CN115356415A - Method for detecting content of 5 components in manna sterilizing pill and application thereof - Google Patents

Method for detecting content of 5 components in manna sterilizing pill and application thereof Download PDF

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CN115356415A
CN115356415A CN202211034613.8A CN202211034613A CN115356415A CN 115356415 A CN115356415 A CN 115356415A CN 202211034613 A CN202211034613 A CN 202211034613A CN 115356415 A CN115356415 A CN 115356415A
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manna
phase
content
pill
components
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CN115356415B (en
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沈丹萍
张正光
温方方
姜鹏
詹常森
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Shanghai Hutchison Pharmaceuticals Ltd
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Abstract

The invention provides a method for detecting the content of 5 components in a manna sterilizing pill and application thereof, comprising the following steps: dissolving the manna disinfectant pill in solvent, ultrasonic extracting, filtering, detecting the obtained test solution by Ultra Performance Liquid Chromatography (UPLC), and determining 5 index components in the test solution: chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin, and wogonoside. The method for detecting the content of the 5 components in the manna disinfectant pill and the application thereof provided by the invention have the advantages of good linear relation, good repeatability, good precision, high accuracy and good stability, can truly reflect the quality difference of various main active components in the manna disinfectant pill, ensure the stability of production process and quality among batches, and comprehensively perfect the quality control system of the manna disinfectant pill.

Description

Method for detecting content of 5 components in manna disinfectant pill and application thereof
Technical Field
The invention belongs to the technical field of traditional Chinese medicine component detection, relates to a detection method and application of the content of 5 components in a manna sterilizing pill, and particularly relates to a manna sterilizing pill, which comprises the following 5 components: a method for detecting contents of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin, and wogonoside, and its application are provided.
Background
The manna toxin-removing pill is composed of 11 medicines, namely wrinkled gianthyssop herb, weeping forsythia, unibract fritillary bulb, virgate wormwood herb, blackberry lily, round cardamom, akebia stem, baical skullcap root, talc, grassleaf sweelflag rhizome and mint, has the characteristics of pungent flavor for opening lung qi, aromatic flavor for eliminating dampness in middle, and light permeability for promoting diuresis in lower, and has the efficacies of clearing heat and removing toxicity, aromatic flavor for eliminating dampness, eliminating turbidity and promoting diuresis in a complete formula. Clinical researches show that the Ganlun Xiaodan has good treatment effect on influenza, epidemic parotitis, atypical pneumonia, respiratory virus infection, hand-foot-and-mouth disease and other infectious diseases, and experiments also prove that the Ganlun Xiaodan has broad-spectrum antiviral effect and has certain curative effects on influenza virus, coxsackie virus, varicella-zoster virus and the like.
Clinical research on the manna disinfectant pill also finds that the manna disinfectant pill is safe and effective to COVID-19. Ganlu Xiaodu Wan, as a famous ancient prescription, is first recorded in the "follow-up medical records" which carries: yongzheng Jieban, epidemic qi epidemic \8230, \8230, seasonal toxic pestilential qi, which must be administered on a day, jieban dampness soil gasification operation \8230, and \8230, so all people with spleen and stomach deficiency should have their pestilential qi, pathogenic factors coming from the oral and nasal skin and dampness, fever, yellow eyes, fullness in chest, erythemas and diarrhea, and when observing their tongue color, or pale, or dry and scorched tongue heart, the damp pathogen is in the qi system, and the treatment with manna disinfection pill. Similar to symptoms of damp-heat accumulation in lung disease in seven versions of diagnosis and treatment schemes, cold-damp exterior syndrome, heat stagnation and body fluid injury type in Shaanxi traditional Chinese medicine treatment scheme, damp-heat accumulation toxicity and lung qi obstruction in Jilin province traditional Chinese medicine treatment scheme and common type COVID-19 patients in Shanghai city 2019 coronavirus disease comprehensive treatment specialist consensus recommend addition or subtraction of Ganlu Disinfection pill.
The manna sterilizing pill has more clinical applications and obvious curative effect, but the manna sterilizing pill lacks the comprehensive quality control at present, is only used for measuring the content of baicalin in the part of 2020 edition pharmacopoeia of the people's republic of China, and other documents are less than the content measuring method reported.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide a method for detecting the content of 5 components in manna sterilizing pills and an application thereof, which provides a certain guarantee for the safe clinical administration of manna sterilizing pills.
In order to achieve the above objects and other related objects, a first aspect of the present invention provides a method for detecting the content of 5 components in a manna disinfectant pill, comprising: dissolving the manna disinfectant pill in solvent, ultrasonic extracting, filtering, detecting the obtained test solution by Ultra Performance Liquid Chromatography (UPLC), and determining 5 index components in the test solution: chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin, and wogonoside.
Preferably, the CAS number of the chlorogenic acid is 327-97-9, the CAS number of the forsythiaside A is 79916-77-1, the CAS number of the 3, 5-O-dicaffeoylquinic acid is 89919-62-0, the CAS number of the baicalin is 21967-41-9, and the CAS number of the wogonoside is 51059-44-0.
Preferably, the manna sterilizing pill is in a powder shape.
Preferably, the solvent is methanol.
Preferably, the ratio of the added mass g of the manna sterilizing pill to the added volume mL of the solvent is 0.1.
Preferably, the time of ultrasonic extraction is 20 to 40 minutes, preferably 30 minutes.
Preferably, the ultrasonic extraction is followed by cooling. And cooling to room temperature, wherein the room temperature is 20-30 ℃.
Preferably, the filtration is a supernatant filtering membrane, and after the primary filtrate is abandoned, a subsequent filtrate is taken.
Preferably, the filter is a 0.22 μm filter.
Preferably, the detection is performed by Ultra Performance Liquid Chromatography (UPLC), comprising the steps of:
1) Preparing a reference solution: adding solvent to control of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin, and wogonoside, dissolving and diluting to desired volume to obtain control solution;
2) Sample detection: respectively detecting the test solution and the reference solution in the step 1) by adopting Ultra Performance Liquid Chromatography (UPLC), comparing the retention time for qualitative determination, and determining the content of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside in the test solution by adopting an external standard method for quantitative determination.
Preferably, in step 1), the control solution is shaken up and filtered, and the filtrate is taken.
Preferably, in step 1), the reference solution is prepared by stepwise dilution. The reference substance stock solution adopted by the stepwise dilution is stored in a refrigerator at 4 ℃ in a dark place.
Preferably, in the step 1), the content range of chlorogenic acid in the reference solution is 1.85-23.16 μ g/mL, the content range of forsythoside A is 8.16-102.00 μ g/mL, the content range of 3, 5-O-dicaffeonic acid acyl quinic acid is 1.82-22.73 μ g/mL, the content range of baicalin is 38.10-476.24 μ g/mL, and the content range of wogonoside is 7.84-98.01 μ g/mL.
More preferably, the content range of chlorogenic acid in the control solution is 5 μ g/mL, the content range of forsythoside A is 20 μ g/mL, the content range of 3, 5-O-dicaffeoylquinic acid is 5 μ g/mL, the content range of baicalin is 100 μ g/mL, and the content range of wogonoside is 20 μ g/mL.
Preferably, in step 1), the solvent is methanol.
Preferably, in the step 2), the detector is a Diode Array Detector (DAD) in the Ultra Performance Liquid Chromatography (UPLC).
Preferably, in the step 2), the chromatographic column in the ultra-high performance liquid chromatography is a T3 chromatographic column.
More preferably, the chromatographic column in the ultra high performance liquid chromatography is a WatersAcquisity UPLC HSS T3 chromatographic column (the column length is 100mm, the inner diameter is 2.1mm, and the packing particle size is 1.8 μm).
Preferably, in the step 2), the detection wavelength in the ultra-high performance liquid chromatography is 320-340 nm. More preferably, the detection wavelength is 330nm.
Preferably, in the step 2), the column temperature in the ultra-high performance liquid chromatography is 30-45 ℃. More preferably, the column temperature in the ultra-high performance liquid chromatography is 40 ℃.
Preferably, in the step 2), the flow rate in the ultra high performance liquid chromatography is 0.1 to 0.5mL/min. More preferably, the flow rate in ultra high performance liquid chromatography is 0.4mL/min.
Preferably, in the step 2), the sample amount in the ultra high performance liquid chromatography is 0.5 to 5 μ L. More preferably, the sample amount in the ultra high performance liquid chromatography is 1 μ L.
Preferably, in the step 2), in the ultra-high performance liquid chromatography, the mobile phase is acetonitrile-0.05-0.15% phosphoric acid aqueous solution, wherein the phase A is acetonitrile, and the phase B is 0.05-0.15% phosphoric acid aqueous solution; the analysis time is 32min; and (4) gradient elution. More preferably, in the ultra-high performance liquid chromatography, the mobile phase is acetonitrile-0.10% phosphoric acid aqueous solution, wherein the phase A is acetonitrile, and the phase B is 0.10% phosphoric acid aqueous solution; the analysis time is 32min; gradient elution.
The 0.05-0.15 percent phosphoric acid aqueous solution is 0.05-0.15 percent phosphoric acid aqueous solution by volume percentage. The 0.10% phosphoric acid aqueous solution is a 0.10% phosphoric acid aqueous solution in volume percentage.
More preferably, the specific procedure for the gradient elution is shown in table 1 as:
0-3min, phase A: the volume ratio of the phase B is 10:90-10:90, respectively;
3-14min, phase A: the volume ratio of the phase B is 10:90-20:80;
14-20min, phase A: the volume ratio of the phase B is 20:80-30:70;
20-23min, phase A: the volume ratio of the phase B is 30:70-95:5;
23-26min, phase A: the volume ratio of the phase B is 95:5-95:5;
26-27min, phase A: the volume ratio of the phase B is 95:5-10:90, respectively;
27-32min, phase A: the volume ratio of the phase B is 10:90-10:90.
TABLE 1
Figure BDA0003818446770000031
Figure BDA0003818446770000041
Preferably, in step 2), the external standard method comprises the following steps:
a) Preparing a series of reference substance solutions with different concentrations according to the step 1), respectively carrying out UPLC detection to obtain linear relations between chromatographic peak areas of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside and contents of corresponding chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeic acid acyl quinic acid, baicalin and wogonoside, drawing corresponding standard working curves, and respectively calculating regression equations of the standard working curves of the chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeic acid acyl quinic acid, baicalin and wogonoside;
b) Carrying out UPLC detection on the test solution, substituting the chromatographic peak areas of the obtained chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside into the regression equation of the standard working curve of the corresponding chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeic acid acyl quinic acid, baicalin and wogonoside in the step A), and calculating to obtain the contents of the chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside in the test solution.
More preferably, in the standard working curve, the chromatographic peak areas of chlorogenic acid, forsythiaside a, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside are taken as vertical coordinates (Y axis), and the contents (i.e. concentrations) of the corresponding chlorogenic acid, forsythiaside a, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside are taken as horizontal coordinates (X axis).
The second aspect of the invention provides an application of a method for measuring the content of various components in the manna disinfectant pill in the quality detection of the manna disinfectant pill.
As described above, the method for detecting the content of 5 ingredients in the manna disinfectant pill and the application thereof provided by the invention adopt the pretreatment of optimized conditions and the instrument detection method to detect the content of 5 active ingredients in the manna disinfectant pill: chlorogenic acid, forsythoside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside are subjected to accurate quantitative and qualitative detection. The method is simple to operate and convenient to control, the standard curves of the 5 measured components have good linear relation in respective ranges, good repeatability, good precision, high accuracy and good stability, can be used for data accumulation of samples of multiple batches, a reasonable content limit is formulated, the quality difference of various main active components in the manna disinfectant pills can be truly reflected, the stability of production process and quality among batches is ensured, and the quality control system of the manna disinfectant pills is comprehensively perfected.
Drawings
FIG. 1 shows chromatograms of the sample and the reference substance of the present invention and the chromatogram for the specificity study of Scutellariae radix, herba Artemisiae Scopariae, and fructus forsythiae.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and equipment used in the following examples are as follows:
1. reagent
Sample preparation: 15 batches of the manna disinfectant pills (batches S1-20201, S2-20202, S3-20220203, S4-20204, S5-20205, S6-20220301, S7-20220311, S8-20220315, S9-20220324, S10-20220325, S11-20220506, S12-20220507, S13-20220509, S14-20220511, S15-20220512) and negative samples (negative for fructus forsythiae, negative for herba Artemisiae Scopariae and negative for radix Scutellariae) were all provided by Shanghai and Huangyao industries, inc.
Comparison products: chlorogenic acid (batch No. 110753-202018, mass fraction 96.1%), forsythiaside A (batch No. 111810-202108, mass fraction 100%), 3, 5-O-dicaffeonic acid acyl quinic acid (batch No. 111782-201807, mass fraction 94.3%), baicalin (batch No. 110715-201821, mass fraction 95.4%) and wogonoside (batch No. 112002-201702, mass fraction 98.5%), all of which were purchased from China food and drug assay institute.
Reagent: anhydrous methanol (analytical pure AR, national institute of chemical and technology, ltd.), acetonitrile (chromatographic pure, TEDIA corporation, usa), phosphoric acid (chromatographic pure, TEDIA corporation, usa), and ultrapure water were prepared by Milli-Q ultrapure water treatment system.
2. Instrument
A Waters Acquity UPLC H-Class ultra high performance liquid chromatograph (equipped with Empower 3 chromatographic workstation, quaternary ultra high pressure solvent manager, autosampler sample manager, column oven, PDAe λ detector, waters corporation, usa); SB-5200DTD ultrasonic cleaning machine (Ningbo Xinzhi Biotech Co., ltd.); analytical electronic balances of models AL204 and XS205 (Metler-Tolydo Meter Shanghai Co., ltd.); milli-QAdvantage A10 ultra pure water system (Merck Millipore, germany).
Example 1
1. Preparation of test solution
Taking 0.1g of the manna disinfectant pill powder sample, precisely weighing, placing in a 50mL centrifuge tube, precisely adding 20mL of methanol, ultrasonically extracting for 30 minutes, cooling, taking the supernatant, filtering with a 0.22 μm filter membrane, and taking the subsequent filtrate to obtain the sample solution # 1.
2. Preparation of control solutions
Precisely weighing control substances of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside, adding methanol, dissolving in 100mL volumetric flask, and adding to constant volume to obtain control substance stock solution. The reference stock solution was stored in a refrigerator at 4 ℃ in the dark.
And diluting the reference substance stock solution step by adopting methanol and fixing the volume to prepare a series of reference substance solutions with different concentrations. In a series of reference solutions with different concentrations, the content range of chlorogenic acid is 1.85-23.16 mug/mL, the content range of forsythiaside A is 8.16-102.00 mug/mL, the content range of 3, 5-O-dicaffeonic acid acyl quinic acid is 1.82-22.73 mug/mL, the content range of baicalin is 38.10-476.24 mug/mL, and the content range of wogonoside is 7.84-98.01 mug/mL. Shaking the control solution, filtering, and collecting the filtrate.
3. Measurement of
Detecting the sample solution 1# and a series of reference substance solutions with different concentrations respectively by Ultra Performance Liquid Chromatography (UPLC), comparing retention time for qualitative determination, and quantifying by external standard method. A series of reference substance solutions with different concentrations are respectively subjected to UPLC detection to obtain linear relations between chromatographic peak areas of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside and concentrations of corresponding chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeic acid acyl quinic acid, baicalin and wogonoside, corresponding standard working curves are drawn, and regression equations of the standard working curves of the chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoic acid acyl quinic acid, baicalin and wogonoside are respectively obtained through calculation. And performing UPLC detection on the test solution 1#, substituting the chromatographic peak areas of the obtained chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside into regression equations of standard working curves of the corresponding chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeic acid acyl quinic acid, baicalin and wogonoside, and calculating to obtain the concentrations of the chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeic acid acyl quinic acid, baicalin and wogonoside in the test solution 1#.
Wherein, the high performance liquid chromatography comprises the following detection conditions:
the detector is a Diode Array Detector (DAD); the chromatographic column is a WatersAcquity UPLC HSS T3 chromatographic column (the column length is 100mm, the inner diameter is 2.1mm, and the particle size of the filler is 1.8 μm); the detection wavelength is 330nm; the column temperature is 40 ℃; the flow rate is 0.4mL/min; the sample injection amount is 1 mu L; the mobile phase is acetonitrile-0.10% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.10% phosphoric acid water solution; the analysis time is 32min; and (4) gradient elution.
The specific procedure for gradient elution was:
0-3min, phase A: the volume ratio of the phase B is 10:90-10:90, respectively;
3-14min, phase A: the volume ratio of the phase B is 10:90-20:80;
14-20min, phase A: the volume ratio of the phase B is 20:80-30:70;
20-23min, phase A: the volume ratio of the phase B is 30:70-95:5;
23-26min, phase A: the volume ratio of the phase B is 95:5-95:5;
26-27min, phase A: the volume ratio of the phase B is 95:5-10:90, respectively;
27-32min, phase A: the volume ratio of the phase B is 10:90-10:90.
example 2
1. Preparation of test solution
Taking 0.1g of the manna disinfectant pill powder sample, precisely weighing, placing in a 50mL centrifuge tube, precisely adding 15mL of methanol, ultrasonically extracting for 20 minutes, cooling, filtering the supernatant with 0.22 μm filter membrane, and collecting the subsequent filtrate to obtain sample solution 2#.
2. Preparation of control solutions
Precisely weighing control substances of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside, adding methanol, dissolving in 100mL volumetric flask, and adding to constant volume to obtain control substance stock solution. The reference stock solution was stored in a refrigerator at 4 ℃ in the dark. And diluting the reference substance stock solution step by adopting methanol and fixing the volume to a certain volume to prepare a series of reference substance solutions with different concentrations.
The concentrations of chlorogenic acid, forsythiaside a, 3, 5-O-dicaffeoylquinic acid, baicalin, and wogonoside in the control stock solution and a series of control solutions of different concentrations were in the same range as in step 2 of example 1.
3. Measurement of
Respectively detecting the sample solution 2# and a series of reference solutions with different concentrations by Ultra Performance Liquid Chromatography (UPLC), comparing retention time for qualitative determination, and quantifying by external standard method to obtain the concentrations of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside in the sample solution 2#. The specific quantitative procedure was the same as in step 3 of example 1.
The high performance liquid chromatography comprises the following detection conditions:
the detector is a Diode Array Detector (DAD); the chromatographic column is a WatersAcquity UPLC HSS T3 chromatographic column (the column length is 100mm, the inner diameter is 2.1mm, and the particle size of the filler is 1.8 μm); the detection wavelength is 340nm; the column temperature is 50 ℃; the flow rate is 0.3mL/min; the sample injection amount is 0.5 mu L; the mobile phase is acetonitrile-0.15% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.15% phosphoric acid water solution; the analysis time is 32min; gradient elution.
The procedure for gradient elution was the same as in step 3 of example 1.
Example 3
1. Preparation of test solution
Taking 0.1g of the manna disinfectant pill powder sample, precisely weighing, placing in a 50mL centrifuge tube, precisely adding 25mL of methanol, ultrasonically extracting for 40 min, cooling, filtering the supernatant with 0.22 μm filter membrane, and collecting the subsequent filtrate to obtain test solution # 3.
2. Preparation of control solutions
Precisely weighing control substances of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside, adding methanol, dissolving in a 100mL volumetric flask, and diluting to constant volume to obtain control substance stock solution. The reference stock solution was stored in a refrigerator at 4 ℃ in the dark. And diluting the reference substance stock solution step by adopting methanol and fixing the volume to prepare a series of reference substance solutions with different concentrations.
The concentrations of chlorogenic acid, forsythiaside a, 3, 5-O-dicaffeoylquinic acid, baicalin, and wogonoside in the control stock solution and a series of control solutions of different concentrations were in the same range as in step 2 of example 1.
3. Measurement of
Respectively detecting the sample solution 3# and a series of reference solutions with different concentrations by ultra-high performance liquid chromatography (UPLC), comparing the retention time for qualitative determination, and quantifying by external standard method to obtain the concentrations of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside in the sample solution 3#. The specific quantitative procedure was the same as in step 3 of example 1.
Wherein, the high performance liquid chromatography comprises the following detection conditions:
the detector is a Diode Array Detector (DAD); the chromatographic column is a WatersAcquity UPLC HSS T3 chromatographic column (the column length is 100mm, the inner diameter is 2.1mm, and the particle size of the filler is 1.8 mu m); the detection wavelength is 320nm; the column temperature is 30 ℃; the flow rate is 0.5mL/min; the sample injection amount is 2 mu L; the mobile phase is acetonitrile-0.05% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.05% phosphoric acid water solution; the analysis time is 32min; gradient elution.
The procedure for gradient elution was the same as in step 3 of example 1.
Example 4
A sample of the manna sterilizing pill powder was taken, and the test solution was prepared in step 1 of example 1. Meanwhile, negative samples without the odor of the manna disinfectant pill, which are obtained by removing the scutellaria baicalensis, the oriental wormwood and the fructus forsythiae, are respectively taken, and a negative test sample solution is prepared by adopting the step 1 in the example 1. In addition, a control solution was prepared in step 2 of example 1, wherein the content of chlorogenic acid in the control solution was 5. Mu.g/mL, the content of forsythoside A was 20. Mu.g/mL, the content of 3, 5-O-dicaffeoylquinic acid was 5. Mu.g/mL, the content of baicalin was 100. Mu.g/mL, and the content of wogonoside was 20. Mu.g/mL.
The test solution, the negative test solution and the reference solution are respectively measured according to the step 3 in the example 1, the retention time is compared and determined qualitatively, and the specific test result is shown in the figure 1. As can be seen from FIG. 1, 5 chromatographic peaks do not have negative interference on the test sample, and the specificity of the method is good.
Example 5
The detection method of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside in the mannurus disinfecting pill powder sample is proved by methodology, and the performance index results are as follows.
1. Linear relation
A proper amount of reference substances of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside are precisely weighed, and methanol is added according to the step 2 in the embodiment 1 to prepare a series of reference substance solutions with different concentrations. According to the chromatographic conditions of the step 3 in the embodiment 1, precisely absorbing 1 μ L of the reference solution, injecting the reference solution into an ultra-high performance liquid chromatograph, drawing a standard curve by taking the concentration of the reference as a horizontal coordinate (x axis) and the peak area of each index component as a vertical coordinate (y axis), and calculating to obtain a standard regression equation, a correlation coefficient, a linear range, a quantitative limit and a detection limit of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside by setting the quantitative limit S/N to be not less than 10 and the detection limit S/N to be not less than 3, wherein the specific results are shown in Table 2.
As can be seen from Table 2, the linear relationship of the 5 index components in the respective sample introduction mass concentration ranges is good, which shows that the method of the invention has wide linear range and high accuracy.
TABLE 2
Figure BDA0003818446770000091
2. Stability of
A test sample solution is prepared according to the step 1 in the example 1 by taking a sample of the mannitol disinfectant pill powder of the batch number 210901, and sample injection analysis is carried out for 2h, 4h, 8h, 12h, 24h and 36h respectively according to the chromatographic conditions of the step 3 in the example 1, and the chromatographic peak area data of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside are recorded. The result shows that the RSD of the chromatographic peak areas of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside within 36h is less than 1.65%, and the test solution has no influence on the result and good stability when being detected within 36 h.
3. Precision degree
Taking any one of the control solutions prepared in the step 2 in the example 1, respectively and continuously injecting and analyzing for 6 times according to the chromatographic conditions of the step 3 in the example 1, and recording peak area data of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside. The result shows that the peak area RSD of continuous 6 times of sample injection of 5 components is less than 1.04 percent, which indicates that the precision of the instrument is good.
4. Repeatability of
A sample of the mannitol disinfectant pill powder of batch No. 20220201 is accurately weighed to 6 parts, 6 parts of test solution are prepared in parallel according to the step 1 in the example 1, the chromatographic conditions in the step 3 in the example 1 are measured, peak area data of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonoside are recorded, and the content is calculated. The result shows that the content RSD of 5 index components is less than 2.46 percent, which indicates that the method has good repeatability.
5. Recovery rate of added standard
Weighing 9 parts of manna sterilizing pill powder sample (batch number 210901) with known concentration, precisely weighing, respectively adding any reference substance solution (respectively corresponding to 50%, 100% and 150% of the original mass fraction) prepared in step 2 in example 1 with low, medium and high mass concentrations, taking 3 parts of each mass concentration, preparing a sample solution according to step 1 in example 1, respectively carrying out sample injection analysis according to the chromatographic conditions of step 3 in example 1, and calculating the sample adding recovery rate and RSD of each component according to the measured amount and the added amount, wherein the results are shown in Table 3. As can be seen from Table 3, the average recovery of 5 components was 97.27-100.76% and the RSD was 1.43-2.94%, indicating that the accuracy of the process was good.
TABLE 3 test results of recovery with added label (n = 3)
Figure BDA0003818446770000101
Figure BDA0003818446770000111
Example 6
15 batches of manna disinfectant pill samples with different batch numbers are taken, a test solution is prepared according to the step 1 in the example 1, and the contents of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside are respectively calculated according to the chromatographic conditions of the step 3 in the example 1 by sample injection analysis, and the results are shown in a table 4.
As can be seen from Table 4, 15 batches of the mannoprotein sterile pill samples are relatively stable from batch to batch, the RSD of 5 components is not more than 10%, and the maximum RSD is only 6.99%.
TABLE 4 results of content measurement of samples
Figure BDA0003818446770000112
Figure BDA0003818446770000121
In conclusion, the method for detecting the content of the 5 components in the manna disinfectant pill and the application thereof provided by the invention have the advantages of good linear relation, good repeatability, good precision, high accuracy and good stability, can truly reflect the quality difference of various main active components in the manna disinfectant pill, ensure the stability of the production process and quality among batches, and comprehensively perfect the quality control system of the manna disinfectant pill. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (10)

1. A method for detecting the content of 5 components in a manna sterilizing pill comprises the following steps: dissolving the manna disinfectant pill in a solvent, performing ultrasonic extraction, filtering, detecting the obtained test solution by using ultra-high performance liquid chromatography, and determining 5 index components in the test solution: chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin, and wogonoside.
2. The method for detecting the content of 5 components in the manna disinfectant pill according to claim 1, wherein the solvent is methanol.
3. The method for detecting the content of the 5 components in the manna disinfectant pill according to claim 1, wherein the ratio of the mass of the manna disinfectant pill to the volume of the solvent is 0.1.
4. The method for detecting the content of 5 components in the manna sterilizing pill as claimed in claim 1, wherein the ultrasonic extraction time is 20-40 min.
5. The method for detecting the content of the 5 components in the manna disinfectant pill according to claim 1, wherein the detection is performed by ultra-high performance liquid chromatography, and comprises the following steps:
1) Preparing a reference solution: dissolving chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoylquinic acid, baicalin, and wogonoside in solvent, and diluting to constant volume to obtain control solution;
2) Sample detection: respectively detecting the test solution and the reference solution in the step 1) by adopting ultra-high performance liquid chromatography, comparing the retention time for qualitative determination, and determining the content of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeonic acid acyl quinic acid, baicalin and wogonoside in the test solution by adopting an external standard method for quantitative determination.
6. The method for detecting the content of 5 components in the manna disinfectant pill according to claim 5, wherein in the step 1), the content range of chlorogenic acid in the reference solution is 1.85-23.16 μ g/mL, the content range of forsythiaside A is 8.16-102.00 μ g/mL, the content range of 3, 5-O-dicaffeoylquinic acid is 1.82-22.73 μ g/mL, the content range of baicalin is 38.10-476.24 μ g/mL, and the content range of wogonoside is 7.84-98.01 μ g/mL; and/or the solvent is methanol.
7. The method for detecting the content of 5 components in the manna sterilizing pill according to claim 5, wherein in the step 2), the detection conditions of the ultra-high performance liquid chromatography are as follows: the detector is a diode array detector; the chromatographic column is a T3 chromatographic column; the detection wavelength is 320-340 nm; the mobile phase is acetonitrile-0.05-0.15% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.05-0.15% phosphoric acid water solution; the analysis time is 32min; and (4) gradient elution.
8. The method for detecting the content of 5 components in the manna sterilizing pill according to claim 5, wherein the detection conditions of the ultra high performance liquid chromatography in the step 2) further comprise: the column temperature is 30-45 ℃; the flow rate is 0.1-0.5 mL/min; the sample injection amount is 0.5-5 mu L.
9. The method for detecting the content of the 5 components in the manna disinfectant pill according to claim 7, wherein the gradient elution is performed by the following specific procedures:
0-3min, phase A: the volume ratio of the phase B is 10:90-10:90, respectively;
3-14min, phase A: the volume ratio of the phase B is 10:90-20:80;
14-20min, phase A: the volume ratio of the phase B is 20:80-30:70;
20-23min, phase A: the volume ratio of the phase B is 30:70-95:5;
23-26min, phase A: the volume ratio of the phase B is 95:5-95:5;
26-27min, phase A: the volume ratio of the phase B is 95:5-10:90;
27-32min, phase A: the volume ratio of the phase B is 10:90-10:90.
10. the use of the method for determining the contents of a plurality of components in the manna sterilizing pill according to any one of claims 1 to 9 in the quality detection of the manna sterilizing pill.
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