Eatablishment of HPLC fingerprint of lichens of Thamnolia subuliformis(Ehrh.)W.Culb.and its UHPLC-Q Exactive Focus MS/MS analysis
Objective:To establish a quality assessment method for lichens of Thamnolia subuliformis(Ehrh.)W.Culb.based on HPLC fingerprint,and qualitative analysis of the chemical constituents by ultra-high performance liquid chromatography coupled with quadrupole/electrostatic field orbital trap high resolution mass spectrometry(UHPLC-Q Exactive Focus MS/MS).Methods:Agilent TC-C18(250 mm × 4.6 mm,5 μm)chromatographic column was used,the mobile phase was methol-0.1％phosphoric acid with gradient elution at the flow rate of 1.0 mL·min-1,the column temperature was 30 ℃,the detection wavelength was 254 nm,and the injection volume was 10 μL.HPLC fingerprints of lichens of Thamnolia subuliformis was established.The soft-ware of similarity calculation for traditional Chinese medicines fingerprints(version 2012)was used to establish the fingerprinting of lichens of Thamnolia subuliformis.The chemical constituents were analyzed by UHPLC-Q Exactive Focus MS/MS,The chromatographic separation was performed on a Waters Acquity UPLC BEH C18(50 mm ×2.1 mm,1.7 μm)column with acetonitrile(A)-0.1％formic acid aqueous solution(B)as mobile phase for gradient elution,the flow rate was 0.3 mL·min-1 and the column temperature was 30 ℃.Mass spec-trometry was performed using an electrospray ionization and data was collected in negative ion modes in the range of m/z 80-1 200.Results:Eight common peaks were identified from fingerprints of 10 batches of samples.The RSD values of relative retention time of 8 common peaks of chromotograms of samples were all below 0.5％and the similarities were above 0.9.4 of the identified peaks were further confirmed by UHPLC-Q Exactive Focus MS/MS as squamatic acid,baeomycesic acid,thamnoliadepsides B,barbatinic acid.Conclusion:The estab-lished method of fingerprint is stable,reliable,and specific,which can be used for quality evaluation of lichens of Thamnolia subuliformis.
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