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Updated: Oct 19, 2025
Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS
Published on: February 24, 2021
UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin
Zhizhen Song1,2, Zeyun Li1,2, Xueqian Wen1,2
1Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People's Republic of China.
Background:
Epimedin C, one of the main active ingredients of Epimedium, has been reported to have potential hepatotoxicity. However, the mechanism of Epimedin C-induced liver injury has not been studied. mRNA methylation, mainly including N6-methyladenosine and N5-methylcytidine, is implicated in the regulation of many biological processes and diseases. The study of quantifying mRNA methylation alterations in Epimedin C-induced liver injury mice may contribute to clarify the mechanism of its hepatotoxicity. Therefore, an analysis method needs to be established to determine nucleoside and methyl-nucleoside levels in liver mRNA.
Methods:
An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously determine six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in liver mRNA. Besides, the Epimedin C-induced liver injury mouse model was studied by intragastrical administration Epimedin C at a daily dose of 10 or 40 mg/kg for 4 weeks. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer.
Results:
In this method, calibration curves of the six nucleosides showed good linearity over their concentration ranges. The linear ranges were 40-20,000 pg/mL for adenosine, cytidine, N6-methyladenosine and N5-methylcytidine, 0.2-100 ng/mL for guanosine, and 2-1000 ng/mL for uridine. Epimedin C-induced liver injury mouse model was successfully established,which could be proved by the elevation of serum aminotransferase levels, and the increased inflammatory cell infiltration as well as vacuolar degeneration in liver. The N6-methyladenosine and N5-methylcytidine levels, and the ratios of N6-methyladenosine to adenosine and N5-methylcytidine to cytidine of the mice liver mRNA were all significantly increased after Epimedin C treatment.
Conclusion:
The established method was successfully applied to the determination of six nucleosides levels in liver mRNA of the Epimedin C-induced liver injury mice model and the control group. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study will facilitate the mechanism research on the hepatotoxicity of Epimedin C.
Insights
Epimedium
Area of Science:
- Pharmacology and Toxicology
- Molecular Biology
- Biochemistry
Background:
- Epimedium contains Epimedin C, a compound with potential liver toxicity.
- The mechanism of Epimedin C-induced liver injury remains unclear.
- mRNA methylation, including N6-methyladenosine (m6A) and N5-methylcytidine (m5C), plays a role in biological processes and diseases.
Purpose of the Study:
- To establish and validate an analytical method for quantifying nucleosides and methyl-nucleosides in liver mRNA.
- To investigate the alterations in mRNA methylation in a mouse model of Epimedin C-induced liver injury.
- To elucidate the potential role of mRNA methylation in Epimedin C hepatotoxicity.
Main Methods:
- Developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of six nucleosides (adenosine, uridine, cytidine, guanosine, m6A, m5C) in liver mRNA.
- Established a mouse model of Epimedin C-induced liver injury through intragastrical administration (10 or 40 mg/kg daily for 4 weeks).
- Analyzed liver mRNA nucleoside levels after enzymatic digestion and UPLC-MS/MS detection.
Main Results:
- The UPLC-MS/MS method demonstrated good linearity for all six nucleosides.
- Epimedin C treatment successfully induced liver injury in mice, evidenced by elevated serum aminotransferase levels and histological changes.
- Significant increases in m6A and m5C levels, as well as their ratios to adenosine and cytidine, were observed in the liver mRNA of treated mice.
Conclusions:
- The developed UPLC-MS/MS method is effective for analyzing nucleoside levels in liver mRNA.
- mRNA methylation alterations are associated with Epimedin C-induced liver injury.
- This study provides insights into the mechanism of Epimedin C hepatotoxicity, facilitating further research.
