阅读说明：本技术 脉络舒通丸hplc指纹图谱的建立方法及其标准图谱 (Method for establishing HPLC fingerprint of Mailuoshutong pill and standard spectrum thereof ) 是由 刘武占 庄会芳 于 2018-08-24 设计创作，主要内容包括：本发明公开了一种脉络舒通丸HPLC指纹图谱的建立方法,该方法包括供试品溶液的制备、HPLC色谱条件的确定及HPLC标准指纹图谱的制作。本发明同时公开了由该方法得到的脉络舒通丸HPLC标准指纹图谱,该指纹图谱有25个共有峰。本发明质量检测方法方法操作简便、稳定性高、重现性好,所得图谱特征峰多,通过标准指纹图谱共有峰的比较,可对脉络舒通丸的质量进行全面评价,有利于稳定产品质量,确保临床用药的安全性、有效性。(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.)

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 peaks_RSequentially 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.

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 t_RSequentially 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.