CN108205022B - Method for measuring contents of ginsenoside Rg1, re and Rb1 in Yihe spring preparation - Google Patents
Method for measuring contents of ginsenoside Rg1, re and Rb1 in Yihe spring preparation Download PDFInfo
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- CN108205022B CN108205022B CN201611180013.7A CN201611180013A CN108205022B CN 108205022 B CN108205022 B CN 108205022B CN 201611180013 A CN201611180013 A CN 201611180013A CN 108205022 B CN108205022 B CN 108205022B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 63
- YURJSTAIMNSZAE-HHNZYBFYSA-N ginsenoside Rg1 Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O YURJSTAIMNSZAE-HHNZYBFYSA-N 0.000 title claims abstract description 56
- GZYPWOGIYAIIPV-JBDTYSNRSA-N ginsenoside Rb1 Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)O[C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GZYPWOGIYAIIPV-JBDTYSNRSA-N 0.000 title claims abstract description 43
- PWAOOJDMFUQOKB-WCZZMFLVSA-N ginsenoside Re Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@](C)(CCC=C(C)C)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O PWAOOJDMFUQOKB-WCZZMFLVSA-N 0.000 title claims abstract description 43
- AOGZLQUEBLOQCI-UHFFFAOYSA-N ginsenoside-Re Natural products CC1OC(OCC2OC(OC3CC4(C)C(CC(O)C5C(CCC45C)C(C)(CCC=C(C)C)OC6OC(CO)C(O)C(O)C6O)C7(C)CCC(O)C(C)(C)C37)C(O)C(O)C2O)C(O)C(O)C1O AOGZLQUEBLOQCI-UHFFFAOYSA-N 0.000 title claims abstract description 43
- YURJSTAIMNSZAE-UHFFFAOYSA-N UNPD89172 Natural products C1CC(C2(CC(C3C(C)(C)C(O)CCC3(C)C2CC2O)OC3C(C(O)C(O)C(CO)O3)O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O YURJSTAIMNSZAE-UHFFFAOYSA-N 0.000 title claims abstract description 37
- CBEHEBUBNAGGKC-UHFFFAOYSA-N ginsenoside Rg1 Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5CC(OC6OC(CO)C(O)C(O)C6O)C34C)C CBEHEBUBNAGGKC-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 32
- UFNDONGOJKNAES-UHFFFAOYSA-N Ginsenoside Rb1 Natural products CC(=CCCC(C)(OC1OC(COC2OC(CO)C(O)C(O)C2O)C(O)C(O)C1O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CC(O)C45C)C UFNDONGOJKNAES-UHFFFAOYSA-N 0.000 title claims abstract description 30
- TXEWRVNOAJOINC-UHFFFAOYSA-N ginsenoside Rb2 Natural products CC(=CCCC(OC1OC(COC2OCC(O)C(O)C2O)C(O)C(O)C1O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CCC45C)C TXEWRVNOAJOINC-UHFFFAOYSA-N 0.000 title claims abstract description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 41
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000013558 reference substance Substances 0.000 claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 36
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 24
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 239000012488 sample solution Substances 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 14
- 238000005303 weighing Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 238000009210 therapy by ultrasound Methods 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- 238000001704 evaporation Methods 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 238000005259 measurement Methods 0.000 claims description 7
- 239000012088 reference solution Substances 0.000 claims description 7
- 239000003643 water by type Substances 0.000 claims description 7
- 239000000945 filler Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 6
- 229920006395 saturated elastomer Polymers 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 239000003513 alkali Substances 0.000 claims description 4
- 239000002021 butanolic extract Substances 0.000 claims description 4
- 239000012467 final product Substances 0.000 claims description 4
- 238000007873 sieving Methods 0.000 claims description 4
- 239000012085 test solution Substances 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims 1
- 229940089161 ginsenoside Drugs 0.000 abstract description 9
- 229930182494 ginsenoside Natural products 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000010829 isocratic elution Methods 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 20
- 238000012360 testing method Methods 0.000 description 15
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 241000208340 Araliaceae Species 0.000 description 9
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 9
- 235000003140 Panax quinquefolius Nutrition 0.000 description 9
- 239000003814 drug Substances 0.000 description 9
- 235000008434 ginseng Nutrition 0.000 description 9
- 238000000605 extraction Methods 0.000 description 6
- 239000013642 negative control Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 4
- 238000013112 stability test Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229940126680 traditional chinese medicines Drugs 0.000 description 2
- 241000427159 Achyranthes Species 0.000 description 1
- 235000018645 Allium odorum Nutrition 0.000 description 1
- 244000003377 Allium tuberosum Species 0.000 description 1
- 235000005338 Allium tuberosum Nutrition 0.000 description 1
- 241000212948 Cnidium Species 0.000 description 1
- 241000804384 Cynomorium songaricum Species 0.000 description 1
- 241000721047 Danaus plexippus Species 0.000 description 1
- 241000893536 Epimedium Species 0.000 description 1
- 241000405414 Rehmannia Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000003056 antler Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000018905 epimedium Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- 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)
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Abstract
The invention provides a method for measuring the content of ginsenoside Rg1, re and Rb1 in Yihe Chun preparation, which comprises the steps of taking ginsenoside Rg1, re and Rb1 reference substances as reference substances, acetonitrile as mobile phase A and water as mobile phase B, and measuring the content of ginsenoside Rg1, re and Rb1 in Yihe Chun preparation by adopting a high performance liquid chromatography combining gradient and isocratic elution. The method has good separation effect, high detection sensitivity and good stability, and can accurately measure ginsenoside Rg in Yihe spring preparation 1 ，Re，Rb 1 The quality of the Yihe spring preparation can be comprehensively and objectively embodied and evaluated, and the preparation has the characteristics of high precision, good stability, good repeatability, good linearity, high recovery rate and the like, can effectively improve the quality control standard of the Yihe spring preparation, and ensures the clinical curative effect of the Yihe spring preparation.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and relates to a method for measuring contents of ginsenoside Rg1, re and Rb1 in Yihe spring preparation by using a high performance liquid chromatography.
Background
The Yihe Chun preparation is prepared with 15 kinds of Chinese medicinal materials, including ginseng, achyranthes root, dog's kidney, prepared rhizome of rehmannia, pilose antler, epimedium, chinese chive seed, cnidium fruit, cynomorium songaricum, etc. and is used mainly in invigorating kidney and strengthening Yang, and has obvious curative effect, less side effect and convenient use. Wherein the personGinseng is a monarch drug, and its effective component is ginsenoside Rg 1 ，Re，Rb 1 In order to ensure the quality of the Yihe spring preparation, ginsenoside Rg in the preparation is needed 1 ，Re，Rb 1 The content is measured to ensure that the content of the effective components is stable and uniform.
High Performance Liquid Chromatography (HPLC) has the advantages of high sensitivity, high accuracy and the like, and has become one of important means for controlling the quality of traditional Chinese medicines, the basic modes of the HPLC are isocratic and gradient elution, and the components can be well separated by combining the two modes. In the prior art, high performance liquid chromatography is often adopted to measure ginsenoside Rg in the ginseng-containing traditional Chinese medicine preparation 1 ，Re，Rb 1 For example, patent CN102435697A, CN104807905A discloses the detection of ginsenoside Rg in a traditional Chinese medicine preparation by adopting an HPLC isocratic and gradient combination mode 1 ，Re，Rb 1 Content detection of (2). However, as the Yihe spring preparation consists of 15 traditional Chinese medicines, the components are more complex (the sample pretreatment is extremely important) than the components of other traditional Chinese medicine preparations; the combination mode of isocratic and gradient elution is difficult to count, the separation mechanism of gradient elution is complex, and the experimental scheme disclosed in the prior literature cannot meet the requirement of ginsenoside Rg in the Yihe spring preparation 1 ，Re，Rb 1 And (3) separation and content detection.
The invention mainly researches pretreatment and HPLC analysis conditions of the Yihe spring preparation and develops an analysis method capable of completely separating ginsenoside Rg1, re and Rb1 in the Yihe spring preparation.
Disclosure of Invention
The invention aims to: the invention aims to provide a method for detecting the content of related substances in a Yichun preparation,
the technical scheme is as follows: the invention provides a method for measuring the content of ginsenoside Rg1, re and Rb1 in Yihe Chun preparation, which comprises the steps of taking ginsenoside Rg1, re and Rb1 reference substances as reference substances, acetonitrile as mobile phase A and water as mobile phase B, and measuring the content of ginsenoside Rg1, re and Rb1 in Yihe Chun preparation by adopting a high performance liquid chromatography combining gradient and isocratic elution.
Preferably, the method for measuring the content of ginsenoside Rg1, re and Rb1 in the Yihe spring preparation comprises the following steps:
(1) Preparing a reference substance: precisely weighing ginsenoside Rg 1 Reference substance, ginsenoside Re reference substance and ginsenoside Rb 1 Adding methanol into the reference substance to dissolve to obtain mixed solution containing Rg1, re and Rb1 0.05mg, 0.25mg and 0.60mg respectively, shaking uniformly to obtain the final product;
(2) Sample solution preparation: precisely weighing YICHU preparation, placing in conical flask, adding alcohol solvent, performing ultrasonic treatment, centrifuging, collecting supernatant, adding alcohol solvent into residue, performing ultrasonic treatment, centrifuging, and mixing the supernatants; concentrating the supernatant under reduced pressure; dissolving the residue in water, and extracting with diethyl ether; removing diethyl ether layer, adding water saturated n-butanol into water layer, and extracting; washing n-butanol layer with sodium hydroxide aqueous solution and n-butanol saturated water sequentially, evaporating n-butanol layer, dissolving residue in methanol, fixing volume, and shaking to obtain the final product;
(3) Content determination of ginsenoside Rg1, re and Rb 1: precisely sucking 10 μl of the sample solution and the reference solution, respectively, and injecting into high performance liquid chromatograph for measurement; the chromatographic conditions are as follows:
filler: octadecylsilane chemically bonded silica gel;
chromatographic column: waters Symmetry C18 250mm 4.6mm,5um;
acetonitrile is a mobile phase A, and water is a mobile phase B;
the detection wavelength is 203nm, the column temperature is 30 ℃, and the flow rate is 1.0ml/min;
the theoretical plate number is 6000 or more, calculated by ginsenoside Rg 1;
elution procedure:
| time (min) | Mobile phase a (acetonitrile) | Mobile phase B (water) |
| 0-35 | 20％ | 80％ |
| 35-55 | 20％—29％ | 80％—71％ |
| 55-70 | 29％ | 71％ |
| 70-100 | 29％—40％ | 71％—60％ |
| 100-110 | 40％—19％ | 60％—81％ |
| 110 | 19％ | 81％ |
In the step (2), the alcohol solvent is methanol.
Wherein, in the step (2), the centrifugal speed is 4000-6000r/min and the centrifugal time is 5-15 minutes.
Wherein, in the step (2), the concentration of the sodium hydroxide aqueous solution is 0.2% -0.6%.
Wherein, in the step (2), the ultrasonic time is 0.5-2h; the reduced pressure concentration temperature is 40-60 ℃; the evaporating temperature is 95-105 ℃.
Wherein the Yihe spring preparation comprises granules, tablets, capsules and oral liquid.
The beneficial effects are that: the book is provided withThe method has good separation effect, high detection sensitivity and good stability, and can accurately measure the ginsenoside Rg in the Yihe spring preparation 1 ，Re，Rb 1 The quality of the Yihe spring preparation can be comprehensively and objectively embodied and evaluated, and the preparation has the characteristics of high precision, good stability, good repeatability, good linearity, high recovery rate and the like, can effectively improve the quality control standard of the Yihe spring preparation, and ensures the clinical curative effect of the Yihe spring preparation.
Drawings
FIG. 1 is a standard curve of ginsenoside Rg 1;
FIG. 2 is a ginsenoside Re standard curve;
fig. 3 is a ginsenoside Rb1 standard curve.
Detailed Description
Reference substance and sample source:
ginsenoside Rg 1 Control: lot number of Chinese food and drug inspection institute: 110703-201529
Ginsenoside Re reference: lot number of Chinese food and drug inspection institute: 110754-201525
Ginsenoside Rb 1 Control: lot number of Chinese food and drug inspection institute: 110704-201424
Formulation for treating Yihe spring: the Jiangsu Su Chinese medicine group Co., ltd, commercially available
The preparation method of the ginseng negative control sample comprises the following steps: the preparation method is operated according to the preparation method of the sample solution, and the ginseng negative control solution (the raw material is Yihe spring preparation which does not comprise ginseng, and the preparation method of the preparation is similar to that of the Yihe spring preparation).
Example 1
(1) Preparation of control solution
Precisely weighing ginsenoside Rg 1 Reference substance, ginsenoside Re reference substance and ginsenoside Rb 1 Adding methanol to dissolve the reference substance to obtain mixed solution containing Rg1, re and Rb1 0.05mg, 0.25mg and 0.60mg, respectively, and shaking.
(2) Preparation of test solution
Precisely weighing 10g of crushed preparation of the first and second hand and spring after sieving with No. four sieve, adding 150ml of methanol into a conical flask, performing ultrasonic treatment for 1 hour, taking out, centrifuging at 4000r/min for 8 minutes, taking out supernatant, adding 150ml of methanol into residue, performing ultrasonic treatment for 40 minutes, centrifuging at 4000r/min for 8 minutes, and combining the two supernatants. The solution was concentrated to dryness under reduced pressure in a 50 ℃ water bath. 40ml of water was added to dissolve the residue and transferred completely to a separating funnel, extraction was performed once with 50ml of diethyl ether, the diethyl ether solution was discarded, extraction was performed 4 times with 40ml of water-saturated n-butanol each time, and the n-butanol extracts were combined. Washing with 0.2% sodium hydroxide solution for 2 times (40 ml each time), discarding alkali liquor, washing with 50ml water saturated with n-butanol for 1 time, discarding water layer, evaporating n-butanol solution in 100deg.C water bath, dissolving residue in methanol, fixing volume to 10ml, and shaking.
(3) Content determination of ginsenoside Rg1, re and Rb 1: precisely sucking 10 μl of the sample solution and the reference solution, respectively, and injecting into high performance liquid chromatograph for measurement;
instrument: the Agilent 1260, high performance liquid chromatograph system includes quaternary pump, column temperature box, ultraviolet detector, chemical chromatography data workstation, etc.;
the chromatographic conditions are as follows:
filler: octadecylsilane chemically bonded silica gel;
chromatographic column: waters Symmetry C18 250mm 4.6mm,5um;
acetonitrile is a mobile phase A, and water is a mobile phase B;
the detection wavelength is 203nm, the column temperature is 30 ℃, and the flow rate is 1.0ml/min;
the theoretical plate number is 6000 or more, calculated by ginsenoside Rg 1;
elution procedure:
| time (min) | Mobile phase a (acetonitrile) | Mobile phase B (water) |
| 0-35 | 20％ | 80％ |
| 35-55 | 20％—29％ | 80％—71％ |
| 55-70 | 29％ | 71％ |
| 70-100 | 29％—40％ | 71％—60％ |
| 100-110 | 40％—19％ | 60％—81％ |
| 110 | 19％ | 81％ |
Example 2
(1) Specificity test:
a ginseng negative control solution was prepared by the method for preparing a sample solution in example 1.
According to the content determination method, ginsenoside Rg1, re, rb1 reference substance solution, test sample solution and ginseng negative reference solution are respectively injected.
The results show that: the ginsenoside Rg1, re and Rb1 are well separated from other peaks, and the ginseng negative control liquid has no interference at corresponding positions. The results are shown in Table 1.
TABLE 1 results of specific experiments
Note that: group 1 (ginseng negative control group) had no peak at the corresponding position.
(2) Linear relation investigation:
precisely weighing ginsenoside Rg 1 Reference substance, ginsenoside Re reference substance and ginsenoside Rb 1 Adding methanol into the reference substance to dissolve to obtain mixed reference substance solution containing Rg1, re and Rb1 0.05mg, 0.25mg and 0.60mg respectively; the sample was precisely measured and injected into a liquid chromatograph at 7.5, 10, 12.5, 15, 17.5 and 20. Mu.l, and the chromatogram was recorded under the same conditions as in example 1.
Linear regression was performed with the amount of sample introduced (X: μg) as the abscissa and the peak area (Y) as the ordinate.
Ginsenoside Rg 1 Regression equation: y=18.71x+9.832 (r=0.998); the results show that: in the sample injection amount range of 0.459-1.224 mug, the linear relation between the peak area and the sample injection amount of the sample is good.
Regression equation of ginsenoside Re: y=57.20x+39.30 (r=0.999); the results show that: in the sample injection amount range of 1.540-4.108 mug, the linear relation between the peak area and the sample injection amount of the sample is good.
Ginsenoside Rb 1 Regression equation: y=131.3x-18.24 (r=0.999); the results show that: in the sample injection amount range of 4.126-11.004 mug, the linear relation between the peak area and the sample injection amount of the sample is good.
The results are shown in Table 2 and FIGS. 1, 2 and 3.
TABLE 2 Linear relation experimental results of ginsenoside Rg1, re, rb1
(3) Precision test
Taking prepared sample solution, continuously sampling for 6 times, and performing chromatographic conditions in the same way as in example 1; recording the chromatogram, and calculating the RSD value, wherein the result shows that the method has good sample injection precision and meets the test requirements, and the sample injection precision is shown in tables 3-5.
TABLE 3 ginsenoside Rg 1 Sample injection precision test result
TABLE 4 results of ginsenoside Re sample injection precision test
TABLE 5 ginsenoside Rb 1 Sample injection precision test result
(4) Stability test:
taking prepared sample solutions, precisely measuring 10 μl in 0, 2, 4, 6, 8, 10 and 12 hours, feeding according to the method, recording chromatogram under the same chromatographic conditions as in example 1, and calculating RSD value with relative content of 100% in 0 hours. The results show that the sample solution is basically stable within 12 hours, as shown in tables 6-8.
TABLE 6 sample solution ginsenoside Rg 1 Stability test results
TABLE 7 test results of ginsenoside Re stability test of sample solution
TABLE 8 sample solution ginsenoside Rb 1 Stability test results
Repeatability:
the prepared sample solution was measured (total 6 parts) in the same manner as in example 1, and the RSD% value was calculated. The results show that the method has good repeatability test and meets the test requirements, and the results are shown in tables 9-11.
TABLE 9 ginsenoside Rg 1 Results of the repeatability test
TABLE 10 results of ginsenoside Re repeatability test
TABLE 11 results of ginsenoside Rb1 repeatability test
(5) Sample adding and recycling:
precisely weighing 10g of Yihe spring preparation, placing into a conical flask, adding 9 parts of 1 st, 2 nd and 3 rd parts of ginsenoside Rg into the mixture respectively 1 1.814mg of ginsenoside Re, 5.184mg of ginsenoside Rb 1 Mixing the reference substances; adding 0.473mg ginsenoside Rg into parts 4, 5 and 6 respectively 1 2.277mg of ginsenoside Re and 6.429mg of ginsenoside Rb 1 Mixing the reference substances; adding 0.567mg ginsenoside Rg into parts 6, 7 and 8 respectively 1 2.733mg ginsenoside Re, 7.714mg ginsenoside Rb 1 Mixing the reference substances, shaking uniformly, operating according to the preparation method of the sample solution in the embodiment 1, measuring the contents of ginsenoside Rg1, re and Rb1 according to the chromatographic conditions under the content measurement items, and calculating the recovery rate according to the addition amount and the measured amount, wherein the result shows that the recovery rate is between 90% and 110%, and the accuracy of the method is good and meets the test requirements, as shown in Table 12.
TABLE 12 sample recovery test results
Example 3
(1) Preparation of control solution
Precisely weighing ginsenoside Rg 1 Reference substance, ginsenoside Re reference substance and ginsenoside Rb 1 Adding methanol to dissolve the reference substance to obtain mixed solution containing Rg1, re and Rb1 0.05mg, 0.25mg and 0.60mg, respectively, and shaking.
(2) Preparation of test solution
Precisely weighing 10g of crushed preparation of the first and second hand and spring after sieving with No. four sieve, adding 150ml of methanol into a conical flask, performing ultrasonic treatment for 1 hour, taking out, centrifuging at a rotation speed of 5000r/min for 15 minutes, taking out supernatant, adding 150ml of methanol into residue, performing ultrasonic treatment for 30 minutes, centrifuging at a rotation speed of 5000r/min for 15 minutes, and combining the two supernatants. The solution was concentrated to dryness under reduced pressure in a 40 ℃ water bath. 40ml of water was added to dissolve the residue and transferred completely to a separating funnel, extraction was performed once with 50ml of diethyl ether, the diethyl ether solution was discarded, extraction was performed 4 times with 40ml of water-saturated n-butanol each time, and the n-butanol extracts were combined. Washing with 0.4% sodium hydroxide solution for 2 times (40 ml each time), discarding alkali liquor, washing with 50ml water saturated with n-butanol for 1 time, discarding water layer, evaporating n-butanol solution in 100deg.C water bath, dissolving residue in methanol, fixing volume to 10ml, and shaking.
(3) Content determination of ginsenoside Rg1, re and Rb 1: precisely sucking 10 μl of the sample solution and the reference solution, respectively, and injecting into high performance liquid chromatograph for measurement;
instrument: the Agilent 1260, high performance liquid chromatograph system includes quaternary pump, column temperature box, ultraviolet detector, chemical chromatography data workstation, etc.;
the chromatographic conditions are as follows:
filler: octadecylsilane chemically bonded silica gel;
chromatographic column: waters Symmetry C18 250mm 4.6mm,5um;
acetonitrile is a mobile phase A, and water is a mobile phase B;
the detection wavelength is 203nm, the column temperature is 30 ℃, and the flow rate is 1.0ml/min;
the theoretical plate number is 6000 or more, calculated by ginsenoside Rg 1;
elution procedure:
| time (min) | Mobile phase a (acetonitrile) | Mobile phase B (water) |
| 0-35 | 20％ | 80％ |
| 35-55 | 20％—29％ | 80％—71％ |
| 55-70 | 29％ | 71％ |
| 70-100 | 29％—40％ | 71％—60％ |
| 100-110 | 40％—19％ | 60％—81％ |
| 110 | 19％ | 81％ |
Example 4
(1) Preparation of control solution
Precisely weighing ginsenoside Rg 1 Reference substance, ginsenoside Re reference substance and ginsenoside Rb 1 Adding methanol to dissolve the reference substance to obtain mixed solution containing Rg1, re and Rb1 0.05mg, 0.25mg and 0.60mg, respectively, and shaking.
(2) Preparation of test solution
Precisely weighing 10g of crushed preparation of the first and second hand and spring after sieving with No. four sieve, adding 150ml of methanol into a conical flask, performing ultrasonic treatment for 1 hour, taking out, centrifuging at 6000r/min for 5 minutes, taking out supernatant, adding 150ml of methanol into residue, performing ultrasonic treatment for 120 minutes, centrifuging at 6000r/min for 5 minutes, and combining the two supernatants. The solution was concentrated to dryness under reduced pressure in a 60 ℃ water bath. 40ml of water was added to dissolve the residue and transferred completely to a separating funnel, extraction was performed once with 50ml of diethyl ether, the diethyl ether solution was discarded, extraction was performed 4 times with 40ml of water-saturated n-butanol each time, and the n-butanol extracts were combined. Washing with 0.6% sodium hydroxide solution for 2 times (40 ml each time), discarding alkali liquor, washing with 50ml water saturated with n-butanol for 1 time, discarding water layer, evaporating n-butanol solution in 100deg.C water bath, dissolving residue in methanol, fixing volume to 10ml, and shaking.
(3) Content determination of ginsenoside Rg1, re and Rb 1: precisely sucking 10 μl of the sample solution and the reference solution, respectively, and injecting into high performance liquid chromatograph for measurement;
instrument: the Agilent 1260, high performance liquid chromatograph system includes quaternary pump, column temperature box, ultraviolet detector, chemical chromatography data workstation, etc.;
the chromatographic conditions are as follows:
filler: octadecylsilane chemically bonded silica gel;
chromatographic column: waters Symmetry C18 250mm 4.6mm,5um;
acetonitrile is a mobile phase A, and water is a mobile phase B;
the detection wavelength is 203nm, the column temperature is 30 ℃, and the flow rate is 1.0ml/min;
the theoretical plate number is 6000 or more, calculated by ginsenoside Rg 1;
elution procedure:
| time (min) | Mobile phase a (acetonitrile) | Mobile phase B (water) |
| 0-35 | 20％ | 80％ |
| 35-55 | 20％—29％ | 80％—71％ |
| 55-70 | 29％ | 71％ |
| 70-100 | 29％—40％ | 71％—60％ |
| 100-110 | 40％—19％ | 60％—81％ |
| 110 | 19％ | 81％ |
Claims (7)
1. The method for measuring the contents of ginsenoside Rg1, re and Rb1 in the Yihe spring preparation is characterized in that: measuring the contents of ginsenoside Rg1, re and Rb1 in the Yihe spring preparation by high performance liquid chromatography with ginsenoside Rg1, re and Rb1 as reference substances, acetonitrile as mobile phase A and water as mobile phase B;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, a chromatographic column is Waters Symmetry C, 250mm multiplied by 4.6mm,5um, the detection wavelength is 203nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min, and the elution procedure is as follows:
Wherein the sample solution is prepared by the following method: precisely weighing YICHU preparation, placing in conical flask, adding methanol solvent, performing ultrasonic treatment, centrifuging, collecting supernatant, adding methanol solvent into residue, performing ultrasonic treatment, centrifuging, and mixing the supernatants; concentrating the supernatant under reduced pressure; dissolving the residue in water, and extracting with diethyl ether; removing diethyl ether layer, adding water saturated n-butanol into water layer, and extracting for 4 times; washing n-butanol layer with 0.2% -0.6% sodium hydroxide aqueous solution for 2 times and n-butanol saturated water for 1 time, evaporating n-butanol layer, dissolving residue with methanol, fixing volume, and shaking.
2. The method for measuring the contents of ginsenoside Rg1, re and Rb1 in the Yihe Chun preparation according to claim 1, which is characterized in that: the reference substance is prepared by the following method: precisely weighing ginsenoside Rg 1 Reference substance, ginsenoside Re reference substance and ginsenoside Rb 1 Adding methanol to dissolve the reference substance to obtain mixed solution containing Rg1, re and Rb1 0.05mg, 0.25mg and 0.60mg, respectively, and shaking.
3. The method for measuring the contents of ginsenoside Rg1, re and Rb1 in the Yihe Chun preparation according to claim 1, which is characterized in that: precisely sucking 10 μl of the sample solution and the reference solution, respectively, and injecting into high performance liquid chromatograph for measurement; wherein the chromatographic column is Waters Symmetry C, 250mm×4.6mm,5um;
the flow rate is 1.0ml/min;
the theoretical plate number is 6000 or more, calculated by ginsenoside Rg 1;
the elution procedure was:
。
4. The method for measuring the contents of ginsenoside Rg1, re and Rb1 in the Yihe Chun preparation according to claim 1, which is characterized in that: the centrifugal speed is 4000-6000r/min, and the centrifugal time is 5-15 minutes.
5. The method for measuring the contents of ginsenoside Rg1, re and Rb1 in the Yihe Chun preparation according to claim 1, which is characterized in that: the ultrasonic time is 0.5-2h; the reduced pressure concentration temperature is 40-60 ℃; the evaporating temperature is 95-105 ℃.
6. The method for measuring the contents of ginsenoside Rg1, re and Rb1 in the Yihe Chun preparation according to claim 1, which is characterized in that: the method comprises the following steps:
(1) Preparation of control solution
Precisely weighing ginsenoside Rg1 reference substance, ginsenoside Re reference substance and ginsenoside Rb1 reference substance, adding methanol to dissolve to obtain mixed solution containing Rg1, re and Rb1 0.05mg, 0.25mg and 0.60mg, respectively, and shaking uniformly to obtain the final product;
(2) Preparation of test solution
Precisely weighing 10g of crushed preparation of the first and second hand and spring after sieving with No. four sieve, adding 150ml of methanol into a conical flask, performing ultrasonic treatment for 1 hour, taking out, centrifuging at 4000r/min for 8 minutes, collecting supernatant, adding 150ml of methanol into residue, performing ultrasonic treatment for 40 minutes, centrifuging at 4000r/min for 8 minutes, and combining the two supernatants; concentrating the solution in water bath at 50deg.C under reduced pressure to dry; adding 40ml of water to dissolve the residue, completely transferring to a separating funnel, extracting with 50ml of diethyl ether once, discarding diethyl ether solution, extracting with water saturated n-butanol for 4 times, 40ml each time, and mixing n-butanol extracts; washing with 0.2% sodium hydroxide solution for 2 times (40 ml each time), discarding alkali liquor, washing with 50ml water saturated with n-butanol for 1 time, discarding water layer, evaporating n-butanol solution in 100deg.C water bath, dissolving residue in methanol, fixing volume to 10ml, and shaking to obtain the final product;
(3) Content determination of ginsenoside Rg1, re and Rb 1: precisely sucking 10 μl of the sample solution and the reference solution, respectively, and injecting into high performance liquid chromatograph for measurement;
the chromatographic conditions are as follows:
filler: octadecylsilane chemically bonded silica gel;
chromatographic column: waters Symmetry C18 250mm 4.6mm,5um;
acetonitrile is a mobile phase A, and water is a mobile phase B;
the detection wavelength is 203nm, the column temperature is 30 ℃, and the flow rate is 1.0ml/min;
the theoretical plate number is 6000 or more, calculated by ginsenoside Rg 1;
elution procedure:
。
7. The method for measuring the contents of ginsenoside Rg1, re and Rb1 in the Yihe Chun preparation according to claim 1, which is characterized in that: the Yihe spring preparation comprises granules, tablets, capsules and oral liquid.
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| CN113295816B (en) * | 2020-03-26 | 2022-12-27 | 苏中药业集团股份有限公司 | Thin layer chromatography detection method for Yihechun preparation |
| CN111983096A (en) * | 2020-08-28 | 2020-11-24 | 地奥集团成都药业股份有限公司 | Method for detecting ginsenoside in compound glutamine enteric-coated capsule |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101614708A (en) * | 2008-06-26 | 2009-12-30 | 贵州益佰制药股份有限公司 | Method for measuring content of ginsenoside in the Aidi preparation |
| CN104807905A (en) * | 2015-05-04 | 2015-07-29 | 中国人民解放军第三七一医院 | Method for determining contents of ginsenosides Rg1 and Re in Xinnaoning tablet |
-
2016
- 2016-12-19 CN CN201611180013.7A patent/CN108205022B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101614708A (en) * | 2008-06-26 | 2009-12-30 | 贵州益佰制药股份有限公司 | Method for measuring content of ginsenoside in the Aidi preparation |
| CN104807905A (en) * | 2015-05-04 | 2015-07-29 | 中国人民解放军第三七一医院 | Method for determining contents of ginsenosides Rg1 and Re in Xinnaoning tablet |
Non-Patent Citations (5)
| Title |
|---|
| 周兰.HPLC 法测定人参须中人参皂苷 Rg1、人参皂苷Re及人参皂苷Rb1的含量.中国民族民间医药.2009,第18卷(第3期),第1-2页. * |
| 李强 等.新编常用中药有效成分手册.中国协和医科大学出版社,2008,第24-25页. * |
| 李静涛 等. HPLC-ELSD法测定西洋参含片中人参皂苷Rg1、Re和Rb1的含量.药物分析杂志.2006,第26卷(第9期),第1289-1291页. * |
| 毕晓黎 等.HPLC同时测定参茸养生酒中人参皂苷Rg1、Re和Rb1的含量.江西中医药.2008,第39卷(第8期),第70-72页. * |
| 胡海波 等.HPLC测定颐和春口服液中人参皂苷Rg1、Re、Rb1的含量.南京医科大学学报(自然科学版).2009,第29卷(第2期),第200-204页. * |
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