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Updated: Apr 19, 2026
Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase
Published on: October 15, 2018
Ultrahigh-performance liquid chromatography/tandem mass spectrometry method for evaluating enzyme activity and
Hongbin Zhu1, Xiaoyu Zhuang, Shu Liu
1National Center of Mass Spectrometry in Changchun & Jilin Province Key Laboratory of Chinese Medicine Chemistry and Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China; University of the Chinese Academy of Sciences, Beijing, 100039, China.
Rationale:
Prostaglandin E2 is an important biomarker in many biological systems. The development of sensitive and reliable analytical methods for monitoring PGE2 contents in various samples is of great interest. Here we developed an improved method for evaluating the enzyme activity and screening COX-2 inhibitors using ultrahigh-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) combined with PGE2 derivatization.
Methods:
Girard's reagent-T was used as the derivatization reagent and the reaction conditions were optimized. The established method was performed to screen the COX-2 inhibitors from effective constituents of herbs and detect the concentration of PGE2 in biological tissue samples (liver and kidney). The IC50 values of celecoxib, rofecoxib, sinomenine, bulleyaconitine A, tetrandrine, fangchinoline, berberine hydrochloride and sophocarpidine towards COX-2 were determined.
Results:
This method improves the quantitative ability for PGE2 , including the linearly dependent coefficient, linearity range and limit of detection. After derivatization, the derivatized PGE2 could be detected in positive ion mode of electrospray ionization (ESI), which improves the detection sensitivity 10-fold compared to that of the direct detection of underivatized PGE2 in negative ESI mode. Besides the positive control, sinomenine (IC50 =113 μM) and bulleyaconitine A (IC50=53 μM) were found to be potent COX-2 inhibitors.
Conclusions:
All the results indicate that the present derivatization quantification method of PGE2 could be used as the detection method of COX-2 enzyme activity and as the screening method for COX-2 inhibitors.
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