CN113960184A - Establishment and quality detection method of blood-replenishing and motherwort-tonifying pill fingerprint spectrum - Google Patents

Establishment and quality detection method of blood-replenishing and motherwort-tonifying pill fingerprint spectrum Download PDF

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CN113960184A
CN113960184A CN202110162382.8A CN202110162382A CN113960184A CN 113960184 A CN113960184 A CN 113960184A CN 202110162382 A CN202110162382 A CN 202110162382A CN 113960184 A CN113960184 A CN 113960184A
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motherwort
blood
pill
fingerprint
enriching
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龚云
吴梦瑶
邹亮
王安齐
张艳
李维
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Qianjin Pharmaceutical Co ltd
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    • 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
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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
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • 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
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • G01N30/8637—Peak shape
    • 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
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components

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Abstract

The invention discloses a method for establishing a fingerprint spectrum of a blood-enriching motherwort pill and detecting the quality of the blood-enriching motherwort pill. The method for establishing the fingerprint of the blood-enriching motherwort pill comprises the preparation of a reference solution and a test solution, the establishment of chromatographic conditions and HPLC detection. The fingerprint spectrum established by the invention can comprehensively reflect the chemical components in the blood-replenishing motherwort pill, and has the advantages of good separation of various spectrum peaks, stable baseline, good peak pattern, good repeatability, good linear relation, precision, stability and recovery rate, and can accurately detect the content of index components in the blood-replenishing motherwort pill, the quality of a sample of the blood-replenishing motherwort pill can be objectively evaluated by the obtained control fingerprint spectrum, and the research standard of quality control is provided for the blood-replenishing motherwort pill so as to ensure the quality and clinical curative effect of the product.

Description

Establishment and quality detection method of blood-replenishing and motherwort-tonifying pill fingerprint spectrum
Technical Field
The invention relates to the field of traditional Chinese medicine analysis and detection, and more particularly relates to a method for establishing a fingerprint spectrum of a blood-enriching motherwort pill and detecting the quality of the blood-enriching motherwort pill.
Background
Pelvic spasm often accompanies with symptoms such as hemorrhage, mood fluctuation, debilitation, etc., and is prone to cause secondary diseases such as anemia, ischemia, etc. Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX-2) inhibitors, such as ibuprofen, naproxen and the like, are commonly used clinically to treat gynecological inflammation, and although the drugs can relieve dysmenorrhea, the drugs are easy to cause severe adverse reactions such as nausea, peptic ulcer, diarrhea and the like. The blood-enriching motherwort pill is a Chinese patent medicine developed and produced in the pharmaceutical industry of Qianjin, shouzhou, Hunan province. The compound is composed of five medicines of angelica, motherwort, astragalus, donkey-hide gelatin and dried orange peel, wherein the angelica-motherwort is the core medicine. The Chinese angelica root-motherwort herb is a classic qi and blood tonifying medicine pair, has unique compatibility significance and is used up to now. Many clinical proved formulas such as blood-enriching motherwort pill/particle, eight-treasure motherwort pill, four-ingredient motherwort pill and the like are prepared by taking the medicine pair as a basic formula. The blood-enriching motherwort pill is widely applied clinically and obtains good curative effect, and clinical observation and pharmacological research find that the pill can promote blood circulation of uterus, improve tolerance of endometrium and obviously improve pregnancy rate by enhancing uterine blood flow supply and endometrium thickness.
At present, in the research literature on the quality control of the blood-enriching motherwort preparation, mass spectrometry is adopted to detect donkey-hide gelatin components (chenhongyu, liwenli, lijinping, and the like) in the blood-enriching motherwort pill, ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is adopted to detect donkey-hide gelatin [ J ] in the blood-enriching motherwort pill, 2016,014(005):540-, the quality control standard is single, and the method is difficult to be used as a comprehensive quality control method of the blood-enriching motherwort preparation. At present, no relevant report exists on a fingerprint spectrum establishment method of the blood-enriching motherwort pill and detection of various index components of the blood-enriching motherwort pill. Therefore, in order to better control the quality of the medicine and ensure the clinical curative effect of the medicine, a method for comprehensively evaluating the quality of the preparation needs to be established, so that the quality control method of the blood-enriching motherwort-tonifying pill has important practical significance.
Disclosure of Invention
The invention aims to provide a method for establishing a fingerprint spectrum of a blood-replenishing motherwort pill and detecting the quality of the blood-replenishing motherwort preparation, aiming at solving the problems that in the prior art, the quality detection method of the blood-replenishing motherwort preparation is not comprehensive enough, and the quality control standard is single and relatively weak, so that the quality of the blood-replenishing motherwort preparation is difficult to accurately evaluate in the market.
The invention aims to provide a method for establishing a fingerprint of a blood-enriching motherwort pill.
The invention further aims to provide a quality detection method of the blood-enriching motherwort-benefiting pills.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides a method for establishing a fingerprint of a blood-enriching motherwort pill, which comprises the following steps:
s1, preparation of a reference substance solution: preparing leonurine, calycosin glucoside, rutin, ferulic acid, and ligustilide as reference substances into mixed reference substance solution;
s2, preparation of a test solution: taking the blood-replenishing motherwort pills, crushing, extracting with acetonitrile with the volume percentage of 75-85%, and preparing a test solution with the mass concentration of 0.05-0.3 g/mL of the blood-replenishing motherwort pills;
s3, HPLC chromatographic conditions: adopting a C18 chromatographic column, wherein the column temperature is 30-35 ℃; the flow rate is 0.9-1.2 mL/min; the detection wavelength is 254-277 nm; acetonitrile is taken as a mobile phase A, formic acid aqueous solution with the volume percentage of 0.05-0.2% is taken as a mobile phase B, and in the gradient elution procedure, the volume percentage of the mobile phase B in a mobile phase system is changed as follows: 0-8 min, and 95% of mobile phase B; reducing the content of the mobile phase B from 95% to 10% in 8-60 min; reducing the content of the mobile phase B from 10% to 2% in 60-63 min; 63-66 min, the mobile phase B rises from 2% to 95%; 66-72 min, and 95% of mobile phase B;
s4, the detection method comprises the following steps: and injecting the mixed reference substance solution and the test solution into a liquid chromatograph, and measuring to obtain the blood-enriching motherwort pill fingerprint.
In the method for establishing the fingerprint, in order to make the operation simpler and more convenient and consume less time, and make each characteristic peak in the fingerprint have better separation degree and peak shape, and simultaneously, the detection time of the fingerprint needs to be reasonably controlled, and the method is very important for the preparation method of the test sample and the selection of gradient elution conditions. Meanwhile, because the chemical components in different Chinese medicinal materials are different, the extraction conditions of different medicinal materials and preparations and the elution conditions of the fingerprint have no reference value, the selection of the preparation conditions of the test sample not only influences the number of peaks of the fingerprint, but also has great influence on the time consumption of the whole fingerprint operation process, and small changes of the proportion of mobile phases can cause great influence on the fingerprint, such as the separation degree and the peak shape of characteristic peaks. Therefore, the inventor conducts a great deal of creative work on the research on the chromatographic conditions and the preparation method of the test sample, and finally determines the preparation method and the gradient elution conditions of the test sample.
Preferably, the volume percentage of acetonitrile in step S2 is 80%.
Preferably, the extraction in step S2 is ultrasonic extraction, and the extraction time is 60-90 min.
More preferably, the time of extraction is 60 min.
Preferably, the mass concentration of the blood-enriching motherwort pill in the test solution in step S2 is 0.05-0.1 g/mL.
More preferably, the mass concentration of the blood-enriching motherwort pill in the test solution in step S2 is 0.1 g/mL.
More preferably, the specific process for preparing the test solution is as follows: pulverizing the blood replenishing and motherwort benefiting pill, performing ultrasonic treatment with water for 10min, adding 80% acetonitrile, performing ultrasonic extraction for 60min, cooling, adding acetonitrile, supplementing weight loss, shaking, filtering with 0.22 μm microporous membrane, and collecting filtrate to obtain test solution.
Preferably, the chromatographic column in step S3 is Symmetry ShieldTMRP18 chromatographic column with column length of 150mm and inner partThe diameter is 3.9mm, and the particle diameter is 5 μm.
Preferably, the column temperature of step S3 is 35 ℃.
The flow rate in step S3 was 1.0 mL/min.
Preferably, the detection wavelength of step S3 is 254 nm.
Preferably, the mobile phase B in step S3 is 0.1% by volume of formic acid solution.
The fingerprint obtained by the method has 36 common peaks, 5 chromatographic peaks are identified according to a reference substance, namely a peak 1 (leonurine), a peak 2 (calycosin glucoside), a peak 3 (rutin), a peak 4 (ferulic acid) and a peak 5 (ligustilide), the relative retention time of the rest peaks is calculated by taking the peak 5 (ligustilide, the retention time is 47.654-47.804 min) as a reference peak, and the relative retention time ranges of the rest peaks are respectively as follows: peak 1 corresponding to leonurine: 0.398 to 0.399; peak No. 2 corresponding to calycosin glucoside: 0.531 to 0.532; peak No. 3 corresponding to rutin: 0.560 to 0.560; peak No. 4 corresponding to ferulic acid: 0.593 to 0.594; peak No. 5 corresponding to ligustilide: 1.000 to 1.000.
The invention also claims a quality detection method of the blood-replenishing and motherwort-benefiting pills, which comprises the following steps: and comparing the HPLC detection spectrum of the sample to be detected with the comparison fingerprint spectrum by using the fingerprint spectrum of the blood-enriching motherwort pill as the comparison fingerprint spectrum, and calculating according to the similarity evaluation system of the traditional Chinese medicine fingerprint spectrum, wherein if the similarity between the fingerprint spectrum of the sample and the comparison fingerprint spectrum is not less than 0.90, the sample is qualified.
Preferably, the leonurine content in the blood-enriching motherwort pill is not lower than 100 mu g/g, the calycosin glucoside content is not lower than 120 mu g/g, the rutin content is not lower than 60 mu g/g, the ferulic acid content is not lower than 150 mu g/g, and the ligustilide content is not lower than 1600 mu g/g in terms of dry products.
A method for measuring the content of index components of a blood-enriching motherwort-benefiting pill comprises the following steps:
s11, diluting the mass concentration of each reference substance in the step S1 by a series of multiples, measuring according to the chromatographic conditions in the step S3, and drawing a standard curve by taking the mass concentration of each reference substance as a horizontal coordinate and a peak area as a vertical coordinate to obtain a regression equation of each component reference substance;
s12, measuring the sample to be measured according to the chromatographic conditions in the step S3, recording the peak area, and substituting the peak area into the regression equation in the step S11 to calculate the content of the component to be measured.
The index components are leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide.
Preferably, the mass concentration of each control is diluted by 0, 2, 4, 8 and 16 times respectively.
Compared with the prior art, the invention has the following beneficial effects:
the method of the invention establishes the fingerprint of the blood-enriching motherwort pill under the same chromatographic condition, and simultaneously measures the content of 5 effective index components in the blood-enriching motherwort pill by combining a multi-index component content measuring method, and the method is simple and convenient and has dual functions of qualitative and quantitative.
The fingerprint measured by the establishing method can comprehensively reflect chemical components in the blood-replenishing motherwort pill, and has the advantages of good separation of various spectrum peaks, stable baseline, good peak type and good repeatability.
Drawings
FIG. 1 shows the effect of different extraction solvents (60% methanol, 80% acetonitrile) on the peak shape and resolution of a chromatographic sample during the preparation of a test solution.
FIG. 2 shows the effect of different mobile phase systems (acetonitrile-0.2% phosphoric acid in water, acetonitrile-0.2% formic acid in water, acetonitrile-0.1% formic acid in water, acetonitrile-0.05% formic acid in water) on the various peaks of the spectra.
FIG. 3 shows the effect of different detection wavelengths (235nm, 254nm, 277nm, 320nm) on the individual chromatographic peaks
FIG. 4 is the results of a systematic adaptation test of chromatographic conditions; wherein, (A) is the chromatogram of each single reference solution and mixed reference solution; (B) is a chromatogram of a mixed reference solution and a blood-enriching motherwort pill test solution.
FIG. 5 shows HPLC fingerprints and comparison fingerprint (R) of 10 blood-tonifying and motherwort-tonifying pills (S1-S10).
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The blood-enriching motherwort pill samples adopted in the following examples have 10 batches, and are all developed and produced by the company of Qianjin pharmaceutical industry, Japan, and the batches are 20200418(S1), 20200104(S2), 20200105 (S3), 20200202(S4), 20200205(S5), 20200306(S6), 20200307(S7), 20200308 (S8), 20200401(S9) and 20200417 (S10).
The experimental equipment and reagents used in the following examples are as follows:
shimadzu high performance liquid chromatograph SHIMADZU LC-16, PDA detector; waters Symmetry ShieldTMRP18 column (150 mm. times.3.9 mm, 5 μm); CPA225D electronic analytical balance (Sartorius, usa); JL-UPT-II-10T ultrapure water machine (Szechwan gold wave science and technology Co., Ltd.); SB-5200DT ultrasonic cleaning machine (Ningbo Xinzhi Biotech Co., Ltd.); the reference leonurine (batch number MUST-20032605), calycosin glucoside (batch number MUST-20031920), and ferulic acid (batch number MUST-20060511) were purchased from Goldmann Biotech limited; the reference rutin (batch number L-001-171216) and ligustilide (batch number G01001909022) are purchased from Donreisi Biotechnology Co., Ltd, and the purity of each reference is not less than 98%. Water is purified water, acetonitrile and methanol are chromatographic grade, and other reagents are analysisAnd (4) purifying.
Example 1 optimization of chromatographic conditions
In the establishing method of the fingerprint, because the chemical components in different Chinese medicinal materials are different, the elution conditions of the fingerprint of different Chinese medicinal materials and preparations have no reference value, and small changes of the proportion of mobile phases can cause great influence on the fingerprint, such as the separation degree and the peak shape of a characteristic peak. In order to make each characteristic peak in the fingerprint have better separation degree and peak shape, the selection of the gradient elution condition is crucial, and in this embodiment, the investigation and optimization are mainly performed on the mobile phase and the detection wavelength.
1. Selection of mobile phase
Taking a proper amount of the blood-replenishing leonurus pills, crushing, precisely weighing about 3.0g into a 50mL measuring flask, adding 10mL of water, performing ultrasonic treatment for 10min, then adding 40mL of acetonitrile, performing ultrasonic treatment for 30min, cooling, adding acetonitrile, fixing the volume to the scale, shaking up, sucking the solution, passing the solution through a 0.22 mu m microporous filter membrane, collecting the subsequent filtrate, performing sample detection on a high performance liquid chromatograph, eluting by 4 different mobile phases of acetonitrile-0.2% phosphoric acid aqueous solution, acetonitrile-0.2% formic acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution and acetonitrile-0.05% formic acid aqueous solution respectively, and inspecting the influence of the 4 different mobile phase systems on various spectral peaks.
As a result, as shown in fig. 2, when an acetonitrile-formic acid aqueous solution was used as a mobile phase, the chromatographic peak separation effect was the best, the number of detected peaks was large, and the peak signal intensity was high; further, as a result of examining the influence of formic acid aqueous solutions (0.2%, 0.1%, 0.05%) having different concentrations on chromatographic peak information, it was found that a preferable result was obtained with 0.2%, 0.1%, 0.05% formic acid aqueous solution as the mobile phase B, and therefore, the mobile phase B was selected to have an intermediate concentration of 0.1% formic acid aqueous solution, and gradient elution was performed using acetonitrile (a) -0.1% formic acid aqueous solution (B) as a mobile phase system, whereby the chromatographic peaks could be well separated.
2. Selection of detection wavelength
Taking a proper amount of the blood replenishing leonurus pills, crushing, precisely weighing about 3.0g into a 50mL measuring flask, adding 10mL of water for ultrasonic treatment for 10min, then adding 40mL of acetonitrile for ultrasonic treatment for 30min, cooling, adding acetonitrile for constant volume to scale, shaking up, sucking the solution through a 0.22 mu m microporous filter membrane, collecting the subsequent filtrate, carrying out sample detection on a high performance liquid chromatograph, carrying out detection at four different detection wavelengths of 235nm, 254nm, 277nm and 320nm respectively, and inspecting the influence of the different detection wavelengths on various spectral peaks.
As a result, as shown in FIG. 3, the detection wavelength of 254nm was selected because the peak shape, the resolution, and the number of peaks were superior under the condition of 254 nm.
3. Determination of chromatographic conditions
From the above observations, the preferred chromatographic conditions were determined to be: waters Symmetry ShieldTMRP18 chromatographic column (column length 150mm, inner diameter 3.9mm, particle size 5 μm); the mobile phase is acetonitrile (A) -0.1% formic acid water solution (B); in the gradient elution procedure, the volume percent change of mobile phase B was: 0-8 min, and 95% of mobile phase B; reducing the content of the mobile phase B from 95% to 10% in 8-60 min; reducing the content of the mobile phase B from 10% to 2% in 60-63 min; 63-66 min, the mobile phase B rises from 2% to 95%; 66-72 min, and 95% of mobile phase B; the column temperature was 35 ℃; the flow rate is 1.0 mL/min; the detection wavelength is 254 nm; the amount of sample was 10. mu.L.
Example 2 optimization of preparation method of blood-enriching motherwort pill test solution
In the method for establishing the fingerprint, in order to make the operation simpler and more convenient, consume less time, make each characteristic peak in the fingerprint have better resolution and peak shape, and simultaneously need reasonable control of the detection time of the fingerprint, the method is of great importance for the investigation and optimization of the preparation method of the test sample, and the method mainly performs the investigation and optimization on the amount of the extracted sample, the extraction solvent and the extraction time in the embodiment.
This optimization was tested under the chromatographic conditions determined in example 1.
1. Examination of the amount of sample extracted
Taking a proper amount of the blood replenishing leonurus pills, crushing, precisely weighing three different sample amounts of 3g, 1g and 0.5g respectively in a 50mL measuring flask, adding 10mL of water for ultrasonic treatment for 10min, then adding 40mL of acetonitrile for ultrasonic treatment for 30min, cooling, adding acetonitrile to a constant volume to scale, shaking up, sucking the solution through a 0.22 mu m microporous filter membrane, collecting subsequent filtrate, carrying out sample detection on a high performance liquid chromatograph, and inspecting the influence of the three different sample amounts of 3g, 1g and 0.5g on the content determination of the component to be detected in the sample.
The results are shown in table 1, according to the extraction content of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide in the sample, when the sample amount is 3g, the components in the blood-replenishing and motherwort-tonifying pill cannot be completely extracted, when the sample amount is 1g and 0.5g, the extraction is sufficient, the peak area of each component to be detected is comprehensively considered, and the preferable 1g is the extraction sample amount of the blood-replenishing and motherwort-tonifying pill.
Table 1. influence of different extracted sample amounts on peak areas and contents of 5 components in blood-enriching motherwort pill
Figure RE-GDA0003018596650000071
2. Examination of extraction solvent
Taking a proper amount of the blood-replenishing leonurus pills, crushing, precisely weighing about 1.0g into a 50mL measuring flask, respectively adding extraction solvents (60% methanol, 80% methanol and 80% acetonitrile) with different volume percentages, carrying out ultrasonic treatment for 30min, cooling, adding acetonitrile to a constant volume to reach a scale, shaking up, absorbing the solution, filtering through a 0.22 mu m microporous filter membrane, collecting subsequent filtrate, carrying out sample injection detection on a high performance liquid chromatograph, and inspecting the influence of three different extraction solvents, namely 60% methanol, 80% methanol and 80% acetonitrile, on various spectral peaks.
As shown in fig. 1, the baseline shift of the sample solution after extraction with methanol is relatively large, the peak shape and the separation degree of the chromatographic peak of each component to be detected are considered comprehensively, and acetonitrile with 80% volume percentage is preferably used as the extraction solvent of the blood-enriching motherwort pill sample solution.
3. Investigation of extraction time
Taking a proper amount of the blood-enriching motherwort pills, crushing, precisely weighing about 1.0g of the blood-enriching motherwort pills in a 50mL measuring flask, adding 10mL of water for ultrasonic treatment for 10min, then adding 40mL of acetonitrile for ultrasonic extraction for different extraction times (30min, 60min and 90min), cooling, adding acetonitrile for constant volume to a scale, shaking up, sucking the solution through a 0.22 mu m microporous filter membrane, collecting the subsequent filtrate, carrying out sample detection on a high performance liquid chromatograph, and inspecting the influence of three different extraction times of 30min, 60min and 90min on the content determination of the component to be detected in the sample.
The results are shown in table 2, and according to the extraction contents of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide in the sample, it is shown that the components in the blood-replenishing and motherwort pill cannot be completely extracted when the extraction time is 30min, the extraction is sufficient when the extraction time is 60min and 90min, the peak area of each component to be detected is comprehensively considered, and the preferred 60min is the extraction time of the test solution of the blood-replenishing and motherwort pill.
Table 2. influence of different extraction time on peak area and content of 5 components in blood-enriching motherwort pill
Figure RE-GDA0003018596650000081
3. Determination of preparation method of test solution
According to the above results, the preferable preparation method of the test solution is determined as follows: taking a proper amount of the blood-replenishing leonurus pills, crushing, precisely weighing 1.0g into a 50mL measuring flask, adding 10mL of water for ultrasonic treatment for 10min, then adding acetonitrile with the volume percentage of 80% for 40mL for ultrasonic treatment for 60min, cooling, adding acetonitrile to a constant volume to scale, shaking up, sucking the solution, filtering through a 0.22 mu m microporous filter membrane, and collecting the subsequent filtrate to obtain the test solution.
Example 3 methodological examination
Preparation of control solutions:
precisely weighing 7.96mg of leonurine, 13.03mg of calycosin glucoside, 3.53mg of rutin, 4.88mg of ferulic acid and 2.71mg of ligustilide reference substances, respectively placing the reference substances into 6 measuring bottles, and adding methanol to prepare single reference substance stock solutions with the mass concentrations of 796.0, 1303.0, 353.0, 488.0 and 2710.0 mu g/mL in sequence. And adding appropriate amount of the 5 reference substance stock solutions into a 10mL measuring flask to obtain mixed reference substance solutions with leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide mass concentrations of 7.96, 5.47, 35.30, 58.56 and 100.27 μ g/mL respectively.
Preparation of a test solution:
the blood-enriching motherwort pill test solution is prepared according to the preferable test solution preparation method determined in example 2.
1. System suitability test
And (3) detecting the single reference substance solution, the mixed reference substance solution and the blood-enriching motherwort pill test solution of each reference substance on a high performance liquid chromatograph according to the preferable chromatographic conditions determined in the example 1, and recording chromatograms.
As shown in FIG. 4, under the preferable chromatographic conditions and the method for preparing the test solution determined in examples 1 and 2, the chromatographic peaks of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide achieve ideal separation effects, the separation degree from the adjacent chromatographic peaks is more than 1.5, and the theoretical plate number is not less than 1 × 10 based on the measured component chromatographic peaks4. Indicating that the chromatographic system used for the analysis under the chromatographic conditions is effective and suitable.
2. Detection limit and quantitative limit determination
The mixed control solution was diluted stepwise, and the measurement was performed under the preferable chromatographic conditions as determined in example 1, and the concentration at a signal-to-noise ratio of about 3 was taken as the limit of detection (LOD) and the concentration at a signal-to-noise ratio of about 10 was taken as the limit of quantitation (LOQ).
As shown in Table 3, the quantitative limits of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide are 0.4975, 0.1712, 0.0182, 3.6600 and 0.6250 μ g/mL respectively, and the detection limits are 0.2488, 0.0171, 0.0091, 0.2288 and 0.1250 μ g/mL respectively.
TABLE 3.5 quantitative and detection limits for the ingredients
Figure RE-GDA0003018596650000091
3. Investigation of linear relationships
Taking the mixed reference substance solution, respectively diluting by 0, 2, 4, 8 and 16 times, determining according to the preferable chromatographic conditions determined in the example 1, and respectively recording the peak areas of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide; and drawing a standard curve by taking the reference mass concentration of each component as an abscissa (X) and taking the peak area as an ordinate (Y) to obtain a regression equation of each component.
The results are shown in Table 4, the linear correlation coefficients of 5 components of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide are good, the linear ranges are respectively 0.50-7.96 mu g/mL, 0.34-5.47 mu g/mL, 2.21-35.30 mu g/mL, 3.66-58.56 mu g/mL and 6.27-100.27 mu g/mL, and the linear correlation coefficients can be used for calculating the content of the 5 components in a sample to be detected.
TABLE 4.5 Linear relationship of ingredients
Figure RE-GDA0003018596650000101
4. Precision test
Taking the same batch of blood-replenishing motherwort pills (S1), preparing a blood-replenishing motherwort pill test solution according to the preferable test solution preparation method determined in the example 2, continuously feeding the samples for 6 times according to the preferable chromatographic conditions determined in the example 1, measuring peak areas of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide in the test solution, and calculating RSD.
As a result, the RSD values of the peak areas of the 5 components were calculated to be 0.94%, 1.40%, 1.30%, 1.49%, and 1.53%, respectively, as shown in Table 5, indicating that the precision of the apparatus was good.
5. Repeatability test
Taking the same batch of blood-replenishing motherwort pills (S1), preparing 6 parts of blood-replenishing motherwort pill test solution in parallel according to the preferable test solution preparation method determined in the example 2, respectively injecting samples according to the preferable chromatographic conditions determined in the example 2, measuring peak areas of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide in the test solution, and calculating RSD.
The results are shown in table 5, and the RSDs of the calculated peak areas of the 5 components were 0.76%, 1.44%, 1.43%, 1.40%, and 1.24%, respectively, indicating that the method is good in reproducibility.
6. Stability test
Taking the same batch of blood-replenishing motherwort pills (S1), preparing a test solution of the blood-replenishing motherwort pills according to the preferable preparation method of the test solution determined in the example 2, respectively placing the test solution for 0, 2, 4, 8, 12 and 24 hours, respectively injecting samples according to the chromatographic conditions of the example 1, measuring the peak areas of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide in the test solution, and calculating RSD.
As a result, the RSD values of the peak areas of the 5 components calculated as shown in Table 5 were 1.31%, 1.29%, 1.28%, 0.60%, and 0.30%, respectively, indicating that the sample solution was stable within 24 hours.
TABLE 5 precision, repeatability and stability of 5 ingredients in BUXUEYIMU pill
Figure RE-GDA0003018596650000111
7. Sample application recovery test
Taking 6 parts of the blood-replenishing and motherwort-benefiting pills (S1) of the same batch, precisely weighing about 0.5g respectively, placing the weighed materials into a 50mL measuring flask, adding 0.084g of leonurine, 0.130g of calycosin glucoside, 0.136g of rutin, 0.154g of ferulic acid and 1.504g of ligustilide reference substances respectively, preparing a blood-replenishing and motherwort-benefiting pill sample solution according to the preferable sample solution preparation method determined in the example 2, injecting samples according to the preferable chromatographic conditions determined in the example 1, determining peak areas of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide in the sample solution, and calculating the sample recovery rate and RSD.
The results are shown in table 6, the average sample recovery rates of the 5 index components are 92.0%, 92.5%, 90.8%, 91.9% and 99.0%, and the RSDs are 1.31%, 0.77%, 1.59% and 1.07%, respectively, which indicates that the method of the present invention is accurate and reliable.
TABLE 6 sample recovery rates of 5 ingredients in BUXUEYIMU pill
Figure RE-GDA0003018596650000121
Example 4 application of fingerprint of blood-enriching motherwort pill
1. Preparation of the solution
(1) Mixed control solutions
Precisely weighing 7.96mg of leonurine, 13.03mg of calycosin glucoside, 3.53mg of rutin, 4.88mg of ferulic acid and 2.71mg of ligustilide reference substances, respectively placing the reference substances into 6 measuring bottles, and adding methanol to prepare reference substance stock solutions with the mass concentrations of 796.0, 1303.0, 353.0, 488.0 and 2710.0 mu g/mL in sequence. Respectively adding appropriate amount of the above 5 reference substance stock solutions into 10mL measuring bottles to obtain mixed reference substance solutions with leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide mass concentrations of 7.96, 5.47, 35.30, 58.56 and 100.27 μ g/mL respectively.
(2) Test solution
10 batches (S1-S10) of the blood-enriching motherwort pill test solution were prepared according to the preferred test solution preparation method identified in example 2.
2. Determination of common peaks
HPLC detection is carried out on 10 batches of the blood-enriching motherwort pill test solution according to the determined and preferred chromatographic conditions in example 1, and an HPLC chromatogram of each sample is recorded and compared with a control sample.
The chromatogram is shown in fig. 5, 36 common peaks are obtained in the fingerprint of 10 batches of blood-replenishing motherwort pills, and compared with a reference substance, the common peaks of 5 index components are identified and respectively named as No. 1 peak (leonurine), No. 2 peak (calycosin glucoside), No. 3 peak (rutin), No. 4 peak (ferulic acid) and No. 5 peak (ligustilide) according to retention time; taking the peak No. 5 (ligustilide) as a reference peak, defining the ratio of the retention time of the fingerprint spectrum common peak of the other index components to the retention time of the reference peak as the relative retention time of the other peaks, and calculating the relative retention time of the fingerprint spectrum common peak of the index components of 10 batches of the blood-enriching motherwort pill and the RSD value thereof, and the results are shown in Table 7.
The relative retention times of the common peaks of the 5 index constituents were: and (3) taking the No. 5 peak (ligustilide, with the retention time of 47.654-47.804 min) as a reference peak, respectively calculating the relative retention time of the rest peaks, wherein the relative retention time ranges of the rest peaks are respectively as follows: peak 1 corresponding to leonurine: 0.398 to 0.399; peak No. 2 corresponding to calycosin glucoside: 0.531 to 0.532; peak No. 3 corresponding to rutin: 0.560 to 0.560; peak No. 4 corresponding to ferulic acid: 0.593 to 0.594; peak No. 5 corresponding to ligustilide: 1.000 to 1.000.
TABLE 7.10 relative retention time of fingerprint spectrum common peak of blood-replenishing and motherwort-tonifying pill batches and its RSD value
Figure RE-GDA0003018596650000131
Figure RE-GDA0003018596650000141
Figure RE-GDA0003018596650000151
4. Evaluation of similarity
The fingerprints of 10 batches of the blood-replenishing and motherwort-tonifying pills are shown in fig. 5, a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012A) is adopted, an S1 chromatogram is set as a reference chromatogram, a median method is utilized, the time window width is set for 0.1min, similarity calculation is carried out, the result is shown in table 8, the similarity of 10 batches of the blood-replenishing and motherwort-tonifying pills is greater than 0.96, the similarity of 10 batches of the blood-replenishing and motherwort-tonifying pills is good, and the established HPLC fingerprint of the blood-replenishing and motherwort-tonifying pills is reasonable and accurate.
TABLE 8 similarity of fingerprint of BUXUEYIMU pill
Figure RE-GDA0003018596650000152
5. Quality control method of blood-enriching motherwort-benefiting pills
And comparing the HPLC detection spectrum of the sample to be detected with the comparison fingerprint spectrum by taking the fingerprint spectrum of the blood-enriching motherwort pill as the comparison fingerprint spectrum, and judging the sample to be qualified if the similarity between the fingerprint spectrum of the sample and the comparison fingerprint spectrum is not less than 0.90 according to the calculation of a traditional Chinese medicine fingerprint spectrum similarity evaluation system.
In conclusion, a large number of experimental tests and analyses are adopted in the embodiment, 36 common peaks are obtained in 10 batches of blood-replenishing motherwort pill samples, the common peaks of 5 index components are identified and respectively include leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide, the 10 batches of medicinal materials have no obvious difference, and the similarity is more than 0.96.
Example 5 content determination of index Components of blood-enriching motherwort pill
From example 4, it can be seen that 5 index active ingredients in the blood-tonifying motherwort pill are leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide, respectively, and therefore, the content of 5 index active ingredients in 10 batches of blood-tonifying motherwort pills is determined according to the preferable chromatographic conditions determined in example 1, so as to evaluate the quality of the blood-tonifying motherwort pills.
Taking a proper amount of 10 batches of blood-enriching motherwort pill samples, preparing 10 batches (S1-S10) of blood-enriching motherwort pill sample solutions according to the preferable sample solution preparation method determined in the example 2, wherein 3 parts of each sample are prepared in parallel, respectively injecting samples according to the chromatographic conditions of the example 1, measuring peak areas of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide in the sample solutions, and substituting the peak areas into the regression equation obtained in the linear relation investigation of the example 3 to calculate the content of 5 components to be measured.
The results are shown in table 9, the content of leonurine, calycosin glucoside, rutin, ferulic acid and ligustilide in 10 batches of blood-enriching motherwort pills is 128.5-168.9, 153.6-303.4, 79.5-306.6, 167.6-280.9 and 1812.2-3325.6 μ g/g respectively, wherein the content of ligustilide is higher.
TABLE 9.10 contents of 5 index components (μ g/g) in the blood-enriching motherwort pills
Figure RE-GDA0003018596650000161
Figure RE-GDA0003018596650000171
The results show that the content of rutin in different batches is greatly different, the rutin source is wide, and the rutin is contained in the angelica, the motherwort, the dried orange peel and the astragalus in the blood-enriching motherwort pill formula and possibly related to the feeding of medicinal materials in different producing areas, different harvesting years and different harvesting periods. Therefore, the content of leonurine in the blood-enriching motherwort pill is not lower than 128.5 mu g/g, the content of calycosin glucoside is not lower than 153.6 mu g/g, the content of rutin is not lower than 79.5 mu g/g, the content of ferulic acid is not lower than 167.6 mu g/g, and the content of ligustilide is not lower than 1812.2 mu g/g in terms of dry products.
The establishing method of the invention has good linear relation, precision, stability and recovery rate, can accurately detect the content of the index components in the blood-enriching motherwort pill, provides the standard of quality monitoring for the blood-enriching motherwort pill and ensures the quality and clinical curative effect of the product.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A method for establishing a fingerprint of a blood-replenishing motherwort pill is characterized by comprising the following steps:
s1, preparation of a reference substance solution: preparing leonurine, calycosin glucoside, rutin, ferulic acid, and ligustilide as reference substances into mixed reference substance solution;
s2, preparation of a test solution: taking the blood-replenishing motherwort pills, crushing, extracting with acetonitrile with the volume percentage of 75-85%, and preparing a test solution with the mass concentration of 0.05-0.3 g/mL of the blood-replenishing motherwort pills;
s3, HPLC chromatographic conditions: adopting a C18 chromatographic column, wherein the column temperature is 30-35 ℃; the flow rate is 0.9-1.2 mL/min; the detection wavelength is 235-277 nm; acetonitrile is used as a mobile phase A, a formic acid water solution with the volume percentage of 0.05-0.2% is used as a mobile phase B, and in the gradient elution procedure, the volume percentage of the mobile phase B in a mobile phase system is changed as follows: 0-8 min, and 95% of mobile phase B; reducing the content of the mobile phase B from 95% to 10% in 8-60 min; reducing the content of the mobile phase B from 10% to 2% in 60-63 min; 63-66 min, the mobile phase B rises from 2% to 95%; 66-72 min, and 95% of mobile phase B;
s4, the detection method comprises the following steps: and respectively injecting the mixed reference substance solution and the test substance solution into a liquid chromatograph, and measuring to obtain the blood-enriching motherwort pill fingerprint.
2. The method for establishing fingerprint of BUXUEYIMU pill according to claim 1, wherein the acetonitrile content in step S2 is 80 vol%.
3. The method for establishing the fingerprint of the blood-enriching motherwort pill according to claim 1, wherein the extraction in step S2 is ultrasonic extraction, and the extraction time is 60-90 min.
4. The method for establishing fingerprint of blood-enriching motherwort pill according to claim 1, wherein the mass concentration of the blood-enriching motherwort pill in the test solution of step S2 is 0.05-0.1 g/mL.
5. The method for establishing fingerprint of BUXUEYIMU pill according to claim 1, wherein the chromatographic column of step S3 is Symmetry ShieldTMRP18 chromatographic column, column length 150mm, internal diameter 3.9mm, particle size 5 μm.
6. The method for establishing fingerprint of BUXUEYIMU pill according to claim 1, wherein the column temperature in step S3 is 35 ℃.
7. The method for establishing fingerprint of BUXUEYIMU pill according to claim 1, wherein the flow rate in step S3 is 1.0 mL/min.
8. The method for establishing fingerprint of BUXUEYIMU pill according to claim 1, wherein the detection wavelength of step S3 is 254 nm.
9. A quality detection method of a blood-enriching motherwort pill is characterized by comprising the following steps: taking the fingerprint established by the method of any one of claims 1 to 8 as a comparison fingerprint, measuring the HPLC fingerprint of the sample to be detected according to the method of any one of claims 1 to 8, comparing the HPLC detection fingerprint of the sample to be detected with the comparison fingerprint, and determining the sample to be qualified if the similarity between the fingerprint of the sample and the comparison fingerprint is not less than 0.90 according to the calculation of a traditional Chinese medicine fingerprint similarity evaluation system.
10. The method for detecting the quality of the blood-enriching motherwort pill as claimed in claim 9, wherein the content of leonurine, the content of calycosin glucoside, the content of rutin, the content of ferulic acid and the content of ligustilide in the blood-enriching motherwort pill are respectively not less than 100 μ g/g, not less than 120 μ g/g, not less than 60 μ g/g, not less than 150 μ g/g and not less than 1600 μ g/g, respectively, on a dry basis.
CN202110162382.8A 2021-02-05 2021-02-05 Establishment and quality detection method of blood-replenishing and motherwort-tonifying pill fingerprint spectrum Pending CN113960184A (en)

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