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

• Published in: Analytical and bioanalytical chemistry Vol. 411; no. 14; pp. 3191 - 3207
• Main Authors: Ma, Shu-Rong, Tong, Qian, Zhao, Zhen-Xiong, Cong, Lin, Yu, Jin-Bo, Fu, Jie, Han, Pei, Pan, Li-Bin, Gu, Randy, Peng, Ran, Zhang, Zheng-Wei, Wang, Yan, Jiang, Jian-Dong
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-05-2019; Springer; Springer Nature B.V
• Subjects: Acetophenones - chemistry; Alkaloids; Analytical Chemistry; Animals; Benzylisoquinolines - analysis; Berberine; Berberine - pharmacology; Biochemistry; Biological properties; Biological samples; Blood; Blood lipids; Chains; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Chromatography; Chromatography, Liquid - methods; Cricetinae; Energy metabolism; Esters; Fatty acids; Fatty Acids, Volatile - analysis; Fatty Acids, Volatile - blood; Fatty Acids, Volatile - chemistry; Fatty Acids, Volatile - standards; Feces; Feces - chemistry; Food Science; Gas chromatography; Gastrointestinal Microbiome; Hamsters; Health; Hyperlipidemia; Identification and classification; In vivo methods and tests; Intestinal microflora; Laboratory Medicine; Limit of Detection; Linearity; Lipid metabolism; Lipids; Liquid chromatography; Low level; Male; Mass spectrometry; Metabolism; Metabolites; Microbiota; Microbiota (Symbiotic organisms); Monitoring/Environmental Analysis; Reagents; Reference Standards; Reproducibility of Results; Research Paper; Saturated fatty acids; Sensitivity analysis; Spectrometry; Stability analysis; Tandem Mass Spectrometry - methods; Tetrahydroisoquinolines - analysis; Tetrandrine; Up-Regulation - drug effects; LC-MS/MS; Berberine; Isoquinoline alkaloids; Derivatization; 2-Bromoacetophenone; Butyrate
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-019-01793-3

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 C
18
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