CN106483226A - A kind of method of high performance liquid chromatography separation LIUSHEN WAN uv absorption composition - Google Patents
A kind of method of high performance liquid chromatography separation LIUSHEN WAN uv absorption composition Download PDFInfo
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- 239000000203 mixture Substances 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000010521 absorption reaction Methods 0.000 title claims abstract description 21
- 238000000926 separation method Methods 0.000 title claims abstract description 19
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 15
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000004458 analytical method Methods 0.000 claims abstract description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 105
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 4
- 238000003809 water extraction Methods 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 25
- 150000001875 compounds Chemical class 0.000 abstract description 9
- HIYGARYIJIZXGW-UHFFFAOYSA-N bufotenidine Chemical compound C1=C([O-])C=C2C(CC[N+](C)(C)C)=CNC2=C1 HIYGARYIJIZXGW-UHFFFAOYSA-N 0.000 abstract description 8
- 238000004587 chromatography analysis Methods 0.000 abstract description 2
- 238000013461 design Methods 0.000 description 24
- 239000000523 sample Substances 0.000 description 22
- 229940079593 drug Drugs 0.000 description 12
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- SCULJPGYOQQXTK-UHFFFAOYSA-N Cinobufagin Natural products CC(=O)OC1C2OC22C3CCC4CC(O)CCC4(C)C3CCC2(C)C1C=1C=CC(=O)OC=1 SCULJPGYOQQXTK-UHFFFAOYSA-N 0.000 description 11
- ATLJNLYIJOCWJE-CWMZOUAVSA-N bufogenin Chemical compound C=1([C@H]2C[C@H]3O[C@@]43[C@H]3[C@@H]([C@]5(CC[C@H](O)C[C@H]5CC3)C)CC[C@@]42C)C=CC(=O)OC=1 ATLJNLYIJOCWJE-CWMZOUAVSA-N 0.000 description 10
- 229950006858 bufogenin Drugs 0.000 description 10
- 239000007791 liquid phase Substances 0.000 description 10
- 239000012071 phase Substances 0.000 description 10
- 229910021642 ultra pure water Inorganic materials 0.000 description 10
- 239000012498 ultrapure water Substances 0.000 description 10
- 239000000843 powder Substances 0.000 description 9
- 238000007639 printing Methods 0.000 description 9
- 238000011160 research Methods 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- KBKUJJFDSHBPPA-UHFFFAOYSA-N 5beta-hydroxylcinobufagin Natural products CC(=O)OC1C2OC22C3CCC4(O)CC(O)CCC4(C)C3CCC2(C)C1C=1C=CC(=O)OC=1 KBKUJJFDSHBPPA-UHFFFAOYSA-N 0.000 description 8
- KBKUJJFDSHBPPA-ZNCGZLKOSA-N cinobufotalin Chemical compound C=1([C@@H]2[C@@]3(C)CC[C@@H]4[C@@]5(C)CC[C@H](O)C[C@@]5(O)CC[C@H]4[C@@]43O[C@@H]4[C@@H]2OC(=O)C)C=CC(=O)OC=1 KBKUJJFDSHBPPA-ZNCGZLKOSA-N 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 7
- 229930190011 Bufogenin Natural products 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 6
- 239000000919 ceramic Substances 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- QEEBRPGZBVVINN-UHFFFAOYSA-N Desacetyl-bufotalin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C1(O)CCC2C=1C=CC(=O)OC=1 QEEBRPGZBVVINN-UHFFFAOYSA-N 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000012982 microporous membrane Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 3
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 3
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- ATLJNLYIJOCWJE-UHFFFAOYSA-N resibufogenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C=1C=CC(=O)OC=1 ATLJNLYIJOCWJE-UHFFFAOYSA-N 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 208000002193 Pain Diseases 0.000 description 2
- QEEBRPGZBVVINN-BMPKRDENSA-N bufalin Chemical compound C=1([C@H]2CC[C@]3(O)[C@H]4[C@@H]([C@]5(CC[C@H](O)C[C@H]5CC4)C)CC[C@@]32C)C=CC(=O)OC=1 QEEBRPGZBVVINN-BMPKRDENSA-N 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
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- 239000000284 extract Substances 0.000 description 2
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- 238000004811 liquid chromatography Methods 0.000 description 2
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- 239000002244 precipitate Substances 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- DTGKSKDOIYIVQL-WEDXCCLWSA-N (+)-borneol Chemical compound C1C[C@@]2(C)[C@@H](O)C[C@@H]1C2(C)C DTGKSKDOIYIVQL-WEDXCCLWSA-N 0.000 description 1
- VOZHMAYHYHEWBW-NVOOAVKYSA-N Bufotalin Chemical compound C=1([C@@H]2[C@@]3(C)CC[C@@H]4[C@@]5(C)CC[C@H](O)C[C@H]5CC[C@H]4[C@@]3(O)C[C@@H]2OC(=O)C)C=CC(=O)OC=1 VOZHMAYHYHEWBW-NVOOAVKYSA-N 0.000 description 1
- VOZHMAYHYHEWBW-UHFFFAOYSA-N Bufotalin Natural products CC(=O)OC1CC2(O)C3CCC4CC(O)CCC4(C)C3CCC2(C)C1C=1C=CC(=O)OC=1 VOZHMAYHYHEWBW-UHFFFAOYSA-N 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
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- 206010017553 Furuncle Diseases 0.000 description 1
- 206010019133 Hangover Diseases 0.000 description 1
- 244000153234 Hibiscus abelmoschus Species 0.000 description 1
- 206010042674 Swelling Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000003088 amphibian venom Substances 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000001430 anti-depressive effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 201000010099 disease Diseases 0.000 description 1
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- 210000003026 hypopharynx Anatomy 0.000 description 1
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- 238000002955 isolation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
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- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
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- 238000002715 modification method Methods 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
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- 239000000047 product Substances 0.000 description 1
- 229910052957 realgar Inorganic materials 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
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Classifications
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- 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
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- 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)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The present invention relates to technical field of chromatographic analysis, more particularly, to a kind of method of high performance liquid chromatography separation LIUSHEN WAN uv absorption composition.The composition in Chinese medicine compound LIUSHEN WAN with uv absorption can be realized efficiently separating by the method, realizes efficiently separating by wherein big polar portion 5 hydroxytryptamine and bufotenidine first；Method according to the present invention is easy and simple to handle, laboratory equlpment is required simple, the loss to chromatographic column is less, repeatable high, and the separation analysis to the big polar substancess in other Chinese medicine compound has reference value.
Description
Technical field
The present invention relates to technical field of chromatographic analysis, more particularly, to a kind of high performance liquid chromatography separation LIUSHEN WAN uv absorption
The method of composition.
Background technology
LIUSHEN WAN is that one kind has more than 100 year history, enjoys great prestige famous Chinese patent medicine both domestic and external.LIUSHEN WAN is by Six-element medical material
Configuration forms, including Calculus Boviss, Margarita, Venenum Bufonis, Realgar, Moschus, Borneolum Syntheticum.It has effect (Lee of heat-clearing and toxic substances removing, reducing swelling and alleviating pain
Water good fortune, HUANGLONG is gloomy, Guo Wenxin. LIUSHEN WAN quality standard and quality problems. precious traditional Chinese medicines research when [J], and 1996,7 (1)：57-58),
It is applied to laryngopharynx swelling and pain, carbuncle and ulcer furuncle etc..In modern Chinese medicine exploitation, LIUSHEN WAN is applied to more diseases such as antiinflammatory, analgesia, resists
Hepatocarcinoma, antitumor etc. (Chen Qi, Li little Jun, Jia Meimei, Li Yusang give Li Jing, Tang Hebin. and the external resisting liver cancer activity of LIUSHEN WAN grinds
Study carefully. [J] Journal of Chinese Hospital Pharmacy .2014,34 (19)：1627-1630).For the analysis of LIUSHEN WAN composition, Cao Yang etc. has relatively
For comprehensive report (Cao Yang, Liang Qionglin, Zhang Hongyang, Wang Yiming, Bi Kaishun, Luo Guoan. many constituents in Chinese medicine compound LIUSHEN WAN
Multidimensional liquid matter system examination and identification. [J] analytical chemistry .2008,36 (1):39-46).For LIUSHEN WAN quality standard research,
Wu Yinsheng etc. done and explored (Wu Yinsheng. LIUSHEN WAN quality standard research. [J] Nanjing University of Traditional Chinese Medicine journal .2002,18 (4):
232-233).5-hydroxy tryptamine is included for polar portion big in LIUSHEN WAN and bufotenidine report is less, and do not set up matter
Amount research on standard.
Most Chinese medicine is all water decoction, and in aqueous solvent extract, big polar substancess are more.Because big polar substancess have
Appearance is fast, retention time is short, separating degree low high performance liquid chromatography separation difficult point, and majority is the scientific research people of Chinese medicine research
Member abandons and this moieties is investigated, and this is incomplete.For example, two kinds of things in big polar portion in LIUSHEN WAN
Matter, 5-hydroxy tryptamine and bufotenidine are kind of indole derivativeses.Content is very in cerebral cortex and nerve synapse for 5-hydroxy tryptamine
Height, it is a kind of inhibitory neurotransmitter, has antidepressant effect.And polar substancess research big in LIUSHEN WAN is rarely reported,
And do not included quality control clearance, this just creates potential safety hazard to the quality control of LIUSHEN WAN.
Currently for the separation of big polar substancess, common method is to add ion-pairing agent or using exchange ion to chromatograph
Post, this two methods are all larger to the damage of chromatographic column, high cost.The present invention intends using conventional C18 chromatographic column, by extending
The retention time of big polar component, 5-hydroxy tryptamine and bufotenidine are that kind of indole derivativeses efficiently separate.This separation side
Method is verified through different compound recipes, all effective.This method is that the separation of the big polar component of Chinese medicine is offered reference；And low cost,
Wide adaptability, experimental facilitiess are required low it is adaptable to most of laboratory.
Content of the invention
For in prior art, incomplete problem is separated to Chinese medicine compound LIUSHEN WAN composition, the invention provides a kind of energy
Carry out detached method by having uv absorption composition in LIUSHEN WAN comprehensively, can will have uv absorption in Chinese medicine compound LIUSHEN WAN
Composition realize efficiently separating, be kind of indole derivativeses particularly by wherein big polar substancess 5-hydroxy tryptamine and bufotenidine
Realization efficiently separates.
To achieve these goals, the present invention adopts the following technical scheme that：
The present invention is with being become using the high performance liquid chromatography separation Chinese medicine compound LIUSHEN WAN uv absorption of conventional C18 chromatographic column
Point, identify its structure using LC-MS.Set up the HPLC method of energy its component content of Accurate Determining, from Chinese medicine compound LIUSHEN WAN
In isolated and purified uv absorption composition, wherein big polar portion 5-hydroxy tryptamine and bufotenidine belong to first from LIUSHEN WAN
In efficiently separate.On this basis, with 5-hydroxy tryptamine, Cinobufotalin, bufotalien, Toadpoison Medicine, cinobufagin, fat toadpoison
The standard substance such as aglucon are comparison, with 5-hydroxy tryptamine, Cinobufotalin, bufotalien, Toadpoison Medicine, China in external standard method LIUSHEN WAN
Bufalin, the content of bufogenin.
A kind of modification method of high performance liquid chromatography separation LIUSHEN WAN composition, its step is as follows：
(1) sample solution preparation：Take LIUSHEN WAN, after processing through water extraction, LIUSHEN WAN water extract is made need testing solution；
It is specially：Take LIUSHEN WAN particulate abrasive to become powder, add in ultra-pure water, supersound process, standing, take supernatant, with rotation
Turn evaporimeter recycle-water and obtain extractum, then lyophilization obtains brown ceramic powder；The brown ceramic powder taking gained is dissolved in ultra-pure water, and constant volume,
With 0.45 μm of filtering with microporous membrane, take filtrate as need testing solution；
(2) each standard solution preparation：Respectively precision weigh 5-hydroxy tryptamine, Cinobufotalin, bufotalien, Toadpoison Medicine,
The standard substance such as cinobufagin, bufogenin, are dissolved in ultra-pure water constant volume respectively, with 0.45 μm of filtering with microporous membrane, standby；
(3) qualitative detection：Using high effective liquid chromatography for measuring sample solution, obtain being completely separating LIUSHEN WAN there is ultraviolet
Absorb the finger printing of composition, Liquid Chromatography/Mass Spectrometry analyzes each composition of LIUSHEN WAN；
(4) detection by quantitative：Using high performance liquid chromatography determination sample solution and standard solution respectively, according to chromatographic peak
Area, determines 5-hydroxy tryptamine in LIUSHEN WAN, Cinobufotalin, bufotalien, Toadpoison Medicine, cinobufagin, fat using external standard method
The content of bufotalin；
The chromatographic condition of described high performance liquid chromatography is：DIONEX ultimate 3000 high performance liquid chromatograph, color
Spectrum post is DIONEX C18 chromatographic column, 250mm × 4.6mm, 5 μm, and mobile phase is acetonitrile -0.1v/v% trifluoroacetic acid solution, stream
Dynamic phase volume flow is 1.0mL/min, and sample size is 10 μ L, and Detection wavelength is 296nm, and column temperature is 30 DEG C, gradient elution program：
0-3min, eluent system 5v/v% acetonitrile, design isocratic elution program is to extend retention time, thus differentiating solvent
Peak and signal peak；
3-13min, eluent system 5-12v/v% acetonitrile, design easy gradient elution program is so that big polar component is preferable
Separate；
13-15min, eluent system 12-20v/v% acetonitrile, design express speed gradient elution program is to shorten the time；
15-25min, eluent system 20-30v/v% acetonitrile, design slow gradient elution program finely progressively to adjust, will
Close peak is separated, and does basis for Fast Field eluting below simultaneously；
25-30min, eluent system 30-51v/v% acetonitrile, design Fast Field elution program is most preferably flat to be rapidly achieved
Weighing apparatus point；
30-50min, eluent system 51v/v% acetonitrile, design isocratic elution program is to join cinobufagin and ester toadpoison
Base separates.
Compared with prior art, the present invention has advantages below and effect：
1) one-step method realizes efficiently separating of LIUSHEN WAN all uv absorption composition, first by big for LIUSHEN WAN polar substancess 5-
Hydroxytryptamine and bufotenidine are realized efficiently separating, be the active ingredient determination of LIUSHEN WAN, quality control, deep development and
Clinical expansion provides new scientific basis and method.
2) this method is easy and simple to handle, experimental facilitiess is required few, repeatable high；Can carry out in common laboratory, right
The loss of chromatographic column is little, cost-effective.
3) this method has reference value to the separation analysis of other Chinese medicine compound compositions.
Brief description
Fig. 1 is that in LIUSHEN WAN sample 1, each composition obtains the high-efficient liquid phase chromatogram efficiently separating；
Fig. 2 is that in LIUSHEN WAN sample 2, each composition obtains the high-efficient liquid phase chromatogram efficiently separating；
Fig. 3 is that sample 2 obtains, with different chromatographic columns, the high-efficient liquid phase chromatogram that LIUSHEN WAN efficiently separates；
Fig. 4 is the high-efficient liquid phase chromatogram of hybrid standard product solution, 1,5-hydroxy tryptamine；10th, bufotalien；11st, magnificent toadpoison
Its spirit；12nd, Toadpoison Medicine；13rd, cinobufagin；14th, bufogenin.
Specific embodiment
With reference to specific embodiment, technical scheme is described in further detail.It should be understood that in following
Rong Buying is any limitation as to protection scope of the present invention by any way.
Embodiment 1：LIUSHEN WAN has the isolation identification of uv absorption composition
1st, sample preparation：Take LIUSHEN WAN granule (Shanghai Leiyun Pharmaceutical Industry Co., Ltd., lot number：090904), pulverize,
Weigh about 90mg LIUSHEN WAN powder and be dissolved in 5mL ultra-pure water；Supersound process 30min, standing, take supernatant.Method described above, will precipitate
Extracted again with 5ml ultra-pure water, supersound process 30min.Merge the supernatant obtaining twice, must be soaked with Rotary Evaporators recycle-water
Cream, then lyophilization obtains brown ceramic powder.Gained brown ceramic powder 6.50mg is taken to be dissolved in ultra-pure water, constant volume, in 5mL volumetric flask, uses 0.45
μm filtering with microporous membrane, obtains sample 1, standby.
2nd, high performance liquid chromatography and Mass Spectrometry Conditions
2.1 high-efficient liquid phase chromatogram condition：DIONEX ultimate 3000 high performance liquid chromatograph, chromatographic column is DIONEX
C18 chromatographic column, 250mm × 4.6mm, 5 μm, mobile phase is acetonitrile -0.1v/v% trifluoroacetic acid, and mobile phase volume flow is
1.0mL/min, sample size is 10 μ L, and Detection wavelength is 296nm, and column temperature is 30 DEG C, elution program：
(5v/v% refers to acetonitrile and accounts for mixed flow phase acetonitrile and 0.1v/v% trifluoroacetic acid total amount 0-3min, 5v/v% acetonitrile
Volume fraction, similarly hereinafter), design isocratic elution program to extend retention time, thus differentiating solvent peak and signal peak；
3-13min, 5-12v/v% acetonitrile, design easy gradient elution program is so that big polar component preferably separates；
13-15min, 12-20v/v% acetonitrile, design express speed gradient elution program is to shorten the time；
15-25min, 20-30v/v% acetonitrile, designs slow gradient elution program finely progressively to adjust, by close peak
Separate, do basis for Fast Field eluting below simultaneously；
25-30min, 30-51v/v% acetonitrile, design Fast Field elution program is to be rapidly achieved optimal balance point；
30-50min, 51%v/v acetonitrile, design isocratic elution program to separate cinobufagin with resibufogenin.
2.2 Mass Spectrometry Conditions：Full scan mass spectrum, mass scan range M/Z 100～800amu, dry gas stream speed 15L/min,
Temperature degree 320 DEG C, capillary voltage 2.6kV, source voltage 4KV, total current 100mA, capillary temperature 320 DEG C, detection side are dried
Formula MSn(n=1～3).
3rd, LIUSHEN WAN has the finger printing of uv absorption composition
Result：Separating degree is all higher than 1.5, and, between 0.90-1.17, theoretical cam curve is equal in terms of each chromatographic peak for tailing factor
More than 10000, it is shown in Table 1.The all the components in LIUSHEN WAN with uv absorption are realized efficiently separating.High performance liquid chromatography is complete
The finger printing of the composition that face separation LIUSHEN WAN has uv absorption is shown in Fig. 1.
The separation of each composition of table 1 sample 1 LIUSHEN WAN
According to 2.2 Mass Spectrometry Conditions, qualitative analyses are carried out to 1-14 peak, by standard substance relative analyses, we determined that 1
Number peak is 5-hydroxy tryptamine, and No. 10 peaks are bufotalien；No. 11 peaks are Cinobufotalin；No. 12 peaks are Toadpoison Medicine；No. 13 peaks are China
Bufalin；14 peaks are bufogenin.The molecular weight at wherein No. 2 peaks is 218, according to molecular weight and retention time and two phases
Close document contrast thus it is speculated that No. 2 peaks be bufotenidine (Cao Yang, Liang Qionglin, Zhang Hongyang, Wang Yiming, Bi Kaishun, Luo Guoan. in
The multidimensional liquid matter system examination of many constituents and identification in recurrence due to taking drug side's LIUSHEN WAN. [J] analytical chemistry .2008,36 (1):39-46；
Zhang Ping. the finger printing research of different base toad venoms. Shenyang Pharmaceutical University Ph.D. Dissertation .2006,45p.).
Embodiment 2：Analysis method with embodiment 1 detects the LIUSHEN WAN finger printing of different batches
1st, sample preparation：Take LIUSHEN WAN granule (Shanghai Leiyun Pharmaceutical Industry Co., Ltd., lot number：140701), pulverize,
Weigh about 90mg LIUSHEN WAN powder and be dissolved in 5mL ultra-pure water；Supersound process 30min, standing, take supernatant.Method described above, will precipitate
Extracted again with 5ml ultra-pure water, supersound process 30min.Merge the supernatant obtaining twice, must be soaked with Rotary Evaporators recycle-water
Cream, then lyophilization obtains brown ceramic powder.Gained brown ceramic powder 6.50mg is taken to be dissolved in ultra-pure water, constant volume, in 5mL volumetric flask, uses 0.45
μm filtering with microporous membrane, obtains sample 2, standby.
2nd, high-efficient liquid phase chromatogram condition
High-efficient liquid phase chromatogram condition：DIONEX ultimate 3000 high performance liquid chromatograph, chromatographic column is DIONEX
C18 chromatographic column, 250mm × 4.6mm, 5 μm, mobile phase is acetonitrile -0.1v/v% trifluoroacetic acid solution, and mobile phase volume flow is
1.0mL/min, sample size is 10 μ L, and Detection wavelength is 296nm, and column temperature is 30 DEG C, elution program：
0-3min, 5v/v% acetonitrile, design isocratic elution program is to extend retention time, thus differentiating solvent peak and signal
Peak；
3-13min, 5-12v/v% acetonitrile, design easy gradient elution program is so that big polar component preferably separates；
13-15min, 12-20v/v% acetonitrile, design express speed gradient elution program is to shorten the time；
15-25min, 20-30v/v% acetonitrile, designs slow gradient elution program finely progressively to adjust, by close peak
Separate, do basis for Fast Field eluting below simultaneously；
25-30min, 30-51v/v% acetonitrile, design Fast Field elution program is to be rapidly achieved optimal balance point；
30-50min, 51v/v% acetonitrile, design isocratic elution program to separate cinobufagin with resibufogenin.
3rd, LIUSHEN WAN has the finger printing of uv absorption composition
Result：Separating degree is all higher than 1.5, and, between 0.90-1.13, theoretical cam curve is equal in terms of each chromatographic peak for tailing factor
More than 10000.It is shown in Table 2, all the components in LIUSHEN WAN with uv absorption are realized efficiently separating.High performance liquid chromatography is complete
The finger printing of the composition that face separation LIUSHEN WAN has uv absorption is shown in Fig. 2.
The each composition separation of table 2 sample 2 LIUSHEN WAN
Embodiment 3：The different impact to LIUSHEN WAN finger printing for the chromatographic column of analysis method detection with embodiment 1
1st, sample preparation：Using sample 2 in embodiment 2；
2nd, high-efficient liquid phase chromatogram condition
High-efficient liquid phase chromatogram condition：DIONEX ultimate 3000 high performance liquid chromatograph, chromatographic column is KROMASIL
C18 chromatographic column, 250mm × 4.6mm, 5 μm, mobile phase is acetonitrile -0.1v/v% trifluoroacetic acid solution, and mobile phase volume flow is
1.0mL/min, sample size is 10 μ L, and Detection wavelength is 296nm, and column temperature is 30 DEG C, elution program：
0-3min, 5v/v% acetonitrile, design isocratic elution program is to extend retention time, thus differentiating solvent peak and signal
Peak；
3-13min, 5-12v/v% acetonitrile, design easy gradient elution program is so that big polar component preferably separates；
13-15min, 12-20v/v% acetonitrile, design express speed gradient elution program is to shorten the time；
15-25min, 20-30v/v% acetonitrile, designs slow gradient elution program finely progressively to adjust, by close peak
Separate, do basis for Fast Field eluting below simultaneously；
25-30min, 30-51v/v% acetonitrile, design Fast Field elution program is to be rapidly achieved optimal balance point；
30-50min, 51v/v% acetonitrile, design isocratic elution program to separate cinobufagin with resibufogenin.
3rd, LIUSHEN WAN has the finger printing of uv absorption composition
Result：After changing chromatographic column, in LIUSHEN WAN, each composition separating effect is almost identical, and separating degree is all higher than 1.5, hangover
The factor between 0.82-1.59, theoretical cam curve in terms of each chromatographic peak all more than 10000, as shown in table 3.Although with not
With chromatographic column test, but its repeatability is preferably, shows experimental result that this analysis test method obtains to the chromatograph meeting condition
The dependency of column type number is relatively low.See Fig. 3.
The separation of different chromatograph post separation sample 2 LIUSHEN WAN of table 3
Embodiment 4：Method with embodiment 1 measures the content of each composition in LIUSHEN WAN
1st, solution preparation
1.1 sample preparation
Method according to embodiment 1-2 prepares sample 1,2.
1.2 standard substance preparations：Precision weighs 5-hydroxy tryptamine hydrochlorate (numbering respectively：111656-200401, Chinese food
Medicine identification research institute), Cinobufotalin (numbering：P24N7F25514, Shanghai Yuan Ye biotechnology Co., Ltd), toad
Its spirit (numbering of poison：P12O7F22619, Shanghai Yuan Ye biotechnology Co., Ltd), Toadpoison Medicine (numbering：W05D6Z7090,
Shanghai Yuan Ye biotechnology Co., Ltd), cinobufagin (numbering：W10O7Z22424, Shanghai source leaf biotechnology has
Limit responsible company), bufogenin (numbering：PJ0727RB13, Shanghai Yuan Ye biotechnology Co., Ltd) etc. standard substance
10.00mg, is dissolved in ultra-pure water respectively, is made into the mother solution of the 1mg/mL of each standard substance, and the equal gradient dilution of six kinds of standard solutions is extremely
0.33rd, 0.11,0.037,0.012,0.004,0.001mg/mL, with 0.45 μm of filtering with microporous membrane, standby.
2nd, liquid-phase condition：With embodiment 1.
3rd, sample size measures
Determination sample 1,2 under above-mentioned chromatographic condition.
Result：As shown in Fig. 4 (standard substance HPLC figure) (result of sample 2 is much like with sample 1, therefore does not enumerate spectrogram).
Record chromatographic peak area, with reference substance concentration as abscissa (x), with peak area as vertical coordinate (y), draws standard curve, carries out
Linear regression analyses, 5-hydroxy tryptamine y=147.7x+0.1858, R2=1；Bufotalien y=176.69x+0.4679, R2=
0.999；Cinobufotalin y=160.27x+1.2218, R2=0.999；Toadpoison Medicine y=150.63x-0.2965, R2=
0.9998；Cinobufagin y=160.27x+1.2218, R2=0.999, bufogenin y=171.73x+0.0584, R2=
1, standard substance linear relationship in the range of 0.001～0.33mg/mL mass concentration is good.According to the linearity curve of standard substance, count
Calculate the compositions such as 5-hydroxy tryptamine in LIUSHEN WAN, Cinobufotalin, bufotalien, Toadpoison Medicine, cinobufagin, bufogenin to divide
Content.The concrete mass fraction of corresponding composition is shown in Table 4.
The mass fraction of corresponding composition in table 4 different batches LIUSHEN WAN
Claims (4)
1. in a kind of high performance liquid chromatography separation LIUSHEN WAN the method for uv absorption composition it is characterised in that chromatographic condition is as follows：
C18 chromatographic column, mobile phase is acetonitrile -0.1 v/v % trifluoroacetic acid；
Gradient elution program：
0-3 min, 5 v/v % acetonitriles；
3-13 min, 5-12 v/v % acetonitrile；
13-15 min, 12-20 v/v % acetonitrile；
15-25 min, 20-30 v/v % acetonitrile；
25-30 min, 30-51 v/v % acetonitrile；
30-50 min, 51 v/v % acetonitriles.
2. method according to claim 1 it is characterised in that：LIUSHEN WAN is made need testing solution after water extraction process.
3. method according to claim 1 and 2 it is characterised in that：Described uv absorption composition includes 5-hydroxy tryptamine and toad
Malicious tryptamines inner salt.
4. in the uv absorption composition in qualitative and/or quantitative analyses LIUSHEN WAN of the method described in claim 1 or 2 or 3 should
With.
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