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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).
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 software 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 spectrometry was performed using an electrospray ionization and data was collected in negative ion modes in the range of m/z 80-1 200.
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.
The established method of fingerprint is stable, reliable, and specific, which can be used for quality evaluation of lichens of Thamnolia subuliformis.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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