Metabolism Analysis·Activity Analysis

Simultaneous determination of glycyrrhizic acid and glycyrrhetinic acid in human plasma by liquid-liquid extraction LC-MS/MS method

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  • Lepu Pharmaceuticals, Inc., Zhengzhou 45000, China

Received date: 2024-03-06

  Online published: 2024-10-17

Abstract

Objective: To establish a liquid-liquid extraction liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of the concentration of glycyrrhizic acid and glycyrrhetinic acid in human plasma. Methods: Glycyrrhizic acid, glycyrrhetinic acid and the internal standard Apixaban-13C-d3(IS) were added to 0.1 mL of human blank plasma. 50 μL of ionization reagent (20% formic acid solution), 490 μL of ethyl acetate and 210 μL of methyl tert-butyl ether were used as extractant. The supernatant was dried by nitrogen, and the residue was dissolved with 200 μL acetonitrile-water (1∶1) containing 0.2% formic acid. And 5 μL of resulting solution was injected to the LC-MS/MS for analysis. Chromatographic conditions: the saparation was performed on a Boston Μni C18(50 mm×2.1 mm,3 μm)column with mobile phase consisting of 0.2% formic acid aqueous solution(mobile phase A)and 0.2% formic acid acetonitrile solution(mobile phase B)by gradient elution. The flow rate was 0.6 mL·min-1, the temperature of column was 40 ℃. The sample volume was 5 μL, and the temperature of the sampler was 4 ℃. Mass spectrometry conditions: multiple reaction montoring(MRM) was performed on a triple quadrupole mass spectrometer equipped with a ESI source in the positive mode. The detection ion pairs were m/z 823.4→453.3(glycyrrhizic acid), m/z 471.4→189.0 (glycyrrhetinic acid)and m/z 464.3→447.1(IS) respectively. Results: The calibration curves were linear over the concentrion ranges of 0.5-80 ng·mL-1 for glycyrrhizic acid and 2-800 ng·mL-1 for glycyrrhetinic acid (r>0.99) in the plasma, the lower limits of quantifications (LLOQ) were 0.5 ng·mL-1(glycyrrhizic acid) and 2 ng·mL-1(glycyrrhetinic acid), respectively. Inter-and intra-batch precisions (RSDs) were less than 6.8%, and the accuracy ranged from 92.3% to 104.2%. The recovery rates of glycyrrhizic acid and glycyrrhetinic acid were about 28.0% and 40.0% separately, and the recoveries of IS were about 65.0%, the precision (RSDs) were less than 7.9%. The normalized matrix factors of glycyrrhizic acid and glycyrrhetinic acid were about 1, and the precision (RSDs) were less than 7.3%. Conclusion: The method is sensitive, accurate, simple, rapid and applicable to simultaneous determination of the concentration of glycyrrhizic acid and glycyrrhetinic acid in human plasma.

Cite this article

LUO Xian-li, CHEN Xiao-ying, LI Yuan, SUN Dong-mei, ZHANG Ming-hui, LIU Shan-qi . Simultaneous determination of glycyrrhizic acid and glycyrrhetinic acid in human plasma by liquid-liquid extraction LC-MS/MS method[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44(9) : 1504 -1512 . DOI: 10.16155/j.0254-1793.2024-0146

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