CN116381099B - Method for measuring contents of seven components in spleen-invigorating pill by adopting one-measurement-multiple-evaluation method - Google Patents
Method for measuring contents of seven components in spleen-invigorating pill by adopting one-measurement-multiple-evaluation method Download PDFInfo
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- CN116381099B CN116381099B CN202310421500.1A CN202310421500A CN116381099B CN 116381099 B CN116381099 B CN 116381099B CN 202310421500 A CN202310421500 A CN 202310421500A CN 116381099 B CN116381099 B CN 116381099B
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- sucrose
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- sinapiyl
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- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000011156 evaluation Methods 0.000 title claims abstract description 20
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- 229930006000 Sucrose Natural products 0.000 claims abstract description 70
- 239000005720 sucrose Substances 0.000 claims abstract description 70
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 65
- 239000013558 reference substance Substances 0.000 claims abstract description 56
- 238000012937 correction Methods 0.000 claims abstract description 53
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- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims abstract description 39
- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 claims abstract description 39
- CUGKULNFZMNVQI-UHFFFAOYSA-N Costunolid I Natural products CC1=CCC=C(/C)CCC2C(C1)OC(=O)C2=C CUGKULNFZMNVQI-UHFFFAOYSA-N 0.000 claims abstract description 39
- HRYLQFBHBWLLLL-AHNJNIBGSA-N costunolide Chemical compound C1CC(/C)=C/CC\C(C)=C\[C@H]2OC(=O)C(=C)[C@@H]21 HRYLQFBHBWLLLL-AHNJNIBGSA-N 0.000 claims abstract description 39
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- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims abstract description 39
- 229940114124 ferulic acid Drugs 0.000 claims abstract description 39
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims abstract description 39
- 235000001785 ferulic acid Nutrition 0.000 claims abstract description 39
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 claims abstract description 39
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 claims abstract description 39
- WKSUCCVMYJRMFR-UHFFFAOYSA-N Dehydrocostus lactone Natural products C12OC(=O)C(=C)C2CCC(=C)C2(C)C1(C)C(=C)CC2 WKSUCCVMYJRMFR-UHFFFAOYSA-N 0.000 claims abstract description 38
- NETSQGRTUNRXEO-XUXIUFHCSA-N dehydrocostus lactone Chemical compound C([C@H]1C(=C)C(=O)O[C@@H]11)CC(=C)[C@H]2[C@@H]1C(=C)CC2 NETSQGRTUNRXEO-XUXIUFHCSA-N 0.000 claims abstract description 38
- VGGSULWDCMWZPO-UHFFFAOYSA-N flavoayamenin Natural products COC1=CC=2OC(C=3C=CC(O)=CC=3)=CC(=O)C=2C(O)=C1C1OC(CO)C(O)C(O)C1OC1OC(CO)C(O)C(O)C1O VGGSULWDCMWZPO-UHFFFAOYSA-N 0.000 claims abstract description 37
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 claims abstract description 36
- 239000004378 Glycyrrhizin Substances 0.000 claims abstract description 35
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 claims abstract description 35
- 229960004949 glycyrrhizic acid Drugs 0.000 claims abstract description 35
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 claims abstract description 35
- 235000019410 glycyrrhizin Nutrition 0.000 claims abstract description 35
- 238000001514 detection method Methods 0.000 claims abstract description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 126
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- 239000012085 test solution Substances 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
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- GSZUGBAEBARHAW-UHFFFAOYSA-N sophoraflavone B Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(C=2OC3=CC(O)=CC=C3C(=O)C=2)C=C1 GSZUGBAEBARHAW-UHFFFAOYSA-N 0.000 claims description 4
- 230000001502 supplementing effect Effects 0.000 claims description 4
- KSDSYIXRWHRPMN-UHFFFAOYSA-N 4'-O-beta-D-Galactopyranoside-6''-p-Coumaroylprunin-4',5,7-Trihydroxyflavanone Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(C2OC3=CC(O)=CC(O)=C3C(=O)C2)C=C1 KSDSYIXRWHRPMN-UHFFFAOYSA-N 0.000 claims description 3
- DEMKZLAVQYISIA-ONJCETCRSA-N Liquiritin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)c1ccc([C@@H]2Oc3c(C(=O)C2)ccc(O)c3)cc1 DEMKZLAVQYISIA-ONJCETCRSA-N 0.000 claims description 3
- DEMKZLAVQYISIA-UHFFFAOYSA-N Liquirtin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(C2OC3=CC(O)=CC=C3C(=O)C2)C=C1 DEMKZLAVQYISIA-UHFFFAOYSA-N 0.000 claims description 3
- DEMKZLAVQYISIA-ZRWXNEIDSA-N liquiritin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([C@H]2OC3=CC(O)=CC=C3C(=O)C2)C=C1 DEMKZLAVQYISIA-ZRWXNEIDSA-N 0.000 claims description 3
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- SMDOOINVMJSDPS-UHFFFAOYSA-N Astragaloside Natural products C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)=C1 SMDOOINVMJSDPS-UHFFFAOYSA-N 0.000 description 2
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- QMNWISYXSJWHRY-XWJCTJPOSA-N astragaloside Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)C4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)CC3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-XWJCTJPOSA-N 0.000 description 2
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- YPGCWEMNNLXISK-UHFFFAOYSA-N hydratropic acid Chemical class OC(=O)C(C)C1=CC=CC=C1 YPGCWEMNNLXISK-UHFFFAOYSA-N 0.000 description 1
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- 150000002596 lactones Chemical class 0.000 description 1
- FURUXTVZLHCCNA-AWEZNQCLSA-N liquiritigenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC=C2C(=O)C1 FURUXTVZLHCCNA-AWEZNQCLSA-N 0.000 description 1
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- 238000007873 sieving Methods 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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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)
- Spectroscopy & Molecular Physics (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a method for measuring seven component contents in a spleen-invigorating pill by adopting a one-measurement-multiple-evaluation method. The method takes 3,6' -sinapiyl sucrose as an internal reference substance, and calculates relative correction factors of the other six components; the content of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone in the spleen-invigorating pill is calculated according to relative correction factors and chromatographic peak positioning by using 3,6' -sinapiyl sucrose as a reference substance; provides a basis for quality control standard of the spleen-invigorating pill, and is convenient for production and detection of finished products of the spleen-invigorating pill.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a method for measuring seven component contents in a spleen-invigorating pill by adopting a one-measurement-multiple-evaluation method.
Background
The preparation of GUIPI pill comprises 11 kinds of medicinal materials including radix Codonopsis, parched Atractylodis rhizoma, radix astragali Preparata, radix Glycyrrhizae Preparata, poria, radix Polygalae Preparata, semen Ziziphi Spinosae Preparata, arillus longan, radix Angelicae sinensis, radix aucklandiae, and fructus Jujubae (core removed). The spleen-invigorating pill has the effects of replenishing qi to invigorate the spleen, nourishing blood and tranquillizing, and is clinically used for treating deficiency of heart and spleen, shortness of breath, palpitation, insomnia and dreaminess, dizziness, tiredness and hypodynamia, inappetence, metrorrhagia and hematochezia. In the formula, the roasted astragalus and the dangshen are monarch drugs, and invigorate spleen qi, so that qi is vigorous and blood is generated; chinese angelica and longan pulp, stir-fried white atractylodes rhizome and honey-fried licorice root are used as ministerial drugs, and Chinese angelica and longan pulp nourish blood and tonify heart; the stir-fried white atractylodes rhizome and the honey-fried licorice root can tonify spleen and qi, and help ginseng and astragalus root to tonify spleen to promote biochemical resource; the fried wild jujube seed and the poria cocos are added, the polygala tenuifolia is processed, the blood is nourished, and the heart is calmed and the spirit is calmed; the costustoot regulates qi and enlivens spleen to make the costustoot complement without stagnation; the jujube regulates the spleen and stomach, the honey-fried licorice root regulates the medicines, and the medicines are combined to play roles of tonifying qi and enriching blood, and strengthening spleen and nourishing heart.
The spleen invigorating pill contains various compounds such as saponins, phenylpropionic acids, furans, flavonoids, polysaccharides, and lactones. The content measurement index specified under the content measurement item of the spleen-invigorating pill of the 2020 edition of Chinese pharmacopoeia is astragaloside, but the chemical components in the spleen-invigorating pill are complex, and the quality current situation of the spleen-invigorating pill is difficult to comprehensively evaluate by simply using the content of astragaloside. Along with the deep clinical application and research, the establishment of a scientific, reasonable, convenient and feasible quality control method has important significance for controlling the quality of the spleen-invigorating pill and guaranteeing the clinical curative effect.
Disclosure of Invention
In order to solve the problems, the method for measuring the contents of seven components in the spleen-invigorating pill by adopting a one-measurement-multiple-evaluation method is provided, and the method takes 3,6' -sinapiyl sucrose as an internal reference substance, so that the contents of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone in the spleen-invigorating pill big honeyed pill can be rapidly detected, a basis is provided for the quality control standard of the spleen-invigorating pill big honeyed pill finished product, and the production and detection of the spleen-invigorating pill big honeyed pill finished product are facilitated.
The invention provides a method for measuring the contents of seven components in a spleen-invigorating pill by adopting a one-measurement-multiple-evaluation method, which comprises the following steps:
a method for measuring the contents of seven components in the spleen-invigorating pill by adopting a multi-evaluation method comprises the following steps:
(1) Preparation of mixed control solution and test solution:
Taking methanol as a solvent to respectively prepare single reference substance solutions of seven reference substances of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosyl sucrose, 3,6' -sinapioyl sucrose, costunolide and dehydrocostuslactone, and mixing a proper amount of the seven single reference substance solutions to prepare a series of mixed reference substance solutions with different concentrations;
Cutting GUIPI pill, adding diatomite, grinding, adding 80% methanol, refluxing, filtering, collecting filtrate, concentrating to near dryness, dissolving with 80% methanol, transferring into a measuring flask, metering volume, shaking, filtering, and collecting filtrate to obtain sample solution;
(2) Determining chromatographic conditions:
Chromatographic column: agilent Poroshell 120EC-C18, specification 4.6X105 mm,4 μm; the column temperature is 33-37 ℃; the detection wavelength is 225nm; the flow rate is 1ml/min; acetonitrile is taken as a mobile phase A, and a 0.1% phosphoric acid solution is taken as a mobile phase B for gradient elution;
(3) Determining a relative correction factor:
Taking mixed reference substance solutions with different concentrations, respectively injecting samples for high performance liquid chromatography analysis, selecting 3,6' -sinapiyl sucrose as an internal reference substance, and respectively calculating relative correction factors of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone;
(4) Determination of the component content of the sample solution:
Preparing a 3,6' -dissinapiyl sucrose reference substance solution from a sample solution, and performing high performance liquid chromatography analysis, wherein the concentration of the 3,6' -dissinapiyl sucrose reference substance solution is close to that of the 3,6' -dissinapiyl sucrose in the sample solution; and (3) calculating the contents of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone according to the relative correction factors by taking the concentration and peak area of the 3,6' -sinapiyl sucrose reference substance as a reference substance.
Preferably, the gradient elution conditions are:
0～15min，A：3％～8％，B：97％～92％；
15～32min，A：8％～12.5％，B：92％～87.5％；
32～45min，A：12.5％，B：87.5％；
45～50min，A：12.5％～15％，B：87.5％～85％；
50～70min，A：15％～21％，B：85％～79％；
70～120min，A：21％～58％，B：79％～42％。
preferably, the calculation formula of the relative correction factor is:
Wherein s is 3,6' -sinapiyl sucrose, k is a component to be detected, and one of six components of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone is sequentially taken; w s is the concentration of 3,6 '-disjuncyl sucrose, W k is the concentration of the component to be detected, A s is the peak area of the 3,6' -disjuncyl sucrose, and A k is the peak area of the component to be detected; f s is the correction factor of 3,6' -sinapiyl sucrose and f k is the correction factor of the component to be tested.
Specifically, the chromatographic peak relative retention time of 5-hydroxymethylfurfural is 0.10, and the relative correction factor is 0.95;
the chromatographic peak relative retention time of ferulic acid was 0.53, and the relative correction factor was 0.66;
The chromatographic peak relative retention time of the liquiritin is 0.65, and the relative correction factor is 0.72;
The chromatographic peak relative retention time of spinosin was 0.78 and the relative correction factor was 1.03;
The chromatographic peak relative retention time of costunolide is 1.71, and the relative correction factor is 1.12;
The chromatographic peak relative retention time of dehydrocostuslactone is 1.74, and the relative correction factor is 1.62;
The chromatographic peak of 3,6' -sinapiyl sucrose had a relative retention time of 1.00 and a relative correction factor of 1.
Preferably, the preparation method of the sample solution specifically comprises the following steps:
cutting spleen-invigorating pill, precisely weighing 9-12g, adding diatomite 4.5-6g, grinding into fine powder, adding 80% methanol 120ml, weighing, heating and refluxing for 60min, cooling, supplementing the reduced weight with 80% methanol, filtering, precisely weighing 60ml filtrate, concentrating to near dryness, adding 80% methanol for dissolving, transferring to 20ml measuring flask, adding methanol to volume to scale, shaking, filtering, and collecting the filtrate to obtain the sample solution.
Preferably, in the step (4), the preparation method of the 3,6' -sinapiyl sucrose reference substance solution comprises the following steps:
3,6' -dissinapiyl sucrose was dissolved in methanol to obtain a3, 6' -dissinapiyl sucrose control solution having a concentration consistent with the concentration of 3,6' -dissinapiyl sucrose in the test solution.
Preferably, the concentrations of the single reference solutions of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6' -sinapiyl sucrose, costunolide and dehydrocostuslactone are respectively as follows: 1.6583mg/ml, 0.8613mg/ml, 0.9491mg/ml, 0.9271mg/ml, 0.9259mg/ml, 0.9401mg/ml, 0.9816mg/ml.
Preferably, in chromatographic conditions, the theoretical plate number is not less than 3000 calculated according to 3,6' -sinapiyl sucrose, and the chromatographic peak is better under the arrangement, so that the quantitative calculation of the content of 7 components is facilitated.
Preferably, the spleen-invigorating pill is in the form of water honeyed pill, big honeyed pill or small honeyed pill.
The beneficial effects of the invention include, but are not limited to:
The method for measuring the contents of seven components in the spleen-invigorating pill by adopting a one-measurement-multiple-evaluation method provided by the invention has the advantages that (1) the seven components which mainly play a pharmacological role in the spleen-invigorating pill can be quantitatively detected, systematic analysis of the spleen-invigorating pill is facilitated, a research direction is provided for pharmacological research of the spleen-invigorating pill, and a basis is provided for quality control of the spleen-invigorating pill so as to improve the quality of products; (2) The quantitative detection of seven components can be realized rapidly and conveniently by adopting the 3,6' -sinapiyl sucrose as a reference substance, so that the detection efficiency is improved; (3) The high performance liquid chromatography has good condition stability, repeatability, precision and reliability, the used chromatographic column has smaller particle diameter, higher column efficiency, short column length, short analysis time and accurate detection result, and is suitable for detecting the spleen-invigorating pill big honeyed pills in a large scale.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a chromatogram of a sample solution obtained by a reflux and ultrasonic extraction method;
FIG. 2 is a chromatogram of a test solution at different wavelengths;
FIG. 3 is a chromatogram of concentrated and unconcentrated test solutions;
FIG. 4 is a chromatogram of a sample solution, a negative control solution, a mixed control solution, and a blank solvent in specificity verification.
Detailed Description
The application is further illustrated below in connection with specific examples, to which the scope of protection of the application is not limited.
The method for measuring the contents of seven components in the spleen-invigorating pill by adopting the one-measurement-multiple-evaluation method provided by the embodiment is specifically as follows:
1. Instrument, reagent and drug:
1.1 instruments
Agilent1260 high performance liquid chromatograph, BSA224S-CW electronic balance (Sidoriko instruments (Beijing) Co., ltd.), XS105 electronic balance (METTLER TOLEDO), KQ-300DE ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.), KMD adjustable temperature electric jacket, HH-8 constant temperature water bath (Jiangsu middle large instrument technologies Co., ltd.).
Chromatographic column: agilent Poroshell 120EC-C18 SN: USHKB21386 (4.6X105 mm,4 μm)
Agilent Poroshell 120EC-C18 SN：USHKB21377(4.6*150mm，4μm)。
1.2 Reagent:
acetonitrile (FISHER CHEMICAL) and phosphoric acid (FISHER SCIENTIFIC) are chromatographic purity, methanol is analytically pure, and water is purified water of the chen type.
1.3 Medicine:
1.3.1 control:
5-hydroxymethylfurfural control (lot number: 111626-202215, national food and drug verification institute, purity 99.5%);
Ferulic acid reference substance (batch number: 110773-201915, purity 99.4% of Chinese food and drug assay institute);
glycyrrhizin reference substance (lot number: 111610-201908, chinese food and drug assay institute, purity 95.0%);
the spinosin reference substance (lot number: 111869-202005, chinese food and drug assay institute, purity 94.8%);
3,6' -bis-sinapiyl sucrose control (lot number: 111884-202006, purity 96.5% of Chinese food and drug assay institute);
costunolide reference substance (lot number: 111524-201911, purity of 99.9% of Chinese food and drug assay institute);
Dehydrocostuslactone reference (lot number: 111525-201912, purity 99.5% of Chinese food and drug inspection institute).
1.3.2 Test pieces
The preparation method of the spleen-invigorating pill comprises the following steps:
Weighing 11 kinds of decoction pieces including 80g of radix codonopsitis, 160g of stir-fried bighead atractylodes rhizome, 80g of stir-fried astragalus root, 40g of stir-fried liquorice, 160g of poria cocos, 160g of prepared polygala tenuifolia, 80g of stir-fried spina date seed, 160g of longan pulp, 160g of angelica, 40g of elecampane and 40g of jujube (stoned), crushing into fine powder, sieving and uniformly mixing. Adding proper amount of water into 25-40 g of refined honey per 100g of powder to prepare pills, drying and preparing the water-honeyed pills; or adding 80-90 g refined honey to make into small honeyed pill or large honeyed pill. The same method is adopted to prepare the spleen-invigorating pill big honeyed pill samples in batches.
Since the effect of the spleen-invigorating pill water honeyed pill, the effect of the small honeyed pill and the effect of the large honeyed pill are the same, the large honeyed pill of the spleen-invigorating pill is taken as an example for research in the embodiment.
2. HPLC (high performance liquid chromatography) chromatographic conditions:
agilent Poroshell 120EC-C18, column length of 150mm, inner diameter of 4.6mm, particle diameter of 4 μm, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution, detection wavelength of 225nm, flow rate of 1ml per minute, and column temperature of 35deg.C. The mobile phase gradient elution conditions are shown in Table 1.
TABLE 1 gradient elution conditions for mobile phases
3. Preparation of control solution
3.1 Preparation of seven control stock solutions:
Dissolving 7 reference substances of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6 '-sinapoyl sucrose, costunolide and dehydrocostuslactone in methanol to obtain a 5-hydroxymethylfurfural reference substance solution with a concentration of 1.6583mg/ml, a ferulic acid reference substance solution with a concentration of 0.8613mg/ml, a glycyrrhizin reference substance solution with a concentration of 0.9491mg/ml, a spinosin reference substance solution with a concentration of 0.9271mg/ml, a 3,6' -sinapoyl sucrose reference substance solution with a concentration of 0.9259mg/ml, a costunolide reference substance solution with a concentration of 0.9401mg/ml and a dehydrocostuslactone single reference substance solution with a concentration of 0.9816 mg/ml.
3.2 Preparation of mixed control solution:
Precisely sucking single reference substance stock solutions of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6' -sinapiyl sucrose, costunolide and dehydrocostuslactone into 3-25 ml measuring flasks, and adding methanol to the measuring flask to obtain mixed reference substance solutions ① with the concentrations of 199.00 mug/ml, 103.36 mug/ml, 113.89 mug/ml, 111.26 mug/ml, 111.11 mug/ml, 112.81 mug/ml and 117.79 mug/ml respectively;
Precisely sucking ① ml of the mixed reference substance solution, placing in a 10ml volumetric flask, adding methanol to scale, shaking, and counting to obtain ②;
Precisely sucking ② ml of the mixed reference substance solution, placing in a 10ml volumetric flask, adding methanol to scale, shaking, and counting to obtain ③;
Precisely sucking ③ ml of the mixed reference substance solution, placing in a 10ml volumetric flask, adding methanol to scale, shaking, and counting to obtain ④;
Accurately sucking up the mixed reference substance solution ④ ml, placing the mixed reference substance solution ④ ml into a 2ml volumetric flask, adding methanol to the scale, shaking uniformly, and counting to obtain the mixed reference substance solution ⑤.
4. Confirmation of chromatographic conditions:
The acquisition wavelength is mainly examined. And recording the absorption spectrum of the sample solution in the range of 190-400 nm by adopting a diode array detector. The research shows that the chromatographic peak at 225nm is more, the baseline noise is low, the response is strong, and the separation degree is good. Thus, the detection wavelength was determined to be 225nm. Representative patterns are shown in FIG. 2.
5. Preparation of test solution:
the sample solution preparation method provided in this example: cutting large honeyed pills of Guipi pills, precisely weighing 9.0g, adding 4.5g of diatomite, grinding into fine powder, placing into a conical flask with a plug, adding 120ml of 80% methanol, weighing, heating and refluxing for 60 minutes, cooling, supplementing the lost weight with 80% methanol, filtering, precisely weighing 60ml of filtrate, concentrating to near dryness, adding 80% methanol for dissolution, shaking to 20ml for uniform volume, filtering, and taking the subsequent filtrate to obtain the sample solution.
In order to determine the optimal extraction conditions of the test sample, the present invention conducted the following extraction method studies.
5.1 Comparison of different extraction solvents
Taking 5 parts of spleen-invigorating pill, each 9.0g of diatomite and 4.5g of diatomite, grinding into fine powder, respectively preparing test sample solutions by adopting water, 20% methanol, 50% methanol, 80% methanol and 100% methanol respectively and performing ultrasonic treatment for 30 minutes, measuring the peak area of the test sample, and calculating the contents of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6' -sinapiyl sucrose, costunolide and dehydrocostuslactone, wherein the results are shown in Table 2, the different extraction solvents have larger influence on each component, the contents of each component to be tested are comprehensively analyzed, and the extraction of each component to be tested is relatively complete when 80% methanol is adopted for extraction, so that the extraction solvent is 80% methanol.
TABLE 2 extraction solvent
5.2 Comparison of different extraction methods
Taking 9.0g of spleen-invigorating pill, 4.5g of kieselguhr, grinding into fine powder, preparing 2 parts, using 100ml of 80% methanol as an extraction solvent, respectively adopting heating reflux and ultrasonic treatment (power is 250W, frequency is 40 kHz) for 30 minutes to prepare a sample solution, injecting the sample solution into a liquid chromatograph for measurement, calculating the contents of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6' -sinapiyl sucrose, costunolide and dehydrocostuslactone, and obtaining the results shown in Table 3 and FIG. 1, wherein the upper graph in FIG. 1 is a chromatogram obtained by detecting the sample solution obtained by a reflux extraction mode, and the lower graph is a chromatogram obtained by detecting the sample solution obtained by an ultrasonic extraction mode; it can be known that the content of each component to be measured is high by heating and reflux extraction, so the extraction mode is selected to be heating and reflux extraction.
TABLE 3 extraction modes
5.3 Comparison of different extraction times
9.0G of Guipi pills and 4.5g of diatomite are taken and ground into fine powder to prepare 4 parts, 100ml of 80% methanol is used as a solvent, and the mixture is respectively subjected to heating reflux treatment for 30 minutes, 45 minutes, 60 minutes and 90 minutes to prepare a sample solution, the peak area of the sample solution is measured, and the contents of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6' -sinapoyl sucrose, costunolide and dehydrocostuslactone are calculated. The results are shown in Table 4, and the content of each component to be measured is high when the heating reflux time is 60 minutes; and there was no significant difference from 90 minutes, the heating reflux time was chosen to be 60 minutes.
TABLE 4 extraction time
5.4 Comparison of different extraction volumes
9.0G of Guipi pills and 4.5g of diatomite are taken and ground into fine powder to prepare 4 parts, 80% methanol is used as an extraction solvent, 80ml, 100ml, 120ml and 150ml are respectively added to prepare sample solutions by heating and refluxing for 60 minutes, the sample solutions are injected into a high performance liquid chromatograph for measurement, the contents of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6' -sinapiyl sucrose, costunolide and dehydrocostuslactone are calculated, the results are shown in table 5, the contents of different extraction volumes have little influence on the contents of all components to be measured, and the final extraction volume is selected to be 120ml.
TABLE 5 extraction volume
5.5 Comparison of different concentration ratios
9.0G of Guipi pills and 4.5g of kieselguhr are taken and ground into fine powder to prepare 2 parts, 120ml of 80% methanol is used as an extraction solvent, the mixture is weighed, heated and refluxed for 60 minutes, cooled, the reduced weight is complemented by 80% methanol, filtered, the mixture is taken as a sample solution 1 (not concentrated), 60ml of filtrate is precisely measured, concentrated to be nearly dry, 80% methanol is added for dissolution, the volumes of the filtrate are respectively fixed to 20ml (concentrated solution 1) and 10ml (concentrated solution 2) for shaking up, filtering is carried out, the subsequent filtrate is taken to obtain a sample solution, and the sample solution is injected into a high performance liquid chromatograph for measurement, and the contents of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6' -sinapoyl sucrose, costunolide and dehydrocostunolide are calculated, and the concentration ratio is 6: unstable flocs appear at 1 and without concentration, the peak area of the chromatography peak of the spinosin is low, so the final concentration ratio is chosen to be 3:1.
TABLE 6 sample unconcentration and concentration of peak areas of the ingredients
TABLE 7 concentration ratio
5.6 Stability test
Sample solutions are taken and respectively injected once in 0h, 4h, 8h, 12h, 18h and 24h, mixed reference solution is adopted as a reference, the content of components to be detected in the samples is measured, RSD is calculated, the result is shown in table 8, the RSD of 5-hydroxymethylfurfural content is 0.94%, the RSD of ferulic acid content is 2.88%, the RSD of glycyrrhizin content is 2.52%, the RSD of spinosin content is 2.13%, the RSD of 3,6' -sinapoyl sucrose content is 1.99%, the RSD of costunolide content is 2.29%, and the RSD of dehydrocostuslactone content is 1.51%, and the sample solutions prepared in the embodiment are proved to have good stability and meet analysis requirements.
Table 8 stability
6. Methodological validation of HPLC detection conditions:
5 μl of each of the mixed reference solutions ①～⑤ μl was precisely aspirated, and injected into a high performance liquid chromatograph, and a standard curve was drawn with reference concentration (μg/mL) as abscissa and peak area as ordinate, and the results are shown in Table 9.
TABLE 9 Linear relationship and Linear Range of seven Components
| Component (A) | Regression equation | Correlation coefficient r | Linear range (μg/ml) |
| 5-Hydroxymethyl Furfural | y＝10.492x+3.4591 | 0.9999 | 12.44-199.00 |
| Ferulic acid | y＝15.938x－11.875 | 0.9998 | 6.46-103.36 |
| Liquiritigenin | y＝14.321x－8.3577 | 0.9998 | 7.12-113.89 |
| Spinosin | y＝10.184x－7.363 | 0.9998 | 6.96-111.26 |
| 3,6' Sinapiyl sucrose | y＝10.344x－5.9892 | 0.9998 | 6.94-111.11 |
| Costunolide | y＝9.1549x－3.4747 | 0.9998 | 7.05-112.81 |
| Dehydrocostuslactone | y＝6.3962x－4.2319 | 0.9998 | 7.36-117.79 |
6.1 Specificity verification
Pulverizing the rest 5 materials except radix Glycyrrhizae Preparata, radix Codonopsis, radix astragali Preparata, poria, arillus longan, fructus Jujubae, and Mel into fine powder, and making into negative sample without 5-hydroxymethylfurfural;
pulverizing the rest 10 materials except radix Angelicae sinensis into fine powder according to 1: adding refined honey according to the powder-honey ratio of 0.8-0.9 to prepare a sample without ferulic acid negative;
pulverizing the rest 10 materials except radix Glycyrrhizae Preparata into fine powder according to the following ratio 1: adding refined honey according to the powder-honey ratio of 0.8-0.9 to prepare a negative sample without liquiritin;
Pulverizing the rest 10 materials except parched semen Ziziphi Spinosae into fine powder according to the ratio of 1: adding refined honey into the mixture according to the powder-honey ratio of 0.8-0.9 to prepare a sample without spinosin negative;
Pulverizing the rest 10 materials except radix Polygalae into fine powder according to 1: adding refined honey into the mixture according to the powder-honey ratio of 0.8-0.9 to prepare a negative sample without 3,6' -sinapiyl sucrose;
Pulverizing the rest 10 medicinal materials except radix aucklandiae into fine powder according to the following ratio of 1: adding refined honey into the powder-honey ratio of 0.8-0.9 to prepare a negative sample which does not contain costunolide and dehydrocostuslactone;
Preparing a negative control solution from the negative sample according to a preparation method of a 2.1.3 sample solution; adopting a mixed reference substance solution as a reference; 80% methanol solvent was used as the blank solvent.
HPLC detection is carried out on the sample solution, the negative control solution, the mixed control solution and the blank solvent to obtain chromatograms, the result is shown in figure 4, and the negative control solution and the blank solvent have no color spectrum peak at the retention time of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6' -sinapiyl sucrose, costunolide and dehydrocostuslactone, so that the specificity of the HPLC detection method is proved to be good.
6.2 Repeatability test
6 Test solutions were prepared in parallel and tested by HPLC, using the mixed control solution as a control, and the results are shown in Table 10. The HPLC detection conditions of the application are proved to have better repeatability, and meet analysis requirements, because the RSD of the 5-hydroxymethylfurfural content is 0.98%, the RSD of the ferulic acid content is 2.45%, the RSD of the glycyrrhizin content is 2.31%, the RSD of the spinosin content is 1.91%, the RSD of the 3,6' -sinapiyl sucrose content is 1.55%, the RSD of the costunolide content is 2.98%, and the RSD of the dehydrocostuslactone content is 2.26%.
Table 10 repeatability test
6.3 Intermediate precision test
6 Parts of test sample solutions are prepared in parallel, different analysts use another high performance liquid chromatograph at different time to perform repeatability test, and the mixed reference sample solution is adopted for comparison, and the results are shown in table 11, wherein the different instruments measure that the RSD of the 5-hydroxymethylfurfural content, the RSD of the ferulic acid content, the RSD of the glycyrrhizin content, the RSD of the spinosin content, the RSD of the 3,6' -bissinapoyl sucrose content, the RSD of the costunolide content, the RSD of the dehydrocostuslactone content and the RSD of the dehydrocostuslactone content are 2.13%, 2.01% respectively, and the analysis requirements are met.
TABLE 11 intermediate precision test
6.4 Recovery test
Cutting a honeyed bolus sample of the Guipi bolus, precisely weighing 4.5g and 2.25g of diatomite, grinding the mixture into fine powder, placing the fine powder into a conical flask with a plug, adding 120ml of 80% methanol, weighing, heating and refluxing for 120 minutes, cooling, supplementing the lost weight with 80% methanol, shaking uniformly, filtering, precisely weighing 60ml of filtrate, concentrating to near dryness, adding 80% methanol for dissolution, shaking uniformly to 20ml of volume, filtering, and taking the subsequent filtrate to obtain the sample solution. The mixed reference solution was used as a control.
And respectively adding proper amounts of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosyl sucrose, 3,6' -sinapioyl sucrose, costunolide and dehydrocostuslactone reference substance into the sample solution, measuring the content of 7 components, calculating the recovery rate, and carrying out 6 groups of parallel experiments on each component.
The results are shown in Table 12-18,5, wherein the recovery rate of the hydroxymethylfurfural is 95.05-101.52%, the recovery rate of the ferulic acid is 97.29-104.95%, the recovery rate of the glycyrrhizin is 90.92-97.07%, the recovery rate of the spinosin is 95.37-98.79%, the recovery rate of the 3,6' -bissinapoyl sucrose is 95.33-100.08%, the recovery rate of the costunolide is 101.64-106.45%, and the recovery rate of the dehydrocostuslactone is 94.09-101.48%, which proves that the HPLC detection conditions of the invention meet the analysis requirements.
The recovery rate was calculated as follows:
Recovery (%) = [ (actual measurement value-amount of measured component contained in sample)/amount of measured component contained in control ] ×100%
TABLE 12 recovery of 5-hydroxymethylfurfural
TABLE 13 Ferulic acid recovery
TABLE 14 recovery of glycyrrhizin
TABLE 15 recovery of spinosin
TABLE 16 recovery of 3,6' -sinapiyl sucrose
Table 17 recovery of costunolide
TABLE 18 dehydrocostuslactone recovery
7. Calculation of relative correction factors and durability investigation
7.1 Determining the relative correction factor:
According to the standard curves and regression equations of seven components in Table 8, relative correction factors of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone are calculated by taking 3,6' -sinapiyl sucrose as an internal reference, wherein the calculation formula of the relative correction factors is shown as the formula (I):
Wherein s is 3,6' -sinapiyl sucrose, k is a component to be detected, and one of six components of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone is sequentially taken; w s is the concentration of 3,6 '-disjuncyl sucrose, W k is the concentration of the component to be detected, A s is the peak area of the 3,6' -disjuncyl sucrose, and A k is the peak area of the component to be detected; f s is the correction factor of 3,6' -sinapiyl sucrose and f k is the correction factor of the component to be tested.
Mixing reference substance solutions ①～⑤ with different concentrations, injecting into liquid chromatograph, detecting, and calculating relative correction factors of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone by using 3,6' -sinapiyl sucrose as internal reference substance, as shown in Table 19.
TABLE 19 relative correction factor
7.2 Effect of different instruments and columns on the relative correction factor
According to the detection conditions of the HPLC and the preparation method of the sample solution in the embodiment, the mixed reference substance solution prepared in the embodiment is used as a reference, different instruments and different chromatographic columns are respectively adopted to measure the relative correction factors, the result is shown in a table 20, the measured relative correction factor RSD value is between 1.00% and 2.31%, and the repeatability of the relative correction factors of the components to be detected between different instruments and different chromatographic columns is good.
TABLE 20 influence of different instruments and different chromatographic columns on the relative correction factors of the components to be tested
7.3 Effect of different flow rates on the relative correction factor
According to the preparation method of the sample solution in this embodiment, the mixed reference solution prepared in this embodiment is used as a control, the relative correction factors are measured by using different flow rates, the other detection conditions of HPLC are the same as those of the detection method provided in this embodiment, the result is shown in table 21, and the measured relative correction factor RSD value is between 0.03% and 1.12%, which indicates that the reproducibility of the relative correction factors of each component to be measured between different flow rates is good.
TABLE 21 influence of different flow rates on the relative correction factors of the components to be tested
7.4 Effect of different column temperatures on the relative correction factor
According to the preparation method of the sample solution in this embodiment, the mixed reference solution prepared in this embodiment is used as a control, different column temperatures are used to measure the relative correction factors, the other detection conditions of HPLC are the same as those in this embodiment, the result is shown in table 22, and the measured relative correction factor RSD value is between 0.07% and 0.98%, which indicates that the reproducibility of the relative correction factors of each component to be measured between different column temperatures is good.
TABLE 22 influence of different column temperatures on the relative correction factors for the components to be tested
8. Positioning chromatographic peaks of components to be detected:
The relative retention time (r) and retention time difference (DeltatR) of the 5-hydroxymethylfurfural control solution, the ferulic acid control solution, the glycyrrhizin control solution, the spinosin control solution, the costunolide control solution, the dehydrocostunolide single control solution and the internal reference 3,6' -sinapiyl sucrose prepared in the embodiment are calculated, the durability between different instruments and different chromatographic columns is examined, the results are shown in tables 23-24, the relative retention time fluctuation is small, and finally + -5% of the average value of the relative retention time of each component to be detected in different instruments and different chromatographic columns is selected as a peak positioning basis.
TABLE 23 relative retention times of the various components to be tested in different instruments and different chromatographic columns
TABLE 24 retention time differences for various components to be tested in different instruments and different chromatographic columns
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9. Determination of the content of the sample solution components:
Taking a test solution, preparing a 3,6' -dissinapiyl sucrose reference substance solution, and performing high performance liquid chromatography analysis, wherein the concentration of the 3,6' -dissinapiyl sucrose reference substance solution is close to that of the 3,6' -dissinapiyl sucrose in the test solution, for example, 70 mug/ml is adopted. And (3) calculating the contents of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone according to the relative correction factors by taking the concentration and peak area of the 3,6' -sinapiyl sucrose reference substance as a reference substance.
10. Comparing the one-test multi-evaluation method with the external standard method
3 Parts of sample solutions are prepared in parallel and are named spleen-invigorating pill-1, spleen-invigorating pill-2 and spleen-invigorating pill-3 respectively.
Multiple evaluation method (QAMS): preparing a3, 6 '-dissinapiyl sucrose reference substance solution with the concentration of 70 mug/ml, and calculating the contents of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide, dehydrocostuslactone and 3,6' -dissinapiyl sucrose in the sample solution according to the average value of relative correction factors.
External Standard Method (ESM): and (3) taking a mixed reference substance solution with the concentration similar to that of seven components in the sample solution as a reference, taking 10 mu l of each of the mixed reference substance solution and the sample solution, and calculating the content of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide, dehydrocostuslactone and 3,6' -sinapiyl sucrose in the sample solution.
The contents of seven components in the sample solution detected by the two methods are shown in a table 24, the contents calculated by the external standard method and the one-measurement-multiple-evaluation method are compared by t test, P is far more than 0.05, no obvious difference exists between the contents detected by the two methods, and the relative deviation between the two groups of contents is less than 3%, so that the one-measurement-multiple-evaluation method established by the application has good accuracy and can be used for measuring the contents of the spleen-invigorating pills. RE in Table 25 represents the relative deviation.
Table 25 one-measurement multiple evaluation method and external standard method for measuring contents of seven components
The above description is only an example of the present application, and the scope of the present application is not limited to the specific examples, but is defined by the claims of the present application. Various modifications and variations of the present application will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.
Claims (6)
1. The method for measuring the contents of seven components in the spleen-invigorating pill by adopting a multi-evaluation method is characterized by comprising the following steps of:
(1) Preparation of mixed control solution and test solution:
Taking methanol as a solvent to respectively prepare single reference substance solutions of seven reference substances of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosyl sucrose, 3,6' -sinapioyl sucrose, costunolide and dehydrocostuslactone, and mixing a proper amount of the seven single reference substance solutions to prepare a series of mixed reference substance solutions with different concentrations;
Cutting GUIPI pill, adding diatomite, grinding, adding 80% methanol, refluxing, filtering, collecting filtrate, concentrating to near dryness, dissolving with 80% methanol, transferring into a measuring flask, metering volume, shaking, filtering, and collecting filtrate to obtain sample solution;
(2) Determining chromatographic conditions:
Chromatographic column: agilent Poroshell 120EC-C18, specification 4.6X105 mm,4 μm; the column temperature is 33-37 ℃; the detection wavelength is 225nm; the flow rate is 1ml/min; acetonitrile is taken as a mobile phase A, and a 0.1% phosphoric acid solution is taken as a mobile phase B for gradient elution;
The gradient elution conditions were:
0～15min，A：3％～8％，B：97％～92％；
15～32min，A：8％～12.5％，B：92％～87.5％；
32～45min，A：12.5％，B：87.5％；
45～50min，A：12.5％～15％，B：87.5％～85％；
50～70min，A：15％～21％，B：85％～79％；
70～120min，A：21％～58％，B：79％～42％；
(3) Determining a relative correction factor:
Taking mixed reference substance solutions with different concentrations, respectively injecting samples for high performance liquid chromatography analysis, selecting 3,6' -sinapiyl sucrose as an internal reference substance, and respectively calculating relative correction factors of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone;
the calculation formula of the relative correction factor is as follows:
wherein s is 3,6' -sinapiyl sucrose, k is a component to be detected, and one of six components of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone is sequentially taken; w s is the concentration of 3,6 '-disjuncyl sucrose, W k is the concentration of the component to be detected, A s is the peak area of the 3,6' -disjuncyl sucrose, and A k is the peak area of the component to be detected; f s is the correction factor of 3,6' -sinapiyl sucrose, and f k is the correction factor of the component to be tested;
The chromatographic peak relative retention time of 5-hydroxymethylfurfural was 0.09, the relative correction factor was 0.95;
the chromatographic peak relative retention time of ferulic acid was 0.51, and the relative correction factor was 0.66;
The chromatographic peak relative retention time of the liquiritin is 0.62, and the relative correction factor is 0.72;
the chromatographic peak relative retention time of spinosin was 0.74 and the relative correction factor was 1.03;
The chromatographic peak relative retention time of costunolide is 1.76, and the relative correction factor is 1.12;
The chromatographic peak relative retention time of dehydrocostuslactone is 1.73, and the relative correction factor is 1.62;
The chromatographic peak relative retention time of 3,6' -sinapiyl sucrose was 1.00, the relative correction factor was 1;
(4) Determination of the component content of the sample solution:
Preparing a sample solution, preparing a 3,6 '-dissinapiyl sucrose reference substance solution, performing high performance liquid chromatography analysis, and calculating the content of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, costunolide and dehydrocostuslactone according to relative correction factors by taking the concentration and peak area of the 3,6' -dissinapiyl sucrose reference substance as a reference.
2. The method for determining contents of seven ingredients in the spleen-invigorating pill by adopting a multi-evaluation method according to claim 1, wherein the preparation method of the sample solution is specifically as follows:
cutting spleen-invigorating pill, precisely weighing 9-12g, adding diatomite 4.5-6g, grinding into fine powder, adding 80% methanol 120ml, weighing, heating and refluxing for 60min, cooling, supplementing the reduced weight with 80% methanol, filtering, precisely weighing 60ml filtrate, concentrating to near dryness, adding 80% methanol for dissolving, transferring to 20ml measuring flask, adding methanol to volume to scale, shaking, filtering, and collecting the filtrate to obtain the sample solution.
3. The method for determining the contents of seven ingredients in the spleen-invigorating pill by adopting a one-test-multiple-evaluation method according to claim 1, wherein the preparation method of the 3,6' -sinapiyl sucrose reference substance solution is as follows:
3,6 '-sinapiyl sucrose was dissolved in methanol to give a3, 6' -sinapiyl sucrose control solution at a concentration of 0.9259 mg/ml.
4. The method for measuring the contents of seven components in the spleen-invigorating pill by adopting a multi-evaluation method according to claim 1, wherein the concentrations of the single reference solutions of 5-hydroxymethylfurfural, ferulic acid, glycyrrhizin, spinosin, 3,6' -dissinapoyl sucrose, costunolide and dehydrocostuslactone are respectively as follows: 1.6583mg/ml, 0.8613mg/ml, 0.9491mg/ml, 0.9271mg/ml, 0.9259mg/ml, 0.9401mg/ml, 0.9816mg/ml.
5. The method for measuring the contents of seven ingredients in the spleen-invigorating pill by a one-test-multiple-evaluation method according to claim 1, wherein the theoretical plate number is not less than 3000 calculated as 3,6' -sinapiyl sucrose in chromatographic conditions.
6. The method for measuring the contents of seven components in the spleen-invigorating pill by adopting a one-measurement-multiple-evaluation method according to claim 1, wherein the spleen-invigorating pill is in the form of a water-honeyed pill, a big honeyed pill or a small honeyed pill.
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| CN101095745A (en) * | 2007-08-13 | 2008-01-02 | 浙江爱生药业有限公司 | Particles for invigorating the spleen, oral liquid and condensed pills and method for preparing the same and the quality control method |
| JP2015140305A (en) * | 2014-01-28 | 2015-08-03 | 国立研究開発法人産業技術総合研究所 | Biological clock adjusting agent |
| CN114354831A (en) * | 2021-12-01 | 2022-04-15 | 四平市食品药品检验所 | Quality detection method of ginseng spleen-invigorating pills |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101095745A (en) * | 2007-08-13 | 2008-01-02 | 浙江爱生药业有限公司 | Particles for invigorating the spleen, oral liquid and condensed pills and method for preparing the same and the quality control method |
| JP2015140305A (en) * | 2014-01-28 | 2015-08-03 | 国立研究開発法人産業技術総合研究所 | Biological clock adjusting agent |
| CN114354831A (en) * | 2021-12-01 | 2022-04-15 | 四平市食品药品检验所 | Quality detection method of ginseng spleen-invigorating pills |
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