CN106501437A - Based on the method that UPLC/LTQ Orbitrap Velos MS detect stem of noble dendrobium alkaloid component - Google Patents
Based on the method that UPLC/LTQ Orbitrap Velos MS detect stem of noble dendrobium alkaloid component Download PDFInfo
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- CN106501437A CN106501437A CN201611221950.2A CN201611221950A CN106501437A CN 106501437 A CN106501437 A CN 106501437A CN 201611221950 A CN201611221950 A CN 201611221950A CN 106501437 A CN106501437 A CN 106501437A
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- stem
- noble dendrobium
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- alkaloid
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- 229930013930 alkaloid Natural products 0.000 title claims abstract description 64
- 240000004638 Dendrobium nobile Species 0.000 title claims abstract description 55
- 150000003797 alkaloid derivatives Chemical class 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000004704 ultra performance liquid chromatography Methods 0.000 title claims abstract description 21
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 81
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 238000000926 separation method Methods 0.000 claims abstract description 23
- 238000012360 testing method Methods 0.000 claims abstract description 14
- 239000012071 phase Substances 0.000 claims description 50
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 29
- 239000000843 powder Substances 0.000 claims description 23
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 20
- 239000000463 material Substances 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 15
- 238000005119 centrifugation Methods 0.000 claims description 14
- 239000003960 organic solvent Substances 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 238000002604 ultrasonography Methods 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- 239000002002 slurry Substances 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 9
- 239000007789 gas Substances 0.000 claims description 8
- 239000007791 liquid phase Substances 0.000 claims description 8
- 239000007921 spray Substances 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 7
- 239000010931 gold Substances 0.000 claims description 7
- 229910052737 gold Inorganic materials 0.000 claims description 7
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 claims description 7
- 238000002525 ultrasonication Methods 0.000 claims description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 238000004451 qualitative analysis Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 238000013467 fragmentation Methods 0.000 claims description 5
- 238000006062 fragmentation reaction Methods 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- 239000003513 alkali Substances 0.000 claims description 4
- 238000001360 collision-induced dissociation Methods 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 4
- 238000001819 mass spectrum Methods 0.000 claims description 4
- 239000002243 precursor Substances 0.000 claims description 4
- 238000005070 sampling Methods 0.000 claims description 4
- 238000013375 chromatographic separation Methods 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 230000007717 exclusion Effects 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims 1
- 239000005864 Sulphur Substances 0.000 claims 1
- 238000007664 blowing Methods 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- -1 alkaloid salt Chemical class 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 239000000401 methanolic extract Substances 0.000 abstract description 2
- 230000000144 pharmacologic effect Effects 0.000 abstract description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 abstract description 2
- 150000002500 ions Chemical class 0.000 description 22
- 238000004458 analytical method Methods 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 5
- 241000026010 Dendrobium candidum Species 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000000498 ball milling Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241000050051 Chelone glabra Species 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 229930003944 flavone Natural products 0.000 description 3
- 235000011949 flavones Nutrition 0.000 description 3
- 238000000227 grinding Methods 0.000 description 3
- PISGDLOMGNKHKP-ROHXPCBUSA-N Luciduline Chemical compound C1[C@@H]2[C@H]3C[C@@H](C)C[C@@H]2N(C)C[C@@H]1C(=O)C3 PISGDLOMGNKHKP-ROHXPCBUSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 2
- 240000007164 Salvia officinalis Species 0.000 description 2
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 150000002212 flavone derivatives Chemical class 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 150000002825 nitriles Chemical class 0.000 description 2
- 238000007781 pre-processing Methods 0.000 description 2
- 235000005412 red sage Nutrition 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000004575 stone Substances 0.000 description 2
- 239000010414 supernatant solution Substances 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100029106 Ethylmalonyl-CoA decarboxylase Human genes 0.000 description 1
- 101710134484 Ethylmalonyl-CoA decarboxylase Proteins 0.000 description 1
- 241000692870 Inachis io Species 0.000 description 1
- STECJAGHUSJQJN-USLFZFAMSA-N LSM-4015 Chemical compound C1([C@@H](CO)C(=O)OC2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-USLFZFAMSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- HYXITZLLTYIPOF-UHFFFAOYSA-N Tanshinone II Natural products O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1C(C)=CO2 HYXITZLLTYIPOF-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229930002995 diterpene alkaloid Natural products 0.000 description 1
- 150000003800 diterpene alkaloid derivatives Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 150000002213 flavones Chemical class 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 description 1
- 229940107698 malachite green Drugs 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930003658 monoterpene Natural products 0.000 description 1
- 150000002773 monoterpene derivatives Chemical class 0.000 description 1
- 235000002577 monoterpenes Nutrition 0.000 description 1
- 229930014716 monoterpenoid indole alkaloid Natural products 0.000 description 1
- 150000003810 morphinanes Chemical class 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-N o-dicarboxybenzene Natural products OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229930000732 piperidine alkaloid Natural products 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 229930002352 pyrrolidine alkaloid Natural products 0.000 description 1
- 150000003235 pyrrolidines Chemical class 0.000 description 1
- LJPZHJUSICYOIX-UHFFFAOYSA-N quinolizidine Chemical compound C1CCCC2CCCCN21 LJPZHJUSICYOIX-UHFFFAOYSA-N 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- AZEZEAABTDXEHR-UHFFFAOYSA-M sodium;1,6,6-trimethyl-10,11-dioxo-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-2-sulfonate Chemical compound [Na+].C12=CC=C(C(CCC3)(C)C)C3=C2C(=O)C(=O)C2=C1OC(S([O-])(=O)=O)=C2C AZEZEAABTDXEHR-UHFFFAOYSA-M 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 229930003352 steroid alkaloid Natural products 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- AIGAZQPHXLWMOJ-UHFFFAOYSA-N tanshinone IIA Natural products C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2C(C)=CO1 AIGAZQPHXLWMOJ-UHFFFAOYSA-N 0.000 description 1
- 229930004668 tropane alkaloid Natural products 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides the method for detecting stem of noble dendrobium alkaloid component based on UPLC/LTQ Orbitrap Velos MS, by ultrasonically treated, and extracted with pure methanol solution, for the chemical property of alkaloid component, methanolic extract is dissolved using sour water, and is further alkalized, alkaloid salt is made to be changed into free alkaloid, extracted using dichloromethane solution, separated the drifting alkaloids testing sample with Ultra Performance Liquid Chromatography, by separation after alkaloid component Mass Spectrometer Method；Unknown metabolin is completed using OSI/SMMS softwares qualitative.The present invention carries out qualitative detection using UPLC/LTQ Orbitrap Velos MS technology to alkaloid component in the stem of noble dendrobium first, 22 kind alkaloid components are identified altogether, and gone out by separation detection in the stem of noble dendrobium first, the type of stem of noble dendrobium alkaloid component is specify that, is to study its pharmacological action to have established theoretical foundation.
Description
Technical field
The invention belongs to detection technique field, and in particular to based on UPLC/LTQ-Orbitrap Velos MS technology to stone
The detection method of alkaloid component in dry measure used in former times.
Background technology
The stem of noble dendrobium is conventional rare traditional Chinese medicine, with nourishing Yin and clearing heat, reinforcing stomach reg fluid, moisten the lung and relieve the cough the effects such as.Alkaloid is stone
One of most important active component in dry measure used in former times medicinal material, has significantly at the aspect such as antitumor, treatment angiocarpy and enterogastric diseases
Curative effect.For clear and definite effective component, deep chemical composition analysis are carried out to alkaloid in the stem of noble dendrobium significant.
At present, it is total alkaloid content to study in the stem of noble dendrobium more, and such as Xu Nin et al. reports and adopts AAS
Total alkaloid content in the stem of noble dendrobium is determined, but the method mainly carries out quantitative determination to alkaloid in the stem of noble dendrobium, and
And be detection that the amount to TA is carried out, it is impossible to distinguish the species of different alkaloids.
UPLC/LTQ-Orbitrap-MS technology be a kind of more efficiently, the technology of reliably analysis of compounds composition,
For example, Xie Tong et al. reports the structural characterization of flavone c-glycoside and flavones carbon in the root of large-flowered skullcap based on UPLC/LTQ-Orbitrap-MS
The differentiation of the isomer of methods of glycosides, the constituent analysis for other Chinese medicines provide reference.In addition, Niu Zengyuan et al. is reported
Antibiotic, hormone, phthalic acid ester, agricultural chemicals and peacock in cosmetics is determined using UPLC-LTQ/Orbitrap MS technology
5 big class such as malachite green and crystal violet totally 89 kinds of compounds, illustrate that the method is easy, quick, reliability is high.But the technical backstopping in
Detected sample through fine preprocessing process, need to remove the analysis knot that the interference of impurity in determinand can just obtain object
Really.Due to UPLC/LTQ-Orbitrap-MS technology very sensitive, if do not gone completely unless mesh in early stage preprocessing process
Mark thing, then easily cause the conclusion that analysis produces error or mistake, and therefore, the early stage pretreatment of sample is particularly significant.
In prior art, although using U PLC/LTQ- after having scholar's report to pre-process the samples such as the red sage root, the root of large-flowered skullcap
Orbitrap-MS technology detected to object, but, identification tanshinone IIA and tanshin polyphenolic acid B, early stage is extracted from the red sage root
Process needs the interference of the material for overcoming carbohydrate lipid；Separation detection flavone c-glycoside from the root of large-flowered skullcap, the purpose thing of extraction is carbohydrate,
Chaff interference is the materials such as albumen lipid, it is seen that the extracting method of purpose thing can not be general.In prior art, also it is not directed to
UPLC/LTQ-Orbitrap-MS technology carries out the report of preprocess method to the species of alkaloid in the stem of noble dendrobium.
Content of the invention
In view of this, it is an object of the invention to provide based on UPLC/LTQ-Orbitrap Velos MS technology to the stem of noble dendrobium
The detection method of middle alkaloid component, make methods described have quick, accurately, batch detection the characteristics of.
In order to realize that foregoing invention purpose, the present invention provide technical scheme below：
The invention provides the method for detecting stem of noble dendrobium alkaloid component based on UPLC/LTQ-Orbitrap Velos MS, bag
Include following steps：
1) toast after complete stem of noble dendrobium testing sample；
2) by the step 1) baking after the stem of noble dendrobium mix with organic solvent after grind, obtain stem of noble dendrobium slurries；Described has
Machine solvent includes methyl alcohol, ether or acetone；
3) by the step 2) stem of noble dendrobium slurries that obtain carry out ultrasonication, after separation of solid and liquid, obtain supernatant；
4) by the step 3) supernatant that obtains concentrated, and obtains extract dry powder；
5) by the step 4) the extract dry powder that obtains mixed with sulfuric acid solution, obtains mixed liquor, adjust the mixing
The alkaline mixed solution for obtaining is stood by the pH value of liquid to 9.5～10.5；
6) by the step 5) stand after alkaline mixed solution mix with dichloromethane, centrifugation, obtain dichloromethane phase；
7) by the step 6) dichloromethane that obtains mutually concentrates, and obtains biological dry alkaline powder；
8) biological dry alkaline powder methyl alcohol is redissolved, after separation of solid and liquid, obtains drifting alkaloids testing sample；
9) separate the drifting alkaloids testing sample with Ultra Performance Liquid Chromatography, by separation after alkaloid component matter
Spectrum detection, obtains alkaloid component material peak；
10) qualitative analysis is carried out to the alkaloid component material peak with OSI/SMMS softwares.
Preferably, the step 1) in the temperature that completes be 100～110 DEG C；The time for completing is 15～25min.
Preferably, the step 1) in baking temperature be 50～65 DEG C；Described it is baked to constant weight.
Preferably, the step 2) in the volume ratio of quality and organic solvent of the stem of noble dendrobium be (50～80) mg：(1～2) mL.
Preferably, the step 3) in ultrasound temperature be 25～30 DEG C；20～the 40min of time of ultrasound；The ultrasound
Power be 100～150W.
Preferably, the step 3) and step 8) described in separation of solid and liquid using centrifugation method；
The rotating speed of the centrifugation is 8000～12000rpm；
The time of the centrifugation is 10～15min.
Preferably, the step 4) and step 7) in concentration stand alone as nitrogen and dry up concentration；
The time of the concentration is 40～100min.
Preferably, the step 5) in the volume ratio of quality and sulfuric acid solution of extract dry powder be (7～11) mg：(1～
2)mL；
The volumetric concentration of the sulfuric acid solution is 8%～12%.
Preferably, the step 5) in stand time be 30～50min.
Preferably, the step 6) volume ratio of neutral and alkali mixed liquor and dichloromethane is 2～3：1～2；
The step 6) described in be centrifuged rotating speed be 3500～5000rpm；
The step 6) described in centrifugation time be 15～20min.
Preferably, the step 9) in chromatographic separation condition include following parameter：
Chromatographic column is Hypersil GOLD liquid-phase chromatographic columns；The specification of the Hypersil GOLD liquid-phase chromatographic columns is
2.1mm×100mm×1.9μm；
Column temperature is 40 DEG C；
Elution system mobile phase A is 0.1% aqueous formic acid of volumetric concentration, and Mobile phase B is 0.1% formic acid second of volumetric concentration
Nitrile solution；
The flow velocity of wash-out is 0.35mL/min；
Sampling volume is 5 μ L；
Eluent gradient elution program：
The volume ratio 19 of (0,1min), mobile phase A and Mobile phase B：1 mixed liquor；
[1min, 24min), mobile phase A is steady state value with the volume ratio of Mobile phase B, and the scope of the steady state value is 1：19
～19：1；
[24min, 28min), mobile phase A is 1 with the volume ratio of Mobile phase B：19；
[28min, 28.1min), mobile phase A is steady state value with the volume ratio of Mobile phase B, and the scope of the steady state value is 1：
19～19：1；
[28.1,30min], mobile phase A are 19 with the volume ratio of Mobile phase B：1.
Preferably, the step 9) in Mass Spectrometer Method condition include following parameter：
Ion gun is electric spray ion source, and positive ion mode is detected；
Sheath gas, 30 free units；Auxiliary gas, 6 free units；
Spray voltage, 3.8kV；
Ion transfer capillary temperature is 350 DEG C, and source heating-up temperature is 350 DEG C；
Precursor scans scope：50-1000m/z, the scan pattern of data dependence gather most strong 5 after each full scan
Individual fragment patterns stored carries out second order mses scanning；
Fragmentation pattern：Collision induced dissociation, normal state energy 30%, q values 0.25, soak time are 30ms, and dynamic is excluded
Time is 5s；
First mass spectrometric resolution ratio in m/z400 is 30000, and second order mses resolution ratio in m/z400 is 30000.
The invention provides the method for detecting stem of noble dendrobium alkaloid component based on UPLC/LTQ-Orbitrap Velos MS, leads to
Cross ultrasonic extraction, meanwhile, soluble in water according to polysaccharide and the characteristics of do not dissolve in methyl alcohol, ether, acetone and other organic solvent, with above-mentioned
Organic solvent is extracted, it is to avoid the leaching of glucide in the stem of noble dendrobium, is more beneficial for the extraction of trace materials alkaloid component.
Chemical property of the present invention for alkaloid component, dissolves methanolic extract using sour water, and further alkalizes, make alkaloid salt
It is changed into free alkaloid, is extracted using dichloromethane solution, removes other water-solubility impurity starch, protein, glues
Liquid matter, tannin, pigment, inorganic salts etc., are more beneficial for the separation and detection of alkaloid component.The present invention is using with extensive generation
Thank to group quick, accurate, OSI/SMMS softwares for batch qualitative analysis, complete unknown metabolin qualitative.The present invention is adopted first
UPLC/LTQ-Orbitrap Velos MS technology detected to alkaloid component in the stem of noble dendrobium and qualitative, identifies 22 kinds of lifes altogether
Alkaloids composition, and gone out by separation detection in the stem of noble dendrobium first, the type of stem of noble dendrobium alkaloid component is further specify that, is research
Theoretical foundation has been established in its pharmacological action.
Description of the drawings
Fig. 1 is total ion current figure under the extract positive ion mode of protocorms of dendrobium candidum in embodiment 1；
Fig. 2 is that m/z 225.19556 extracts ion stream chromatogram in embodiment 2；
Fig. 3 is 225.19556 first mass spectrometric figures of m/z in embodiment 2；
Fig. 4 is 225.19556 second order mses figures of m/z in embodiment 2.
Specific embodiment
The invention provides the method for detecting stem of noble dendrobium alkaloid component based on UPLC/LTQ-Orbitrap Velos MS, bag
Include following steps：
1) toast after complete stem of noble dendrobium testing sample；
2) by the step 1) baking after the stem of noble dendrobium mix with organic solvent after grind, obtain stem of noble dendrobium slurries；Described has
Machine solvent includes methyl alcohol, ether or acetone；
3) by the step 2) stem of noble dendrobium slurries that obtain carry out ultrasonication, after separation of solid and liquid, obtain supernatant；
4) by the step 3) supernatant that obtains concentrated, and obtains extract dry powder；
5) by the step 4) the extract dry powder that obtains mixed with sulfuric acid solution, obtains mixed liquor, adjust the mixing
The alkaline mixed solution for obtaining is stood by the pH value of liquid to 9.5～10.5；
6) by the step 5) stand after alkaline mixed solution mix with dichloromethane, centrifugation, obtain dichloromethane phase；
7) by the step 6) dichloromethane that obtains mutually concentrates, and obtains biological dry alkaline powder；
8) biological dry alkaline powder methyl alcohol is redissolved, after separation of solid and liquid, obtains drifting alkaloids testing sample；
9) separate the drifting alkaloids testing sample with Ultra Performance Liquid Chromatography, by separation after alkaloid component matter
Spectrum detection, obtains alkaloid component material peak；
10) qualitative analysis is carried out to the alkaloid component material peak with OSI/SMMS softwares.
The present invention is toasted after stem of noble dendrobium testing sample is completed.
In the present invention, the kind of the stem of noble dendrobium is preferably dendrobium candidum, Huoshan rice dry measure used in former times or Dendrobium Moniliforme.The stem of noble dendrobium is preferred
Edible stem of noble dendrobium protocorm.
In the present invention, the method for completing is not particularly limited, using the side of completing well-known to those skilled in the art
Method.The temperature for completing is preferably 100～110 DEG C, more preferably 105 DEG C；The time for completing be preferably 15～
25min, more preferably 20min.The purpose for completing is the enzyme activity that destroys in fresh stem of noble dendrobium material, so as to retain effect material.
In the present invention, the temperature of the baking is preferably 50～65 DEG C, more preferably 60 DEG C；The time of the baking does not have
Specifically limited, the degree of baking is preferably baked to constant weight.
After the stem of noble dendrobium after being toasted, the present invention is ground after the stem of noble dendrobium after the baking is mixed with organic solvent, is obtained
Stem of noble dendrobium slurries；Described organic solvent includes methyl alcohol, ether or acetone.
In the present invention, the quality of the stem of noble dendrobium is preferably (50～80) mg with the volume ratio of organic solvent：(1～2) mL, more
Preferably (50～65) mg：(1～1.5) mL.The mass concentration of the methyl alcohol, ether or acetone is preferably 100%.
In the present invention, the method for grinding is not particularly limited, using the side of grinding well-known to those skilled in the art
Method.The degree for grinding preferably is milled to 100～300 mesh.In the embodiment of the present invention, grind and ground using ball milling instrument.
The frequency of the ball milling instrument is 60～70HZ, and the time that grinds of the ball milling instrument is 3～5min, more preferably 4min.
After obtaining stem of noble dendrobium slurries, the stem of noble dendrobium slurries are carried out ultrasonication by the present invention, after separation of solid and liquid, obtain supernatant
Liquid.
In the present invention, the method for the ultrasound is not particularly limited the skill using ultrasound well-known to those skilled in the art
Art scheme.The temperature of the ultrasound is preferably 25～30 DEG C.The time of ultrasound is preferably 20～40min, more preferably
30min.The power of the ultrasound is preferably 100～150W, more preferably 120W.
In the present invention, the method for the separation of solid and liquid is not particularly limited using solid-liquid well-known to those skilled in the art
Detached technical scheme.In the present invention, the separation of solid and liquid is preferably using the method for centrifugation.The rotating speed of the centrifugation is preferred
For 8000～12000rpm, most preferably more preferably 9000～11000rpm, 10000rpm.In the present invention, the centrifugation
Time be preferably 10～15min, more preferably 12min.
In the present invention, the stem of noble dendrobium residue that obtains after the separation of solid and liquid is ground after preferably being mixed with organic solvent again, is obtained
To stem of noble dendrobium slurries carry out ultrasonication again, collect supernatant.The supernatant that this supernatant for obtaining was obtained with last time is closed
And enter lower step process.In the present invention, the mixing ratio of the stem of noble dendrobium residue for mixing with organic solvent again and organic solvent
Example is identical with such scheme.The parameter of the ultrasonication and the parameter all same of above-mentioned steps ultrasonication, here is not done superfluous
State.
After obtaining supernatant, the supernatant is concentrated by the present invention, obtains extract dry powder.
In the present invention, the concentration is preferably nitrogen and dries up concentration.The nitrogen dry up concentration time be 40～
100min, more preferably 60min.In the present invention, the nitrogen dries up concentration and preferably dries up instrument using nitrogen and carry out.The nitrogen
The source for drying up instrument is not particularly limited, and dries up instrument using nitrogen known in the art.In the embodiment of the present invention, described
Nitrogen dries up instrument purchased from Hangzhou Ao Sheng Instrument Ltd..
After obtaining extract dry powder, the extract dry powder is mixed by the present invention with sulfuric acid solution, obtains mixed liquor, is adjusted
The alkaline mixed solution for obtaining is stood by the pH value of the mixed liquor to 9.5～10.5.
In the present invention, the method that the extraction dry powder is mixed with sulfuric acid solution is not particularly limited, using art technology
The technical scheme of the mixing known to personnel.The volume ratio of quality and sulfuric acid solution of the mixing according to extract dry powder
Preferably (7～11) mg：(1～2) mL, more preferably (8～9) mg：(1～1.5) mL.The volumetric concentration of the sulfuric acid solution is excellent
Elect 8%～12%, more preferably 10% as.
In the present invention, the pH value of the adjustment mixed liquor is preferably to 10.The solution of the adjustment pH is preferably ammoniacal liquor.Described
The mass fraction of ammoniacal liquor is preferably 20～30%, more preferably 25%.
In the present invention, the time of the standing is preferably 30～50min, more preferably 40min.The purpose of the standing is
Alkaloid salt is made more fully to be changed into drifting alkaloids.
After the standing, the alkaline mixed solution after the standing is mixed by the present invention with dichloromethane, is centrifuged, is obtained dichloro
Methane phase.
In the present invention, the alkaline mixed solution is preferably (2～3) mL with the volume ratio of dichloromethane：(1～2) mL, more excellent
Elect 2～2.5 as：1～1.5.
In the present invention, described be centrifuged to obtain rotating speed be preferably 3500～5000rpm, more preferably 4000rpm.Institute in the present invention
The time for stating centrifugation is preferably 15～20min, more preferably 18min.
After centrifugation, in careful collection dichloromethane to new centrifuge tube.In the present invention, the method for the collection is preferably adopted
With pipettor absorption dichloromethane phase transfer into new centrifuge tube.
In the present invention, after obtaining dichloromethane phase, remaining extract and absolute dichloromethane are mixed by preferred repeat the above steps
Close, after separation of solid and liquid, lower floor's dichloromethane is collected in new centrifuge tube, merge lower floor's solution twice.The mixing is preferably
Vortex methods.The time of the vortex is preferably 30～40s.
After obtaining dichloromethane phase, the dichloromethane is mutually concentrated by the present invention, obtains biological dry alkaline powder.
In the present invention, the concentration is preferably nitrogen and dries up concentration.The nitrogen dry up concentration time be 40～
100min, more preferably 60min.In the present invention, the nitrogen dries up concentration and preferably dries up instrument using nitrogen and carry out.The nitrogen
The source for drying up instrument is not particularly limited, and dries up instrument using nitrogen known in the art.In the embodiment of the present invention, described
Nitrogen dries up instrument purchased from Hangzhou Ao Sheng Instrument Ltd..
After obtaining biological dry alkaline powder, biological dry alkaline powder absolute methanol is redissolved by the present invention, is obtained after separation of solid and liquid
Drifting alkaloids testing sample.
The quality of the biological dry alkaline powder is preferably (0.6～1.2) mg with absolute methanol volume ratio：(500～800) μ L,
More preferably (0.8～1.0) mg：(500～600) μ L.
Liquid will preferably be redissolved to be filtered after obtaining the solution for redissolving.Described filtration preferably adopt membrane filtration.The filtration
The aperture of film is preferably 0.22 μm.
The drifting alkaloids testing sample for filtering is obtained, the present invention separates the drifting alkaloids with Ultra Performance Liquid Chromatography
Testing sample, by separation after alkaloid component Mass Spectrometer Method, obtain alkaloid component material peak.
In the present invention, the chromatographic separation condition preferably includes following parameter：
Chromatographic column is Hypersil GOLD liquid-phase chromatographic columns；The specification of the Hypersil GOLD liquid-phase chromatographic columns is
2.1mm×100mm×1.9μm；
Column temperature is 40 DEG C；
Elution system mobile phase A is 0.1% aqueous formic acid of volumetric concentration, and Mobile phase B is 0.1% formic acid second of volumetric concentration
Nitrile solution；
The flow velocity of wash-out is 0.35mL/min；
Sampling volume is 5 μ L；
Eluent gradient elution program：The volume ratio 19 of (0,1min), mobile phase A and Mobile phase B：1 mixed liquor；
[1min, 24min), mobile phase A is steady state value with the volume ratio of Mobile phase B, and the scope of the steady state value is 1：19
～19：1；
[24min, 28min), mobile phase A is 1 with the volume ratio of Mobile phase B：19；
[28min, 28.1min), mobile phase A is steady state value with the volume ratio of Mobile phase B, and the scope of the steady state value is 1：
19～19：1；
[28.1,30min], mobile phase A are 19 with the volume ratio of Mobile phase B：1.
In the present invention, the Mass Spectrometer Method condition preferably includes following parameter：
Ion gun be electric spray ion source (ESI), positive ion mode detect；
Sheath gas, 30 free units；Auxiliary gas, 6 free units；
Spray voltage, 3.8kV；
Ion transfer capillary temperature is 350 DEG C, and source heating-up temperature is 350 DEG C；
Precursor scans scope：50-1000m/z, the scan pattern of data dependence gather most strong 5 after each full scan
Individual fragment patterns stored carries out second order mses scanning；
Fragmentation pattern：Collision induced dissociation, normal state energy 30%, q values 0.25, soak time are 30ms, and dynamic is excluded
Time is 5s；
First mass spectrometric resolution ratio in m/z400 is 30000, and second order mses resolution ratio in m/z400 is 30000.
Alkaloid component material peak is obtained, it is fixed that present invention OSI/SMMS softwares are carried out to the alkaloid component material peak
Property analysis.
In the present invention, the qualitative analysis preferred version step is：(1) material is extracted from mass spectrum total ion current figure
Peak, obtains accurate molecular weight；(2) one-level according to compound peaks, second order mses fragment ion information search database, obtain
The ms fragment information matched with the material of screening in database；(3) by when adduct ion classification, molecular weight error, reservation
Between, the Fragmentation information binding analysis that obtain in pertinent literature and step (2), obtain the structure of compound.
In the present invention, the material peak is the peak that mass spectrum is able to detect that.
In the present invention, described database include the network data bases such as HMDB, Metlin, Lipid maps, MMCD and from
Build database.
In the present invention, the adduct ion classification includes M+H, M+Na, M+NH4 etc..
In the present invention, the molecular weight error is that absolute value is less than 10mmu.
Given birth to based on the UPLC/LTQ-Orbitrap Velos MS detection stems of noble dendrobium with reference to what embodiment was provided to the present invention
The method of alkaloids composition is described in detail, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
1st, instrument and reagent
1.1 instrument：UHPLC liquid phase systems Ultimate 3000 (Thermo Fisher Scientific, Waltham,
MA, USA) and mass spectrum LTQ Orbitrap Velos Pro (Thermo Fisher Scientific, Waltham, MA, USA).
1.2 reagent：Protocorms of dendrobium candidum material is provided by Agricultural University Of Anhui's Life Science College, methyl alcohol, acetonitrile
For chromatographic grade, other reagents are that analysis is pure.
The pretreatment of stem of noble dendrobium material
Take stem of noble dendrobium protocorms to complete in 105 DEG C of baking ovens after 20min, dry to constant weight in 60 DEG C of baking ovens.
The extraction of stem of noble dendrobium composition of alkaloids
(1) 50～80mg of stem of noble dendrobium sample of drying is taken in 2mL centrifuge tubes, use ball milling instrument after adding the pure methanol solutions of 1mL
Grind.
(2) 25～30 DEG C of ultrasonic extractions 30min, 11000rpm be centrifuged 10～15min, take 700～800 μ L of supernatant of supernatant in
In new centrifuge tube.
(3) repeat the above steps, add the pure methanol solutions of 1mL in precipitation, and be vortexed 30～45s, and 25～30 DEG C of ultrasounds are carried
30min (ultrasonic power 120w) is taken, and 800～900 μ L of supernatant is taken in new centrifuge tube, is merged supernatant solution twice.
(4) after supernatant solution nitrogen is dried up, 1～2mL10% sulfuric acid solutions (v/v) are added, is used after the 1～2min that is vortexed
25% ammoniacal liquor (m/m) adjusts pH to 10, stands 30～50min under room temperature.
(5) 1mL absolute dichloromethane solution is added, after the 30～40s that is vortexed, 4000rpm is centrifuged 15～20min, removes layer two
700～800 μ L of chloromethanes phase solution are in new centrifuge tube.
(6) repeat the above steps, add 1mL absolute dichloromethane solution in remaining extract, after the 30～40s that is vortexed,
4000rpm is centrifuged 15～20min, removes layer 800～900 μ L of dichloromethane phase solution in new centrifuge tube, under merging twice
Layer solution.
(7) after dichloromethane phase solution nitrogen is dried up, after adding the pure methanol solutions of 500～600 μ L to redissolve, 11000rpm
10～15min of centrifugation, can be analyzed after crossing 0.22 μm of filter membrane.
4. the separation and detection of sample
Chromatographic condition：Chromatographic column is Hypersil GOLD liquid-phase chromatographic columns (C18,2.1mm × 100mm × 1.9 μm)；Post
Temperature is 40 DEG C；Elution system is that mobile phase A is 0.1% (v/v) aqueous formic acid under positive ion mode, and Mobile phase B is 0.1%
(v/v) formic acid acetonitrile solution；Flow velocity is 0.35mL/min；5 μ L of sampling volume；
Gradient elution program is as shown in table 1
1 eluent gradient elution parameters of table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 1 | 95 | 5 |
| 24 | 0 | 95 |
| 28 | 0 | 95 |
| 28.1 | 95 | 5 |
| 30 | 95 | 5 |
Mass Spectrometry Conditions：Ion gun is electric spray ion source, and positive ion mode is detected；Sheath gas, 30 free units；Auxiliary gas, 6
Free unit；Spray voltage, 3.8kV；Ion transfer capillary temperature is 350 DEG C, and source heating-up temperature is 350 DEG C, using front warp
Standard correction liquid is corrected.Precursor scans scope：50-1000m/z, the scan pattern of data dependence are gathered after each full scan
5 most strong fragment patterns storeds carry out second order mses scanning.Fragmentation pattern：Collision induced dissociation, normal state energy 30%, q values
0.25, soak time is 30ms, and the dynamic exclusion time is 5s.MS1 in m/z400 resolution ratio be 30000, MS2 in m/z400
Resolution ratio is 30000.
5. the qualitative analysis of stem of noble dendrobium new type alkaloid component
Metabolism micromolecular compound Rapid identification analysis system (OSI/SMMS) using qualitative system at many levels is to biology
Alkali composition material peak carries out qualitative.22 kinds of alkaloid components are isolated and identified, including 2 kinds of tropane alkaloid, monoterpene is biological
1 kind of alkali, 2 kinds of monoterpenoid indole alkaloid, 3 kinds of pyrrolidine alkaloid, 4 kinds of morphinane alkaloid, a kind of diterpene alkaloid, in quinoline promise
3 kinds of western pyridine type alkaloid, a kind of piperidine alkaloid, 2 kinds of steroid alkaloid, 3 kinds of related alkaloids.Concrete outcome is as shown in table 2.
By taking quinolizidine type alkaloid Luciduline as an example, from total ion current figure, (Fig. 1) extracts m/z225.19556 (figures
2).In the positive-ion mode, first mass spectrometric quasi-molecular ion peak is m/z225.19556 [M+NH4]+(Fig. 3), deposit in second order mses
In m/z 100.11213,143.11813 plasma fragments (Fig. 4) of m/z.According to molecular weight, fragment ion information and compound
Database speculates the compound for Luciduline, and its molecular formula is C13H21NO.
New type alkaloid component in 2 protocorms of dendrobium candidum of table
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. the method for detecting stem of noble dendrobium alkaloid component based on UPLC/LTQ-Orbitrap Velos MS, comprises the following steps：
1) toast after complete stem of noble dendrobium testing sample；
2) by the step 1) baking after the stem of noble dendrobium mix with organic solvent after grind, obtain stem of noble dendrobium slurries；Described is organic molten
Agent includes methyl alcohol, ether or acetone；
3) by the step 2) stem of noble dendrobium slurries that obtain carry out ultrasonication, after separation of solid and liquid, obtain supernatant；
4) by the step 3) supernatant that obtains concentrated, and obtains extract dry powder；
5) by the step 4) the extract dry powder that obtains mixed with sulfuric acid solution, obtains mixed liquor, adjust the mixed liquor
The alkaline mixed solution for obtaining is stood by pH value to 9.5～10.5；
6) by the step 5) stand after alkaline mixed solution mix with dichloromethane, centrifugation, obtain dichloromethane phase；
7) by the step 6) dichloromethane that obtains mutually concentrates, and obtains biological dry alkaline powder；
8) biological dry alkaline powder methyl alcohol is redissolved, after separation of solid and liquid, obtains drifting alkaloids testing sample；
9) separate the drifting alkaloids testing sample with Ultra Performance Liquid Chromatography, by separation after alkaloid component examined with mass spectrum
Survey, obtain alkaloid component material peak；
10) qualitative analysis is carried out to the alkaloid component material peak with OSI/SMMS softwares.
2. detection method according to claim 1, it is characterised in that the step 1) in the temperature that completes be 100～110
℃；The time for completing is 15～25min.
3. detection method according to claim 1, it is characterised in that the step 1) in the temperature of baking be 50～65
℃；Described it is baked to constant weight.
4. detection method according to claim 1, it is characterised in that the step 2) in the stem of noble dendrobium quality and organic solvent
Volume ratio be (50～80) mg：(1～2) mL.
5. detection method according to claim 1, it is characterised in that the step 3) in the temperature of ultrasound be 25～30
℃；20～the 40min of time of ultrasound；The power of the ultrasound is 100～150W.
6. detection method according to claim 1, it is characterised in that the step 4) and step 7) in concentration stand alone as nitrogen
The dry concentration of air-blowing；
The time of the concentration is 40～100min.
7. detection method according to claim 1, it is characterised in that the step 5) in extract dry powder quality and sulphur
The volume ratio of acid solution is (7～11) mg：(1～2) mL；
The volumetric concentration of the sulfuric acid solution is 8%～12%.
8. detection method according to claim 1, it is characterised in that the step 6) neutral and alkali mixed liquor and dichloromethane
Volume ratio be 2～3：1～2；
The step 6) described in be centrifuged rotating speed be 3500～5000rpm；
The step 6) described in centrifugation time be 15～20min.
9. detection method according to claim 1, it is characterised in that the step 9) in chromatographic separation condition include as follows
Parameter：
Chromatographic column is Hypersil GOLD liquid-phase chromatographic columns；The specification of the Hypersil GOLD liquid-phase chromatographic columns is 2.1mm
×100mm×1.9μm；
Column temperature is 40 DEG C；
Elution system mobile phase A is 0.1% (v/v) aqueous formic acid, and Mobile phase B is 0.1% (v/v) formic acid acetonitrile solution；
The flow velocity of wash-out is 0.35mL/min；
Sampling volume is 5 μ L；
Eluent gradient elution program：
The volume ratio 19 of (0,1min), mobile phase A and Mobile phase B：1 mixed liquor；
[1min, 24min), mobile phase A is steady state value with the volume ratio of Mobile phase B, and the scope of the steady state value is 1：19～19：
1；
[24min, 28min), mobile phase A is 1 with the volume ratio of Mobile phase B：19；
[28min, 28.1min), mobile phase A is steady state value with the volume ratio of Mobile phase B, and the scope of the steady state value is 1：19～
19：1；
[28.1,30min], mobile phase A are 19 with the volume ratio of Mobile phase B：1.
10. detection method according to claim 1, it is characterised in that the step 9) in Mass Spectrometer Method condition include with
Lower parameter：
Ion gun is electric spray ion source, and positive ion mode is detected；
Sheath gas, 30 free units；Auxiliary gas, 6 free units；
Spray voltage, 3.8kV；
Ion transfer capillary temperature is 350 DEG C, and source heating-up temperature is 350 DEG C；
Precursor scans scope：50-1000m/z, the scan pattern of data dependence gather most strong 5 broken after each full scan
Piece collection of illustrative plates carries out second order mses scanning；
Fragmentation pattern：Collision induced dissociation, normal state energy 30%, q values 0.25, soak time are 30ms, the dynamic exclusion time
For 5s；
First mass spectrometric resolution ratio in m/z400 is 30000, and second order mses resolution ratio in m/z400 is 30000.
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