Simultaneous determination of glycyrrhizic acid and glycyrrhetinic acid in human plasma by liquid-liquid extraction LC-MS/MS method
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 superna-tant 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 Mni 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(mo-bile 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 glycyrrhet-inic 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 glycyrrhet-inic acid in human plasma.
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