CN111175428A - Method for detecting fingerprint of kidney-tonifying pregnancy-assisting granules - Google Patents
Method for detecting fingerprint of kidney-tonifying pregnancy-assisting granules Download PDFInfo
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- 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
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The invention discloses a method for detecting a fingerprint of a kidney-tonifying pregnancy-assisting granule, which comprises the following steps of 1, preparing a test solution of the kidney-tonifying pregnancy-assisting granule; step 2, preparation of a mixed reference solution: step 3, respectively and precisely sucking the mixed reference solution and the test solution to be injected into a liquid chromatograph, and recording a chromatogram; step 4, guiding out the kidney-tonifying and pregnancy-assisting granule fingerprint instrument, guiding the kidney-tonifying and pregnancy-assisting granule fingerprint instrument into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and selecting chromatographic peaks existing in chromatograms of different batches of kidney-tonifying and pregnancy-assisting granules as common peaks; generating a control fingerprint of the kidney tonifying and pregnancy promoting granule by using an average value calculation method; calculating the relative retention time and the relative peak area of each common peak; comparing the finger print of the granule for tonifying kidney and promoting pregnancy with the mixed standard substance, and identifying the peak of the main component. The fingerprint spectrum of the kidney-tonifying pregnancy-assisting granule provided by the invention can comprehensively and objectively represent the quality of the kidney-tonifying pregnancy-assisting granule. And the detection method has the advantages of simple and stable method, high precision, good reproducibility and the like.
Description
Technical Field
The invention relates to a detection method of traditional Chinese medicine, in particular to a detection method of a finger print of kidney tonifying and pregnancy promoting granules.
Background
The kidney-tonifying pregnancy-assisting particle formula is a proved formula of professor from national doctor and master of Xia Gui adult in traditional Chinese medicine of Jiangsu province, consists of 8 medicines of semen cuscutae, fried white paeony root, wine cornus, salvia miltiorrhiza, Chinese yam, vinegar radix bupleuri, cornu cervi degelatinatum and amethyst, has the effects of enabling yang to grow to be heavy, soothing the heart and liver qi activity and stabilizing the heart and liver spirit, is clinically used for treating kidney deficiency and yang bias and liver depression type corpus luteum insufficiency infertility, is applied for years, has long-term clinical experience, and reveals curative effect through a plurality of pharmacological tests. However, no preparation approval document exists at present, so pharmaceutical research including preparation process, quality standard, stability test and the like for the research of medical institution preparations and new drugs is necessary. The traditional Chinese medicine fingerprint is a comprehensive and quantifiable quality analysis method, can better reflect the overall quality of the traditional Chinese medicine preparation, and is widely applied to the quality control of the traditional Chinese medicine preparation. In order to better control the preparation quality of the kidney-tonifying and pregnancy-assisting granules, the invention adopts an HPLC method to establish a characteristic map, and simultaneously establishes an external standard method to determine the contents of gallic acid, 5-hydroxymethyl furfural, morroniside, swertisin, loganin, paeoniflorin, hyperoside, astragalin, rosmarinic acid and salvianolic acid B. Has important significance for ingredient identification, quality evaluation and quality standard establishment of the kidney-tonifying and pregnancy-assisting granules.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art and provides a fingerprint detection method of kidney-tonifying and pregnancy-assisting granules, which can objectively, comprehensively and accurately evaluate the quality of the kidney-tonifying and pregnancy-assisting granules and has important significance for controlling the quality of the kidney-tonifying and pregnancy-assisting granules and ensuring the clinical curative effect.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:
a detection method of a finger print of kidney-tonifying pregnancy-assisting granules comprises the following steps:
accurately weighing kidney tonifying and pregnancy promoting granules of different batches respectively, grinding, accurately weighing, placing in a conical flask with a plug, adding methanol water solution, ultrasonic treating, filtering, and collecting the subsequent filtrate;
step 2, preparation of mixed reference solution:
precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid, and salvianolic acid B reference substances, respectively, and adding methanol to obtain mixed reference substance solution;
step 3, precisely absorbing the test solution obtained in the step 1 and the mixed reference solution obtained in the step 2 respectively, injecting the solutions into a high performance liquid chromatograph for analysis, and recording a chromatogram;
step 4, exporting the fingerprint of the kidney-tonifying pregnancy-assisting particle test solution obtained in the step 3, and importing the fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of different batches of kidney-tonifying and pregnancy-assisting granules as common peaks; generating a control fingerprint of the kidney-tonifying and pregnancy-assisting granules by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; and marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed comparison product solution chromatogram.
A detection method of a finger print of kidney-tonifying pregnancy-assisting granules comprises the following steps:
accurately weighing kidney tonifying and pregnancy promoting granules of different batches respectively, grinding, accurately weighing, placing in a conical flask with a plug, adding methanol water solution, ultrasonic treating, filtering, and collecting the subsequent filtrate;
step 2, preparation of mixed reference solution:
precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid, and salvianolic acid B reference substances, respectively, and adding methanol to obtain mixed reference substance solution;
step 3, preparation of negative sample solution of missing medicinal materials
Respectively taking kidney-tonifying and pregnancy-promoting granules of medicinal materials of wine-deficient cornus pulp, semen cuscutae, salvia miltiorrhiza, fried white paeony root or wine-deficient cornus pulp-fried white paeony root, grinding, precisely weighing, placing in a conical bottle with a plug, adding a methanol water solution, carrying out ultrasonic treatment, filtering, and taking subsequent filtrate to obtain negative sample solution of each medicinal material;
step 4, precisely sucking the test solution in the step 1, mixing the reference solution in the step 2 and the negative sample solution without the medicinal materials in the step 3, injecting the mixed solution into a high performance liquid chromatograph for analysis, and recording a chromatogram;
step 4, leading out the fingerprints of the test solution of the kidney-tonifying pregnancy-assisting particles of each batch obtained in the step 3, and introducing the fingerprints into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of the kidney-tonifying and pregnancy-assisting granules in each batch as common peaks; generating a control fingerprint of the kidney-tonifying and pregnancy-assisting granules by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed reference solution chromatogram;
and comparing the comparison fingerprint spectrum with the chromatogram of the negative sample solution with the missing medicinal materials, and identifying the correlation between the common peak in the comparison fingerprint spectrum and the medicinal materials.
A detection method of a finger print of kidney-tonifying pregnancy-assisting granules comprises the following steps:
accurately weighing kidney tonifying and pregnancy promoting granules of different batches respectively, grinding, accurately weighing, placing in a conical flask with a plug, adding methanol water solution, ultrasonic treating, filtering, and collecting the subsequent filtrate;
step 2, preparation of mixed reference solution:
precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid, and salvianolic acid B reference substances, respectively, and adding methanol to obtain mixed reference substance solution;
step 3, establishing a linear regression equation
Precisely sucking the mixed reference substance solution obtained in the step (2), diluting the mixed reference substance solution into mixed reference substance solutions with the volume of 1, 2, 5, 10, 20 and 40 times respectively, precisely sucking 10 mu L of each mixed reference substance solution, injecting the solution into a high performance liquid chromatograph for analysis, and performing linear regression by using a least square method according to the measured response signal peak and the sample concentration of the measured component to obtain a linear regression equation and a linear range of each component;
step 4, detecting the test solution
Precisely sucking 10 mu L of the test solution prepared in the step (1), injecting the test solution into a high performance liquid chromatography for detection, and detecting the content of each chemical component in the test solution through the linear regression equation obtained in the step (3).
As a preferred scheme, the above method for detecting the fingerprint of the kidney tonifying and pregnancy promoting granule comprises the following steps of 1: taking kidney-tonifying and pregnancy-assisting particles, grinding, precisely weighing 0.8g, placing in a conical flask with a plug, precisely adding 10mL of methanol water with volume concentration of 70%, sealing the plug, weighing, carrying out ultrasonic treatment at 200W and 40kHz for 30min, placing to room temperature, weighing again, complementing the loss amount with methanol water with volume concentration of 70%, shaking up, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition.
As a preferred scheme, the method for detecting the fingerprint of the kidney-tonifying pregnancy-assisting granule comprises the following steps of 2, preparation of a mixed reference substance solution: precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid and salvianolic acid B reference substances, and adding methanol to obtain solutions with concentration of 0.540 mg/mL-1、1.600mg·mL-1、0.637mg·mL-1、0.225mg·mL-1、 0.623mg·mL-1、1.053mg·mL-1、0.638mg·mL-1、0.480mg·mL-1、0.365mg·mL-1、0.877 mg·mL-1Filtering with 0.45 μm microporous membrane to obtain the final product.
Preferably, in the method for detecting the fingerprint of the kidney-tonifying and pregnancy-assisting granule, the chromatographic conditions analyzed by the high performance liquid chromatograph are as follows:
the chromatographic column is as follows: hedera ODS-2C18 chromatographic column with specification of 250mm × 4.6mm, 5 μm, acetonitrile as mobile phase A, 0.2% phosphoric acid water solution as mobile phase B, gradient elution with flow rate of 1.0 mL/min-1The detection wavelength is 237nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L. The gradient elution procedure was: 0-15 min, 5% A → 7% A; 15-30 min, 7% A → 9.5% A; 30-31 min, 9.5% A → 11% A; 31-55 min, 11% A; 55-65 min, 11% A → 20% A; 65-95 min, 20% A → 22% A; 95-100 min, 22% A → 5% A.
The fingerprint spectrum of the kidney-tonifying pregnancy-assisting granule has 23 peaks in total. Wherein peak 2 is gallic acid, peak 3 is 5-hydroxymethylfurfural, peak 6 is morroniside, peak 8 is swertioside, peak 9 is loganin, peak 12 is paeoniflorin, peak 16 is hyperoside, peak 19 is astragalin, peak 21 is rosmarinic acid, and peak 23 is salvianolic acid B.
The invention relates to a detection method of fingerprint of kidney-tonifying pregnancy-assisting granules, wherein peaks 1, 2, 3, 4, 5, 6, 8, 9 and 20 are ingredients in cornus wine decoction pieces; 7. 13, 14, 15, 16, 17 and 19, which are ingredients in the semen cuscutae decoction pieces; 2. no. 10, No. 11, No. 12 and No. 18 are ingredients in the fried white paeony root decoction pieces; 20. 21, 22 and 23 peaks are the components in the salvia miltiorrhiza decoction pieces; wherein the peak 2 is common component of Corni fructus (processed with wine) and radix Paeoniae alba (parched); no. 20 peak is the common component of alcohol pulp cornus and dodder.
The invention relates to a method for detecting a fingerprint of kidney-tonifying pregnancy-assisting particles, which comprises the following steps of:
| composition (I) | Regression equation | Linear Range/(μ g. mu.mL)-1) | r |
| Gallic acid | Y＝8 145.2X+27 742 | 13.50～540.00 | 0.9999 |
| 5-hydroxymethylfurfural | Y＝8 251X+86 250 | 40.02～1600.84 | 0.9999 |
| Morroniside | Y＝17 063X+39 070 | 15.92～636.80 | 0.9998 |
| Swertisin | Y＝15 767X-11 585 | 5.02～200.80 | 0.9998 |
| Loganin A. Loganin A | Y＝17 806X-35 604 | 15.59～623.60 | 0.9997 |
| Paeoniflorin | Y＝12 710X-195 305 | 26.32～1 052.84 | 0.9996 |
| Hyperoside A | Y＝17 290X-24 182 | 15.96～638.42 | 0.9998 |
| Astragalin | Y＝17 653X-49 093 | 12.00～480.07 | 0.9995 |
| Rosmarinic acid | Y＝13 862X-40 387 | 9.12～364.81 | 0.9998 |
| Salvianolic acid B | Y＝11 925Xx+118 000 | 21.92～876.80 | 0.9999 |
。
Optimizing fingerprint detection conditions:
1.1 selection of detection wavelength
The invention carries out Waters2998PDA full-wavelength scanning on the mixed reference substance, finds that the target components have better absorption at about 237nm, and the peak shape of the target peak in the chromatogram is also better. The morroniside is an important active ingredient of the pulp of dogwood, has high content, stable property and good separation degree, and the maximum absorption wavelength is 237nm, so 237nm is selected as the detection wavelength.
1.2 selection of the Mobile phase
The invention inspects different mobile phases (acetonitrile-0.2% phosphoric acid aqueous solution, methanol-0.2% phosphoric acid aqueous solution and acetonitrile-water), and the result adopts an acetonitrile-0.2% phosphoric acid aqueous system, compared with the methanol-0.2% phosphoric acid aqueous solution, the peak speed is high, the theoretical plate number of paeoniflorin is slightly reduced but has no great influence; compared with acetonitrile-water, the morroniside, loganin, hyperoside and salvianolic acid B have better peak shapes and separation degrees and more stable base lines, so that the morroniside, loganin, hyperoside and salvianolic acid B are selected as the mobile phase.
1.3 selection of gradient of mobile phase
Since most of the components measured in this experiment were polar components, the influence of the gradient change on the separation effect was examined by determining that the mobile phase was selected from acetonitrile (a) -0.2% phosphoric acid aqueous solution (B). In the preparation, the interference peaks near 5-hydroxymethylfurfural, loganin, paeoniflorin and hyperin are more and difficult to separate; therefore, isocratic elution or low-flow phase change elution is selected in the time period, and the separation degree of peaks is ensured to meet the requirement. The gradient of 15-30 min part also screens various conditions of 7% A isocratic elution, 8% A isocratic elution, 7% A → 9% A gradient elution and 7% A → 10% A gradient elution, and the morroniside separation degree is better when the gradient is 7% A → 9.5% A, and the subsequent chromatographic peak is not influenced; a 31-55 min part, wherein the peak can be formed by using 12% A isocratic elution for about 20min, and the long-time isocratic elution of 11% A is delayed compared with the short-time isocratic elution of 12% A, so that the separation degree of loganin and paeoniflorin is better, and the theoretical plate number is higher; 11% A → 20% A mobile phase portion has fewer peaks but takes longer to transition in order to smooth the baseline; gradient 20% A isocratic elution, 20% A → 21% A, 20% A → 22A% gradient elution of 65-95 min part can separate target peak, except that the peak emergence time is different, the difference is smaller, so 20% A → 22% A with faster peak emergence time is selected. In summary, the HPLC method was finally determined to be a gradient elution with acetonitrile (a) -0.2% phosphoric acid in water (B) as mobile phase: 0-15 min, 5% A → 7% A; 15-30 min, 7% A → 9.5% A; 30-31 min, 9.5% A → 11% A; 31-55 min, 11% A; 55-65 min, 11% A → 20% A; 65-95 min, 20% A → 22% A; 95-100 min, 22% A → 5% A.
1.4 selection of column temperature
The column temperature also has an influence on the separation degree of morroniside, loganin, swertisin and hyperin in the gradient condition screened out by the invention, and the invention examines different column temperatures (25, 30 and 35 ℃). The column temperature in the method for measuring morroniside and loganin in wine dogwood fruit is 35 ℃ in pharmacopoeia of the people's republic of China. When the column temperature is 30 ℃, the separation degree of the morroniside and the loganin can meet the requirement, and the separation degrees of other peaks are good; when the column temperature is 35 ℃, the separation degree of the drug glycoside is reduced, and the separation degree of the other components is good; when the column temperature is 25 ℃, the separation degree of the drug glycoside is better, but the separation degree of the morroniside and the hyperin is reduced. In general, 30 ℃ was selected as the column temperature.
The invention relates to a detection method of a fingerprint of a kidney-tonifying pregnancy-assisting granule, which comprises the following steps: weighing 10 parts of antler, 15 parts of semen cuscutae, 15 parts of fried white paeony root, 15 parts of wine cornus, 15 parts of salvia miltiorrhiza, 15 parts of Chinese yam, 6 parts of vinegar radix bupleuri and 10 parts of amethyst;
firstly, 10 parts of antler and 10 parts of amethyst are taken, 8 times of water is added, the mixture is decocted for 0.5 hour, and the filtrate is filtered for standby;
mixing the residues with semen Cuscutae 15 parts, radix Paeoniae alba 15 parts, Corni fructus 15 parts, Saviae Miltiorrhizae radix 15 parts, rhizoma Dioscoreae 15 parts, and bupleuri radix 6 parts, decocting with water twice, adding water 8 times the volume of the decoction pieces, soaking for 0.5 hr, decocting for 0.5 hr, and filtering; adding water with volume 8 times of the weight of the decoction pieces for the second time, decocting for 0.5h, and filtering. Mixing the above filtrates, concentrating under reduced pressure to relative density of about 1.10 (60 deg.C), and centrifuging with a straight tube type high speed centrifuge (rotation speed: 16000r/min, flow rate: 4-6L/min). Taking a proper amount of dextrin, placing the dextrin into a fluidized bed, setting the air inlet temperature to be 100 ℃, starting to feed liquid when the temperature of the materials rises to 70 ℃, and controlling the liquid feeding speed to be 80-150 r/min and the atomization pressure to be 0.2MPa outside and 0.15MPa inside. And (5) after spraying is finished, continuously drying for 1 hour at 70 ℃ to obtain the product.
Has the advantages that:
1. according to the structural property characteristics of active ingredients contained in the kidney-tonifying and pregnancy-assisting granules, the best mobile phase composition is screened out through a large number of experiments, and analysis conditions such as gradient elution procedures, flow rate, detection wavelength, chromatographic column, column temperature and the like are verified through a plurality of experiments.
2. The fingerprint of the kidney-tonifying and pregnancy-assisting granule established by the method provided by the invention can effectively represent the quality of the kidney-tonifying and pregnancy-assisting granule, objectively reflect the front and back sequence and the mutual relation of characteristic peaks of each formed fingerprint, avoid the one-sidedness of judging the quality of the kidney-tonifying and pregnancy-assisting granule due to the determination of individual chemical components, and reduce the possibility of manual treatment for reaching the quality standard.
3. The method for detecting the fingerprint of the kidney-tonifying pregnancy-assisting particle can also measure the content of 10 effective components in the kidney-tonifying pregnancy-assisting particle, and has the advantages of simple method, good stability, high precision, good reproducibility and the like.
Drawings
Fig. 1 is a comparison fingerprint of a kidney-tonifying pregnancy-assisting granule sample of the present invention.
FIG. 2 is a fingerprint of 8 batches of samples of the kidney-tonifying and pregnancy-promoting granule of the present invention.
FIG. 3 is a chromatogram of a mixed control of the present invention.
FIG. 4 is a chromatogram of a negative sample solution lacking the drug substance.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not specified, according to conventional conditions or conditions recommended by manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The following examples used the following instruments and reagents:
1 Instrument and reagent
1.1 instruments
Waters e2695HPLC, Waters2998PDA detector; sartorius BP-211D electronic analytical balance (Sartorius); an SK6200H ultrasonic instrument (shanghai koku qiao ultrasonic instruments ltd).
1.2 reagents
Acetonitrile (Tedia, pure chromatography), water as ultrapure water, and the remaining reagents were analytically pure.
1.3 control and sample
The reference substances of salvianolic acid B, hyperoside, astragalin, morroniside, swertisin and rosmarinic acid are purchased from Nanjing Polykang pharmaceutical and chemical Co Ltd (the batches are 160508, 181025, 180924, 160928, 181211 and 161110 respectively, the mass fractions are all more than or equal to 98%), the gallic acid, loganin and paeoniflorin are purchased from China food and drug testing research institute (the batches are 110831-201204, 111640-201005 and 110736-201337 respectively, the concentrations are respectively calculated by 89.9%, 99.2% and 94.9%), and the 5-hydroxymethylfurfural is purchased from Nanjing Ningqi pharmaceutical Co Ltd (the batch is 130911, the mass fraction is more than or equal to 98%).
Salvia miltiorrhiza (batch No. 20180101-01, Guizhou of origin), Vinegar Bupleurum root (batch No. 20181002-01, Shaanxi of origin) were purchased from Tongde pharmaceutical Co., Ltd of Guizhou, Jiu Yu (batch No. 190201, Henan of origin), Yam (batch No. 190101, Henan of origin) were purchased from Hequan Chinese herbal pieces Co., Ltd of Maanshan, dodder (batch No. 181101, produced Indomon), Zingiber officinale (batch No. 181101, produced Jiansu) were purchased from Hequan Chinese herbal pieces Co., Ltd of Anhui, antler pieces (batch No. 190101, produced Jilin) were purchased from Wan Sheng Chinese herbal pieces Co., Ltd of Anhui, Paeonia lactiflora (batch No. 19010712, produced Anhui) were purchased from Bujing pharmaceutical Co., Ltd, dried root of Buchi DC of Buchi, Umbelliferae, dried root and dried root of Buchi, dried mature pulp of Cornus officinalis Sieb. et Zucc. of Cornaceae, dried rhizome of Dioscorea opposita Thunb. of Dioscoreaceae, dried mature seed of Cuscuta chinensis Lam. of Convolvulaceae, dried fluorite mineral fluorite family mainly containing calcium fluoride, ossified horn of Cervus elaphus Linnaeus of Cervidae, and dried root of Paeonia lactiflora pall. of Ranunculaceae. 15 batches of kidney-tonifying and pregnancy-assisting granules with numbers of S1-S15 are provided by traditional Chinese medicine academy of Jiangsu province (the preparation process is the same as the previous batch), and the batches are 171011, 171012, 171015, 171026, 171027, 171028, 171203, 171205, 171208, 171221, 171225, 180110, 180112, 180113 and 180320 respectively. The negative sample of the alcohol-deficient cornus, the negative sample of the dodder, the negative sample of the salvia miltiorrhiza, the negative sample of the parched white paeony root and the negative sample of the alcohol-deficient cornus-parched white paeony root are prepared by laboratories according to the same preparation process.
Embodiment 1 a method for detecting a fingerprint of a granule for tonifying kidney and helping pregnancy, comprising the following steps:
respectively precisely weighing the 15 batches of the kidney-tonifying and pregnancy-assisting granules, grinding, precisely weighing 0.8g, placing in a conical flask with a plug, precisely adding 10mL of methanol water with the volume concentration of 70%, sealing the plug, weighing, carrying out ultrasonic treatment at 200W and 40kHz for 30min, standing to room temperature, weighing again, complementing the loss amount by using methanol water with the volume concentration of 70%, shaking uniformly, filtering by using a 0.45 mu m microporous membrane, and taking the subsequent filtrate to obtain the kidney-tonifying and pregnancy-assisting granules
Step 2, preparation of mixed reference solution:
precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid and salvianolic acid B reference substances, and adding methanol to obtain solutions with concentration of 0.540 mg/mL-1、1.600mg·mL-1、0.637mg·mL-1、0.225mg·mL-1、0.623mg·mL-1、 1.053mg·mL-1、0.638mg·mL-1、0.480mg·mL-1、0.365mg·mL-1、0.877mg·mL-1Filtering with 0.45 μm microporous membrane to obtain the final product.
Step 3, precisely absorbing the 15 batches of test solution in the step 1 and the mixed reference solution in the step 2 respectively, injecting the solutions into a high performance liquid chromatograph for analysis, and recording a chromatogram; the chromatographic conditions are as follows: the chromatographic column is as follows: hedera ODS-2C18 chromatographic column with specification of 250mm × 4.6mm, 5 μm, acetonitrile as mobile phase A, 0.2% phosphoric acid water solution as mobile phase B, and gradient elution (0-15 min, 5% A → 7% A, 15-30 min, 7% A → 9.5% A, 30-31 min, 9.5% A → 11% A, 31-55 min, 11% A, 55-65 min, 11% A → 20% A, 65-95 min, 20% A → 22% A, 95-100 min, 22% A → 5% A), flow rate of 1.0mL min-1The detection wavelength is 237nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.
Step 4, leading out the fingerprints of the 15 batches of kidney-tonifying and pregnancy-assisting granule test solution obtained in the step 3, and introducing the fingerprints into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of 15 batches of kidney-tonifying and pregnancy-assisting granules as common peaks (as shown in figure 1); generating a control fingerprint (as shown in figure 2) of the kidney tonifying and pregnancy promoting granule by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; and labeling the chemical components of the peaks in the control fingerprint according to the retention time of the mixed control solution chromatogram (as shown in figure 3).
The fingerprint spectrum obtained has 23 peaks. Wherein peak 2 is gallic acid, peak 3 is 5-hydroxymethylfurfural, peak 6 is morroniside, peak 8 is swertioside, peak 9 is loganin, peak 12 is paeoniflorin, peak 16 is hyperoside, peak 19 is astragalin, peak 21 is rosmarinic acid, and peak 23 is salvianolic acid B. And calculating the similarity between the characteristic maps of 15 batches of samples and the control characteristic map by taking the morroniside peak as a reference peak, wherein the similarity between S1 and S15 is respectively 0.995, 0.984, 0.991, 0.998, 0.997, 0.995, 0.983, 0.998, 0.984, 0.997, 0.996, 0.997 and 0.997, and the similarity between the 15 batches of samples is more than 0.98.
Example 2
A detection method of a finger print of kidney-tonifying pregnancy-assisting granules comprises the following steps:
respectively precisely weighing the 15 batches of the kidney-tonifying and pregnancy-assisting granules, grinding, precisely weighing 0.8g, placing in a conical flask with a plug, precisely adding 10mL of methanol water with the volume concentration of 70%, sealing the plug, weighing, carrying out ultrasonic treatment at 200W and 40kHz for 30min, standing to room temperature, weighing again, complementing the loss amount by using methanol water with the volume concentration of 70%, shaking uniformly, filtering by using a 0.45 mu m microporous membrane, and taking the subsequent filtrate to obtain the kidney-tonifying and pregnancy-assisting granules
Step 2, preparation of mixed reference solution:
precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid and salvianolic acid B reference substances, and adding methanol to obtain solutions with concentration of 0.540 mg/mL-1、1.600mg·mL-1、0.637mg·mL-1、0.225mg·mL-1、0.623mg·mL-1、 1.053mg·mL-1、0.638mg·mL-1、0.480mg·mL-1、0.365mg·mL-1、0.877mg·mL-1Filtering with 0.45 μm microporous membrane to obtain the final product.
Step 3, preparation of negative sample solution of missing medicinal materials
Respectively taking kidney-tonifying pregnancy-assisting granules without medicinal materials of wine dogwood, semen cuscutae, salvia miltiorrhiza, fried white paeony root or medicinal materials of wine dogwood and fried white paeony root, grinding, precisely weighing 0.8g, placing in a conical flask with a plug, precisely adding 10mL of methanol water with volume concentration of 70%, sealing the plug, weighing, carrying out ultrasonic treatment at 200W and 40kHz for 30min, placing to room temperature, weighing again, complementing loss amount by using methanol water with volume concentration of 70%, shaking up, filtering by using a 0.45 mu m microporous membrane, and taking subsequent filtrate to obtain a negative sample solution without the medicinal materials;
step 4, precisely sucking the test solution in the step 1, mixing the reference solution in the step 2 and the negative sample solution without the medicinal materials in the step 3, injecting the mixed solution into a high performance liquid chromatograph for analysis, and recording a chromatogram; the chromatographic conditions are as follows: the chromatographic column is as follows: HederaODS-2C 18 chromatographic column with specification of 250mm × 4.6mm, 5 μm, acetonitrile as mobile phase A, 0.2% phosphoric acid water solution as mobile phase B, and gradient elution (0-15 min, 5% A → 7% A, 15-30 min, 7% A → 9.5% A, 30-31 min, 9.5% A → 11% A, 31-55 min, 11% A, 55-65 min, 11% A → 20% A, 65-95 min, 20% A → 22% A, 95-100 min, 22% A → 5% A), flow rate of 1.0mL min-1The detection wavelength is 237nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.
Step 4, leading out the fingerprints of the test solution of the kidney-tonifying pregnancy-assisting particles of each batch obtained in the step 3, and introducing the fingerprints into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of the kidney-tonifying and pregnancy-assisting granules in each batch as common peaks; generating a control fingerprint of the kidney-tonifying and pregnancy-assisting granules by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed reference solution chromatogram; the fingerprint spectrum obtained has 23 peaks. Wherein peak 2 is gallic acid, peak 3 is 5-hydroxymethylfurfural, peak 6 is morroniside, peak 8 is swertioside, peak 9 is loganin, peak 12 is paeoniflorin, peak 16 is hyperoside, peak 19 is astragalin, peak 21 is rosmarinic acid, and peak 23 is salvianolic acid B.
And comparing the comparison fingerprint spectrum with the chromatogram of the negative sample solution with the missing medicinal materials, and identifying the correlation between the common peak in the comparison fingerprint spectrum and the medicinal materials. As shown in fig. 4 (chromatogram of lack of wine fructus Corni negative sample (C), lack of semen Cuscutae negative sample (D), lack of radix Salviae Miltiorrhizae negative sample (E), lack of parched radix Paeoniae alba negative sample (F) and lack of wine fructus Corni-parched radix Paeoniae alba negative sample (G)), wherein peaks 1, 2, 3, 4, 5, 6, 8, 9, and 20 are components in wine fructus Corni decoction pieces; 7. 13, 14, 15, 16, 17 and 19, which are ingredients in the semen cuscutae decoction pieces; 2. no. 10, No. 11, No. 12 and No. 18 are ingredients in the fried white paeony root decoction pieces; 20. 21, 22 and 23 peaks are the components in the salvia miltiorrhiza decoction pieces; wherein the peak 2 is common component of Corni fructus (processed with wine) and radix Paeoniae alba (parched); no. 20 peak is the common component of alcohol pulp cornus and dodder.
Embodiment 3 a method for detecting fingerprint of kidney-tonifying pregnancy-assisting granules, which comprises the following steps:
respectively precisely weighing the 15 batches of the kidney-tonifying and pregnancy-assisting granules, grinding, precisely weighing 0.8g, placing in a conical flask with a plug, precisely adding 10mL of methanol water with the volume concentration of 70%, sealing the plug, weighing, carrying out ultrasonic treatment at 200W and 40kHz for 30min, standing to room temperature, weighing again, complementing the loss amount by using methanol water with the volume concentration of 70%, shaking uniformly, filtering by using a 0.45 mu m microporous membrane, and taking the subsequent filtrate to obtain the kidney-tonifying and pregnancy-assisting granules
Step 2, preparation of mixed reference solution:
precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid and salvianolic acid B reference substances, and adding methanol to obtain solutions with concentration of 0.540 mg/mL-1、1.600mg·mL-1、0.637mg·mL-1、0.225mg·mL-1、0.623mg·mL-1、 1.053mg·mL-1、0.638mg·mL-1、0.480mg·mL-1、0.365mg·mL-1、0.877mg·mL-1Filtering with 0.45 μm microporous membrane to obtain the final product.
Step 3, establishing a linear regression equation
Precisely sucking the mixed reference substance solution obtained in the step (2), diluting the mixed reference substance solution into mixed reference substance solutions with the volume of 1, 2, 5, 10, 20 and 40 times respectively, precisely sucking 10 mu L of each mixed reference substance solution, injecting the solution into a high performance liquid chromatograph for analysis, and performing linear regression by using a least square method according to the measured response signal peak and the sample concentration of the measured component to obtain a linear regression equation and a linear range of each component; as in table 1 below.
Table 110 component linear regression equation
Step 4, detecting the test solution
Precisely sucking 10 mu L of the 15 batches of test solution prepared in the step (1), injecting into a high performance liquid chromatography for detection, and detecting the content of each chemical component in the test solution through the linear regression equation obtained in the step (3). As shown in table 2.
TABLE 215 measurement of the content of 10 components in the sample batch (mg. g)-1)
。
Example 4 methodological Studies
1. Precision test
10. mu.L of the same mixed control solution of example 1 was precisely pipetted and subjected to continuous sampling 6 times. According to the calculation of the peak area, the RSD of gallic acid, 5-hydroxymethyl furfural, morroniside, swertisin, loganin, paeoniflorin, hyperoside, astragalin, rosmarinic acid and salvianolic acid B are respectively 1.1%, 1.6%, 1.9%, 2.5%, 2.7%, 2.6%, 3.0%, 2.9%, 1.6% and 2.7%, and the result shows that the precision of the instrument is good.
2. Repeatability test
171011 the same batch of kidney-tonifying and pregnancy-assisting granules 6 parts are taken, the test solution is prepared according to the preparation method of the test solution in the example 1, and the peak area is measured by sample injection. According to peak area calculation, the contents of the gallic acid, 5-hydroxymethyl furfural, morroniside, swertisin, loganin, paeoniflorin, hyperoside, astragalin, rosmarinic acid and salvianolic acid B are respectively 1.3%, 0.45%, 1.1%, 3.0%, 1.4%, 2.6%, 2.9%, 1.4%, 1.2% and 1.3%, and the result shows that the method has good repeatability.
3. Stability test
0.8g of an S1 sample with the batch number of 171011 is taken, a test sample solution is prepared according to the preparation method of the test sample solution in the example 1, the sample injection is respectively carried out for 0 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours for determination, and the peak area is determined. According to peak area calculation, the RSD of gallic acid, 5-hydroxymethyl furfural, morroniside, swertisin, loganin, paeoniflorin, hyperoside, astragalin, rosmarinic acid and salvianolic acid B are respectively 1.8%, 1.9%, 2.0%, 1.8%, 1.1%, 2.7%, 2.4%, 2.2%, 3.2% and 2.1%, and the result shows that the method has good repeatability.
4. Sample application recovery test
171011 particles with known content for tonifying kidney and helping pregnancy are taken, about 0.4g of each particle is taken, methanol water with volume concentration of 70% is precisely added into each particle to prepare gallic acid with concentration of 1.60, 1.80, 1.84, 0.49, 1.05, 1.00, 1.44, 1.60, 1.20 and 1.18mg/ml, 5-hydroxymethyl furfural, morroniside, swertide, loganin, paeoniflorin, hyperoside, astragalin, rosmarinic acid, salvianolic acid B0.25, 0.50, 0.10, 0.25, 1.0, 0.25, 0.20, 0.40 and 2.5ml respectively, and a sample solution for sample supply is prepared according to the preparation method of the sample solution in the embodiment 1, sample injection measurement and sample addition calculation recovery rate are shown in a table 3. Test results show that the method has good accuracy.
Table 3 sample adding and recovering test results of kidney tonifying and pregnancy promoting granule (n ═ 6)
The experimental results show that the fingerprint spectrum detection method for the kidney-tonifying and pregnancy-assisting granules provided by the invention has the advantages of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of the kidney-tonifying and pregnancy-assisting granules, and has important significance for ensuring the clinical curative effect.
The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.
Claims (11)
1. A detection method of a finger print of kidney tonifying and pregnancy promoting granules is characterized by comprising the following steps:
step 1, preparation of a test solution of kidney-tonifying and pregnancy-assisting particles:
accurately weighing kidney tonifying and pregnancy promoting granules of different batches respectively, grinding, accurately weighing, placing in a conical flask with a plug, adding methanol water solution, ultrasonic treating, filtering, and collecting the subsequent filtrate;
step 2, preparation of mixed reference solution:
precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid, and salvianolic acid B reference substances, respectively, and adding methanol to obtain mixed reference substance solution;
step 3, precisely absorbing the test solution obtained in the step 1 and the mixed reference solution obtained in the step 2 respectively, injecting the solutions into a high performance liquid chromatograph for analysis, and recording a chromatogram;
step 4, exporting the fingerprint of the kidney-tonifying pregnancy-assisting particle test solution obtained in the step 3, and importing the fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of different batches of kidney-tonifying and pregnancy-assisting granules as common peaks; generating a control fingerprint of the kidney-tonifying and pregnancy-assisting granules by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; and marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed comparison product solution chromatogram.
2. A detection method of a finger print of kidney tonifying and pregnancy promoting granules is characterized by comprising the following steps:
step 1, preparation of a test solution of kidney-tonifying and pregnancy-assisting particles:
accurately weighing kidney tonifying and pregnancy promoting granules of different batches respectively, grinding, accurately weighing, placing in a conical flask with a plug, adding methanol water solution, ultrasonic treating, filtering, and collecting the subsequent filtrate;
step 2, preparation of mixed reference solution:
precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid, and salvianolic acid B reference substances, respectively, and adding methanol to obtain mixed reference substance solution;
step 3, preparation of negative sample solution of missing medicinal materials
Respectively taking kidney-tonifying and pregnancy-promoting granules of medicinal materials of wine-deficient cornus pulp, semen cuscutae, salvia miltiorrhiza, fried white paeony root or wine-deficient cornus pulp-fried white paeony root, grinding, precisely weighing, placing in a conical bottle with a plug, adding a methanol water solution, carrying out ultrasonic treatment, filtering, and taking subsequent filtrate to obtain negative sample solution of each medicinal material;
step 4, precisely sucking the test solution in the step 1, mixing the reference solution in the step 2 and the negative sample solution without the medicinal materials in the step 3, injecting the mixed solution into a high performance liquid chromatograph for analysis, and recording a chromatogram;
step 4, leading out the fingerprints of the test solution of the kidney-tonifying pregnancy-assisting particles of each batch obtained in the step 3, and introducing the fingerprints into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of the kidney-tonifying and pregnancy-assisting granules in each batch as common peaks; generating a control fingerprint of the kidney-tonifying and pregnancy-assisting granules by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed reference solution chromatogram;
and comparing the comparison fingerprint spectrum with the chromatogram of the negative sample solution with the missing medicinal materials, and identifying the correlation between the common peak in the comparison fingerprint spectrum and the medicinal materials.
3. A detection method of a finger print of kidney tonifying and pregnancy promoting granules is characterized by comprising the following steps:
step 1, preparation of a test solution of kidney-tonifying and pregnancy-assisting particles:
accurately weighing kidney tonifying and pregnancy promoting granules of different batches respectively, grinding, accurately weighing, placing in a conical flask with a plug, adding methanol water solution, ultrasonic treating, filtering, and collecting the subsequent filtrate;
step 2, preparation of mixed reference solution:
precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid, and salvianolic acid B reference substances, respectively, and adding methanol to obtain mixed reference substance solution;
step 3, establishing a linear regression equation
Precisely sucking the mixed reference substance solution obtained in the step (2), diluting the mixed reference substance solution into mixed reference substance solutions with the volume of 1, 2, 5, 10, 20 and 40 times respectively, precisely sucking 10 mu L of each mixed reference substance solution, injecting the solution into a high performance liquid chromatograph for analysis, and performing linear regression by using a least square method according to the measured response signal peak and the sample concentration of the measured component to obtain a linear regression equation and a linear range of each component;
step 4, detecting the test solution
Precisely sucking 10 mu L of the test solution prepared in the step (1), injecting the test solution into a high performance liquid chromatography for detection, and detecting the content of each chemical component in the test solution through the linear regression equation obtained in the step (3).
4. The method for detecting the fingerprint of the kidney tonifying and pregnancy promoting granule according to claims 1 to 3, wherein the preparation method of the test solution of the kidney tonifying and pregnancy promoting granule in step 1 is: taking kidney-tonifying and pregnancy-assisting particles, grinding, precisely weighing 0.8g, placing in a conical flask with a plug, precisely adding 10mL of methanol water with volume concentration of 70%, sealing the plug, weighing, carrying out ultrasonic treatment at 200W and 40kHz for 30min, placing to room temperature, weighing again, complementing the loss amount with methanol water with volume concentration of 70%, shaking up, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition.
5. The method for detecting fingerprint of kidney tonifying and pregnancy promoting granule according to claims 1 to 3, wherein the step 2 of preparing the mixed reference solution comprises: precisely weighing gallic acid, 5-hydroxymethyl furfural, morroniside, loganin, swertisin, paeoniflorin, hyperoside, astragalin, rosmarinic acid and salvianolic acid B reference substances, and adding methanol to obtain solutions with concentration of 0.540 mg/mL-1、1.600mg·mL-1、0.637mg·mL-1、0.225mg·mL-1、0.623mg·mL-1、1.053mg·mL-1、0.638mg·mL-1、0.480mg·mL-1、0.365mg·mL-1、0.877mg·mL-1Filtering with 0.45 μm microporous membrane to obtain the final product.
6. The method for detecting fingerprint of kidney tonifying and pregnancy promoting granule as claimed in claims 1 to 3, wherein the chromatographic conditions of HPLC analysis are as follows:
the chromatographic column is as follows: hedera ODS-2C18 chromatographic column with specification of 250mm × 4.6mm, 5 μm, acetonitrile as mobile phase A, 0.2% phosphoric acid water solution as mobile phase B, gradient elution with flow rate of 1.0 mL/min-1The detection wavelength is 237nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.
7. The method for detecting the fingerprint of the kidney tonifying and pregnancy promoting granule as claimed in claim 6, wherein the gradient elution procedure is as follows: 0-15 min, 5% A → 7% A; 15-30 min, 7% A → 9.5% A; 30-31 min, 9.5% A → 11% A; 31-55 min, 11% A; 55-65 min, 11% A → 20% A; 65-95 min, 20% A → 22% A; 95-100 min, 22% A → 5% A.
8. The method for detecting fingerprint of kidney tonifying and pregnancy promoting granule according to claim 1 or 2, wherein there are 23 peaks in the fingerprint.
9. The method for detecting fingerprint of particles for tonifying kidney and assisting pregnancy as claimed in claim 8, wherein the 23 common peaks include gallic acid at peak 2, 5-hydroxymethylfurfural at peak 3, morroniside at peak 6, swertisin at peak 8, loganin at peak 9, paeoniflorin at peak 12, hyperoside at peak 16, astragalin at peak 19, rosmarinic acid at peak 21, and salvianolic acid B at peak 23.
10. The method for detecting fingerprint of granule for tonifying kidney and assisting pregnancy as claimed in claim 8, wherein Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 8, Peak 9, Peak 20 are ingredients of decoction pieces of Corni fructus; 7. 13, 14, 15, 16, 17 and 19, which are ingredients in the semen cuscutae decoction pieces; 2. no. 10, No. 11, No. 12 and No. 18 are ingredients in the fried white paeony root decoction pieces; 20. 21, 22 and 23 peaks are the components in the salvia miltiorrhiza decoction pieces; wherein the peak 2 is common component of Corni fructus (processed with wine) and radix Paeoniae alba (parched); no. 20 peak is the common component of alcohol pulp cornus and dodder.
11. The method for detecting the fingerprint of the kidney tonifying and pregnancy promoting granule as claimed in claim 3, wherein the linear regression equation of each compound is as follows:
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