Background: Sodium 9-dehydro-17-hydro-andrographolide-19-yl sulfate (DHAS) is the active ingredient of Xiyanping injection, a traditional Chinese medicine in clinical use. However, there has been no report about the metabolic rate and metabolites of DHAS in vitro.
Materials and methods: In this article, DHAS was incubated with rat liver S9, and liquid chromatography/mass spectrometry (LC/MS) was used for the metabolism study. The residual concentrations of substrate were determined by ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the metabolic rate study of DHAS in liver S9. Metabolites were identified by the (UPLC-TOF-MS(E)) Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry method.
Results: The calibration curves of DHAS were linear over the concentration range from 0.75 μM to 75.22 μM with correlation coefficients >0.99. The lower limit of quantification was 0.150 μM for DHAS. The determination recoveries of DHAS were in the range of 84.9-90.6%. The t½ and CLint of DHAS in rat liver S9 were 98.6 ± 2.1 min and 3.5 ± 0.1 mL/min/g, respectively. Five metabolites were preliminarily identified based on the high resolution mass spectrum data in comparison with related references. These metabolites were mainly the products of dehydration and hydrogenation of DHAS.
Conclusion: The present in vitro metabolic study of DHAS provided valuable information about the metabolic rate and potential metabolites of DHAS, which are important for future in vivo metabolism studies of DHAS and the discovery of more active andrographolide derivatives.
Summary: In this paper, sodium 9-dehydro-17-hydro-andrographolide-19-yl sulfate (DHAS) metabolism in vitro has been investigated with rat liver S9 using liquid chromatography-mass spectrometry (LC-MS). The result of quantitative analysis showed that DHAS had a long t1/2, which indicated its high metabolic stability. Five metabolites of DHAS were identified in the incubation system based on the high resolution mass spectrum data in comparison with related references, particularly dehydrated and hydrogenated products. The results would provide certain references to screen out more active andrographolide derivative for pre-clinically. Abbreviations used: MRM: Multiple reaction monitoring, DHAS: Sodium 9-dehydro-17-hydro-andrographolide-19-yl sulfate, IS: Internal standard.
Keywords: Liquid chromatography-mass spectrometry; metabolic rate; metabolites; rat liver S9; sodium 9-dehydro-17-hydro-andrographolide-19-yl sulfate.