| Objective1.To establish the UPLC fingerprints of Antrodia camphorata from wild.2.To establish a comparative analysis and qualitative assessment method of the chemical constituents of Antrodia camphorata from wild,basswood cultivation,solid culture and plate culture based on LCMS-IT-TOF technology.3.To establish the method for determinations of Antcin A,antcin C,antcin H and antcin K in Antrodia camphorata by UPLC-DAD.Methods1.With Antcin A,antcin C,antcin H and antcin K as a reference peak,the UPLC fingerprints of 10 batches of Antrodia camphorata from wild samples were established.The analysis of Antrodia camphorata from wild was performed on a 25℃ Shim-pack GISS-C18(2.1×100 mm,1.9 μm),with the mobile phase comprising of acetonitrile-0.1%formic acid flowing at 0.20 mL/min in a gradient elution manner,and the detection wavelength was set at 254 nm.2.Liquid chromatography-ion trap-time-of-flight-mass spectrometry(LCMS-IT-TOF)was performed on Shim-pack GISS-C18(2.1×100 mm,1.9 μm),mobile phase was 0.1%formic acid aqueous solution(A)-acetonitrile(B)for gradient elution(0-2.0 min,5%B;2.0～2.5 min,5%～36%B;2.5～7.5 min,36%B;7.5～35.0 min,36%～40%B;35.0～45.0 min,40%～58%B;45.0～60.0 min,58%B),the column temperature was 25℃ and the flow rate was 0.20 mL/min,volume of sample injection was 1.0 μL;electrospray ionization(ESI)was applied for mass spectrometric analysis under positive and negative ion mode,the scanning ranges were m/z 100～1000.3.The four different cultivation methods of Antrodia camphorata samples using ethanol ultrasonic extraction were separated on a Shim-pack GISS-C18(2.1×100 mm,1.9μm)and using 0.1%formic acid water-acetonitrile as mobile phase gradient elution,the detection wavelength was set at 254 nm,the flow rate was 0.20 mL/min,and the column temperature was 25 0C.LCMS-IT-TOF was used to qualitatively analyze the main chemical constituents of the ethanol extract of these samples.4.The UPLC-DAD assay was performed on a Shim-pack GISS-C18(2.1×100 mm,1.9μm)with water(containing 0.1%formic acid)-acetonitrile as mobile phase by gradient elution at a flow rate of 0.20 mL/min.The column temperature was set at 25℃,the detection wavelength was set at 254 nm.Resu ts1.The UPLC fingerprint was established and four components were identified(Antcin A,antcin C,antcin H and antcin K).The similarity of 10 batches of preparations were at least 0.927.2.The 39 chemical components were preliminarily identified of the Antrodia camphorata from wild via referring to literatures and analyzing MS/MS data.3.In this study,Sixty-three constituents of the four different cultivation methods of Antrodia camphorata were qualitatively analyzed by IT-TOF MS.And twelve components name among those were identified,including methyl antcinate B,dexamethasone,antrocinnamomin G,antcin K,antcin N,antcin D,lanosta-8,24-dien-3β,15a,21-triol,antroquinonol B,antcin C,antcin H,antrocinnamomins B,dehydrosulphurenic acid,methyl-3,11-dioxo-4a-methylergost-8,24(28)-dien-26-oate,a-Tocospiro B,antcin A and dehydroeburicoic acid.4.The antcin K,antcin C,antcin H and antcin A showed good linear relationships within the ranges of 5.00～200.00 μg/mL(r>0.999 7),13.75～550.00 μg/mL(r>0.999 8),5.50～220.00 μg/mL(r>0.999 7),and 6.25～250.00 pg/mL(r>0.999 7),whose average recoveries(RSD)were 98.16%(1.44%),103.42%(1.69%),97.82%(2.44%),and 104.78%(1.82%),respectively.The content of antcin K in the 19 batches of preparations was between 0.01 and 1.96 mg/g,the content of antcin C was between 1.08 and 12.40 mg/g,the content of antcin H was between 0.06 and 11.68 mg/g,and the content of antcin A was between 0.21 and 3.42 mg/g.Conclusion1.The method is accurate,simple,stable,and reliable,and can be used for quality control of Antrodia camphorata from wild.2.The qualitative analysis by LCMS-IT-TOF which provides a feasible method of quality control for Antrodia camphorata.3.This method is reliable,accurate,which offers advantages of high sensitivity and precision.It can be used for the overall assessment of the quality of Antrodia camphorata. |
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