial support of the National Natural Science Foundation of China (81073027), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1071), and the Shanghai Science and Technology Development Foundation (12QH1402200) are gratefully acknowledged.

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Authors and Affiliations

  1. The Ministry of Education (MOE) Key Laboratory for Standardization of Chinese Medicines, Institute of Traditional Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201210, China

    Han Han, Ai-Zhen Xiong, Chun-Yong He, Qing Liu, Li Yang & Zheng-Tao Wang

  2. The State Administration of TCM (SATCM) Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Traditional Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201210, China

    Han Han, Ai-Zhen Xiong, Chun-Yong He, Qing Liu, Li Yang & Zheng-Tao Wang

Authors
  1. Han Han
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  2. Ai-Zhen Xiong
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  3. Chun-Yong He
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  4. Qing Liu
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  5. Li Yang
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Correspondence to Li Yang or Zheng-Tao Wang.

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Han, H., Xiong, AZ., He, CY. et al. Combination of UHPLC/Q-TOF-MS, NMR spectroscopy, and ECD calculation for screening and identification of reactive metabolites of gentiopicroside in humans. Anal Bioanal Chem 406, 1781–1793 (2014). https://doi.org/10.1007/s00216-013-7572-z

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  • DOI: https://doi.org/10.1007/s00216-013-7572-z

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