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Proteomics and UHPLC-DAD-Q/Orbitrap-MS used to identify impurities in andrographolide
Shuangbing Deng1,2, Yawen Zhang1, Juan Chen3
1School of Pharmacy, Nanjing University of Chinese Medicine, Jiangsu Collaborative Innovation Centre of Chinese Medicinal Resources Industrialisation, and Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing, China.
Introduction:
Andrographolide active pharmaceutical ingredient (API) is a semidiurnal diterpene lactone with significant antipyretic, antiviral, anti-inflammatory and anticancer activities. A large amount of andrographolide API could only be obtained by extraction from Andrographis paniculata. Therefore, there may be related compounds, plant proteins and other impurities in andrographolide API.
Objective:
In order to improve the safety of andrographolide related preparations, it was necessary to clarify the impurities and improve the quality standard of andrographolide API.
Methods:
The related compounds were identified by ultrahigh-performance liquid chromatography with diode-array detector quadrupole Orbitrap mass spectrometry (UHPLC-DAD-Q/Orbitrap-MS), and the residual proteins were determined by ultrafiltration combined with proteomics. The proteomics method included protein extraction, content determination, digestion, desalination and nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) analysis. Then, MS results were compared with Andrographis paniculata protein database by Peaks Studio.
Results:
The results showed that 32 related compounds were putatively identified, of which 30 impurities were identified for the first time. Seven residual proteins together with 11 highly suspected proteins were uniquely identified, including the T1UNN5_ANDPA protein with the highest intensity.
Conclusions:
This study will provide useful information on the composition of andrographolide API, which is important for the quality control and clinical safety assurance of API and related prescriptions. Reasonable guidance will also be provided on the necessity of ultrafiltration in the production process of related injections.
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