CN115144507B - Method for simultaneously measuring active ingredients in Sang Ren lung-heat clearing oral liquid - Google Patents
Method for simultaneously measuring active ingredients in Sang Ren lung-heat clearing oral liquid Download PDFInfo
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- CN115144507B CN115144507B CN202210892889.3A CN202210892889A CN115144507B CN 115144507 B CN115144507 B CN 115144507B CN 202210892889 A CN202210892889 A CN 202210892889A CN 115144507 B CN115144507 B CN 115144507B
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- lung
- volume fraction
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- heat clearing
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- 239000007788 liquid Substances 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 48
- 239000004480 active ingredient Substances 0.000 title claims abstract description 14
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- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 claims abstract description 24
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- 239000001685 glycyrrhizic acid Substances 0.000 claims abstract description 16
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 claims abstract description 15
- KFFCKOBAHMGTMW-LGQRSHAYSA-N Forsythin Chemical compound C1=C(OC)C(OC)=CC=C1[C@H]1[C@@H](CO[C@@H]2C=3C=C(OC)C(O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)=CC=3)[C@@H]2CO1 KFFCKOBAHMGTMW-LGQRSHAYSA-N 0.000 claims abstract description 15
- QUQPHWDTPGMPEX-UHFFFAOYSA-N Hesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(COC4C(C(O)C(O)C(C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-UHFFFAOYSA-N 0.000 claims abstract description 15
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- IKIIZLYTISPENI-ZFORQUDYSA-N baicalin Chemical compound O1[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IKIIZLYTISPENI-ZFORQUDYSA-N 0.000 claims abstract description 15
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- APSNPMVGBGZYAJ-GLOOOPAXSA-N clematine Natural products COc1cc(ccc1O)[C@@H]2CC(=O)c3c(O)cc(O[C@@H]4O[C@H](CO[C@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)cc3O2 APSNPMVGBGZYAJ-GLOOOPAXSA-N 0.000 claims abstract description 15
- 229940025878 hesperidin Drugs 0.000 claims abstract description 15
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 claims abstract description 15
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 claims abstract description 15
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 claims abstract description 15
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- 241001254606 Peucedanum praeruptorum Species 0.000 claims abstract description 13
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 13
- QIMGUQIHCNDUKU-UHFFFAOYSA-N 2-[[6-[2-(3,4-dihydroxyphenyl)ethoxy]-3,4,5-trihydroxyoxan-2-yl]methoxy]-6-methyloxane-3,4,5-triol Chemical compound OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OCCC=2C=C(O)C(O)=CC=2)O1 QIMGUQIHCNDUKU-UHFFFAOYSA-N 0.000 claims abstract description 12
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- 229930189432 forsythoside Natural products 0.000 claims abstract description 12
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- KSDSYIXRWHRPMN-UHFFFAOYSA-N 4'-O-beta-D-Galactopyranoside-6''-p-Coumaroylprunin-4',5,7-Trihydroxyflavanone Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(C2OC3=CC(O)=CC(O)=C3C(=O)C2)C=C1 KSDSYIXRWHRPMN-UHFFFAOYSA-N 0.000 claims abstract description 4
- DEMKZLAVQYISIA-ONJCETCRSA-N Liquiritin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)c1ccc([C@@H]2Oc3c(C(=O)C2)ccc(O)c3)cc1 DEMKZLAVQYISIA-ONJCETCRSA-N 0.000 claims abstract description 4
- DEMKZLAVQYISIA-UHFFFAOYSA-N Liquirtin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(C2OC3=CC(O)=CC=C3C(=O)C2)C=C1 DEMKZLAVQYISIA-UHFFFAOYSA-N 0.000 claims abstract description 4
- DEMKZLAVQYISIA-ZRWXNEIDSA-N liquiritin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([C@H]2OC3=CC(O)=CC=C3C(=O)C2)C=C1 DEMKZLAVQYISIA-ZRWXNEIDSA-N 0.000 claims abstract description 4
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Classifications
-
- 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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for simultaneously measuring active ingredients in Sang Ren lung-heat clearing oral liquid, which comprises the following steps: performing ultrasonic extraction, centrifugation and filtration on a Sang Ren lung-heat clearing oral liquid sample by adopting an extractant to obtain an extract; detecting liquiritin, glycyrrhizic acid, peucedanum praeruptorum first, baicalin, wogonin, forsythin, forsythoside and hesperidin simultaneously by using ultra-high performance liquid chromatography; the chromatographic column is an HSS T3 chromatographic column; mobile phase a was acetonitrile +0.2% phosphoric acid and mobile phase B was 0.2% phosphoric acid, and the gradient elution procedure was explored. The method is simple, convenient and quick, has good separation effect, high measurement precision, good result reproducibility, easy observation, strong specificity and good chromatographic peak shape, not only meets the requirements of Chinese veterinary pharmacopoeia, but also saves time and reagents.
Description
Technical Field
The invention relates to a method for simultaneously determining active ingredients in Sang Ren lung-heat clearing oral liquid, and belongs to the field of detection of active ingredients in veterinary medicines.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art. Sang Ren the lung-heat clearing oral liquid is a Chinese veterinary medicine prescription preparation, is received in a Chinese medicine roll p233-234 of the national standard for veterinary medicine (2017 edition), and has the prescription: 100g of white mulberry root-bark, 80g of rhizoma anemarrhenae, 80g of bitter apricot seed, 100g of radix peucedani, 120g of gypsum, 120g of fructus forsythiae, 60g of loquat leaf, 40g of pumice, 60g of liquorice, 100g of exocarpium citri rubrum and 140g of radix scutellariae; decocting the above materials with water for 2 times, mixing decoctions, filtering, concentrating the filtrate under reduced pressure to relative density of 1.02, refrigerating for 24 hr, filtering, adding appropriate amount of benzoic acid into the filtrate, adding water to 1000mL, stirring, packaging, and sterilizing to obtain brown-yellow to brown-brown liquid. Has effects of clearing lung heat, relieving cough and asthma, and can be used for treating cough and asthma due to lung heat. Is widely applied to intensive poultry enterprises.
Sang Ren the quality standard of the oral liquid for clearing lung-heat contains 11 medicines, and is mainly prepared by performing thin-layer chromatography identification on liquorice (ammonium glycyrrhizate), baicalin, fructus forsythiae (forsythin) and peucedanum root (peucedanum praeruptorum dunn A) and performing content measurement on the baicalin.
In the thin-layer chromatography detection method of the national standard method, spots are greatly influenced by environmental temperature and humidity and manual operation, the spots are often unclear, the result is not easy to judge, and the time and Fei Shiji are wasted, and the toxicity of the developing agent for chromatography is found to be large in the actual detection work, for example: petroleum ether, ethyl acetate, butanone, methanol, chloroform and the like have great harm to experimental staff. And the operation is complicated and the time is more.
In addition, the preparation of the Sang Ren lung-heat clearing oral liquid in the early stage of thin-layer chromatography detection is complex, multiple steps such as spreading, airing, checking and checking results are needed, time and labor are wasted, interference ratio by factors such as environment temperature and humidity is large, edge effect and the like are caused, and thin-layer spots are sometimes difficult to distinguish by the method, and reproducibility is poor.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method for simultaneously measuring active ingredients in Sang Ren lung-heat clearing oral liquid.
In order to achieve the above object, the present invention is realized by the following technical scheme:
a method for simultaneously measuring active ingredients in Sang Ren lung-heat clearing oral liquid comprises the following steps: performing ultrasonic extraction, centrifugation and filtration on a Sang Ren lung-heat clearing oral liquid sample by adopting an extractant to obtain an extract; detecting liquiritin, glycyrrhizic acid, peucedanum praeruptorum first, baicalin, wogonin, forsythin, forsythoside and hesperidin simultaneously by using ultra-high performance liquid chromatography;
the detection wavelength of the ultra-high performance liquid chromatography is 205nm, 229nm, 252nm, 277nm and 348nm; the chromatographic column is an HSS T3 chromatographic column; mobile phase a is acetonitrile +0.2% phosphoric acid, mobile phase B is 0.2% phosphoric acid;
the gradient elution procedure was: 0-2min, the volume fraction of A is maintained at 10%;2-3min, the volume fraction of A is changed from 10% to 12%;3-9min, the volume fraction of A is maintained at 12%;9-12min, the volume fraction of A is changed from 12% to 20%;12-20min, the volume fraction of A is maintained at 20%;20-26min, wherein the volume fraction of A is changed from 20% to 30%;26-30min, the volume fraction of A is maintained at 30%;30-33min, the volume fraction of A is changed from 30% to 38%;33-36min, the volume fraction of A is maintained at 38%;36-38min, wherein the volume fraction of A is changed from 38% to 45%;38-42min, wherein the volume fraction of A is changed from 45% to 65%;42-44min, the volume fraction of A is maintained at 65%;44-45min, wherein the volume fraction of A is changed from 65% to 10%;45-48min, the volume fraction of A was maintained at 10%.
The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:
1. according to the invention, 9 main components of glycyrrhizin and glycyrrhizic acid in Sang Ren lung-heat clearing oral liquid, baicalin, wogonin and baicalin in baicalein, forsythia suspensa, forsythin and forsythoside in peucedanum praeruptorum, peucedanum praeruptorum first, hesperidin in exocarpium citri rubrum and the like are measured at one time by a liquid chromatography method, and the method is simple, convenient and rapid, good in separation effect, high in measurement precision, good in result reproducibility, easy to observe, strong in specificity, good in chromatographic peak shape, and capable of meeting the requirements of Chinese beast pharmacopoeia, saving time and saving reagents.
2. According to the determination method, absolute ethyl alcohol can be selected as an extraction solvent to extract a test sample, so that the toxicity is low, the peak area response value of the compound is moderate, the sample is simple and convenient to process, the reagent and the time are saved, the used solvent is low in toxicity, the operation is safe and environment-friendly, and the harm to people and the environment is low.
3. The determination method of the invention uses the high performance liquid chromatograph, presents the result in a chromatogram and spectrogram mode, is convenient and quick, has visual result, is easy to judge, has low detection limit, greatly shortens the detection time (the detection can be completed in about 2 hours from sample processing to on-machine detection of the determination result of the sample, and the thin layer detection method needs about 8-10 hours for one sample), and improves the detection efficiency, the detection sensitivity and the detection effect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a spectrum chart of the comparison products of glycyrrhizin, glycyrrhizic acid, peucedanin, baicalin, wogonin, forsythin, forsythoside and hesperidin in example 1 of the present invention; the arrangement sequence in the spectrogram is as follows from top to bottom: peak 1: glycyrrhizin; peak 2: forsythoside; peak 3: hesperidin; peak 4: baicalin; peak 5: forsythin; peak 6: baicalein; peak 7: wogonin; peak 8: glycyrrhizic acid; peak 9: peucedanum praeruptorum dunn A;
FIG. 2 shows a graph 229nm of a Sang Ren lung-heat clearing oral liquid mixture control in example 1, with a sample injection amount of 2. Mu.L;
FIG. 3 shows a graph of a Sang Ren lung-heat clearing oral liquid mixed control chromatogram of 225nm and a sample injection amount of 2 mu L in the embodiment 1 of the invention;
FIG. 4 shows a graph 277nm of a mixed control chromatogram of Sang Ren lung-heat clearing oral liquid in example 1, with a sample injection amount of 2. Mu.L;
FIG. 5 shows a graph of a Sang Ren lung-heat clearing oral liquid mixed control chromatogram of 324nm and a sample injection amount of 2 mu L in the embodiment 1 of the invention;
FIG. 6 shows a graph of a Sang Ren lung-heat clearing oral liquid mixed control chromatogram of 330nm and a sample injection amount of 2 mu L in the embodiment 1 of the invention;
FIG. 7 shows a graph of a Sang Ren lung-heat clearing oral liquid mixed control chromatogram of 348nm and a sample injection amount of 2 mu L in the embodiment 1 of the invention;
FIG. 8 shows a graph of a Sang Ren lung-heat clearing oral liquid mixed control chromatogram at 252nm with a sample injection amount of 2 μL in example 1 of the present invention;
FIG. 9 shows a graph of a Sang Ren lung-heat clearing oral liquid mixed control chromatogram of 205nm in an embodiment 1 of the invention, wherein the sample injection amount is 2 mu L;
FIG. 10 shows a graph 229nm of absolute ethyl alcohol 2/10 treatment chromatogram of a Baiming sample of Sang Ren lung-heat clearing oral liquid in example 1, with a sample injection amount of 1 μL;
FIG. 11 shows a methanol 5/10 treatment chromatogram 229nm of a Baiming sample of Sang Ren lung-heat clearing oral liquid in example 1, with a sample injection amount of 1 μL;
FIG. 12 shows a graph of a sample size of 229nm in 1. Mu.L of absolute ethyl alcohol 5/10 treatment chromatogram of a Baiming sample of Sang Ren lung-heat-clearing oral liquid in example 1 of the present invention;
FIG. 13 shows a graph 229nm of absolute ethyl alcohol 5/10 treatment chromatogram of a Sang Ren lung-heat clearing oral liquid Huajun sample in example 1, with a sample injection amount of 1 μL;
FIG. 14 shows a sample size of 1. Mu.L of a 229nm sample size of a Sang Ren lung-heat clearing oral liquid and a Meixin sample absolute ethyl alcohol 5/10 treatment chromatogram in example 1 of the present invention;
FIG. 15 shows a graph 229nm of absolute ethyl alcohol 5/10 treatment chromatogram of a sample of Sang Ren lung-heat clearing oral liquid cloud lake in example 1, with a sample injection amount of 1 μL;
FIG. 16 is a spectrum chart of the control substances of glycyrrhizin, glycyrrhizic acid, peucedanin, baicalin, wogonin, forsythin, forsythiaside and hesperidin in example 2 according to the invention;
FIG. 17 is a liquid chromatogram of a control sample in example 2 of the present invention when the gradient elution procedure is changed;
FIG. 18 is a liquid chromatogram of a Wanjun sample according to example 2 of the present invention;
FIG. 19 is a liquid chromatogram of a sample of a cloud lake in example 2 of the present invention;
FIG. 20 is a liquid chromatogram of a comparative example of the present invention using petroleum ether as the extractant.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
A method for simultaneously measuring active ingredients in Sang Ren lung-heat clearing oral liquid comprises the following steps: performing ultrasonic extraction, centrifugation and filtration on a Sang Ren lung-heat clearing oral liquid sample by adopting an extractant to obtain an extract; detecting liquiritin, glycyrrhizic acid, peucedanum praeruptorum first, baicalin, wogonin, forsythin, forsythoside and hesperidin simultaneously by using ultra-high performance liquid chromatography;
the detection wavelength of the ultra-high performance liquid chromatography is 225-229nm; the chromatographic column is an HSS T3 chromatographic column; mobile phase a is acetonitrile +0.2% phosphoric acid, mobile phase B is 0.2% phosphoric acid;
the gradient elution procedure was: 0-2min, the volume fraction of A is maintained at 10%;2-3min, the volume fraction of A is changed from 10% to 12%;3-9min, the volume fraction of A is maintained at 12%;9-12min, the volume fraction of A is changed from 12% to 20%;12-20min, the volume fraction of A is maintained at 20%;20-26min, wherein the volume fraction of A is changed from 20% to 30%;26-30min, the volume fraction of A is maintained at 30%;30-33min, the volume fraction of A is changed from 30% to 38%;33-36min, the volume fraction of A is maintained at 38%;36-38min, wherein the volume fraction of A is changed from 38% to 45%;38-42min, wherein the volume fraction of A is changed from 45% to 65%;42-44min, the volume fraction of A is maintained at 65%;44-45min, wherein the volume fraction of A is changed from 65% to 10%;45-48min, the volume fraction of A was maintained at 10%.
Ultra-high performance liquid chromatography detection wavelength selection: 225-229nm, due to: 205nm, the chromatographic response of peucedanum praeruptorum A is higher; the chromatographic response of the 229nm forsythin is higher, the chromatographic response of 7 types of glycyrrhizic acid and the like at 252nm is higher, but the chromatographic response of the forsythin and the hesperidin are not higher, and the chromatographic response of the 277nm baicalin, wogonin, glycyrrhizin and hesperidin is higher; the chromatographic response of the forsythoside at 330nm is higher; in order to find the common detection wavelength, the detection method is tried to observe that baicalin, baicalein, wogonin, forsythoside and peucedanin A have response at 324nm, 330nm and 348nm, and the forsythin, hesperidin and ammonium glycyrrhizate have no response, and the response of the glycyrrhizin is extremely low or no response; however, the responses of all the 9 compounds are 225-229nm, and the chromatographic response is relatively good when other compounds are compatible, and the detection of the imperatorin by thin layer chromatography is difficult.
The mobile phase A is acetonitrile+0.2% phosphoric acid, the mobile phase B is 0.2% phosphoric acid, the aqueous phase and the organic phase both contain the same amount of phosphoric acid, the mobile phase is stable in pH no matter how the proportion is changed, the chromatographic baseline is stable, and the stability and the detection accuracy of the detection process are improved more easily.
In some embodiments, the extractant is absolute ethanol or methanol.
The Sang Ren lung-heat clearing oral liquid has a large variety of substances, so that the substances to be detected are seriously interfered, and are difficult to directly detect. Through repeated experiments, absolute ethyl alcohol and methanol can be used for completely and better selectively extracting 9 substances to be detected such as glycyrrhizin, glycyrrhizic acid, peucedanin, baicalin, wogonin, forsythin, forsythoside, hesperidin and the like in a Sang Ren lung-heat-clearing oral liquid sample, and the simultaneous separation and detection of the 9 substances to be detected are facilitated.
Preferably, the volume ratio of the Sang Ren lung-heat clearing oral liquid sample to the extractant is 1:1-4.
Preferably, the time of ultrasonic extraction is 20-30min. The ultrasonic extraction mode can effectively improve the extraction efficiency and the extraction effect.
Further preferably, the ultrasonic power is 35-45KHz.
Preferably, the rotational speed of the centrifugation is 8000-15000rpm/min, and the centrifugation time is 3-18min; it is further preferable that the rotational speed of centrifugation is 10000rpm/min and the centrifugation time is 8-13min.
In some embodiments, the detector for ultra performance liquid chromatography is a photodiode array detector, the column temperature is 30-40deg.C, the flow rate of mobile phase is 0.36ml/min, and the sample injection amount is 0.5-5 μl.
Preferably, the sample amount is 1-2. Mu.L.
The invention is further illustrated below with reference to examples.
Example 1
Test article: for enterprise report samples, the samples in this example were supplied by ataxia Bai Chun biopharmaceutical Co., shandong Jun biopharmaceutical Co., shandong Yunhua biological pharmaceutical Co., shandong Hu biological pharmaceutical Co., shandong and Meixin animal pharmaceutical Co., respectively.
The phosphoric acid is of high-grade purity, ethanol and methanol chromatographic purity, and the reference substance is mainly purchased from China institute of veterinary medicine and food verification, china institute of biological products.
The instrument used; BP211D analytical balance (saidolis, germany); waters Acquity TM Ultra performance liquid chromatography (Waters, USA), PDA detector, column HSS T3 column (2.1 mm. Times.100 mm,1.8 μm).
The method for simultaneously measuring 9 effective components in Sang Ren lung-heat clearing oral liquid comprises the following steps of:
a. preparation of control stock solution and mixing control stock solution
Respectively taking glycyrrhizin, glycyrrhizic acid, peucedanum praeruptorum A, baicalin, wogonin, forsythin, forsythoside and hesperidin reference substances, dissolving with ethanol, and fixing the volume to prepare respective reference stock solutions; the control stock solutions were mixed in appropriate volumes to give mixed control stock solutions, see in particular table 1 below.
TABLE 1 control configuration information
b. Preparing a sample solution to be tested: precisely measuring Sang Ren 1-5 mL of a lung-heat clearing oral liquid sample, placing the sample in a 10mL volumetric flask, dissolving the sample in a proper solvent, fixing the volume to a scale, ultrasonically extracting the sample for 20min, precisely measuring 5mL of the sample, centrifuging the sample at 10000rpm/min for 5min, filtering the sample, and measuring the filtrate by liquid chromatography;
c. the used instrument is an ultra-high performance liquid chromatograph, the detector is a photodiode array detector, the chromatographic column is an HSS T3 chromatographic column, the column temperature is 30-40 ℃, 229nm is optimally selected as the detection wavelength of 9 effective components, the mobile phase A is acetonitrile, and the mobile phase B is 0.4% phosphoric acid; flow rate: 0.35mL/min; sample injection amount: 0.5-5. Mu.L;
the PDA detector is selected to collect multiple wavelengths simultaneously, channels such as 205nm, 225nm, 229nm, 252nm, 277nm, 324nm, 330nm and 348nm are selected in a multi-channel scanning mode, detection wavelengths are compared, and 9 compounds are in 225-229nm positions, so that chromatographic peak response is good, and separation degree is good.
The mobile phase gradient elution procedure is shown in table 2:
TABLE 2
| Time | A％ | B％ | Curve |
| Initial initiation | 10 | 90 | Initial initiation |
| 2 | 10 | 90 | 6 |
| 3 | 12 | 88 | 6 |
| 9 | 12 | 88 | 6 |
| 12 | 20 | 80 | 6 |
| 20 | 20 | 80 | 6 |
| 26 | 30 | 70 | 6 |
| 30 | 30 | 70 | 6 |
| 33 | 38 | 62 | 6 |
| 36 | 38 | 62 | 6 |
| 38 | 45 | 55 | 6 |
| 42 | 65 | 35 | 6 |
| 44 | 65 | 35 | 6 |
| 45 | 10 | 90 | 6 |
| 48 | 10 | 90 | 6 |
d. Comparison of different sample injection amounts: c, injecting 0.5 mu L,1 mu L and 2 mu L of the mixed control stock solution into a high performance liquid chromatograph, and carrying out gradient elution and detection under the chromatographic condition of the step c so as to carry out qualitative identification on retention time and spectrogram; the chromatographic parameter information such as the sample injection amount and the peak area of the reference substances are shown in tables 3 to 5, and the data show that when the sample injection amount is 0.5 mu L and 1 mu L, the response of hesperidin is low due to the fact that the sample injection amount is too small, the integral is ignored, and the sample injection amount is optimized and selected to be 2 mu L.
TABLE 3 chromatographic data for Mixed control Table 0.5. Mu.L
Table 4 chromatographic data for mixed control table 1 μl
Table 5 chromatographic data for mixed control table 2 μl
TABLE 6 chromatographic data for Baiming sample-2/10 absolute ethanol extraction
Remarks: 2/10 means that the volume of the oral liquid sample is 2ml, and absolute ethyl alcohol is added to 10ml.
TABLE 7 chromatographic data for Baiming sample-5/10 absolute ethanol extraction
Remarks: 5/10 means that the volume of the oral liquid sample is 5ml, and absolute ethyl alcohol is added to 10ml.
TABLE 8 chromatographic data for Baiming sample Table-5/10 methanol extraction
Remarks: 5/10 means that the volume of the oral liquid sample is 5ml, and the volume of the methanol is added to 10ml.
Table 9 chromatographic data table for Jun sample-5/10 absolute ethanol extraction
Remarks: 5/10 means that the volume of the oral liquid sample is 5ml, and the volume of the absolute ethyl alcohol is 5ml.
Table 9 and Meixin sample chromatographic data table-5/10 absolute ethanol extraction
Remarks: 5/10 means that the volume of the oral liquid sample is 5ml, and absolute ethyl alcohol is added to 10ml.
TABLE 10 cloud lake sample chromatographic data table-5/10 absolute ethanol extraction
Remarks: 5/10 means that the volume of the oral liquid sample is 5ml, and absolute ethyl alcohol is added to 10ml.
The data of different factories show that the detected number of chromatographic peaks and the area of the chromatographic peaks are different, and the detected number of the chromatographic peaks and the area of the chromatographic peaks are related to the production process of enterprises and the used medicinal material base sources. The content of the medicinal material compound is different due to the differences of growth environment, climate, harvesting time and processing of medicinal materials purchased by different enterprises, so that chromatographic inspection is very necessary to improve the monitoring efficiency and the production level.
From the above examples, it was found from the data that the extraction amounts of each compound of methanol were slightly higher than those of absolute ethanol in terms of peak area, but methanol was more toxic than ethanol, and absolute ethanol was preferably selected as the extractant.
e. Analysis of results: and c, filling the filtrate obtained in the step b into a high performance liquid chromatograph, performing gradient elution and detection under the chromatographic condition of the step c, and measuring the peak area of each target in the filtrate so as to obtain retention time and spectrogram characterization. According to the chromatogram, the same chromatographic peak appears in the chromatogram of the positive sample of the test sample at the position corresponding to the chromatogram of the 9 reference substances, as shown in figures 1-15, the peak shape is good, and the separation degree reaches the standard, which indicates that the target medicinal material can be detected.
Chromatographic column tolerance test
The mixed control solution was tested for retention time and stability of separation with minor changes in temperature and flow rate. The flow rate was changed by 0.355mL/min and 0.365mL/min, and the column temperature was 34℃or 36℃or 35℃was maintained. The retention time is not more than 0.5min up and down, the separation degree is good, and the experimental requirement is met.
Under the existing quality standard of 2017 edition national veterinary drugs, the quality of Sang Ren lung-heat clearing oral liquid is difficult to control by thin layer measurement, and by using the method, accurate and rapid measurement can be performed, thus laying a solid foundation for formulating 2025 edition national veterinary drug quality standard.
Example 2
The control configuration ratio is shown in Table 11, the gradient elution procedure is shown in Table 12, and the other is unchanged. Compared with example 1, the gradient change is slow, the retention time of the intermediate component is delayed, the gradient elution program is rapidly pulled up in the later stage, the later components are separated as soon as possible, and the separation degree can be as shown in fig. 16 and 17, and the problems are that: wogonin and glycyrrhizic acid are very difficult to separate, but the separation time is long, and the interference of other impurity peaks cannot be ensured.
The difficulty of gradient elution is that the sample components are more, other impurity peaks interfere too much, better separation is needed, and interference is reduced so as to ensure the detection accuracy.
TABLE 11 control configuration information
TABLE 12 gradient elution procedure
Table 13 chromatographic data table-5/10 gradient elution procedure for absolute ethanol extraction of Jun sample method 2
Remarks: 5/10 means that the volume of the oral liquid sample is 5ml, and the volume of the absolute ethyl alcohol is 5ml.
The number of theoretical plates was not as high as in example 1 for the early-peak components, but rather high for the late-peak components, as shown in FIG. 18.
TABLE 14 cloud lake sample chromatographic data table-5/10 absolute ethanol extraction
Remarks: 5/10 means that the volume of the oral liquid sample is 5ml, and absolute ethyl alcohol is added to 10ml.
The chromatogram of the cloud lake sample is shown in fig. 19.
Table 15 chromatographic data for 9 mixed controls table 2 μl
Comparative example
The solubility of the 9 compounds to different extraction solvents, especially the solubility of peucedanum praeruptorum A and glycyrrhizic acid to the extraction solvents is too different, and detection is difficult.
Water is also used as an extracting agent of the Sang Ren lung-heat clearing oral liquid, 2mL of a Sang Ren lung-heat clearing oral liquid test sample is precisely measured, the test sample is placed in a centrifuge tube, 8mL of water is added for ultrasonic treatment for 25 minutes, 10000rpm/min is adopted for centrifugation for 5 minutes, an upper layer solution filter membrane is taken for filtration, the filtrate is used for ultra-high performance liquid chromatography determination, and a liquid chromatography detection method is as in example 1. Since peucedanin is less soluble in water, peucedanin was also not detected. The method is finally abandoned.
The peucedanum praeruptorum dunn A has certain solubility in ethyl acetate, ethyl acetate is used as an extraction solvent in the test, but the peucedanum praeruptorum dunn A has too great toxicity to stimulate the respiratory system, is too great to harm human body, and finally the method is abandoned.
In addition, the test (petroleum ether 60-90 ℃) is also carried out as an extractant of Sang Ren lung-heat clearing oral liquid, 10mL of a Sang Ren lung-heat clearing oral liquid test sample is precisely measured, the sample is placed in a separating funnel, 40mL of the extractant is transversely rotated for 5 minutes, 5mL of an upper layer solution is taken, the upper layer solution is added into an evaporation dish and evaporated to dryness, 5mL of methanol is added into residues for dissolution, filtration is carried out, the filtrate is subjected to ultra-high performance liquid chromatography measurement, and a liquid chromatography detection method is as in example 1. The result has poor repeatability, and is easy to emulsify and difficult to delaminate during extraction, so that pretreatment fails and detection fails, which means that the detection failure caused by the thin layer treatment method is caused by the fact that emulsification is difficult to extract. The method is finally abandoned.
The method is also tested (petroleum ether 60-90 ℃) as an extracting agent of Sang Ren lung-heat clearing oral liquid, the extracting method of the thin-layer chromatography is finely adjusted, 2mL of Sang Ren lung-heat clearing oral liquid sample is precisely measured, the sample is placed in a centrifuge tube, 8mL of extracting agent is added for ultrasonic treatment for 25 minutes, 10000rpm/min is adopted for centrifugation for 5 minutes, 5mL of upper solution is taken, the upper solution is added into an evaporation dish for evaporation to dryness, 5mL of absolute ethyl alcohol is added into residues for dissolution, the filtration is carried out, the filtrate is used for ultra-high performance liquid chromatography determination, and the liquid chromatography detection method is as in example 1. Only very small amounts of wogonin, hesperidin (extraction is too small, the original integration method cannot integrate but there is a chromatographic peak), peucedanin (extraction is too small, the original integration method cannot integrate but there is a chromatographic peak), and other substances such as glycyrrhizin, glycyrrhizic acid, forsythin, forsythoside and the like are not detected. Finally, the method is abandoned, see fig. 20.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A method for simultaneously measuring active ingredients in Sang Ren lung-heat clearing oral liquid is characterized by comprising the following steps of: the method comprises the following steps: performing ultrasonic extraction, centrifugation and filtration on a Sang Ren lung-heat clearing oral liquid sample by adopting an extractant to obtain an extract; detecting liquiritin, glycyrrhizic acid, peucedanum praeruptorum first, baicalin, wogonin, forsythin, forsythoside and hesperidin simultaneously by using ultra-high performance liquid chromatography;
the chromatographic column is an HSS T3 chromatographic column; mobile phase a is acetonitrile +0.2% phosphoric acid, mobile phase B is 0.2% phosphoric acid;
the gradient elution procedure was: 0-2min, the volume fraction of A is maintained at 10%;2-3min, the volume fraction of A is changed from 10% to 12%;3-9min, the volume fraction of A is maintained at 12%;9-12min, the volume fraction of A is changed from 12% to 20%;12-20min, the volume fraction of A is maintained at 20%;20-26min, wherein the volume fraction of A is changed from 20% to 30%;26-30min, the volume fraction of A is maintained at 30%;30-33min, the volume fraction of A is changed from 30% to 38%;33-36min, the volume fraction of A is maintained at 38%;36-38min, wherein the volume fraction of A is changed from 38% to 45%;38-42min, wherein the volume fraction of A is changed from 45% to 65%;42-44min, the volume fraction of A is maintained at 65%;44-45min, wherein the volume fraction of A is changed from 65% to 10%;45-48min, the volume fraction of A is maintained at 10%;
the detection wavelength of the ultra-high performance liquid chromatography is 225-229nm;
the extractant is absolute ethyl alcohol;
the detector of the ultra-high performance liquid chromatography is a photodiode array detector, the column temperature is 30-40 ℃, the flow rate of the mobile phase is 0.36ml/min, and the sample injection amount is 1-2 mu L.
2. The method for simultaneously determining the active ingredients in Sang Ren lung-heat clearing oral liquid according to claim 1, wherein the method comprises the following steps: sang Ren the volume ratio of the lung-heat clearing oral liquid sample to the extractant is 1:1-4.
3. The method for simultaneously determining the active ingredients in Sang Ren lung-heat clearing oral liquid according to claim 1, wherein the method comprises the following steps: the ultrasonic extraction time is 20-30min.
4. The method for simultaneously measuring active ingredients in Sang Ren lung-heat clearing oral liquid according to claim 3, wherein the method comprises the following steps: the ultrasonic power is 35-45KHz.
5. The method for simultaneously determining the active ingredients in Sang Ren lung-heat clearing oral liquid according to claim 1, wherein the method comprises the following steps: the rotational speed of the centrifugation is 8000-15000rpm/min, and the centrifugation time is 3-18min.
6. The method for simultaneously determining the active ingredients in Sang Ren lung-heat clearing oral liquid according to claim 1, wherein the method comprises the following steps: the rotational speed of centrifugation is 10000rpm/min, and the centrifugation time is 8-13min.
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