CN115356415B - Method for detecting content of 5 components in manna disinfection pill and application - Google Patents

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

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CN115356415B
CN115356415B CN202211034613.8A CN202211034613A CN115356415B CN 115356415 B CN115356415 B CN 115356415B CN 202211034613 A CN202211034613 A CN 202211034613A CN 115356415 B CN115356415 B CN 115356415B
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manna
content
pill
components
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CN115356415A (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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    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N30/06—Preparation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
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    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
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Abstract

The invention provides a method for detecting the content of 5 components in a manna disinfection pill and application thereof, comprising the following steps: adding solvent into the manna disinfection pill, dissolving, performing ultrasonic extraction, filtering, detecting the obtained test solution by adopting ultra-high performance liquid chromatography (UPLC), and determining 5 index components in the test solution: chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin, and wogonin. The detection method and application of the content of 5 components in the manna disinfection pill 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 disinfection pill, ensure the stability of the production process and quality among batches, and comprehensively perfect the quality control system of the manna disinfection pill.

Description

Method for detecting content of 5 components in manna disinfection pill and application
Technical Field
The invention belongs to the technical field of traditional Chinese medicine component detection, relates to a detection method and application of 5 component contents in a manna disinfection pill, and in particular relates to 5 components in the manna disinfection pill: a method for detecting content of chlorogenic acid, forsythiaside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin and application thereof are provided.
Background
The manna sterilizing pill is composed of 11 medicines, namely wrinkled gianthyssop herb, weeping forsythia, unibract fritillary bulb, virgate wormwood herb, blackberry lily, cardamom, akebia stem, baical skullcap root, talcum, grassleaf sweetflag rhizome and peppermint, has the characteristics of opening lung qi upwards, aromatic resolving dampness in middle energizer and light excreting dampness in lower, and is combined with the functions of clearing heat and detoxicating, aromatic resolving light excreting dampness and resolving turbidity and excreting dampness. Clinical researches show that the manna disinfection pill has good treatment effect on infectious diseases such as influenza, mumps, atypical pneumonia, respiratory tract virus infection, hand-foot-mouth disease and the like, and experiments also prove that the manna disinfection pill has broad-spectrum antiviral effect and has certain curative effects on influenza viruses, coxsackie viruses, varicella-zoster viruses and the like.
Clinical studies on the manna disinfection pill also found that the manna disinfection pill is safe and effective on the COVID-19. The manna sterilizing pill is used as an ancient prescription and is firstly carried in 'continuous famous medical cases', wherein 'Yongzhengfuzhu and epidemic febrile qi are toxic when epidemic qi is popular in … …', and the damp-earth qi of the semen tsumadai is required to be transformed into … … so that people with spleen and stomach deficiency should have pestilence qi, evil entering from the skin and hair of the mouth and nose, and from the damp-people, fever, yellow eyes, chest fullness, dan rash and diarrhea, and the tongue color, pale tongue or dry and burnt tongue heart should be observed when the people are examined, and the damp evil is hesitant to qi component and the manna sterilizing pill is treated. Similar to the symptoms of damp-heat accumulated in lung in seven-edition diagnosis and treatment scheme, cold-dampness binding watch and heat Yu Jin injury in the traditional Chinese medicine treatment scheme of Shanxi, the common type COVID-19 patients in the general medical treatment expert consensus of the 2019 coronavirus diseases in Shanghai and the Ji Lin Sheng traditional Chinese medicine treatment scheme are recommended to use the modified manna disinfection pill.
The manna disinfection pill has more clinical application and obvious curative effect, but the quality of the manna disinfection pill is not fully controlled at present, the content of baicalin is only measured in one part of pharmacopoeia of the people's republic of China of 2020 edition, and other documents are less than reports on the content measuring method.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a detection method and application of 5 component contents in the manna disinfection pill, and provides a certain guarantee for clinical safety medication of the manna disinfection pill.
To achieve the above and other related objects, a first aspect of the present invention provides a method for detecting contents of 5 ingredients in a manna disinfection pill, comprising: adding solvent into the manna disinfection pill, dissolving, performing ultrasonic extraction, filtering, detecting the obtained test solution by adopting ultra-high performance liquid chromatography (UPLC), and determining 5 index components in the test solution: chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin, and wogonin.
Preferably, the CAS number of chlorogenic acid is 327-97-9, the CAS number of forsythoside A is 79916-77-1, the CAS number of 3, 5-O-dicaffeoylquinic acid is 89919-62-0, the CAS number of baicalin is 21967-41-9, and the CAS number of wogonin is 51059-44-0.
Preferably, the manna disinfection pill is in powder form.
Preferably, the solvent is methanol.
Preferably, the ratio of the mass g added to the solvent added to the volume mL of the manna disinfection pill is 0.1:15-25, preferably 0.1:20.
Preferably, the time of the ultrasonic extraction is 20 to 40 minutes, preferably 30 minutes.
Preferably, the ultrasonic extraction is followed by cooling. The cooling is carried out to room temperature, and the room temperature is 20-30 ℃.
Preferably, the filtering is to take a supernatant filtering membrane, and take a subsequent filtrate after discarding the primary filtrate.
Preferably, the filter is a 0.22 μm filter.
Preferably, the detection by Ultra Performance Liquid Chromatography (UPLC) comprises the following steps:
1) Preparing a reference substance solution: dissolving chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin reference substances in a solvent, and fixing the volume to prepare reference substance solution;
2) Sample detection: and (2) respectively detecting the sample solution and the reference substance solution in the step (1) by adopting an ultra-high performance liquid chromatography (UPLC), comparing the retention time for qualitative determination, and quantifying by adopting an external standard method to determine the contents of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin in the sample solution.
Preferably, in step 1), the reference solution is shaken up and filtered, and the subsequent filtrate is taken.
Preferably, in step 1), the reference solution is prepared by stepwise dilution. The reference substance stock solution adopted by progressive dilution is stored in a refrigerator at the temperature of 4 ℃ in a dark place.
Preferably, in the step 1), the content of chlorogenic acid in the reference solution ranges from 1.85 to 23.16 mug/mL, the content of forsythoside A ranges from 8.16 to 102.00 mug/mL, the content of 3, 5-O-dicaffeoyl quinic acid ranges from 1.82 to 22.73 mug/mL, the content of baicalin ranges from 38.10 to 476.24 mug/mL, and the content of wogonin ranges from 7.84 to 98.01 mug/mL.
More preferably, the control solution has a chlorogenic acid content ranging from 5 mug/mL, forsythoside A content ranging from 20 mug/mL, 3, 5-O-dicaffeoyl quinic acid content ranging from 5 mug/mL, baicalin content ranging from 100 mug/mL, and wogonin content ranging from 20 mug/mL.
Preferably, in step 1), the solvent is methanol.
Preferably, in step 2), the detector in the ultra high performance liquid chromatography (UPLC) is a Diode Array Detector (DAD).
Preferably, in step 2), the chromatographic column in the ultra-high performance liquid chromatography is a T3 chromatographic column.
More preferably, the column in the ultra-high performance liquid chromatography is a WatersAcquity UPLC HSS T3 column (column length is 100mm, inner diameter is 2.1mm, and filler particle diameter is 1.8 μm).
Preferably, in step 2), the detection wavelength in the ultra 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 performance liquid chromatography is 40 ℃.
Preferably, in the step 2), the flow rate in the ultra performance liquid chromatography is 0.1-0.5 mL/min. More preferably, the flow rate in the ultra performance liquid chromatography is 0.4mL/min.
Preferably, in the step 2), the sample injection amount in the ultra-high performance liquid chromatography is 0.5-5 μl. More preferably, the sample injection amount in the ultra performance liquid chromatography is 1 μl.
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 A phase is acetonitrile and the B phase is 0.05-0.15% phosphoric acid aqueous solution; the analysis time is 32min; gradient elution. More preferably, in the ultra performance liquid chromatography, the mobile phase is acetonitrile-0.10% phosphoric acid aqueous solution, wherein the A phase is acetonitrile and the B phase is 0.10% phosphoric acid aqueous solution; the analysis time is 32min; gradient elution.
The 0.05-0.15% phosphoric acid aqueous solution is 0.05-0.15% phosphoric acid aqueous solution by volume percent. The 0.10% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.10.
More preferably, the specific procedure of the gradient elution is shown in table 1, and is:
0-3 min, phase A: the volume ratio of the phase B is 10:90-10:90;
3-14 min, phase A: the volume ratio of the phase B is 10:90-20:80;
14-20 min, phase A: the volume ratio of the phase B is 20:80-30:70;
20-23 min, phase A: the volume ratio of the phase B is 30:70-95:5, a step of;
23-26 min, phase A: the volume ratio of the phase B is 95:5-95:5, a step of;
26-27 min, phase A: the volume ratio of the phase B is 95:5-10:90;
27-32 min, phase A: the volume ratio of the phase B is 10:90-10:90.
TABLE 1
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 performing UPLC detection to obtain linear relations between chromatographic peak areas of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin and contents of corresponding chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin, drawing corresponding standard working curves, and respectively calculating regression equations of the standard working curves of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin;
b) Performing UPLC detection on the sample solution, substituting chromatographic peak areas of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin into regression equations of standard working curves of the chlorogenic acid, the forsythoside A, the 3, 5-O-dicaffeoyl quinic acid, the baicalin and the wogonin corresponding to the step A), and calculating to obtain contents of the chlorogenic acid, the forsythoside A, the 3, 5-O-dicaffeoyl quinic acid, the baicalin and the wogonin in the sample solution.
More preferably, in the standard working curve, the chromatographic peak areas of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin are taken as an ordinate (Y axis), and the contents (i.e. the concentrations) of the chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin are taken as an abscissa (X axis).
The second aspect of the invention provides an application of a method for measuring the content of various components in the manna disinfection pill in quality detection of the manna disinfection pill.
As described above, the method for detecting the content of 5 components in the manna disinfection pill and the application thereof provided by the invention adopt pretreatment and instrument detection methods with optimized conditions to detect 5 active components in the manna disinfection pill: chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin are accurately quantitatively and qualitatively detected. The method is simple to operate and convenient to control, the measured standard curves of 5 components have good linear relation in respective ranges, good repeatability, good precision, high accuracy and good stability, can be used for accumulating multiple batches of sample data, makes reasonable content limits, can truly reflect the quality differences of various main active components in the manna disinfection pill, ensures the stability of the production process and quality among batches, and comprehensively perfects the quality control system of the manna disinfection pill.
Drawings
FIG. 1 shows the chromatograms of the samples and the reference substances in the invention and the chromatograms of the specificity investigation of the baikal skullcap root, the capillary artemisia and the weeping forsythia.
Detailed Description
The invention is further illustrated below in connection with specific examples, which are to be understood as being illustrative of the invention and not limiting the scope of the invention.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The reagents and instrumentation used in the following examples were as follows:
1. reagent(s)
Sample: batch 15 (lot numbers S1-20220201, S2-20220202, S3-20220203, S4-20220204, S5-20220205, S6-20220301, S7-20220311, S8-20220315, S9-20220324, S10-20220325, S11-20220506, S12-20220507, S13-20220509, S14-20220511, S15-20220512), negative samples (Forsythia-deficient negative, artemisiae capillaris-deficient negative and Scutellaria-deficient negative) were all provided by Shanghai and Huang-medical limited.
Control: chlorogenic acid (lot No. 110753-202018, mass fraction 96.1%), forsythoside A (lot No. 111810-202108, mass fraction 100%), 3, 5-O-dicaffeoylquinic acid (lot No. 111782-201807, mass fraction 94.3%), baicalin (lot No. 110715-201621, mass fraction 95.4%) and wogonin (lot No. 112002-201702, mass fraction 98.5%) were all purchased from China food and drug inspection institute.
Reagent: anhydrous methanol (analytically pure AR, national pharmaceutical chemicals limited), acetonitrile (chromatographic purity, TEDIA, usa), phosphoric acid (chromatographic purity, TEDIA, usa), ultrapure water was prepared by Milli-Q ultrapure water treatment system.
2. Instrument for measuring and controlling the intensity of light
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 lambda detector, waters, USA); SB-5200DTD ultrasonic cleaner (Ningbo Xinzhi biotechnology Co., ltd.); AL204 and XS205 analytical electronic balances (meltrehler-tolidom METTLER tolio instruments Shanghai limited); milli-QAdvantage A10 ultra pure water system (Merck, germany).
Example 1
1. Preparation of test solutions
Taking 0.1g of manna sterilizing pill powder sample, precisely weighing, placing in a 50mL centrifuge tube, precisely adding 20mL of methanol, performing ultrasonic extraction for 30 minutes, cooling, taking supernatant, passing through a 0.22 μm filter membrane, and taking subsequent filtrate to obtain sample solution 1#.
2. Preparation of control solution
Taking reference substances of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonin, precisely weighing, adding methanol, dissolving in a 100mL volumetric flask to constant volume, and preparing a reference substance stock solution. The control stock solution was stored in a refrigerator at 4℃in the dark.
And then the reference substance stock solution is diluted step by adopting methanol and the volume is fixed, so as to prepare a series of reference substance solutions with different concentrations. In a series of reference substance solutions with different concentrations, the content range of chlorogenic acid is 1.85-23.16 mug/mL, the content range of forsythoside A is 8.16-102.00 mug/mL, the content range of 3, 5-O-dicaffeoyl 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 wogonin is 7.84-98.01 mug/mL. Shaking the reference solution, filtering, and collecting filtrate.
3. Measurement
And (3) respectively detecting the sample solution 1# and a series of reference substance solutions with different concentrations by adopting an ultra-high performance liquid chromatography (UPLC), comparing the retention time for qualitative determination, and quantifying by adopting an external standard method. Respectively carrying out UPLC detection on a series of reference substance solutions with different concentrations to obtain linear relations between chromatographic peak areas of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin and concentrations of the chlorogenic acid, the forsythoside A, the 3, 5-O-dicaffeoyl quinic acid, the baicalin and the wogonin, drawing corresponding standard working curves, and respectively calculating regression equations of the standard working curves of the chlorogenic acid, the forsythoside A, the 3, 5-O-dicaffeoyl quinic acid, the baicalin and the wogonin. And then carrying out UPLC detection on the sample solution 1# and substituting chromatographic peak areas of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin into regression equations of standard working curves of the corresponding chlorogenic acid, the forsythoside A, the 3, 5-O-dicaffeoyl quinic acid, the baicalin and the wogonin to calculate the concentration of the chlorogenic acid, the forsythoside A, the 3, 5-O-dicaffeoyl quinic acid, the baicalin and the wogonin in the sample 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 WatersAcquity UPLC HSS T3 chromatographic column (column length is 100mm, inner diameter is 2.1mm, and filler particle diameter is 1.8 μm); the detection wavelength is 330nm; 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 aqueous solution, wherein the A phase is acetonitrile, and the B phase is 0.10% phosphoric acid aqueous solution; the analysis time is 32min; gradient elution.
The specific procedure of gradient elution is:
0-3 min, phase A: the volume ratio of the phase B is 10:90-10:90;
3-14 min, phase A: the volume ratio of the phase B is 10:90-20:80;
14-20 min, phase A: the volume ratio of the phase B is 20:80-30:70;
20-23 min, phase A: the volume ratio of the phase B is 30:70-95:5, a step of;
23-26 min, phase A: the volume ratio of the phase B is 95:5-95:5, a step of;
26-27 min, phase A: the volume ratio of the phase B is 95:5-10:90;
27-32 min, phase A: the volume ratio of the phase B is 10:90-10:90.
example 2
1. Preparation of test solutions
Taking 0.1g of manna sterilizing pill powder sample, precisely weighing, placing in a 50mL centrifuge tube, precisely adding 15mL of methanol, performing ultrasonic extraction for 20 minutes, cooling, taking supernatant, passing through a 0.22 μm filter membrane, and taking subsequent filtrate to obtain sample solution No. 2.
2. Preparation of control solution
Taking reference substances of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonin, precisely weighing, adding methanol, dissolving in a 100mL volumetric flask to constant volume, and preparing a reference substance stock solution. The control stock solution was stored in a refrigerator at 4℃in the dark. And then the reference substance stock solution is diluted step by adopting methanol and the volume is fixed, so as to prepare a series of reference substance solutions with different concentrations.
In the control stock solution and a series of control solutions with different concentrations, the concentration ranges of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin are the same as in the step 2 in the example 1.
3. Measurement
And (3) respectively detecting the sample solution 2# and a series of reference substance solutions with different concentrations by adopting an ultra-high performance liquid chromatography (UPLC), comparing the retention time for qualitative determination, and quantifying by adopting an external standard method to obtain the concentrations of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonin in the sample solution 2#. 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 WatersAcquity UPLC HSS T3 chromatographic column (column length is 100mm, inner diameter is 2.1mm, and filler particle diameter is 1.8 μm); the detection wavelength is 340nm; 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 aqueous solution, wherein the A phase is acetonitrile, and the B phase is 0.15% phosphoric acid aqueous solution; the analysis time is 32min; gradient elution.
The procedure for gradient elution was as in step 3 of example 1.
Example 3
1. Preparation of test solutions
Taking 0.1g of manna sterilizing pill powder sample, precisely weighing, placing in a 50mL centrifuge tube, precisely adding 25mL of methanol, performing ultrasonic extraction for 40 minutes, cooling, taking supernatant, passing through a 0.22 μm filter membrane, and taking subsequent filtrate to obtain sample solution 3#.
2. Preparation of control solution
Taking reference substances of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonin, precisely weighing, adding methanol, dissolving in a 100mL volumetric flask to constant volume, and preparing a reference substance stock solution. The control stock solution was stored in a refrigerator at 4℃in the dark. And then the reference substance stock solution is diluted step by adopting methanol and the volume is fixed, so as to prepare a series of reference substance solutions with different concentrations.
In the control stock solution and a series of control solutions with different concentrations, the concentration ranges of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin are the same as in the step 2 in the example 1.
3. Measurement
And (3) respectively detecting the solution 3# of the sample and a series of reference substance solutions with different concentrations by adopting an ultra-high performance liquid chromatography (UPLC), comparing the retention time for qualitative determination, and quantifying by adopting an external standard method to obtain the concentrations of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonin in the solution 3# of the sample. 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 WatersAcquity UPLC HSS T3 chromatographic column (column length is 100mm, inner diameter is 2.1mm, and filler particle diameter is 1.8 μm); 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 aqueous solution, wherein the A phase is acetonitrile, and the B phase is 0.05% phosphoric acid aqueous solution; the analysis time is 32min; gradient elution.
The procedure for gradient elution was as in step 3 of example 1.
Example 4
Taking a manna disinfection pill powder sample, and preparing a test solution by adopting the step 1 in the example 1. Meanwhile, respectively taking odor-deficient negative samples of the manna sterilizing pills except for the baikal skullcap root, the capillary artemisia and the weeping forsythia, and adopting the step 1 in the example 1 to prepare a negative test sample solution. In addition, a control solution was prepared in step 2 of example 1, in which the content of chlorogenic acid 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 wogonin 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 for qualitative determination, and the specific test result is shown in figure 1. As can be seen from FIG. 1, 5 chromatographic peaks have no negative interference to the test sample, and the method has good specificity.
Example 5
The detection method of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin in the manna disinfection pill powder sample is subjected to methodological verification, and the performance index results are as follows.
1. Linear relationship
Precisely weighing appropriate amounts of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonin reference substances, and adding methanol to obtain a series of reference substance solutions with different concentrations according to step 2 in example 1. According to the chromatographic conditions of step 3 in example 1, precisely sucking 1 μl of reference substance solution, injecting into an ultra-high performance liquid chromatograph, taking the reference substance concentration as abscissa (x-axis), taking the peak area of each index component as ordinate (y-axis), drawing a standard curve, making the quantitative limit S/N be equal to or greater than 10 and the detection limit S/N be equal to or greater than 3, and calculating to obtain the standard regression equation, correlation coefficient, linear range, quantitative limit and detection limit of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogoniside, with specific results shown in Table 2.
As can be seen from Table 2, the 5 index components have good linear relation in the respective sample mass concentration ranges, which indicates that the method has wide linear range and high accuracy.
TABLE 2
2. Stability of
Taking powder samples of manna sterilizing pill of batch No. 210901, preparing a sample solution according to the step 1 in the example 1, respectively carrying out sample injection analysis at 2h, 4h, 8h, 12h, 24h and 36h according to the chromatographic conditions of the step 3 in the example 1, and recording chromatographic peak area data of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogoniside. The results show that the chromatographic peak areas of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin are less than 1.65% in 36h, and the test sample solution has no influence on the detection result in 36h and has good stability.
3. Precision of
Taking any control solution prepared in step 2 in example 1, continuously sampling and analyzing for 6 times according to the chromatographic conditions in step 3 in example 1, and recording peak area data of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin. The result shows that the peak area RSD of the continuous 6-time sampling of 5 components is less than 1.04%, which indicates that the precision of the instrument is good.
4. Repeatability of
Taking a manna sterilizing pill powder sample of lot number 20220201, precisely weighing 6 parts, preparing 6 parts of sample solution in parallel according to the step 1 in the example 1, measuring according to the chromatographic condition of the step 3 in the example 1, recording the peak area data of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogoniside, and calculating the content. The results show that the content RSD of 5 index components is less than 2.46%, and the method has good repeatability.
5. Recovery rate of adding mark
9 parts of a powder sample (batch No. 210901) of the mannose disinfection pill with known concentration is weighed, any reference substance solution (respectively corresponding to 50%, 100% and 150% of the original mass fraction) prepared in the step 2 in the example 1 with low, medium and high mass concentrations is respectively added, 3 parts of each mass concentration is taken, a sample solution is prepared according to the step 1 in the example 1, sample injection analysis is respectively carried out according to the chromatographic condition of the step 3 in the example 1, and the sample injection recovery rate and RSD of each component are calculated according to the measured amount and the addition amount, and the result is shown in Table 3. As is clear from Table 3, the average recovery rate of 5 components was 97.27 to 100.76%, and the RSD was 1.43 to 2.94%, indicating that the accuracy of the method was good.
Table 3 labeled recovery test results (n=3)
Example 6
Samples of 15 batches of mannose disinfection pills with different batch numbers are taken, a sample solution is prepared according to the step 1 in the example 1, and the contents of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin are respectively calculated by sample injection analysis according to the chromatographic conditions of the step 3 in the example 1, and the results are shown in Table 4.
As can be seen from Table 4, 15 batches of manna disinfection pill samples are relatively stable from batch to batch, the RSD of 5 ingredients is not more than 10%, and the maximum RSD is only 6.99%.
TABLE 4 sample content determination results
In conclusion, the detection method and application of the content of 5 components in the manna disinfection pill 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 disinfection pill, ensure the stability of the production process and quality among batches, and comprehensively perfect the quality control system of the manna disinfection pill. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims (7)

1. A method for detecting the content of 5 components in a manna disinfection pill comprises the following steps: adding solvent into the manna disinfection pill, dissolving, performing ultrasonic extraction, filtering, detecting the obtained test solution by adopting an ultra-high performance liquid chromatography, and determining 5 index components in the test solution: chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin, wogonin content;
the detection by adopting the ultra-high performance liquid chromatography comprises the following steps:
1) Preparing a reference substance solution: adding reference substances of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoyl quinic acid, baicalin and wogonin into a solvent for dissolution and constant volume to prepare a reference substance solution;
2) Sample detection: detecting the sample solution and the reference solution in the step 1) respectively by adopting an ultra-high performance liquid chromatography, comparing the retention time for qualitative determination, and quantifying by adopting an external standard method to determine the contents of chlorogenic acid, forsythoside A, 3, 5-O-dicaffeoylquinic acid, baicalin and wogonin in the sample solution;
in the step 2), the detection conditions of the ultra 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 330nm; the mobile phase is acetonitrile-0.05-0.15% phosphoric acid aqueous solution, wherein the A phase is acetonitrile, and the B phase is 0.05-0.15% phosphoric acid aqueous solution; the analysis time is 32min; gradient elution;
the specific procedure of the gradient elution is as follows:
0-3 min, phase A: the volume ratio of the phase B is 10:90-10:90;
3-14 min, phase A: the volume ratio of the phase B is 10:90-20:80;
14-20 min, phase A: the volume ratio of the phase B is 20:80-30:70;
20-23 min, phase A: the volume ratio of the phase B is 30:70-95:5, a step of;
23-26 min, phase A: the volume ratio of the phase B is 95:5-95:5, a step of;
26-27 min, phase A: the volume ratio of the phase B is 95:5-10:90;
27-32 min, phase A: the volume ratio of the phase B is 10:90-10:90.
2. the method for detecting the content of 5 components in a manna sterile pill according to claim 1, wherein the solvent is methanol.
3. The method for detecting the content of 5 components in the manna disinfection pill according to claim 1, wherein the ratio of the added mass of the manna disinfection pill to the added volume of a solvent is 0.1:15-25 g/mL.
4. The method for detecting the content of 5 components in the manna disinfection pill according to claim 1, wherein the ultrasonic extraction time is 20-40 minutes.
5. The method for detecting the content of 5 ingredients in the manna sterile pill according to claim 1, wherein in the step 1), the content of chlorogenic acid in the reference solution ranges from 1.85 to 23.16mg/mL, the content of forsythoside A ranges from 8.16 to 102.00mg/mL, the content of 3, 5-O-dicaffeoylquinic acid ranges from 1.82 to 22.73mg/mL, the content of baicalin ranges from 38.10 to 476.24mg/mL, and the content of wogonin ranges from 7.84 to 98.01mg/mL; and/or the solvent is methanol.
6. The method for detecting the content of 5 components in the manna sterile pill according to claim 1, wherein in the step 2), the detection conditions of the ultra-high performance liquid chromatography 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.
7. Use of a method for determining the content of 5 ingredients in a manna-disinfectant pill according to any one of claims 1-6 in quality detection of manna-disinfectant pill.
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