CN104833755B - Method for determining content of diester-type alkaloids in ephedra-monkshood-radix glycyrrhizae medicament - Google Patents
Method for determining content of diester-type alkaloids in ephedra-monkshood-radix glycyrrhizae medicament Download PDFInfo
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- 229930013930 alkaloid Natural products 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 60
- 239000003814 drug Substances 0.000 title claims abstract description 29
- 239000013558 reference substance Substances 0.000 claims abstract description 115
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 60
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 30
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000010828 elution Methods 0.000 claims abstract description 20
- 239000000945 filler Substances 0.000 claims abstract description 20
- 238000011084 recovery Methods 0.000 claims abstract description 19
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 10
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 210
- 239000000243 solution Substances 0.000 claims description 192
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 60
- 239000012071 phase Substances 0.000 claims description 55
- 238000012360 testing method Methods 0.000 claims description 55
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 53
- 239000011259 mixed solution Substances 0.000 claims description 47
- 235000009508 confectionery Nutrition 0.000 claims description 41
- 239000000835 fiber Substances 0.000 claims description 35
- 238000001914 filtration Methods 0.000 claims description 35
- XFSBVAOIAHNAPC-XTHSEXKGSA-N 16-Ethyl-1alpha,6alpha,19beta-trimethoxy-4-(methoxymethyl)-aconitane-3alpha,8,10alpha,11,18alpha-pentol, 8-acetate 10-benzoate Chemical compound O([C@H]1[C@]2(O)C[C@H]3[C@@]45C6[C@@H]([C@@]([C@H]31)(OC(C)=O)[C@@H](O)[C@@H]2OC)[C@H](OC)[C@@H]4[C@]([C@@H](C[C@@H]5OC)O)(COC)CN6CC)C(=O)C1=CC=CC=C1 XFSBVAOIAHNAPC-XTHSEXKGSA-N 0.000 claims description 33
- XFSBVAOIAHNAPC-UHFFFAOYSA-N Aconitin Natural products CCN1CC(C(CC2OC)O)(COC)C3C(OC)C(C(C45)(OC(C)=O)C(O)C6OC)C1C32C4CC6(O)C5OC(=O)C1=CC=CC=C1 XFSBVAOIAHNAPC-UHFFFAOYSA-N 0.000 claims description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 229940039750 aconitine Drugs 0.000 claims description 33
- STDXGNLCJACLFY-UHFFFAOYSA-N aconitine Natural products CCN1CC2(COC)C(O)CC(O)C34C5CC6(O)C(OC)C(O)C(OC(=O)C)(C5C6OC(=O)c7ccccc7)C(C(OC)C23)C14 STDXGNLCJACLFY-UHFFFAOYSA-N 0.000 claims description 33
- 239000007788 liquid Substances 0.000 claims description 32
- 238000010521 absorption reaction Methods 0.000 claims description 30
- 239000011550 stock solution Substances 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- DPMGVDIWDTYPMP-UHFFFAOYSA-N Hypaconitine Natural products COCC12CCC(OC)C3(CN(C)C1)C4CC5(O)C(OC)C(O)C(CC(OC)C23)(OC(=O)C)C4C5OC(=O)c6ccccc6 DPMGVDIWDTYPMP-UHFFFAOYSA-N 0.000 claims description 26
- FIDOCHXHMJHKRW-GKVQVCCJSA-N hypaconitine Chemical compound O([C@H]1[C@]2(O)C[C@H]3[C@]45[C@@H](OC)CC[C@@]6([C@H]4[C@@H](OC)[C@H]([C@@](OC(C)=O)([C@H]31)[C@@H](O)[C@H]2OC)[C@H]5N(C)C6)COC)C(=O)C1=CC=CC=C1 FIDOCHXHMJHKRW-GKVQVCCJSA-N 0.000 claims description 26
- 239000000706 filtrate Substances 0.000 claims description 25
- GQRPJUIKGLHLLN-VSTHTWNCSA-N mesaconine Chemical compound COC[C@]12CN(C)C3[C@@H]4[C@H](OC)[C@H]1[C@]3([C@@H]1C[C@@]3(O)[C@H](O)[C@@H]1[C@]4(O)[C@@H](O)[C@@H]3OC)[C@H](C[C@H]2O)OC GQRPJUIKGLHLLN-VSTHTWNCSA-N 0.000 claims description 25
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 23
- 239000003480 eluent Substances 0.000 claims description 22
- 239000002904 solvent Substances 0.000 claims description 22
- 230000006837 decompression Effects 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 17
- 239000003795 chemical substances by application Substances 0.000 claims description 14
- 239000012467 final product Substances 0.000 claims description 13
- 238000005341 cation exchange Methods 0.000 claims description 11
- 238000007689 inspection Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 238000010971 suitability test Methods 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 150000002825 nitriles Chemical class 0.000 claims description 4
- 239000007791 liquid phase Substances 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 239000012085 test solution Substances 0.000 claims description 2
- 241000173529 Aconitum napellus Species 0.000 claims 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims 1
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 1
- 229910052700 potassium Inorganic materials 0.000 claims 1
- 239000011591 potassium Substances 0.000 claims 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 abstract description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 abstract description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 abstract description 9
- WGZCUXZFISUUPR-UHFFFAOYSA-N acetonitrile;oxolane Chemical compound CC#N.C1CCOC1 WGZCUXZFISUUPR-UHFFFAOYSA-N 0.000 abstract 1
- 238000003556 assay Methods 0.000 description 18
- 241000227129 Aconitum Species 0.000 description 17
- 239000002775 capsule Substances 0.000 description 13
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 12
- 241001465251 Ephedra sinica Species 0.000 description 9
- 238000004587 chromatography analysis Methods 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 239000013256 coordination polymer Substances 0.000 description 6
- 239000008187 granular material Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000003513 alkali Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000006187 pill Substances 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 3
- 150000003797 alkaloid derivatives Chemical class 0.000 description 3
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- 239000012535 impurity Substances 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
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- 235000019698 starch Nutrition 0.000 description 3
- 229920001353 Dextrin Polymers 0.000 description 2
- 239000004375 Dextrin Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 235000019425 dextrin Nutrition 0.000 description 2
- -1 esters Alkaloid Chemical class 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
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- 235000020985 whole grains Nutrition 0.000 description 2
- 206010063659 Aversion Diseases 0.000 description 1
- 241000218671 Ephedra Species 0.000 description 1
- 208000015220 Febrile disease Diseases 0.000 description 1
- 206010019133 Hangover Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention provides a method for determining the content of diester-type alkaloids in an ephedra-monkshood-radix glycyrrhizae medicament. The method is implemented by using high performance liquid chromatography, and a chromatographic column takes octadecylsilane chemically bonded silica as a filler; and by taking acetonitrile-tetrahydrofuran as a mobile phase A and a phosphoric acid solution (including potassium dihydrogen phosphate) as a mobile phase B, programmed gradient elution is performed by time, and determination is performed by using a sample-reference substance mixed method, the separating degree, peak shape and tailing factor of a target component are significantly improved, and the specificity, recovery, precision and durability of the target component are good.
Description
Technical field
The present invention relates to medicine, and in particular to a kind of content assaying method of the attached sweet medicine Radix Aconiti Lateralis Preparata alkaloid of fiber crops, belongs to medicine
Technical field.
Technical background
The attached sweet preparation of fiber crops is the abbreviation that preparation is made by Mahuang Fuzi Gancuo Tang Decoction of Ephedra side, is made up of monkshood, Chinese ephedra, Radix Glycyrrhizae, at it
Side, for treatise on Febrile Diseases, has relieving exterior syndrome and dispelling cold, consolidates the effect activated yang, and cures mainly shaoyin disease, and aversion to cold body aches, lossless, slight fever, arteries and veins
Heavy micro- person.
Alkaloid contained by monkshood mainly has dibasic acid esters Alkaloid, monoesters Alkaloid and amido alkaloid.Modern times grind
Study carefully and show that diester-type alkaloids (mesaconine, Hypaconitine, aconitine) are toxic components, existing disclosed mensure diester-type is biological
The content assaying method of alkali is a lot, but is unsuitable for mensure in the attached sweet medicine of this fiber crops it is impossible to effective in production and use
Ensure the security of product.
In the high effective liquid chromatography for measuring monkshood medicinal material adopting in " Chinese Pharmacopoeia " version in 2010, diester-type is biological
Alkali, finds through repetition test, and this method measures diester-type alkaloids content in " the attached sweet medicine of fiber crops ", and impurity separating effect is poor,
Mesaconine, Hypaconitine, aconitine hangover are higher, and negative presence substantially interferes with.
Prior art diester-type alkaloids content assaying method also has:
Thin-layered chromatography: use ultraviolet or radiation of visible light lamellae under certain wavelength, to ultraviolet on lamellae
Or the material that visible ray has absorption will produce corresponding spot, it is scanned, scans the collection of illustrative plates obtaining and integrated value can
It is used as the quality examination of medicinal material.This method can analyze multiple samples simultaneously, and solvent consumption is few, it is many to select, and sample treatment is simple；Lack
Point is that precision is low, and limit handling sensitivity is low, poor repeatability.
High performance capillary electrophoresis: this method has that disengaging time is short, impurity disturbs less, using organic solvent is few, operation letter
Just the advantages of；Separating power is weaker, requires higher, poor reproducibility to ph value.
Content of the invention
The invention aims to providing a kind of content assaying method of the attached sweet medicine diester-type alkaloids of fiber crops, the present invention
Impurity and diester-type alkaloids good separating effect in monkshood, negative noiseless, mesaconine, Hypaconitine, aconitine separating degree
It is all higher than 1.5, linear, test limit, durability are all good.
The purpose of the present invention is achieved through the following technical solutions:
A kind of content assaying method of the attached sweet medicine diester-type alkaloids of fiber crops of the present invention it is characterised in that: with high
Effect liquid phase chromatogram method measures diester-type alkaloids content in monkshood, and mobile phase is temporally carried out gradient elution, entered using sample-adding method
Row measures.
A kind of content assaying method of the attached sweet medicine diester-type alkaloids of fiber crops of the present invention it is characterised in that: with second
Nitrile: oxolane (20:20 to 30:10) is mobile phase a, with the 0.01-0.05mol/l biphosphate containing 0.02%-0.3% phosphoric acid
Potassium solution is mobile phase b, temporally carries out gradient elution, and by 90% to 70%, Detection wavelength is for 0 minute to 80 minutes mobile phase b
222-242nm, number of theoretical plate is calculated by aconitine peak and should be not less than 8000.
Prepared by reference substance solution: take mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurate
Weighed, respectively plus acetonitrile make every 1ml contain 0.05-0.5mg solution, as reference substance stock solution, precision measures above-mentioned three kinds
The each 5ml of reference substance stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product.
Solid-phase extraction column system suitability: precision measures each 3ml of reference substance stock solution, mixes, and reduced pressure at room temperature reclaims
To doing, residue is accurate to add 0.1mol/l hydrochloric acid solution 50ml to make dissolving, and precision measures 5ml, is placed in the SPE handled well
On post, with mixed type cation exchange reverse phase absorption agent as filler, 150-200mg, capacity is 4-10ml, uses second in advance successively
The each 6ml of nitrile, water elutes, and successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discards eluent, places 5 points
Clock, continues and uses acetonitrile: the mixed solution 10ml wash-out of strong ammonia solution (90:10), collects eluent, molten in less than 40 DEG C recovered under reduced pressure
Agent is to dry, residue accurate addition acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution (30:70) makes dissolving, filtration, takes subsequent filtrate
As solid-phase extraction column system suitability solution.
Accurate absorption above-mentioned solid phase extraction column system suitability solution and reference substance solution each 5-30 μ l respectively, injection
Liquid chromatograph, mensure, computing system employment and suitability test (E ＆ ST) solution and each corresponding Component peak area ratio of reference substance solution, must not be little
In 0.95.
The preparation of need testing solution: take inspection product, weigh 0.5-5g, put in conical flask with cover, plus acetonitrile: strong ammonia solution (90:
10) mixed solution 25ml, ultrasonically treated 10-50 minute, filtration, filtrate is in less than 40 DEG C decompression and solvent recoveries to dry, residue
Plus 0.1mol/l hydrochloric acid solution 10ml makes dissolving, filtration, according to the method under solid-phase extraction column system suitability item, certainly " put
In on the solid-phase extraction column handled well ", operate in accordance with the law, obtain need testing solution, measure reference substance solution and join with need testing solution
Make and contain mesaconine, Hypaconitine, the mixed solution of aconitine 2-18 μ g/ml respectively.
Determination method: accurate absorption reference substance solution and mixed solution each 5-30 μ l respectively, injects liquid chromatograph, measures,
Obtain final product.
A kind of content assaying method of the attached sweet medicine diester-type alkaloids of fiber crops of the present invention it is characterised in that: with second
Nitrile: oxolane (20:20 to 30:10) is mobile phase a, with the 0.01-0.05mol/l biphosphate containing 0.02%-0.3% phosphoric acid
Potassium solution is mobile phase b, temporally carries out gradient elution, and 0 minute to 80 minutes mobile phase b is by 90% to 70%.
A kind of content assaying method of the attached sweet medicine diester-type alkaloids of fiber crops of the present invention it is characterised in that: used
Solid phase extraction column stuffing is mixed type cation exchange reverse phase absorption agent, and specification is 100-200mg, and volume is 4-8ml, and in advance
Acetonitrile, water each 6ml wash-out is used to process successively.
A kind of content assaying method of the attached sweet medicine diester-type alkaloids of fiber crops of the present invention it is characterised in that: inspection product
After solution is added on solid-phase extraction column, successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent,
Place 5 minutes, continue and use acetonitrile: the mixed solution 10ml wash-out of strong ammonia solution (90:10), collect eluent.
A kind of content assaying method of the attached sweet medicine diester-type alkaloids of fiber crops of the present invention it is characterised in that: solid phase
In extraction column system suitability, testing liquid must not with the ratio of three kinds of diester-type alkaloids peak areas in reference substance solution
Less than 0.95.
A kind of content assaying method of the attached sweet medicine diester-type alkaloids of fiber crops of the present invention it is characterised in that: mixing
Reference substance solution in solution: the ratio of need testing solution is 10%:90% to 90%:10%.
The invention provides the assay side according to diester-type alkaloids in the attached sweet medicine of fiber crops of specific prescription preparation
Method；Difference with the prior art of the present invention is to employ different gradient elution program, eliminates negative biological to diester-type
The interference that alkali measures, its target component separating degree, peak shape, tailing factor all have and significantly improve, and specificity, linear, durable
Property is all good.
In order to be better understood from the present invention, the methodological study below by way of content assaying method of the present invention further illustrates
Beneficial effects of the present invention.Methodology is intended to further illustrate the effect of the present invention, and the restriction of non-invention.
First, prepare sample
1st, prepare the attached sweet capsule of fiber crops
Take monkshood 20kg, Chinese ephedra 13.34kg, Radix Glycyrrhizae 13.34g, add water to cook secondary, add water 8 times amount for the first time, decoct 3
Hour, add water 6 times amount for the second time, decocts 2 hours, filtration, and filtrate merges, and is condensed into paste, drying under reduced pressure below 80 DEG C,
Pulverize, mix with appropriate amount of auxiliary materials, load capsule, obtain the capsule of numb attached sweet medicine.
2nd, the negative capsule without monkshood for the preparation
Take Chinese ephedra 200g, Radix Glycyrrhizae 200g, the method according to the attached sweet capsule of preparation fiber crops makes the negative capsule without monkshood.
2nd, CP method and method assay applicability of the present invention compare
Monkshood diester-type alkaloids assay under " Chinese Pharmacopoeia " version the second enlarged edition mural nodules item in 2010
Method (hereinafter referred to as " CP method ") and monkshood diester-type alkaloids content determination of the present invention, double for the attached sweet capsule monkshood of fiber crops
The comparison of ester alkaloid assay.
1. " CP method " measures the attached sweet capsule diester-type alkaloids content employment and suitability test (E ＆ ST) of fiber crops
Chromatographic condition is with octadecylsilane chemically bonded silica as filler；With acetonitrile: oxolane (25:15) is for flowing
Phase a, with the 0.03mol/l potassium dihydrogen phosphate containing 0.1% phosphoric acid for mobile phase b；Regulation according to the form below carries out gradient and washes
De-；Detection wavelength is 232nm.
Table 1 " CP method " gradient elution table
| Time (min) | Mobile phase a (%) | Mobile phase b (%) |
| 0~38 | 15→26 | 85→74 |
| 38~39 | 26→35 | 74→65 |
| 39~49 | 35 | 65 |
| 49~50 | 35→15 | 65→85 |
Reference substance solution preparation takes mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurate
Weighed, respectively plus acetonitrile make every 1ml contain 0.2mg solution, as reference substance stock solution.Precision measures above-mentioned three kinds of reference substances
The each 5ml of stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product.
The preparation of need testing solution takes this product content, mixes, and precision weighs 1.0009g, puts in conical flask with cover, plus
Acetonitrile: the mixed solution 25ml of strong ammonia solution (90:10), ultrasonically treated 30 minutes, filtration, filtrate is in less than 40 DEG C recovered under reduced pressure
To doing, residue adds 0.1mol/l hydrochloric acid solution 10ml makes dissolving to solvent, filtration, is placed in the solid-phase extraction column handled well (to mix
Type cation exchange reverse phase absorption agent is filler, 150mg, and capacity is 6ml, uses acetonitrile, water each 6ml wash-out in advance successively) on,
Successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent, place 5 minutes, continue and use acetonitrile: dense ammonia
The mixed solution 10ml wash-out of test solution (90:10), collects eluent, in less than 40 DEG C decompression and solvent recoveries to dry, residue precision
Add acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution (30:70) makes dissolving, filtration, take subsequent filtrate as need testing solution.
The preparation of negative solution takes negative preparation to prepare negative solution with need testing solution preparation method.
The accurate absorption each 10 μ l of above-mentioned solution, inject liquid chromatograph, measure, obtain final product respectively.
Result of the test shows, negative solution with reference substance solution first and third chromatographic peak relevant position near all have chromatogram
Peak, shows that feminine gender measures diester-type alkaloids to this law and there is interference.Therefore in " CP method ", fiber crops are not suitable for using method attached
The assay of sweet medicine diester-type alkaloids.
2. the inventive method measures the attached sweet capsule diester-type alkaloids content employment and suitability test (E ＆ ST) of fiber crops
Chromatographic condition is with octadecylsilane chemically bonded silica as filler；With acetonitrile: oxolane (25:15) is for flowing
Phase a, with the 0.03mol/l potassium dihydrogen phosphate containing 0.1% phosphoric acid for mobile phase b；Regulation according to the form below carries out gradient and washes
De-；Detection wavelength is 232nm.
Table 2 gradient elution table
| Time (min) | Mobile phase a (%) | Mobile phase b (%) |
| 0~75 | 15→20 | 85→80 |
| 75~77 | 20→35 | 80→65 |
| 77~87 | 35 | 65 |
| 87~89 | 35→15 | 65→85 |
Reference substance solution preparation takes mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurate
Weighed, respectively plus acetonitrile make every 1ml contain 0.2mg solution, as reference substance stock solution.Precision measures above-mentioned three kinds of reference substances
The each 5ml of stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product.
Solid-phase extraction column system suitability precision measures each 3ml of reference substance stock solution, mixes, and reduced pressure at room temperature reclaims
To doing, residue is accurate to add 0.1mol/l hydrochloric acid solution 50ml to make dissolving, and precision measures 5ml, is placed in the SPE handled well
Post (with mixed type cation exchange reverse phase absorption agent as filler, 150mg, capacity is 6ml, uses acetonitrile, water each in advance successively
6ml elutes) on, successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent, place 5 minutes, continue
With acetonitrile: the mixed solution 10ml of strong ammonia solution (90:10) elutes, collect eluent, in less than 40 DEG C decompression and solvent recoveries extremely
Dry, residue is accurate to add acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution (30:70) makes dissolving, filtration, takes subsequent filtrate conduct
Solid-phase extraction column system suitability solution.
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 10 μ l of reference substance solution, inject liquid respectively
Chromatography, mensure, computing system employment and suitability test (E ＆ ST) solution and each corresponding Component peak area ratio of reference substance solution, cannot be less than
0.95.
The preparation of need testing solution takes this product content, mixes, and precision weighs 1.0004g, puts in conical flask with cover, plus
Acetonitrile: the mixed solution 25ml of strong ammonia solution (90:10), ultrasonically treated 30 minutes (power 200w, frequency 40khz), filtration, filter
In less than 40 DEG C decompression and solvent recoveries to doing, residue adds 0.1mol/l hydrochloric acid solution 10ml makes dissolving to liquid, and filtration, according to SPE
Method under column system employment and suitability test (E ＆ ST) item, from " being placed on the solid-phase extraction column handled well ", operates in accordance with the law, obtains test sample molten
Liquid.Precision measures reference substance solution, each 1ml of need testing solution, mixes, obtains mixed solution.
The preparation of negative solution takes negative preparation to prepare with need testing solution preparation method.
Determination method is accurate respectively to draw reference substance solution and each 10 μ l of mixed solution, injects liquid chromatograph, measures, that is,
?.
Table 3 system suitability result (n=5)
| Project | Number of theoretical plate | Separating degree | Tailing factor | Repeatability |
| Mesaconine | >21000 | —— | 0.97～0.99 | 0.24% |
| Hypaconitine | >25000 | 6 | 0.95～0.98 | 0.63% |
| Aconitine | >27000 | 3 | 0.99-1.03 | 0.74% |
| Diester-type alkaloids | —— | —— | —— | 0.45% |
It is good that result shows that this law measures diester-type alkaloids system suitability.
Table 4 solid-phase extraction column system suitability result
| Project | Cross long-pending before post | Cross long-pending after post | Ratio |
| Mesaconine | 312362.6 | 310633.5 | 0.994 |
| Hypaconitine | 328493.0 | 327140.75 | 0.996 |
| Aconitine | 301359.0 | 300054.25 | 0.996 |
Solid-phase extraction column system suitability result shows that solid-phase extraction column used by this law mensure is given birth to three kinds of diester-types
The attached rate of alkaloids Adsorption and desorption, all more than 95%, shows the survey of selected solid-phase extraction column diester-type alkaloids be applied to this law
Fixed.
Specificity result of the test display need testing solution is having chromatographic peak with Hypaconitine chromatographic peak corresponding position, negative
Solution with mesaconine, Hypaconitine, aconitine chromatographic peak relevant position all no chromatographic peaks, show that this law measures negative no dry
Disturb, specificity is strong.
Dibasic acid esters content relatively low (0.0012%) in this product, and in Chinese medicine, the more very difficult thoroughly exclusion of various composition is all kinds of micro
The interference to result for the composition, in mensure, slightly chromatographic peak can produce impact to measuring, and causes collection of illustrative plates to be unable to reach efficient liquid
Phase chromatography test requirements document, therefore add quantitative diester-type alkaloids reference substance in need testing solution, beneficial to methodological study examination
Test the control of middle indices, also can get accurate result.
Result of the test shows and records diester-type alkaloids using test sample and reference substance mixing method and directly record result phase
Closely, three kinds of diester-type alkaloids all can efficiently separate with other peaks adjacent, and showing can be biological to diester-type in this product using this law
Alkali is measured.
Compare with " CP method ", chromatographic peak peak shape is obviously improved, and tailing factor is less, each component can efficiently separate, and shows
This method is applied to the assay of the attached sweet medicine diester-type alkaloids of fiber crops.
3rd, diester-type alkaloids content assaying method verification process of the present invention
1. linear test
Every 1ml is taken to contain mesaconine, Hypaconitine, the mixed reference substance solution of each 20 μ g of aconitine respectively, as comparison
The linear stock solution of product.Prepare variable concentrations linear solvent respectively.Accurate each absorption linear solvent 10 μ l, injects liquid chromatogram respectively
Instrument, measures, obtains final product.
Table 5 mesaconine linear test result
| Mesaconine (μ g/ml) | 0.9877 | 1.9754 | 3.9508 | 9.8769 | 19.7538 |
| Peak area | 14637 | 29982 | 62607 | 160145 | 325364 |
With the concentration of mesaconine reference substance as abscissa, peak area is ordinate, carries out linear regression, obtains regression equation
For: y=165787x-2616.6, r=1.0000.Result shows that this law measures mesaconine sample size in 0.01～0.2 μ g range
Interior is in good linear relationship with peak area.
Table 6 Hypaconitine linear test result
| Hypaconitine (μ g/ml) | 1.1058 | 2.2116 | 4.4231 | 11.0578 | 22.1157 |
| Peak area | 15191 | 30873 | 65154 | 168223 | 343857 |
With the concentration of Hypaconitine reference substance as abscissa, peak area is ordinate, carries out linear regression, obtains regression equation
For: y=15674x-3598.6, r=0.9999.Result shows that this law measures Hypaconitine sample size in 0.01～0.2 μ g range
It is in good linear relationship with peak area.
Table 7 aconitine linear test result
| Aconitine (μ g/ml) | 1.0621 | 2.1242 | 4.2484 | 10.6210 | 21.2420 |
| Peak area | 13553 | 27602 | 59771 | 153193 | 316987 |
With the concentration of aconitine reference substance as abscissa, peak area is ordinate, carries out linear regression, obtains regression equation
For: y=15057x-4122.7, r=0.9999.Result show this law measure aconitine sample size in 0.01～0.2 μ g range with
Peak area is in good linear relationship.
2. test limit test
Take diester-type alkaloids reference substance solution dilution sample introduction, recording s/n is 2.5:1, and diester-type alkaloids test limit is divided
It is not: mesaconine 0.49ng, Hypaconitine 0.55ng, aconitine 0.53ng.
3. solution stability testing
Take need testing solution room temperature to place, drew 10 μ l respectively at the 0th, 10,20 hours, inject liquid chromatograph, measure,
Result see table.
Table 8 solution stability testing result
| Project | 0h | 10h | 20h | Averagely | rsd |
| Mesaconine | 158827 | 156839 | 157442 | 157702.7 | 0.65% |
| Hypaconitine | 198547 | 198123 | 197536 | 198068.7 | 0.26% |
| Aconitine | 155464 | 155013 | 154693 | 155056.7 | 0.25% |
| Diester-type alkaloids | 512838 | 509985 | 509691 | 510828 | 0.34% |
It is good that result display need testing solution places 20 hours internal stabilities in room temperature.
4. chromatographic column serviceability test
Chromatographic column using different labels is measured, and result see table.
The different chromatographic column serviceability test of table 9
Result shows that this law measures diester-type alkaloids content to different label chromatographic column good tolerances.
Specific embodiment
Embodiment 1: the attached sweet granule diester-type alkaloids assay of fiber crops
1) prepare granule:
Take monkshood 6kg, Chinese ephedra 2kg, Radix Glycyrrhizae 2kg, add water to cook twice, for the first time plus 8 times amount water, decoct 2 hours, second
Secondary plus 6 times amount water, decoct 1 hour, merge decocting liquid twice, filtration, are concentrated into paste, and drying under reduced pressure below 80 DEG C is pulverized,
Mix with appropriate dextrin, make particle, be dried, whole grain, obtain the granule of numb attached sweet medicine.
2) assay:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With acetonitrile: tetrahydrochysene furan
Mutter (20:20) for mobile phase a, with the 0.01mol/l potassium dihydrogen phosphate containing 0.02% phosphoric acid for mobile phase b；According to the form below
Regulation carries out gradient elution；Detection wavelength is 222nm；Number of theoretical plate is calculated as 26695 by aconitine peak.
Table 10 embodiment 1 gradient elution
| Time (min) | Mobile phase a(%) | Mobile phase b(%) |
| 0~75 | 10→25 | 90→75 |
| 75~77 | 25→35 | 75→65 |
| 77~87 | 35 | 65 |
Prepared by reference substance solution: take mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurate
Weighed, respectively plus acetonitrile make every 1ml contain 0.05mg solution, as reference substance stock solution, precision measure above-mentioned three kinds comparison
The each 5ml of product stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product.
Solid-phase extraction column system suitability: precision measures each 3ml of reference substance stock solution, mixes, and reduced pressure at room temperature reclaims
To doing, residue is accurate to add 0.1mol/l hydrochloric acid solution 50ml to make dissolving, and precision measures 5ml, is placed in the SPE handled well
Post (with mixed type cation exchange reverse phase absorption agent as filler, 150mg, capacity is 4ml, uses acetonitrile, water each in advance successively
6ml elutes) on, successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent, place 5 minutes, continue
With acetonitrile: the mixed solution 10ml of strong ammonia solution (90:10) elutes, collect eluent, in less than 40 DEG C decompression and solvent recoveries extremely
Dry, residue is accurate to add acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution (30:70) makes dissolving, filtration, takes subsequent filtrate conduct
Solid-phase extraction column system suitability solution；
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 5 μ l of reference substance solution, inject liquid respectively
Chromatography, measures, and calculates, and system suitability solution and each corresponding Component peak area ratio of reference substance solution are more than
0.95.
The preparation of need testing solution: take inspection product, weigh 5g, put in conical flask with cover, plus acetonitrile: strong ammonia solution (90:10)
Mixed solution 25ml, ultrasonically treated 50 minutes, filtration, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds filtrate
0.1mol/l hydrochloric acid solution 10ml makes dissolving, filtration, according to the method under solid-phase extraction column system suitability item, certainly " is placed in
On the solid-phase extraction column handled well " rise, operate in accordance with the law, obtain need testing solution, measure reference substance solution 0.1ml and need testing solution
0.9ml, mixes, obtains mixed solution.
Determination method: accurate absorption reference substance solution and each 5 μ l of mixed solution respectively, inject liquid chromatograph, measure, that is,
?.
Result: 3 diester-type alkaloids theoretical cam curves, separating degree, tailing factors all reach requirement, attached sweet of this batch of fiber crops
Granula diester-type alkaloids content is 5.9 g/ bags.
Embodiment 2: the attached sweet granule diester-type alkaloids assay of fiber crops
1) prepare granule:
Take monkshood 12kg, Chinese ephedra 10kg, Radix Glycyrrhizae 10kg, add water to cook twice, add 10 times amount water for the first time, decoct 2.5 little
When, second plus 8 times amount water, decoct 1.5 hours, merge decocting liquid twice, filtration, be concentrated into paste, reduce pressure below 80 DEG C
Dry, pulverize, mix with appropriate dextrin, make particle, be dried, whole grain, obtain the granule of numb attached sweet medicine.
2) assay:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With acetonitrile: tetrahydrochysene furan
Mutter (30:10) for mobile phase a, with the 0.05mol/l potassium dihydrogen phosphate containing 0.3% phosphoric acid for mobile phase b；According to the form below
Regulation carries out gradient elution；Detection wavelength is 242nm；Number of theoretical plate is calculated as 25013 by aconitine peak.
Table 11 embodiment 2 gradient elution
| Time (min) | Mobile phase a(%) | Mobile phase b(%) |
| 0~80 | 15→25 | 85→75 |
| 80~85 | 25→35 | 75→65 |
| 85~95 | 35 | 65 |
Prepared by reference substance solution: take mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurate
Weighed, respectively plus acetonitrile make every 1ml contain 0.5mg solution, as reference substance stock solution, precision measures above-mentioned three kinds of reference substances
The each 5ml of stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product.
Solid-phase extraction column system suitability: precision measures each 3ml of reference substance stock solution, mixes, and reduced pressure at room temperature reclaims
To doing, residue is accurate to add 0.1mol/l hydrochloric acid solution 50ml to make dissolving, and precision measures 5ml, is placed in the SPE handled well
Post (with mixed type cation exchange reverse phase absorption agent as filler, 150mg, capacity is 5ml, uses acetonitrile, water each in advance successively
6ml elutes) on, successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent, place 5 minutes, continue
With acetonitrile: the mixed solution 10ml of strong ammonia solution (90:10) elutes, collect eluent, in less than 40 DEG C decompression and solvent recoveries extremely
Dry, residue is accurate to add acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution (30:70) makes dissolving, filtration, takes subsequent filtrate conduct
Solid-phase extraction column system suitability solution；
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 20 μ l of reference substance solution, inject liquid respectively
Chromatography, measures, and calculates, and system suitability solution and each corresponding Component peak area ratio of reference substance solution are more than
0.95.
The preparation of need testing solution: take inspection product, weigh 1g, put in conical flask with cover, plus acetonitrile: strong ammonia solution (90:10)
Mixed solution 25ml, ultrasonically treated 50 minutes, filtration, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds filtrate
0.1mol/l hydrochloric acid solution 10ml makes dissolving, filtration, according to the method under solid-phase extraction column system suitability item, certainly " is placed in
On the solid-phase extraction column handled well " rise, operate in accordance with the law, obtain need testing solution, measure reference substance solution 0.9ml and need testing solution
0.1ml, mixes, obtains mixed solution.
Determination method: accurate absorption reference substance solution and each 20 μ l of mixed solution respectively, inject liquid chromatograph, measure, that is,
?.
Result: 3 diester-type alkaloids theoretical cam curves, separating degree, tailing factors all reach requirement, attached sweet of this batch of fiber crops
Granula diester-type alkaloids content is 5.1 g/ bags.
Embodiment 3: the attached sweet capsule diester-type alkaloids assay of fiber crops
1) prepare capsule:
Take monkshood 10kg, Chinese ephedra 6.67kg, Radix Glycyrrhizae 6.67kg, add water to cook secondary, add 8 times amount for the first time, decoct 3 little
When, second plus 6 times amount, decoct 2 hours, decocting liquid filters, filtrate merges, and is concentrated into paste, drying under reduced pressure below 80 DEG C,
Pulverize, mix with appropriate amount of starch, load capsule, obtain the capsule of numb attached sweet medicine.
2) assay:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With acetonitrile: tetrahydrochysene furan
Mutter (26:14) for mobile phase a, with the 0.04mol/l potassium dihydrogen phosphate containing 0.1% phosphoric acid for mobile phase b；According to the form below
Regulation carries out gradient elution；Detection wavelength is 230nm；Number of theoretical plate is calculated as 26487 by aconitine peak.
Table 12 embodiment 3 gradient elution
| Time (min) | Mobile phase a(%) | Mobile phase b(%) |
| 0~79 | 15→21 | 85→79 |
| 79~85 | 21→35 | 79→65 |
| 85~95 | 35 | 65 |
Prepared by reference substance solution: take mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurate
Weighed, respectively plus acetonitrile make every 1ml contain 0.3mg solution, as reference substance stock solution, precision measures above-mentioned three kinds of reference substances
The each 5ml of stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product.
Solid-phase extraction column system suitability: precision measures each 3ml of reference substance stock solution, mixes, and reduced pressure at room temperature reclaims
To doing, residue is accurate to add 0.1mol/l hydrochloric acid solution 50ml to make dissolving, and precision measures 5ml, is placed in the SPE handled well
Post (with mixed type cation exchange reverse phase absorption agent as filler, 200mg, capacity is 10ml, uses acetonitrile, water each in advance successively
6ml elutes) on, successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent, place 5 minutes, continue
With acetonitrile: the mixed solution 10ml of strong ammonia solution (90:10) elutes, collect eluent, in less than 40 DEG C decompression and solvent recoveries extremely
Dry, residue is accurate to add acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution (30:70) makes dissolving, filtration, takes subsequent filtrate conduct
Solid-phase extraction column system suitability solution；
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 10 μ l of reference substance solution, inject liquid respectively
Chromatography, measures, and calculates, and system suitability solution and each corresponding Component peak area ratio of reference substance solution are more than
0.95.
The preparation of need testing solution: take inspection product, weigh 1.5g, put in conical flask with cover, plus acetonitrile: strong ammonia solution (90:
10) mixed solution 25ml, ultrasonically treated 40 minutes, filtration, in less than 40 DEG C decompression and solvent recoveries to doing, residue adds filtrate
0.1mol/l hydrochloric acid solution 10ml makes dissolving, filtration, according to the method under solid-phase extraction column system suitability item, certainly " is placed in
On the solid-phase extraction column handled well " rise, operate in accordance with the law, obtain need testing solution, measure reference substance solution 0.9ml and need testing solution
0.1ml, mixes, obtains mixed solution.
Determination method: accurate absorption reference substance solution and each 10 μ l of mixed solution respectively, inject liquid chromatograph, measure, that is,
?.
Result: 3 diester-type alkaloids theoretical cam curves, separating degree, tailing factors all reach requirement, the attached sweet glue of this batch of fiber crops
Wafer diester-type alkaloids content is 0.2 g/ grain.
Embodiment 4: the attached sweet tablet diester-type alkaloids assay of fiber crops
1) prepare tablet:
Take monkshood 10kg, Chinese ephedra 5kg, Radix Glycyrrhizae 5kg, add water to cook three times, for the first time plus 8 times amount, decoct 2 hours, second,
Add 6 times amount respectively three times, decoct 1 hour respectively, decocting liquid filters, filtrate merges, and is concentrated into paste, reduce pressure below 80 DEG C and do
Dry, pulverize, mix with appropriate amount of starch, 70% ethanol is made wetting agent and pelletized, plus appropriate magnesium stearate, mix, compressing tablet, obtain numb attached sweet
The tablet of medicine.
2) assay:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With acetonitrile: tetrahydrochysene furan
Mutter (23:17) for mobile phase a, with the 0.02mol/l potassium dihydrogen phosphate containing 0.08% phosphoric acid for mobile phase b；According to the form below
Regulation carries out gradient elution；Detection wavelength is 235nm；Number of theoretical plate is calculated as 9086 by aconitine peak.
Table 13 embodiment 4 gradient elution
| Time (min) | Mobile phase a(%) | Mobile phase b(%) |
| 0~75 | 15→22 | 85→78 |
| 75~77 | 22→35 | 78→65 |
| 77~87 | 35 | 65 |
Prepared by reference substance solution: take mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurate
Weighed, respectively plus acetonitrile make every 1ml contain 0.15mg solution, as reference substance stock solution, precision measure above-mentioned three kinds comparison
The each 5ml of product stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product.
Solid-phase extraction column system suitability: precision measures each 3ml of reference substance stock solution, mixes, and reduced pressure at room temperature reclaims
To doing, residue is accurate to add 0.1mol/l hydrochloric acid solution 50ml to make dissolving, and precision measures 5ml, is placed in the SPE handled well
Post (with mixed type cation exchange reverse phase absorption agent as filler, 150mg, capacity is 6ml, uses acetonitrile, water each in advance successively
6ml elutes) on, successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent, place 5 minutes, continue
With acetonitrile: the mixed solution 10ml of strong ammonia solution (90:10) elutes, collect eluent, in less than 40 DEG C decompression and solvent recoveries extremely
Dry, residue is accurate to add acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution (30:70) makes dissolving, filtration, takes subsequent filtrate conduct
Solid-phase extraction column system suitability solution；
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 15 μ l of reference substance solution, inject liquid respectively
Chromatography, measures, and calculates, and system suitability solution and each corresponding Component peak area ratio of reference substance solution are more than
0.95.
The preparation of need testing solution: take inspection product, weigh 2g, put in conical flask with cover, plus acetonitrile: strong ammonia solution (90:10)
Mixed solution 25ml, ultrasonically treated 30 minutes, filtration, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds filtrate
0.1mol/l hydrochloric acid solution 10ml makes dissolving, filtration, according to the method under solid-phase extraction column system suitability item, certainly " is placed in
On the solid-phase extraction column handled well " rise, operate in accordance with the law, obtain need testing solution, measure reference substance solution 0.4ml and need testing solution
0.6ml, mixes, obtains mixed solution.
Determination method: accurate absorption reference substance solution and each 15 μ l of mixed solution respectively, inject liquid chromatograph, measure, that is,
?.
Result: 3 diester-type alkaloids theoretical cam curves, separating degree, tailing factors all reach requirement, attached sweet of this batch of fiber crops
Agent diester-type alkaloids content is 3.5 g/ pieces.
Embodiment 5: the attached sweet oral solutions diester-type alkaloids assay of fiber crops
1) prepare oral solutions:
Take monkshood 9kg, Chinese ephedra 7kg, Radix Glycyrrhizae 7kg, add water to cook twice, for the first time plus 8 times amount water, decoct 2 hours, second
Secondary plus 6 times amount water, decoct 1 hour, and decocting liquid filters, and filtrate merges, and is concentrated into paste, lets cool, and add ethanol to reach to alcohol content
70%, stand 24 hours, filtration, filtrate recycling ethanol, it is concentrated into appropriate, add Icing Sugar and appropriate bacteriostatic agent, standing, filtration, fill
Envelope, sterilizing, obtain the oral solutions of numb attached sweet medicine.
2) assay:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With acetonitrile: tetrahydrochysene furan
Mutter (22:18) for mobile phase a, with the 0.03mol/l potassium dihydrogen phosphate containing 0.12% phosphoric acid for mobile phase b；According to the form below
Regulation carries out gradient elution；Detection wavelength is 232nm；Number of theoretical plate is calculated as 21077 by aconitine peak.
Table 14 embodiment 5 gradient elution
| Time (min) | Mobile phase a(%) | Mobile phase b(%) |
| 0~75 | 14→20 | 86→80 |
| 75~77 | 20→35 | 80→65 |
| 77~87 | 35 | 65 |
Prepared by reference substance solution: take mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurate
Weighed, respectively plus acetonitrile make every 1ml contain 0.2mg solution, as reference substance stock solution, precision measures above-mentioned three kinds of reference substances
The each 5ml of stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product.
Solid-phase extraction column system suitability: precision measures each 3ml of reference substance stock solution, mixes, and reduced pressure at room temperature reclaims
To doing, residue is accurate to add 0.1mol/l hydrochloric acid solution 50ml to make dissolving, and precision measures 5ml, is placed in the SPE handled well
Post (with mixed type cation exchange reverse phase absorption agent as filler, 150mg, capacity is 6ml, uses acetonitrile, water each in advance successively
6ml elutes) on, successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent, place 5 minutes, continue
With acetonitrile: the mixed solution 10ml of strong ammonia solution (90:10) elutes, collect eluent, in less than 40 DEG C decompression and solvent recoveries extremely
Dry, residue is accurate to add acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution (30:70) makes dissolving, filtration, takes subsequent filtrate conduct
Solid-phase extraction column system suitability solution；
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 10 μ l of reference substance solution, inject liquid respectively
Chromatography, measures, and calculates, and system suitability solution and each corresponding Component peak area ratio of reference substance solution are more than
0.95.
The preparation of need testing solution: take inspection product, weigh 2g, put in conical flask with cover, plus acetonitrile: strong ammonia solution (90:10)
Mixed solution 25ml, ultrasonically treated 10 minutes, filtration, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds filtrate
0.1mol/l hydrochloric acid solution 10ml makes dissolving, filtration, according to the method under solid-phase extraction column system suitability item, certainly " is placed in
On the solid-phase extraction column handled well " rise, operate in accordance with the law, obtain need testing solution, measure reference substance solution 0.3ml and need testing solution
0.7ml, mixes, obtains mixed solution.
Determination method: accurate absorption reference substance solution and each 10 μ l of mixed solution respectively, inject liquid chromatograph, measure, that is,
?.
Result: 3 diester-type alkaloids theoretical cam curves, separating degree, tailing factors all reach requirement, the attached Gan Kou of this batch of fiber crops
Taking liquor diester-type alkaloids content is 0.8 g/ml.
Embodiment 6: the attached sweet pill diester-type alkaloids assay of fiber crops
1) prepare pill:
Take monkshood 8kg, Chinese ephedra 5kg, Radix Glycyrrhizae 5kg, add water to cook twice, for the first time plus 8 times amount water, decoct 2 hours, second
Secondary plus 8 times amount water, decoct 2 hours, and decocting liquid filters, and filtrate merges, and are concentrated into paste, add starch appropriate, and pill is dried,
Polishing, obtains the pill of numb attached sweet medicine.
2) assay:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With acetonitrile: tetrahydrochysene furan
Mutter (22:18) for mobile phase a, with the 0.03mol/l potassium dihydrogen phosphate containing 0.12% phosphoric acid for mobile phase b；According to the form below
Regulation carries out gradient elution；Detection wavelength is 232nm；Number of theoretical plate is calculated as 11090 by aconitine peak.
Table 16 embodiment 6 gradient elution
| Time (min) | Mobile phase a(%) | Mobile phase b(%) |
| 0~80 | 13→20 | 87→80 |
| 80~85 | 20→35 | 80→65 |
| 85~90 | 35 | 65 |
Prepared by reference substance solution: take mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurate
Weighed, respectively plus acetonitrile make every 1ml contain 0.2mg solution, as reference substance stock solution, precision measures above-mentioned three kinds of reference substances
The each 5ml of stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product.
Solid-phase extraction column system suitability: precision measures each 3ml of reference substance stock solution, mixes, and reduced pressure at room temperature reclaims
To doing, residue is accurate to add 0.1mol/l hydrochloric acid solution 50ml to make dissolving, and precision measures 5ml, is placed in the SPE handled well
Post (with mixed type cation exchange reverse phase absorption agent as filler, 150mg, capacity is 6ml, uses acetonitrile, water each in advance successively
6ml elutes) on, successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent, place 5 minutes, continue
With acetonitrile: the mixed solution 10ml of strong ammonia solution (90:10) elutes, collect eluent, in less than 40 DEG C decompression and solvent recoveries extremely
Dry, residue is accurate to add acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution (30:70) makes dissolving, filtration, takes subsequent filtrate conduct
Solid-phase extraction column system suitability solution；
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 10 μ l of reference substance solution, inject liquid respectively
Chromatography, measures, and calculates, and system suitability solution and each corresponding Component peak area ratio of reference substance solution are more than
0.95.
The preparation of need testing solution: take inspection product, weigh 2g, put in conical flask with cover, plus acetonitrile: strong ammonia solution (90:10)
Mixed solution 25ml, ultrasonically treated 40 minutes, filtration, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds filtrate
0.1mol/l hydrochloric acid solution 10ml makes dissolving, filtration, according to the method under solid-phase extraction column system suitability item, certainly " is placed in
On the solid-phase extraction column handled well " rise, operate in accordance with the law, obtain need testing solution, measure reference substance solution 0.7ml and need testing solution
0.3ml, mixes, obtains mixed solution.
Determination method: accurate absorption reference substance solution and each 10 μ l of mixed solution respectively, inject liquid chromatograph, measure, that is,
?.
Result: 3 diester-type alkaloids theoretical cam curves, separating degree, tailing factors all reach requirement, the attached sweet ball of this batch of fiber crops
Agent diester-type alkaloids content is 0.4 g/ ball.
Claims (2)
1. a kind of attached sweet medicine diester-type alkaloids of fiber crops content assaying method it is characterised in that: surveyed with high performance liquid chromatography
Determine diester-type alkaloids content in monkshood, mobile phase temporally carries out gradient elution, is measured using standard addition method；
Chromatographic condition: with octadecylsilane chemically bonded silica as filler, specification is 250 × 4.6mm to chromatographic column, 5 μm；With second
Nitrile: oxolane=20:20 to 30:10 is mobile phase a, with the 0.01-0.05mol/l di(2-ethylhexyl)phosphate containing 0.02%-0.3% phosphoric acid
Hydrogen potassium solution is mobile phase b, temporally carries out gradient elution, mobile phase b, by 90% to 70%, detects ripple within 0 minute to 80 minutes
A length of 222-242nm, number of theoretical plate is calculated by aconitine peak and is not less than 8000；
Prepared by reference substance solution: take mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance appropriate, accurately weighed,
Respectively plus acetonitrile make every 1ml contain 0.05-0.5mg solution, as reference substance stock solution, precision measures above-mentioned three kinds of reference substances
The each 5ml of stock solution, puts in 50ml measuring bottle, plus 0.1% phosphoric acid solution is diluted to scale, shakes up, obtains final product；
Solid-phase extraction column system suitability: precision measures each 3ml of reference substance stock solution, mix, reduced pressure at room temperature be recycled to dry,
Residue is accurate to add 0.1mol/l hydrochloric acid solution 50ml to make dissolving, and precision measures 5ml, is placed on the solid-phase extraction column handled well,
Successively with 0.1mol/l hydrochloric acid solution, methyl alcohol, acetonitrile each 5ml wash-out, discard eluent, place 5 minutes, continue and use acetonitrile: dense ammonia
The mixed solution 10ml wash-out of test solution=90:10, collects eluent, in less than 40 DEG C decompression and solvent recoveries to dry, residue precision
Add acetonitrile: the mixed solution 3ml of 0.1% phosphoric acid solution=30:70 makes dissolving, filtration, take subsequent filtrate as solid-phase extraction column
System suitability solution；Described solid-phase extraction column with mixed type cation exchange reverse phase absorption agent as filler, 150-
200mg, capacity is 4-10ml, uses acetonitrile, water each 6ml wash-out in advance successively；
Accurate absorption above-mentioned solid phase extraction column system suitability solution and reference substance solution each 5-30 μ l, inject liquid phase respectively
Chromatograph, mensure, computing system employment and suitability test (E ＆ ST) solution and each corresponding Component peak area ratio of reference substance solution, cannot be less than
0.95；
The preparation of need testing solution: take inspection product, weigh 0.5-5g, put in conical flask with cover, plus acetonitrile: strong ammonia solution=90:10
Mixed solution 25ml, ultrasonically treated 10-50 minute, filtration, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds filtrate
0.1mol/l hydrochloric acid solution 10ml makes dissolving, filtration, according to the method under solid-phase extraction column system suitability item, certainly " is placed in
On the solid-phase extraction column handled well " rise, operate in accordance with the law, obtain need testing solution, measure reference substance solution and prepare with need testing solution
Become and contain mesaconine, Hypaconitine, the mixing prepare liquid of aconitine 2-18 μ g/ml respectively；
Determination method: precision is drawn reference substance solution and mixed prepare liquid each 5-30 μ l respectively, injects liquid chromatograph, measures, that is,
?.
2. a kind of attached sweet medicine diester-type alkaloids of fiber crops according to claim 1 content assaying method it is characterised in that: mixing
Reference substance solution in prepare liquid: the ratio of need testing solution is 10%:90% to 90%:10%.
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