CN106501417A - The quick method for determining protodioscin content in Rhizoma Dioscoreae Zingiberensiss - Google Patents
The quick method for determining protodioscin content in Rhizoma Dioscoreae Zingiberensiss Download PDFInfo
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- CN106501417A CN106501417A CN201611135243.1A CN201611135243A CN106501417A CN 106501417 A CN106501417 A CN 106501417A CN 201611135243 A CN201611135243 A CN 201611135243A CN 106501417 A CN106501417 A CN 106501417A
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- protodioscin
- rhizoma dioscoreae
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- LVTJOONKWUXEFR-UEZXSUPNSA-N protodioscin Chemical compound O([C@@H]1[C@@H](CO)O[C@H]([C@@H]([C@H]1O)O[C@H]1[C@@H]([C@H](O)[C@@H](O)[C@H](C)O1)O)O[C@@H]1CC2=CC[C@H]3[C@@H]4C[C@@H]5O[C@]([C@H]([C@@H]5[C@@]4(C)CC[C@@H]3[C@@]2(C)CC1)C)(O)CC[C@@H](C)CO[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O LVTJOONKWUXEFR-UEZXSUPNSA-N 0.000 title claims abstract description 35
- MHKGPHKABOLURA-JNVLQWCMSA-N protodioscin Natural products C[C@@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](O)[C@H]3O)[C@H](O[C@H]4CC[C@]5(C)[C@H]6CC[C@@]7(C)[C@@H](C[C@@H]8O[C@](O)(CCCCO[C@@H]9O[C@H](CO)[C@@H](O)[C@H](O)[C@H]9O)[C@@H](C)[C@H]78)[C@@H]6CC=C5C4)O[C@@H]2CO)[C@H](O)[C@H](O)[C@H]1O MHKGPHKABOLURA-JNVLQWCMSA-N 0.000 title claims abstract description 33
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000013558 reference substance Substances 0.000 claims abstract description 17
- 238000012360 testing method Methods 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 12
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 11
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 claims abstract description 9
- MPDGHEJMBKOTSU-UHFFFAOYSA-N Glycyrrhetinsaeure Natural products C12C(=O)C=C3C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C1(C)CCC1C2(C)CCC(O)C1(C)C MPDGHEJMBKOTSU-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229960003720 enoxolone Drugs 0.000 claims abstract description 9
- WNIFXKPDILJURQ-UHFFFAOYSA-N stearyl glycyrrhizinate Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C)CCC(C(=O)OCCCCCCCCCCCCCCCCCC)(C)CC5C4=CC(=O)C3C21C WNIFXKPDILJURQ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 5
- 238000010812 external standard method Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000010829 isocratic elution Methods 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 101100348341 Caenorhabditis elegans gas-1 gene Proteins 0.000 claims description 4
- 101100447658 Mus musculus Gas1 gene Proteins 0.000 claims description 4
- 101100447665 Mus musculus Gas2 gene Proteins 0.000 claims description 4
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000012085 test solution Substances 0.000 claims description 3
- 239000008186 active pharmaceutical agent Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 238000002604 ultrasonography Methods 0.000 claims description 2
- 239000012071 phase Substances 0.000 claims 2
- 238000002347 injection Methods 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
- 239000007791 liquid phase Substances 0.000 claims 1
- 238000012544 monitoring process Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 4
- 230000002349 favourable effect Effects 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 13
- 238000010828 elution Methods 0.000 description 6
- 229930182490 saponin Natural products 0.000 description 6
- 150000007949 saponins Chemical class 0.000 description 6
- 235000017709 saponins Nutrition 0.000 description 6
- 238000003556 assay Methods 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 4
- 241001678283 Dioscorea zingiberensis Species 0.000 description 3
- 235000004360 Dioscorea zingiberensis Nutrition 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- -1 steroid saponin compound Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 244000281702 Dioscorea villosa Species 0.000 description 2
- 235000000504 Dioscorea villosa Nutrition 0.000 description 2
- 241000234272 Dioscoreaceae Species 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000011017 operating method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000013441 quality evaluation Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 241000256844 Apis mellifera Species 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 235000005903 Dioscorea Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
- 208000026137 Soft tissue injury Diseases 0.000 description 1
- DWCSNWXARWMZTG-UHFFFAOYSA-N Trigonegenin A Natural products CC1C(C2(CCC3C4(C)CCC(O)C=C4CCC3C2C2)C)C2OC11CCC(C)CO1 DWCSNWXARWMZTG-UHFFFAOYSA-N 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 230000000703 anti-shock Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000004879 dioscorea Nutrition 0.000 description 1
- WQLVFSAGQJTQCK-VKROHFNGSA-N diosgenin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)CC4=CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 WQLVFSAGQJTQCK-VKROHFNGSA-N 0.000 description 1
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 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
-
- 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
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
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)
- Steroid Compounds (AREA)
Abstract
The invention discloses in a kind of quick measure Rhizoma Dioscoreae Zingiberensiss protodioscin content method, belong to pharmaceutical technology field.Step is 1）Prepared by reference substance, inner mark solution；2）Prepared by need testing solution；3）HPLC QqQ MS combined instruments are detected that reaction of high order detection pattern is scanned, and lock protodioscin structure, calculate protodioscin content with external standard method.The on-line checking time shorten to 8 minutes.Ionic reaction for quantitative analyses is m/z 1047.6 → 901.4（Protodioscin）、m/z 469.3→425.3（External standard enoxolone）.The present invention gropes quick method, simplicity, accurate, favorable reproducibility, can be used for the control of Rhizoma Dioscoreae Zingiberensiss extract quality.
Description
Technical field
The invention belongs to pharmaceutical technology field, is related to the triple level Four bar matter spectrum quick tests of high performance liquid chromatography series connection
The method of protodioscin content in Rhizoma Dioscoreae Zingiberensiss.
Background technology
Rhizoma Dioscoreae Zingiberensiss are commonly called as Dioscorea zingiberensis, Dioscorea zingiberensis C. H. Wright, are Dioscoreaceae（Dioscoreaceae）Wild yam（Dioscorea）Plant
The root stock of Rhizoma Dioscoreae Zingiberensiss Dioscorea zingiberensis C. H. Wright grows wild, nearly cylinder.Entered with root stock
Medicine, sweet, bitter, flat.Removing toxic substances and promoting subsidence of swelling, for carbuncle furuncle early stage non-ulceration, skin acute festering type infects, and soft tissue injury, honeybee sting worm
Sting.Rhizoma Dioscoreae Zingiberensiss rhizome is rich in saponin component, and because of its complicated components, and separation process difficulty is larger, it is difficult to quantitative, at present only
Can be with total saponins through acid hydrolysis, the content for obtaining diosgenin controls the quality of Rhizoma Dioscoreae Zingiberensiss for testing index.Modern age medicine
Pharmacological research shows that Rhizoma Dioscoreae Zingiberensiss total saponins have antitumor, antithrombus formation, antiallergic, antiviral and Antishock function.And shield
In leaf Rhizoma Dioscoreae, the content of protodioscin affects its quality and action effect.At present, the assay of protodioscin mostly is
HPLC methods, extracting method are complicated, time-consuming, and which only has weaker uv absorption at nearly 200nm, is also easy to produce interference.To shield leaf
In Rhizoma Dioscoreae, protodioscin is qualitative and Rapid Quantification there is not been reported.
Using MRM methods, solve steroid saponin compound does not have conjugated structure to the present invention, and absorbing wavelength is in ultraviolet
End absorption, conventional HPLC detect unstability of base line, and sensitivity is relatively low, the problem for being difficult to determine.Meanwhile, change in Rhizoma Dioscoreae Zingiberensiss rhizome
Complicated component is learned, and saponin chemical constitution is more close, polarity is larger, HPLC methods are determined and need to adopt prolonged gradient elution journey
Sequence.This method is not high to the separation requirement of composition to be measured, elution time is greatly reduced therefore, simplifies operating procedure, is shield
The component analyses of leaf Rhizoma Dioscoreae, quality evaluation provide directive significance and scientific basic.
Content of the invention
The present invention establishes HPLC-QqQ-MS methods, with enoxolone as control, is monitored using reaction of high order（MRM）Method, surveys
Determine the content of protodioscin in Rhizoma Dioscoreae Zingiberensiss.It is using SHIMADZU GL Inertsil ODS-3 C18（150 mm×
4.6 m, 5 μm）Chromatographic column, mobile phase：Acetonitrile：Water（70：30）, isocratic elution：8min；Sample size：10μL；Column temperature：40℃；Stream
Fast 0.5mL/min；Scan mode is monitored for reaction of high order（MRM）Pattern, ion source are ESI（Turbo Spray）Source, anion
Change mode detects that ion source voltage is 5.5kV；Heated capillary temperature is 400 DEG C；CAD is 5；Curtain gas is 10；Gas1 is 45；
Gas2 is 45；It is -114V to remove cluster voltage.Ionic reaction for quantitative analyses is m/z 1047.6 → 901.4（Former Rhizoma Dioscoreae soap
Glycosides）、m/z 469.3→425.3（External standard enoxolone）.As a result：Measured using Liquid Chromatography/Mass Spectrometry, protodioscin quality is dense
Degree is in 8.8～66 μ g mL-1Interior, linear relationship is good, R2=0.9993, mean sample recovery rate is 93.4%.Conclusion:Built
Cube method is quick, easy, accurate, favorable reproducibility, can be used for the control of Rhizoma Dioscoreae Zingiberensiss extract quality.
For achieving the above object, the invention discloses following technology contents：
A kind of method of protodioscin content in measure Rhizoma Dioscoreae Zingiberensiss, it is characterised in that connected using high performance liquid chromatography triple
Level Four bar mass spectrography, the quick content for determining protodioscin in Rhizoma Dioscoreae Zingiberensiss, is carried out as follows：
（1）Prepared by reference substance, inner mark solution：Weigh protodioscin reference substance and each 5.0mg of enoxolone reference substance is put respectively
In 5mL volumetric flasks, plus methanol dissolves and is diluted to scale, is configured to the storing solution that concentration is 1mg/mL, and 4 DEG C save backup；
（2）Prepared by need testing solution：Take Rhizoma Dioscoreae Zingiberensiss dry rhizome powder 1g accurately weighed.Add methanol 25mL, ultrasound wave
It is centrifuged after processing 30min.Supernatant crosses microporous filter membrane, and filtrate is the test solution that HPLC-MS/MS methods are determined；
（3）Determine：The accurate aspiration step of difference（1）The each 10 μ L of the reference substance solution that obtains, step（2）The need testing solution of extraction
10 μ L, inject HPLC-MS/MS combined instruments, determine.Reaction of high order detection pattern is scanned, and locks protodioscin structure, uses
External standard method calculates protodioscin content.
In the present invention, more detailed preparation method and assay method are as follows：
1. instrument and reagent
1.1 instrument LC-20AD liquid chromatographic systems, are equipped with SIL-20AC automatic sample handling systems and CTO-20A column ovens（Japan
Shimadzu Corporation）；4000 triple level Four bar detectors of API, operation software are Analyst 1.5.1（U.S.A. applied biosystem
Company limited）；AL104-1C a ten thousandth electronic balances (Mettler Tolido companies of Switzerland)；Ten a ten thousandths of AB 135-S
Electronic balance（Mettler Tolido companies of Switzerland）；KQ-100DE type numerical control ultrasonic cleaners（Supermarket of city of Kunshan instrument has
Limit company）；Direct-Q3 water purification machines (U.S. Millipore).
1.2 reagent reference substance protodioscins（Lot number：1-0422-50）It is purchased from Beijing Xin Rong Science and Technology Ltd.s pure
Degree>98% reference substance enoxolone（Lot number：Z110723）It is purchased from Shanghai Yuan Ye bio tech ltd purity>98% second
Nitrile, methanol are（Chromatographically pure, TEDIA）Ultra-pure water is prepared through Milli-Q systems purification.
2. method and result
2.1 chromatographs and Mass Spectrometry Conditions chromatographic column：SHIMADZU GL Inertsil ODS-3 C18 posts（Japanese Shimadzu Corporation produces
Product, specification：150 mm × 4.6 mm, column internal diameter：5μm）.Mobile phase：Acetonitrile：Water（70：30）, isocratic elution：8min.Sample introduction
Amount：10μL；Column temperature：40℃；Flow velocity 0.5mL/min.Scan mode is monitored for reaction of high order（MRM）Pattern, ion source ESI
（Turbo Spray）Detect that ion source voltage is 5.5kV in source, negative ionization mode；Heated capillary temperature is 400 DEG C；CAD
For 5；Curtain gas is 10；Gas1 is 45；Gas2 is 45；It is -114V to remove cluster voltage.Composition to be measured and internal standard ion pair mass number and touch
Hit and energy-optimised the results are shown in Table 1.
1 protodioscin of table and outer target MRM parameter
The preparation of 2.2 solution
2.2.1 the preparation of reference substance solution：Difference assay precision weighs protodioscin reference substance and enoxolone reference substance is each
5.0mg is respectively placed in 5mL volumetric flasks, plus methanol dissolves and is diluted to scale, be configured to concentration be 1mg/mL storing solution, 4
DEG C save backup.
2.2.2 the preparation of need testing solution：Take Rhizoma Dioscoreae Zingiberensiss dry rhizome powder 1g accurately weighed.Methanol 25mL is added,
42kHz ultrasonic Treatment 30min, 5000r min-1Centrifugation 5min.Supernatant crosses 0.45 μm of microporous filter membrane filtration, and filtrate is
For the test solution that HPLC-MS/MS methods are determined.
2.3 methodological study
2.3.1 the range of linearity investigates accurate absorption protodioscin reference substance solution, puts in 10mL volumetric flasks, with methanol dilution extremely
Each concentration（Respectively 8.8,17.6,33.0,35.2,66.0 μ g mL-1).Precision draws 10 μ L of each concentration, successively sample introduction,
With sample introduction concentration (μ g mL-1) it is abscissa, product to be tested chromatographic peak area is vertical coordinate with internal standard peak area ratio, draws mark
Directrix curve, protodioscin regression equation are shown in Table 2.
The linear investigation of 2 protodioscin of table
As a result show, protodioscin regression equation linear relationship is good, the range of linearity is 9～66 μ g mL-1.
2.3.2 the measure of quantitative limit and test limit takes 1 mL of protodioscin reference substance solution under 2.2.1 items, dilute step by step
Release, and 10 μ L of sample introduction successively, with the test limit that S/N=3 tries to achieve each component, with the quantitative limit that S/N=10 tries to achieve each component,
As a result table 2 is seen.
2.3.3 precision test takes sample under 2.2.2 items, and continuous sample introduction 6 times determines peak area meansigma methodss, calculates accurate
Degree RSD values.
2.3.4 repeatability weighs 6 parts of Rhizoma Dioscoreae Zingiberensiss medicinal powder, by legal system available test sample solution below 2.2.2 items, surveys
Determine peak area, calculate content meansigma methodss and RSD values.
2.3.5 stability takes obtained need testing solution under 2.2.2 items, puts 4 DEG C of refrigerator storages.Respectively at 0,6,12,
18,24 h sample introductions are analyzed, and determine peak area, computational stability RSD value.
2.3.6 average recovery is investigated precision and weighs 6 parts of Rhizoma Dioscoreae Zingiberensiss medicinal powder, adds reference substance protodioscin
3.2mg, prepares solution by method below 2.2.2 items, and sample introduction is analyzed, and it is 2.0% for 93.4%, RSD to calculate the response rate.
2.4 sample determinations are shown in Fig. 1 by assay, MRM mass spectruies is carried out under 1.2 to sample.Calculate by external standard method
The content of protodioscin, the results are shown in Table 3.
3 protodioscin assay of table
3. mobile phase and Mass Spectrometry detection method are selected：
3.1 saponin component polarity are bigger than normal, therefore this effects methanol-water, acetonitrile-water, methanol-sour water and second
The different solvent system of nitrile-sour water, and 60%, 70%, 80%, 90% 4 kind of ratio isocratic elution and gradient elution.As a result table
The separating effect of bright 80% acetonitrile-water system is preferable so that peak shape is good, and appearance time is moderate, relative to gradient elution more
Stable, easy.Therefore this experimental selection ladder 80% acetonitrile-water system degree type of elution.
3.2 experiments adopt MRM methods, and solving steroid saponin compound does not have conjugated structure, absorbing wavelength to be in
Ultraviolet end absorption, conventional HPLC detect unstability of base line, and sensitivity is relatively low, the problem for being difficult to determine.Meanwhile, Rhizoma Dioscoreae Zingiberensiss rhizome
Middle complex chemical composition, and saponin chemical constitution is more close, polarity is larger, and HPLC methods are determined and need to be washed using prolonged gradient
De- program.This method is not high to the separation requirement of composition to be measured, elution time is greatly reduced therefore, simplifies operating procedure.
In measure Rhizoma Dioscoreae Zingiberensiss disclosed by the invention, the advantage of protodioscin content method is：
（1）Through groping the high performance liquid chromatography series connection second order mses for establishing protodioscin（HPLC-QqQ-MS）Algoscopy.Grind
Study carefully and show, from the method, solving steroid saponin compound does not have conjugated structure, absorbing wavelength to inhale in ultraviolet end
Receive, conventional HPLC detects unstability of base line, sensitivity is relatively low, the problem for being difficult to determine.
（2）Prove through methodological study, this method is easy to operate, accurate, favorable reproducibility, can be used for the composition of Rhizoma Dioscoreae Zingiberensiss
Analysis and quality evaluation.
Claims (5)
1. a kind of determine Rhizoma Dioscoreae Zingiberensiss in protodioscin content method, it is characterised in that using high performance liquid chromatography connect three
Weight level Four bar (atmospheric pressure ionization) mass spectrography（HPLC-QqQ-MS）(API）, protodioscin in quick measure Rhizoma Dioscoreae Zingiberensiss
Content, carry out as follows：
1）Prepared by reference substance, inner mark solution：Weigh protodioscin reference substance and each 5.0mg of enoxolone reference substance is respectively placed in
In 5mL volumetric flasks, plus methanol dissolves and is diluted to scale, is configured to the storing solution that concentration is 1mg/mL, and 4 DEG C save backup；
2）Prepared by need testing solution：Take Rhizoma Dioscoreae Zingiberensiss dry rhizome powder 1g accurately weighed, add methanol 25mL, 42kHz ultrasounds
Ripple processes 30min, 5000r min-1After centrifugation 5min, supernatant crosses 0.45 μm of microporous filter membrane, and filtrate is HPLC-QqQ-MS methods
The test solution for determining；
3）Determine：The accurate aspiration step 1 of difference）The reference substance solution for obtaining and step 2）The each 10 μ L of the need testing solution of extraction,
Injection HPLC-MS/MS combined instruments, determine, and reaction of high order detection pattern is scanned, and calculate protodioscin with external standard method and contain
Amount；
4）Chromatograph and Mass Spectrometry Conditions chromatographic column：SHIMADZU GL Inertsil ODS-3 C18 posts, mobile phase：Acetonitrile：Water
（70：30）, isocratic elution：8min, sample size：10 μ L, column temperature：40 DEG C, flow velocity 0.5mL/min, scan mode are reaction of high order
Monitoring（MRM）Pattern, ion source ESI（Turbo Spray）Negative ionization mode detects that ion source voltage is 5.5kV, heats hair
Capillary temperature is 400 DEG C, and CAD is 5, curtain gas be 10, Gas1 be 45, Gas2 be 45, remove cluster voltage for -114V.
2. as claimed in claim 1, it is characterised in that the liquid phase chromatogram condition, chromatographic column：SHIMADZU GL Inertsil
ODS-3 C18 posts, mobile phase：Acetonitrile：Water（70：30）, isocratic elution：8min, sample size：10 μ L, column temperature：40 DEG C, flow velocity
0.5mL/min.
3. as claimed in claim 1, it is characterised in that Mass Spectrometry Conditions are ion source ESI（Turbo Spray）Source, negative ionization side
Formula detects that ion source voltage is 5.5kV, and heated capillary temperature is 400 DEG C, and CAD is 5, and curtain gas is 45, Gas2 for 10, Gas1
For 45, it is -114V to remove cluster voltage.
4. as claimed in claim 1, it is characterised in that enoxolone is that external standard determines protodioscin content.
5. as claimed in claim 1, it is characterised in that reaction of high order detection pattern（MRM）And its parameter is set up：Protodioscin
Collision energy -56eV, ionic reaction select 1047.6 → 901.4m/z；Enoxolone collision energy -60eV, ionic reaction are selected
469.3→425.3m/z.
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