Determination of berberine-upregulated endogenous short-chain fatty acids through derivatization by 2-bromoacetophenone

  • Shu-Rong Ma
  • Qian Tong
  • Zhen-Xiong Zhao
  • Lin Cong
  • Jin-Bo Yu
  • Jie Fu
  • Pei Han
  • Li-Bin Pan
  • Randy Gu
  • Ran Peng
  • Zheng-Wei Zhang
  • Yan WangEmail author
  • Jian-Dong JiangEmail author
Research Paper


Short-chain fatty acids (SCFAs) are a major group of endogenous metabolites generated by the gut microbiota and have been reported to play an important role in physical health, such as improving energy metabolism. Here, using 2-bromoacetophenone as the derivatization reagent (BP, 10 mg/mL, 40 °C for 20 min), a sensitive liquid chromatography–tandem mass spectrometric method was established for the quantitative determination of seven short-chain fatty acids in plasma and feces. The analyses were performed on a C18 column in positive multiple reaction monitoring mode. Specificity, linearity, accuracy, precision, recovery, and stability were observed within the quantitative limits of biological sample analysis. The established method has largely improved the sensitivity by 200- to 2000-fold than that in gas chromatography (GC). Especially for butyrate, the lower quantitative limit of 1 ng/mL, 1600-fold higher in sensitivity than that of GC (1.6 μg/mL), ensured the accurate determination of its low level in blood or feces (88 ± 29 ng/mL in blood, 176 ± 18 μg/g in feces). Then, the validated method was applied for therapeutic studies of berberine in hyperlipidemia hamsters in vivo and screening of 13 compounds (including five metabolites of berberine and eight typical isoquinoline alkaloids) in vitro. After berberine treatment (oral, 200 mg/kg, 2 weeks) to hyperlipidemia hamsters, the levels of butyrate were significantly upregulated in blood (77 ± 10 ng/mL vs. 117 ± 13 ng/mL, *P < 0.05) and feces (132 ± 11 μg/g vs. 547 ± 57 μg/g, ***P < 0.001), which further verified butyrate as an active endogenous metabolite in coordination with berberine to lower the blood lipids. Additionally, the berberine metabolites (M1, M2, M3), as well as two isoquinoline alkaloids (tetrandrine and dauricine), could also obviously induce the production of SCFAs (butyrate, etc.) in gut microbiota. In total, we have successfully established a new derivative LC-MS/MS method for the targeted quantitative determination of seven SCFAs in biological samples.

Graphical abstract


Derivatization LC-MS/MS Butyrate Berberine Isoquinoline alkaloids 2-Bromoacetophenone 









Electrospray ionization


Electronic Supplementary Material


Free fatty acids


Fasting blood glucose


Gas chromatography


Gas chromatography–mass spectrometry


High-fat diet


High-performance liquid chromatography


High-concentration quality control


Internal standard


Liquid chromatography–tandem mass spectrometry


Low-density lipoprotein cholesterol


Lower limit of quantification


Low-concentration quality control


Medium-concentration quality control


Multiple reaction monitoring mode


Nuclear magnetic resonance


No significance


Quality control


Relative standard deviation


Short-chain fatty acids


Standard deviation





We would like to thank Shimadzu (China) Co., Ltd. for technological support.

Funding information

This work was financially supported by the CAMS Innovation Fund for Medical Sciences (CIFMS, No. 2016-I2M-3-011), the National Natural Science Foundation of China (No. 81573493), the Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD study (No. Z141102004414062), and the Drug Innovation Major Project (Nos. 2018ZX09711001-002-002 and 2018ZX09302015).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving animals

All the research complied with the Institutional Guidelines & Ethics and was approved by the Laboratory Institutional Animal Care and Use Committees of the Chinese Academy of Medical Sciences & Peking Union Medical College.

Supplementary material

216_2019_1793_MOESM1_ESM.pdf (1.5 mb)
ESM 1 (PDF 1498 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Shu-Rong Ma
    • 1
  • Qian Tong
    • 2
  • Zhen-Xiong Zhao
    • 1
  • Lin Cong
    • 1
  • Jin-Bo Yu
    • 1
  • Jie Fu
    • 1
  • Pei Han
    • 1
  • Li-Bin Pan
    • 1
  • Randy Gu
    • 1
  • Ran Peng
    • 1
  • Zheng-Wei Zhang
    • 1
  • Yan Wang
    • 1
    Email author
  • Jian-Dong Jiang
    • 1
    Email author
  1. 1.State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia MedicaChinese Academy of Medical Sciences/Peking Union Medical CollegeBeijingChina
  2. 2.The First Hospital of Jilin UniversityChangchunChina

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