Simultaneous Determination of Multiple Active Constituents in Taxilli Herba by UFLC£­QTRAP£­MS/MS

KeyWord:Taxilli Herba  ultra-fast liquid chromatography£­triple quadrupole£­linear ion trap mass spectrometry  independent samples t-test  correlations  Box£­Behnken response surface methodology  active constituents

Abstract:

      An ultra-fast liquid chromatography£­triple quadrupole£­linear ion trap mass spectrometry £¨UFLC£­QTRAP£­MS/MS£© method was developed for the simultaneous determination of 33 active constituents consisting of flavonoids£¬organic acids£¬nucleosides£¬and amino acids in Taxilli Herba£®The stems branches and leaves of Taxilli Herba from different hosts were also compared and analyzed£®The optimum conditions for the preparation of the test solution were selected by the Box£­Behnken response surface method£¨BBD£­RSM£©£®The target constituents were separated on an XBridge? C18£¨4.6 mm ¡Á 100 mm£¬3.5 ¦Ìm£© analytical column by gradient elution using 0.1% formic acid aqueous solution and methanol as mobile phases£¬then analyzed by mass spectrometry with an electrospray ion source£¨ESI£© in multi reaction monitoring£¨MRM£© mode£¬and finally quantified by the external standard method£®The correlation analysis and independent sample t-test were performed for the contents of the 33 target constituents£®The results showed that the optimal extraction conditions were as follows£ºa methanol volume fraction of 70%£¬a liquid to material ratio £¨mL¡Ãg£© of 31¡Ã1 and an extraction time of 32 min£¬under which the extraction yield for quercetrin reached 3.77 mg/g£®The standard curves for 33 target constituents showed good linearity in the corresponding range with their correlation coefficients£¨r£© not less than 0.999 0£®The limits of detection£¨LODs£© and limits of quantitation£¨LOQs£© were in the range of 0.13£­66.85 ng/mL and 0.42£­222.83 ng/mL£¬respectively£®The precision£¬repeatability and stability of this method were excellent£®The average recoveries at low£¬medium and high spiked levels in real samples ranged from 98.0% to 101%£¬with the relative standard deviations£¨RSDs£© all less than 4.0%£®The correlation analysis revealed that the contents of active constituents were associated to a certain extent£®A t-test revealed that the contents of various active constituents in the stems branches and leaves of Taxilli Herba differed significantly£¬and the contents of each constituent£¬such as quercetin-3-O-glucuronide£¬hyperin£¬rutin£¬isoquercitrin£¬quercetrin and auicularin£¬were significantly higher in the leaves than in the stems and branches£®The developed method is accurate and stable£¬which could provide a new method reference for the comprehensive evaluation and overall control on the quality of Taxilli Herba£¬and a certain reference basis for its rational exploitation£®