CN110857933A - Method for establishing HPLC fingerprint of Mailuoshutong pill and standard spectrum thereof - Google Patents
Method for establishing HPLC fingerprint of Mailuoshutong pill and standard spectrum thereof Download PDFInfo
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Abstract
The invention discloses a method for establishing an HPLC fingerprint of a Mailushutong pill, which comprises the steps of preparing a test solution, determining HPLC chromatographic conditions and manufacturing an HPLC standard fingerprint. The invention also discloses an HPLC standard fingerprint of the Mailuoshutong pill obtained by the method, and the fingerprint has 25 common peaks. The quality detection method has the advantages of simple operation, high stability, good reproducibility and more characteristic peaks of the obtained spectrum, can comprehensively evaluate the quality of the Mailuoshutong pills by comparing common peaks of standard fingerprint spectrums, is favorable for stabilizing the product quality and ensures the safety and the effectiveness of clinical medication.
Description
Technical Field
The invention relates to a method for establishing an HPLC fingerprint of a compound traditional Chinese medicine Mailuoshutong pill and an HPLC standard fingerprint thereof, belonging to the field of analysis of traditional Chinese medicine preparations.
Background
The Mailuoshutong homologous product comprises Mailuoshutong pills (concentrated pills)), Mailuoshutong granules and Mailuoshutong granules (sugar-free type and other dosage forms, wherein the prescription is prepared by refining twelve traditional Chinese medicines of astragalus, honeysuckle, golden cypress, rhizoma atractylodis, coix seeds, figwort roots, angelica sinensis, white paeony roots, liquorice, leeches, centipedes and scorpions, has the effects of clearing heat and detoxicating, removing blood stasis and dredging collaterals, and eliminating dampness and reducing swelling, and is mainly used for treating thrombotic superficial phlebitis caused by damp-heat stasis in venation, and lower limb swelling, pain and dull red skin color or with stringy objects caused by non-acute deep vein thrombosis.
The prescription of the Mailusutong homologous product is derived from clinical proved formulas of Chinese medical master and mr. from Tang ancestor, is derived by combining the prescription of angelica sinensis blood-enriching decoction, peony and licorice decoction, Simiao Yongan decoction, antispasmodic powder and the like on the basis of ancestral proved formulas, inherits the advantages of all the prescriptions, makes up the defects of the original prescription, brings out the best in each other and gives consideration to both the left and the right. The formula takes the astragalus and the honeysuckle as monarch drugs, and has the effects of tonifying qi, promoting diuresis, clearing away heat and toxic materials, purging pathogenic fire from yin, relieving muscle toxicity, removing dirt and treating accumulated toxin; cortex phellodendri, rhizoma atractylodis, semen coicis, angelica sinensis, radix paeoniae alba and radix scrophulariae are ministerial drugs, and have the effects of clearing heat, eliminating dampness, enriching blood, nourishing blood, relieving spasm, harmonizing nutrient and relieving pain, softening hardness to dissipate stagnation, reducing swelling, detoxifying and smoothening veins; leeches, scorpios and centipedes are adjuvant drugs and have the effects of promoting blood circulation, removing blood stasis, counteracting toxic substances, dissipating binds, dredging collaterals and relieving pain; the liquorice is used as a guiding drug to coordinate the effects of the other drugs in the recipe. The medicines are compatible, the effects of clearing heat and removing toxicity, removing blood stasis and dredging collaterals, and inducing diuresis and reducing edema are achieved, the medicine is powerful, and a better effect is achieved.
The Mailuoshutong pills (concentrated pills) obtain a medicine registration lot in 2009, the Mailuoshutong granules obtain a new medicine certificate (third class of traditional Chinese medicines) in 1998, and the Mailuoshutong granules (sugar-free type) obtain a medicine supplement application lot in 2012, are exclusively produced in China, and have independent intellectual property rights of core technologies such as prescriptions, processes, purposes and the like. The product fills the market blank of the traditional Chinese medicine for treating damp-heat stasis vein at home and abroad, is the first Chinese patent medicine approved for the targeted treatment of superficial thrombophlebitis, is still the only medicine approved for the symptomatic treatment of superficial thrombophlebitis in the domestic medicine market at present, is suitable for the long-term treatment of superficial thrombophlebitis, has definite curative effect and is well-received by doctors and patients in the clinical application, and plays an important role in treating clinical venous thrombosis and superficial thrombophlebitis.
The quality control method of the Mailuoshutong pill in the first tonic book of the 'Chinese pharmacopoeia' 2015 edition executed in 1 month and 1 day in 2019 mainly comprises identification and content measurement, wherein the identification item adopts a microscopic identification method to identify scorpion and centipedes in a preparation, and adopts a thin-layer chromatography method to respectively identify medicinal materials such as angelica sinensis, phellodendron amurense, figwort root and leech in the preparation, and the content measurement item adopts HPLC to measure the content of astragaloside in astragalus and the content of chlorogenic acid in honeysuckle. The prescription composition shows that the Mailuoshutong pill is prepared from 12 traditional Chinese medicines of astragalus, honeysuckle, phellodendron, rhizoma atractylodis, semen coicis, radix scrophulariae, angelica, white paeony root, liquorice, leech, centipede and scorpion, but the quality standard of the Mailuoshutong pill only identifies a plurality of raw medicinal materials by a microscopic identification method or a thin-layer chromatography, and the content of one index component in the astragalus and the honeysuckle is detected by HPLC (high performance liquid chromatography), so that the quality of the Mailuoshutong pill is controlled to be incomplete and accurate.
The traditional Chinese medicine fingerprint spectrum is used as a quality control technology, can comprehensively control the quality of the medicine, and has the characteristics of systematicness and integrity. At present, the quality of the Mailuoshutong pills is controlled by fingerprint, and no patent publication and literature report exists at home and abroad. The invention discloses a method for establishing an HPLC fingerprint of a Mailuoshutong pill and an HPLC standard fingerprint of the Mailuoshutong pill prepared by the method. Under the condition of evaluating the similarity, the quality of the Mailuoshutong pill can be comprehensively evaluated and controlled by using the standard fingerprint spectrum, so that the stability of the product quality and the effectiveness and the safety of clinical medication are ensured.
Disclosure of Invention
The invention aims to provide a method for establishing an HPLC fingerprint of a Mailuoshutong pill and an HPLC standard fingerprint obtained by the method, aiming at the defects of the existing quality control method of the Mailuoshutong pill. The method is characterized in that the Mailuoshutong pills are prepared into a test solution, and HPLC separation detection is carried out to obtain the Mailuoshutong pill HPLC standard fingerprint, so that reliable basis is provided for authenticity identification and internal quality of the Mailuoshutong pills.
The invention relates to a method for establishing an HPLC fingerprint of a Mailuoshutong pill, which comprises the following steps:
1) preparing a test solution: adding methanol into the vein dredging pill, performing ultrasonic treatment, cooling, filtering, and collecting the filtrate to obtain a sample solution;
2) carrying out HPLC chromatographic analysis on the test solution, and recording a chromatogram;
3) and (5) standard fingerprint spectrum preparation.
Preferably, 1) preparation of a test solution: placing the Mailuoshutong pill in a conical bottle with a plug, adding 40-70% methanol, performing ultrasonic treatment for 20-50 min, cooling, filtering, and collecting the filtrate to obtain a test solution;
2) HPLC chromatographic conditions: precisely sucking a test solution, and separating and detecting by HPLC (high performance liquid chromatography), wherein the mobile phase comprises acetonitrile: 0.05 to 0.2 percent of phosphoric acid solution, and gradient elution is adopted;
3) making a fingerprint spectrum: analyzing the Mailuoshutong pill sample according to the chromatographic conditions in the step 2) to obtain an HPLC chromatogram, and comparing the chromatogram to obtain the Mailuoshutong pill HPLC standard fingerprint consisting of the common characteristic peaks of the sample.
Further preferably, the test solution is prepared by the following steps: precisely weighing 1.0g of MAILUOSHUTONG pill, placing into a conical flask with a plug, precisely adding 25ml of 50% methanol, ultrasonic treating for 40min, cooling, filtering, and collecting the filtrate to obtain the sample solution.
The HPLC chromatographic conditions are as follows: kromasil C18A column, wherein the grain diameter of a filler is 5 mu m, the length of the column is 250mm, and the inner diameter of the column is 4.6 mm; mobile phase: acetonitrile is used as a mobile phase A, 0.1% phosphoric acid solution is used as a mobile phase B, and a gradient elution mode is adopted:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-40 | 5％→20％ | 95％→80％ |
| 40-60 | 20％→40％ | 80％→60％ |
Detection wavelength: 224 nm; flow rate: 1 ml/min; column temperature: at 30 ℃.
According to the chromatographic conditions, 2 or more batches of Mailuoshutong pill samples are analyzed, in the invention, 12 batches of Mailuoshutong pill samples are analyzed to obtain HPLC chromatograms of 12 batches of samples, and the chromatograms of 12 batches of samples are analyzed and compared to obtain the Mailuoshutong pill HPLC standard fingerprint formed by common characteristic peaks of the 12 batches of samples.
The invention also provides a Mailuoshutong pill HPLC standard fingerprint obtained by the method, which comprises the specific steps of preparing 12 batches of Mailuoshutong pill samples into a test solution according to the method, separating and detecting by HPLC, and analyzing by using software recommended by the State pharmacopoeia Committee, namely 'Chinese medicine chromatogram fingerprint similarity evaluation system 2004 edition', to obtain the Mailuoshutong pill HPLC standard fingerprint.
The Mailuoshutong pill has 25 common peaks in HPLC standard fingerprint spectrum and relative retention time tRSequentially and respectively: 0.119,0.131,0.144,0.188,0.321,0.671,0.801,1.000,1.051,1.073,1.206,1.280,1.349,1.398,1.497,1.529,2.044,2.168,2.241,2.313,2.387,2.404,2.680,2.744,2.790.
Compared with the prior art, the technical scheme of the invention has unexpected technical effects. The HPLC fingerprint spectrum establishing method of the Mailuoshutong pill provided by the invention has high precision and good repeatability, can perform overall process evaluation on the quality of the Mailuoshutong pill by comparing the existence of common peaks in the obtained fingerprint spectrum, effectively ensures the quality of finished products, and can overcome the defects that the prior art has single detection index and cannot reflect the inherent quality. In addition, each characteristic chromatographic peak under the chromatographic condition of the invention realizes good baseline separation, has good stability and more characteristic peaks, can comprehensively and accurately evaluate the quality of the Mailuoshutong pills, and is suitable for identifying the authenticity of the Mailuoshutong pills and controlling the product quality.
Drawings
FIG. 1 is HPLC standard fingerprint of Mailuoshutong pill (25 common peaks in 1-25);
FIG. 2 is an overlay of HPLC fingerprints of 12 batches of Mailushutong pills.
Detailed Description
The invention is further illustrated by the following specific examples.
Example 1 establishment of HPLC standard fingerprint of Mailuoshutong pill
1 Instrument and reagent
1.1 instruments
Agilent 1100 high performance liquid chromatograph (usa): DAD detector, quaternary low pressure gradient pump, AgilentOpen Lab chromatographic workstation.
1.2 reagent
The mailuoshutong pills are provided by the pharmaceutical limited, wannan pachu, see table 1; acetonitrile is chromatographically pure, water is double distilled water, and the rest reagents are analytically pure.
TABLE 1 Mailuoshutong pill test sample batch number
2 methods and results
2.1 chromatographic conditions: a chromatographic column: tianjin Denna Kromasil C18(4.6x250mm, 5um) column; mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume percentage content of 0.1 percent is used as a mobile phase B, and gradient elution is carried out according to the following table:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-40 | 5％→20％ | 95％→80％ |
| 40-60 | 20％→40％ | 80％→60％ |
Detection wavelength: 224 nm; flow rate: 1 ml/min; column temperature: 30 ℃; sample introduction volume: 10 μ l.
2.2 preparation of test solution: precisely weighing 1.0g of MAILUOSHUTONG pill, placing into a conical flask with a plug, precisely adding 25ml of 50% methanol, ultrasonic treating for 40min, cooling, filtering, and collecting the filtrate to obtain the sample solution.
2.3 creation of fingerprint
Measuring HPLC finger prints of 12 batches of MAILUOSHUTONG pill, analyzing and comparing to obtain standard HPLC finger prints of MAILUOSHUTONG pill (see figures 1 and 2), wherein the standard finger print has 25 common peaks and relative retention time t (with chlorogenic acid peak number 8 as reference peak) calculated by calculationRRespectively: 0.119,0.131,0.144,0.188,0.321,0.671,0.801,1.000,1.051,1.073,1.206,1.280,1.349,1.398,1.497,1.529,2.044,2.168,2.241,2.313,2.387,2.404,2.680,2.744,2.790. Wherein, the No. 8 peak is chlorogenic acid, the No. 15 peak is paeoniflorin, and the No. 23 peak is berberine.
Introducing HPLC fingerprints of 12 batches of the Mailuoshutong pills into software recommended by pharmacopoeia committee, namely 2004 edition of Chinese medicinal chromatographic fingerprint similarity evaluation system, for chromatographic peak matching, determining 25 common peaks as characteristic peaks forming the Mailuoshutong pills by taking a No. 8 chlorogenic acid peak as reference, wherein the relative retention time of the common peaks of the samples is shown in a table 2, and the relative peak area of the common peaks of 20 batches of the Mailuoshutong pills is shown in a table 3. The similarity calculation results of 12 batches of the Mailuoshutong pills and the standard fingerprint spectrum sequentially comprise: 0.987,0.992,0.978,0.993,0.976,0.989,0.997,0.969,0.972,0.995,0.996,0.986.
TABLE 212 relative retention time (t) of common peaks of MAILUOSHUTONG pills in batchesR)
Relative peak area (S) of common peaks of 312 Mailuoshutong pills in Table
2.4 methodological considerations
2.4.1 precision test
07217001 batches of samples are taken, a sample solution to be tested is prepared according to the method under item 2.2, continuous sample injection is carried out for 6 times, the No. 8 peak is taken as a reference peak, the relative retention time of the No. 1 to No. 25 common peak and the RSD value of the relative peak area are both calculated to be less than 3 percent, and simultaneously, similarity evaluation software is used for calculating that the similarity of each chromatogram fingerprint is greater than 0.99, which indicates that the instrument is stable and has good precision.
2.4.2 stability test
07217001 batches of samples are taken, a sample solution to be tested is prepared according to the method under item 2.2, sample injection is carried out for 0 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours respectively, the No. 8 peak is taken as a reference peak, the relative retention time of the No. 1-25 common peak and the RSD value of the relative peak area are both calculated to be less than 3 percent, and simultaneously, similarity of each spectrum fingerprint is calculated to be more than 0.99 by using similarity evaluation software, which indicates that the sample solution is stable within 24 hours.
2.4.3 reproducibility test
07217001 batches of samples are taken, 6 parts of samples are respectively and precisely weighed, a sample solution is prepared according to the method under item 2.2, samples are respectively injected, the relative retention time of the No. 1-25 common peak and the RSD value of the relative peak area are calculated to be less than 3% by taking the No. 8 peak as a reference peak, and simultaneously, similarity of each chromatogram fingerprint is calculated to be more than 0.99 by using similarity evaluation software, which indicates that the method has good reproducibility.
Example 2 establishment of HPLC standard fingerprint of Mailuoshutong pill
1 Instrument and reagent
1.1 instruments
Agilent 1100 high performance liquid chromatograph (usa): DAD detector, quaternary low pressure gradient pump, AgilentOpen Lab chromatographic workstation.
1.2 reagent
The mailuoshutong pills were provided by the lunnan pach pharmaceuticals limited, see table 4; acetonitrile is chromatographically pure, water is double distilled water, and the rest reagents are analytically pure.
TABLE 4 MAILUOSHUTONG pill test sample batch number
2 methods and results
2.1 chromatographic conditions: a chromatographic column: tianjin Denna Kromasil C18(4.6x250mm, 5um) column; mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume percentage content of 0.05 percent is used as a mobile phase B, and gradient elution is carried out according to the following table:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-40 | 5％→20％ | 95％→80％ |
| 40-60 | 20％→40％ | 80％→60％ |
Detection wavelength: 224 nm; flow rate: 1 ml/min; column temperature: 30 ℃; sample introduction volume: 10 μ l.
2.2 preparation of test solution: precisely weighing 1.0g of MAILUOSHUTONG pill, placing into a conical flask with a plug, precisely adding 25ml of 40% methanol, ultrasonically treating for 20min, cooling, filtering, and collecting the filtrate to obtain the sample solution.
2.3 creation of fingerprint
Determination of 12 batches of venationHPLC fingerprint of SHUTONG pill, and analyzing and comparing to obtain standard HPLC fingerprint composed of common characteristic peaks, wherein the standard fingerprint has 25 common peaks and relative retention time tRRespectively: 0.120,0.132,0.143,0.187,0.320,0.672,0.799,1.000,1.052,1.074,1.207,1.281,1.350,1.399,1.496,1.528,2.043,2.167,2.240,2.312,2.386,2.403,2.680,2.743,2.791. Wherein, the No. 8 peak is chlorogenic acid, the No. 15 peak is paeoniflorin, and the No. 23 peak is berberine.
Example 3 establishment of HPLC Standard fingerprint of Mailuoshutong pill
1 Instrument and reagent
1.1 instruments
Agilent 1100 high performance liquid chromatograph (usa): DAD detector, quaternary low pressure gradient pump, AgilentOpen Lab chromatographic workstation.
1.2 reagent
The mailuoshutong pills are provided by the lunan pachu pharmaceuticals limited, see table 5; acetonitrile is chromatographically pure, water is double distilled water, and the rest reagents are analytically pure.
TABLE 5 MAILUOSHUTONG pill test sample batch number
2 methods and results
2.1 chromatographic conditions: a chromatographic column: tianjin Denna Kromasil C18(4.6x250mm, 5um) column; mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume percentage content of 0.2 percent is used as a mobile phase B, and gradient elution is carried out according to the following table:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-40 | 5％→20％ | 95％→80％ |
| 40-60 | 20％→40％ | 80％→60％ |
Detection wavelength: 224 nm; flow rate: 1 ml/min; column temperature: 30 ℃; sample introduction volume: 10 μ l.
2.2 preparation of test solution: precisely weighing 1.0g of MAILUOSHUTONG pill, placing into a conical flask with a plug, precisely adding 25ml of 70% methanol, ultrasonically treating for 50min, cooling, filtering, and collecting the filtrate to obtain the sample solution.
2.3 creation of fingerprint
Measuring HPLC fingerprints of 12 batches of Mailuoshutong pills, analyzing and comparing to obtain the HPLC standard fingerprint of Mailuoshutong pills consisting of common characteristic peaks, wherein the relative retention time t of 25 common peaks of the standard fingerprint is calculated by taking the chlorogenic acid peak No. 8 as a reference peakRRespectively: 0.118,0.129,0.144,0.187,0.320,0.669,0.803,1.000,1.054,1.076,1.205,1.281,1.350,1.399,1.496,1.528,2.045,2.169,2.240,2.312,2.386,2.404,2.681,2.742,2.789. Wherein, the No. 8 peak is chlorogenic acid, the No. 15 peak is paeoniflorin, and the No. 23 peak is berberine.
Example 4 establishment of HPLC Standard fingerprint of Mailuoshutong pill
1 Instrument and reagent
1.1 instruments
Agilent 1100 high performance liquid chromatograph (usa): DAD detector, quaternary low pressure gradient pump, AgilentOpen Lab chromatographic workstation.
1.2 reagent
The mailuoshutong pills are provided by lunan pachu pharmaceuticals, ltd, see table 6; acetonitrile is chromatographically pure, water is double distilled water, and the rest reagents are analytically pure.
TABLE 6 MAILUOSHUTONG pill test sample batch number
2 methods and results
2.1 chromatographic conditions: a chromatographic column: tianjin Denna Kromasil C18(4.6x250mm, 5um) column; mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume percentage content of 0.17 percent is used as a mobile phase B, and gradient elution is carried out according to the following table:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-40 | 5％→20％ | 95％→80％ |
| 40-60 | 20％→40％ | 80％→60％ |
Detection wavelength: 224 nm; flow rate: 1 ml/min; column temperature: 30 ℃; sample introduction volume: 10 μ l.
2.2 preparation of test solution: precisely weighing 1.0g of MAILUOSHUTONG pill, placing into a conical flask with a plug, precisely adding 25ml of 55% methanol, ultrasonically treating for 36min, cooling, filtering, and collecting the filtrate to obtain the sample solution.
2.3 creation of fingerprint
Measuring HPLC fingerprints of 12 batches of Mailuoshutong pills, analyzing and comparing to obtain the HPLC standard fingerprint of Mailuoshutong pills consisting of common characteristic peaks, wherein the relative retention time t of 25 common peaks of the standard fingerprint is calculated by taking the chlorogenic acid peak No. 8 as a reference peakRRespectively: 0.119,0.130,0.146,0.187,0.320,0.670,0.803,1.000,1.048,1.072,1.205,1.281,1.350,1.399,1.496,1.527,2.043,2.165,2.240,2.311,2.385,2.402,2.679,2.742,2.789. Wherein, the No. 8 peak is chlorogenic acid, the No. 15 peak is paeoniflorin, and the No. 23 peak is berberine.
Example 5 establishment of HPLC Standard fingerprint of Mailuoshutong pill
1 Instrument and reagent
1.1 instruments
Agilent 1100 high performance liquid chromatograph (usa): DAD detector, quaternary low pressure gradient pump, AgilentOpen Lab chromatographic workstation.
1.2 reagent
The mailuoshutong pills were provided by the lunnan pach pharmaceuticals limited, see table 7; acetonitrile is chromatographically pure, water is double distilled water, and the rest reagents are analytically pure.
TABLE 7 MAILUOSHUTONG pill test sample batch number
2 methods and results
2.1 chromatographic conditions: a chromatographic column: tianjin Denna Kromasil C18(4.6x250mm, 5um) column; mobile phase: acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume percentage content of 0.14 percent is used as a mobile phase B, and gradient elution is carried out according to the following table:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-40 | 5％→20％ | 95％→80％ |
| 40-60 | 20％→40％ | 80％→60％ |
Detection wavelength: 224 nm; flow rate: 1 ml/min; column temperature: 30 ℃; sample introduction volume: 10 μ l.
2.2 preparation of test solution: precisely weighing 1.0g of MAILUOSHUTONG pill, placing into a conical flask with a plug, precisely adding 25ml of 62% methanol, ultrasonically treating for 45min, cooling, filtering, and collecting the filtrate to obtain the sample solution.
2.3 creation of fingerprint
Measuring HPLC fingerprints of 12 batches of Mailuoshutong pills, analyzing and comparing to obtain the HPLC standard fingerprint of Mailuoshutong pills consisting of common characteristic peaks, wherein the relative retention time t of 25 common peaks of the standard fingerprint is calculated by taking the chlorogenic acid peak No. 8 as a reference peakRRespectively: 0.117,0.130,0.142,0.189,0.322,0.672,0.803,1.000,1.054,1.074,1.205,1.279,1.348,1.397,1.496,1.527,2.046,2.167,2.243,2.312,2.386,2.402,2.682,2.742,2.788. Wherein, the No. 8 peak is chlorogenic acid, the No. 15 peak is paeoniflorin, and the No. 23 peak is berberine.
The foregoing is merely a preferred embodiment of the invention, which is described in some detail and with some particularity, and is not to be construed as limiting the scope of the invention. It should be noted that various modifications and improvements can be made by those skilled in the art without departing from the principle and spirit of the invention, and these modifications and improvements should also fall into the scope of the invention.
Claims (9)
1. A method for establishing an HPLC fingerprint of a Mailuoshutong pill is characterized by comprising the following steps:
1) preparing a test solution: adding methanol into the vein dredging pill, performing ultrasonic treatment, cooling, filtering, and collecting the filtrate to obtain a sample solution;
2) carrying out HPLC chromatographic analysis on the test solution, and recording a chromatogram;
3) and (5) standard fingerprint spectrum preparation.
2. The method of claim 1,
step 1) preparation of a test solution: adding 40-70% methanol into the Mailuoshutong pill, performing ultrasonic treatment for 20-50 min, cooling, filtering, and collecting filtrate to obtain test solution;
step 2) HPLC analysis, the chromatographic conditions are as follows: kromasil C18The column, acetonitrile is mobile phase A, 0.05% -0.2% phosphoric acid solution is mobile phase B, adopt the gradient elution mode to elute, detect the wavelength: 224nm, flow rate: 1.0ml/min, column temperature: 30 ℃;
step 3), standard fingerprint spectrum making: analyzing and comparing the Mailuoshutong pill test solution according to the chromatographic conditions in the step 2) to obtain a Mailuoshutong pill HPLC standard fingerprint spectrum formed by common characteristic peaks of the samples.
3. The method of claim 2, wherein the test solution of step 1) is prepared by the steps of: precisely weighing the Mailuoshutong pill, precisely adding 50% methanol, performing ultrasonic treatment for 40min, cooling, filtering, and collecting the filtrate to obtain the test solution.
4. The method of claim 2, wherein the mobile phase B of step 2) is a 0.1% phosphoric acid solution.
5. The method of claim 2, wherein step 2) the gradient elution is:
6. the method of any one of claims 1-5, wherein the Mailuoshutong pill HPLC standard fingerprint is subjected to similarity evaluation using software of traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2004 edition.
7. The method of any one of claims 1-5, wherein 2 or more of the choroid dredging pellets are taken to prepare the test solution according to step 1), the HPLC chromatographic analysis is performed according to step 2), and the standard fingerprint is prepared according to step 3).
8. A standard fingerprint created by the method of any one of claims 1 to 5 wherein the standard fingerprint has 25 peaks in common.
9. The standard fingerprint of claim 8 wherein the relative retention time t of the peaksRSequentially and respectively: 0.119,0.131,0.144,0.188,0.321,0.671,0.801,1.000,1.051,1.073,1.206,1.280,1.349,1.398,1.497,1.529,2.044,2.168,2.241,2.313,2.387,2.404,2.680,2.744,2.790.
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| CN107655989A (en) * | 2017-09-18 | 2018-02-02 | 鲁南制药集团股份有限公司 | The method for building up and its standard diagram of Yinzhihuang" granula HPLC finger-prints |
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2018
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| CN107655989A (en) * | 2017-09-18 | 2018-02-02 | 鲁南制药集团股份有限公司 | The method for building up and its standard diagram of Yinzhihuang" granula HPLC finger-prints |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115184490A (en) * | 2022-06-28 | 2022-10-14 | 鲁南制药集团股份有限公司 | Establishment method and application of HPLC standard fingerprint of leech and leech collateral-dredging capsule |
| CN115184490B (en) * | 2022-06-28 | 2023-10-24 | 鲁南制药集团股份有限公司 | Establishment method and application of HPLC standard fingerprint of Hirudo vein relaxing capsule |
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