CN116818963A - One-measurement-multiple-evaluation-level detection method for five chemical components in corydalis saxicola bunting - Google Patents
One-measurement-multiple-evaluation-level detection method for five chemical components in corydalis saxicola bunting Download PDFInfo
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- RLQYRXCUPVKSAW-UHFFFAOYSA-M 2,3,9,10-tetramethoxy-5,6-dihydroisoquinolino[2,1-b]isoquinolin-7-ium;chloride Chemical compound [Cl-].COC1=C(OC)C=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=C1 RLQYRXCUPVKSAW-UHFFFAOYSA-M 0.000 claims abstract description 18
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- 239000007788 liquid Substances 0.000 claims abstract description 7
- 239000002775 capsule Substances 0.000 claims description 29
- 239000000523 sample Substances 0.000 claims description 26
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- 238000012360 testing method Methods 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 5
- 239000012085 test solution Substances 0.000 claims description 5
- 238000006356 dehydrogenation reaction Methods 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
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- CXEGAUYXQAKHKJ-NSBHKLITSA-N emamectin B1a Chemical class C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](NC)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 CXEGAUYXQAKHKJ-NSBHKLITSA-N 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
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- IHNKNZSIZUBHCB-UHFFFAOYSA-N isoapocavidine Natural products CC1C2=CC=C3OCOC3=C2CN2C1C(C=C(C(=C1)O)OC)=C1CC2 IHNKNZSIZUBHCB-UHFFFAOYSA-N 0.000 description 1
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- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 1
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- 239000000843 powder Substances 0.000 description 1
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- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—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
- G01N30/74—Optical detectors
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a detection method of dehydromethylcavidine, dehydroisopycavidine, dehydrocavidine, palmatine hydrochloride and berberine hydrochloride in corydalis saxicola bunting, which comprises the following steps: step 1, preparing a reference substance solution; step 2, preparing a sample solution; and 3, respectively precisely sucking the reference substance solution and the sample solution, injecting the reference substance solution and the sample solution into a liquid chromatograph, measuring by an ultraviolet-visible light detector, and simultaneously carrying out methodology investigation. The detection method established by the invention has the advantages of quick and accurate analysis, stronger specificity, good reproducibility and stability, and the obtained chromatogram has better chromatographic peak type, and can more effectively provide a certain reference for detecting the content of the corydalis saxicola bunting.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a one-measurement-multiple-evaluation-content detection method for five chemical components in corydalis saxicola bunting, which can be used for detecting the content measurement of the five chemical components in corydalis saxicola bunting, corydalis saxicola bunting medicinal materials and corydalis saxicola bunting total alkali capsules.
Background
Corydalis saxicola bunting (Corydalis saxicola Bunting, CSB) is the whole herb of corydalis saxicola bunting of the genus corydalis of the family Papaveraceae, is mainly distributed in Guangxi, guizhou, yunnan and other places in China, has the effects of clearing heat and detoxicating, promoting diuresis, relieving pain and stopping bleeding and the like, and is one of the common traditional Chinese medicines for clinically treating hepatitis, liver fibrosis and liver cirrhosis at present. The chemical components of corydalis saxicola bunting with pharmacological action are mainly alkaloid components, which are collectively called corydalis saxicola bunting total alkaloids (Corydalis saxicola Bunting total alkaloids, CSBTA). Modern pharmacological research shows that CSBTA has wide pharmacological effects of protecting liver, easing pain, resisting tumor, resisting bacteria and the like, and has wide development and application prospects. The corydalis saxicola bunting total alkali capsule has the effects of clearing heat and detoxicating, promoting diuresis, relieving pain and stopping bleeding, and is mainly used for treating non-alcoholic steatohepatitis with damp-heat accumulation syndrome and acute and chronic hepatitis with liver and gall damp-heat syndrome.
A multi-evaluation method (quantitative analysis of multicomponents by single marker, QAMS) is a new mode of multi-component synchronous quality control in the traditional Chinese medicine field, and is characterized in that by utilizing the inherent functional relationship and proportional relationship of the effective components of the traditional Chinese medicine, the synchronous measurement of a plurality of components (the reference substances are difficult to obtain or supply) is realized by measuring only one of the stable, cheap and easily available components, so that the problems of more reference substances, high cost, difficult supply and the like in the multi-index quality control process of the traditional Chinese medicine can be effectively solved, and the method is the development direction of multi-component synchronous quantification of the traditional Chinese medicine.
The reported method for evaluating the quality of the corydalis saxicola bunting and the preparation thereof has the problems of few detection components, more standard varieties, complicated operation steps, long analysis period, high cost and the like. Therefore, the patent provides a new idea for establishing the stable, controllable, low-cost, accurate and efficient corydalis saxicola bunting and the quality control method of the preparation thereof.
Disclosure of Invention
The invention aims to detect the contents of five monomer alkaloids in corydalis saxicola bunting and a preparation thereof by using a one-test-multiple-evaluation method, and the detection method can objectively, comprehensively and accurately detect the contents of dehydromethylcavidine, dehydroiso-apocavidine, dehydrocavidine, palmatine hydrochloride and berberine hydrochloride, thereby providing a certain data reference for quality research of the corydalis saxicola bunting and the preparation thereof.
A method for detecting one-measurement-multiple-evaluation content of five chemical components in corydalis saxicola bunting, comprising the following steps:
s1: preparing reference substance solution
Dissolving the first product Ding Duizhao, the second product Ding Duizhao, the third product Ding Duizhao, the palmatine hydrochloride reference and the berberine hydrochloride reference with methanol to obtain reference solution;
s2: preparing test solution
The sample is selected from corydalis saxicola bunting medicinal materials, corydalis saxicola bunting total alkaloids and corydalis saxicola bunting total alkaloids capsules;
when the sample is corydalis saxicola bunting, adding 75% ethanol into the corydalis saxicola bunting, performing ultrasonic treatment, filtering with microporous membrane, and collecting subsequent filtrate;
when the sample is corydalis saxicola bunting total alkali, adding 75% ethanol into the corydalis saxicola bunting total alkali, performing ultrasonic treatment, filtering with a microporous filter membrane, and collecting the subsequent filtrate;
when the sample is the corydalis saxicola bunting total alkali capsule, adding 1% methanol hydrochloride into the content of the corydalis saxicola bunting total alkali capsule, performing ultrasonic treatment, filtering by a microporous filter membrane, and taking a subsequent filtrate to obtain the corydalis saxicola bunting total alkali capsule;
s3: high performance liquid chromatography detection
Precisely sucking the reference substance solution and the sample solution respectively, injecting into a liquid chromatograph, and calculating the content by adopting an external standard method and a QAMS method; the chromatographic conditions are as follows:
chromatographic column: octadecylsilane chemically bonded silica is used as a filler;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.01mol/L potassium dihydrogen phosphate solution, and the gradient elution is carried out;
detection wavelength: 345nm;
column temperature: 30 ℃;
flow rate: 0.8-1.2mL/min, preferably, flow rate: 1mL/min;
sample injection amount: 10uL;
the theoretical plate number is not lower than 3000 according to dehydrogenation Kav Ding Feng;
the gradient elution conditions were:
TABLE 1 gradient elution conditions
Further, the concentration of dehydromethylcavidine, dehydroisopsevidin, dehydrocavidine, palmatine hydrochloride, berberine hydrochloride in the control solution in S1 is 100ug/mL, 40ug/mL, 160ug/mL, 100ug/mL, 20ug/mL.
Further, the ultrasonic wave in the step S2 is ultrasonic wave with the power of 500W and the frequency of 40KHz for 15 minutes.
Further, in S2, when the test sample is a corydalis saxicola bunting drug, the concentration of the test sample solution is 4mg/mL according to the corydalis saxicola bunting drug;
further, in S2, when the test sample is total alkaloids of corydalis saxicola bunting, the concentration of the test sample solution is 0.5mg/mL based on the total alkaloids of corydalis saxicola bunting;
further, when the test product is the corydalis saxicola bunting total alkali capsule in the S2, the concentration of the test product solution is 1.4mg/mL based on the content of the corydalis saxicola bunting total alkali capsule.
Advantageous effects
The one-measurement-multiple-evaluation detection method of corydalis saxicola bunting can be used for detecting the content measurement of 5 chemical components in corydalis saxicola bunting medicinal materials, corydalis saxicola bunting total alkaloids and corydalis saxicola bunting total alkaloids capsules. And the relative retention time and relative correction factors between the dehydrocavidine and the dehydrocavidine, the dehydroisopsoralen, the palmatine hydrochloride and the berberine hydrochloride are established by taking the low-cost and easily-obtained dehydrocavidine as an internal reference for one-measurement and multiple-evaluation, the content of the dehydrocavidine, the dehydroisopsoralen, the dehydrocavidine, the palmatine hydrochloride and the berberine hydrochloride in the corydalis saxicola bunting is calculated through the relative correction factors, and the ultra-high performance liquid chromatography is adopted for measurement.
The method can simultaneously and effectively detect the contents of 5 components of dehydro-emamectin benzoate, dehydro-iso-apocavidine, dehydro-emamectin benzoate, palmatine hydrochloride and berberine hydrochloride in the corydalis saxicola bunting by relative correction factors and chromatographic peak positioning calculation index component contents, can save cost, simplify operation, improve efficiency, has high detection sensitivity and good stability, and has great significance for quality control of the corydalis saxicola bunting and ensuring clinical curative effects of the corydalis saxicola bunting.
The sample detection method established by the invention has the advantages of quick and accurate analysis, strong specificity, good reproducibility and stability, and meanwhile, based on the high-performance liquid chromatography technology, the obtained chromatogram has better peak-to-peak type, and is combined with a one-measurement-multiple-evaluation method.
Drawings
FIG. 1 is a chromatogram of a corydalis saxicola bunting mixed control;
FIG. 2 is a chromatogram of a sample of corydalis saxicola bunting;
FIG. 3 is a chromatogram of a sample of corydalis saxicola bunting total alkaloids;
FIG. 4 is a chromatogram of a test sample of corydalis saxicola bunting total alkaloids capsule;
peaks 1-5 marked in the figure are respectively: dehydromecavidine, dehydroisopsoravidine, dehydrocavidine, palmatine hydrochloride, berberine hydrochloride.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the invention in any way.
The instruments and reagents used in the examples were as follows:
instrument: the high performance liquid chromatograph of the Shimadzu corporation comprises a binary pump system LC-40D, a full-automatic sample injection system SIL-40C, an ultraviolet visible light detector SPD-M40 and an automatic Wen Kongzhu incubator CTO-40S; ML104/02 electronic analytical balance (Mettler Toledo); XSR105DU/A electronic analytical balance (Mettler Toledo); KH-500E ultrasonic cleaner (Kunshan He Chuang ultrasonic instruments Co., ltd.).
Reagent: the source of the corydalis saxicola bunting samples is shown in table 2; dehydrogenated emamectin, dehydrogenated iso-apocavidine, dehydrogenated emamectin, and berberine hydrochloride are purchased from the national food and drug verification institute; methanol (analytically pure); ethanol (analytically pure); acetonitrile (chromatographic purity); hydrochloric acid (analytically pure); ultrapure water.
TABLE 2 information table of corydalis saxicola bunting samples
Example 1 a method for measuring the multiple scores of 5 chemical components in corydalis saxicola bunting and its preparation comprises the following steps:
step 1:
preparing a reference substance solution: weighing 5mg of dehydromethylcarbavin, 2mg of dehydroisopsordine, 8mg of dehydrocarbavin, 5mg of palmatine hydrochloride and 1mg of berberine hydrochloride, precisely weighing, placing in a 50mL measuring flask, dissolving in methanol and fixing volume to ensure that the concentration of dehydromethylcarbavin, dehydroisopsoravidine, dehydrocarbavin, palmatine hydrochloride and berberine hydrochloride is 100ug/mL, 40ug/mL, 160ug/mL, 100ug/mL and 20ug/mL.
Step 2:
preparing a medicinal material test solution: weighing 0.2g of corydalis saxicola bunting, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of 75% ethanol, weighing, performing ultrasonic treatment for 15 minutes (power 500W, frequency 40 KHz), cooling, adding 75% ethanol to make up weight loss, shaking, filtering with microporous membrane (0.22 μm), and collecting filtrate.
Preparing a total alkali test sample solution: weighing corydalis saxicola bunting total alkali 25mg, precisely weighing, placing in conical flask with plug, precisely adding 75% ethanol 50mL, weighing, ultrasound for 15 min (power 500W, frequency 40 KHz), cooling, 75% ethanol supplementing weight loss, shaking, filtering with microporous membrane (0.22 μm), and collecting filtrate.
Preparing a capsule test solution: weighing 70mg of the content of the corydalis saxicola bunting total alkali capsule, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of 1% methanol hydrochloride, weighing, performing ultrasonic treatment for 15 minutes (power 500W, frequency 40 KHz), cooling, supplementing the weight loss with 1% methanol hydrochloride, shaking uniformly, filtering with a microporous filter membrane (0.22 μm), and collecting the subsequent filtrate.
Step 3:
precisely sucking 10 mu L of the reference substance solution in the step 1 and 9 batches of the sample solution in the step 2 respectively, injecting into a liquid chromatograph, and taking octadecylsilane chemically bonded silica as a filler; acetonitrile (A) -0.01mol/L potassium dihydrogen phosphate solution (B) is taken as a mobile phase for gradient elution, and the table 3 is shown; the detection wavelength is 345nm; column temperature is 30 ℃ and flow rate is 1mL/min; the theoretical plate number is not less than 3000 calculated according to dehydrogenation Kav Ding Feng.
TABLE 3 gradient elution conditions
The chromatograms of the reference substance, the corydalis saxicola bunting medicinal material, the corydalis saxicola bunting total alkaloids and the corydalis saxicola bunting total alkaloids capsule are shown in figures 1 to 4, and peaks 1 to 5 marked in the figures are respectively: dehydromecavidine, dehydroisopsoravidine, dehydrocavidine, palmatine hydrochloride, berberine hydrochloride.
The contents of dehydromecavidine, dehydroisopycavidine, dehydrocavidine, palmatine hydrochloride and berberine hydrochloride in corydalis saxicola bunting, total alkaloids and capsules were determined by external standard method and QAMS method and are shown in Table 4.
The results showed no significant difference in the amounts measured by the 2 methods.
TABLE 4 content of 5 Compounds in corydalis saxicola bunting drug, total alkali, capsule
EXAMPLE 2 methodology investigation
1. Linear relationship investigation
Taking mixed reference substance solution, precisely transferring 1, 2, 4, 6, 8 and 10ml, respectively placing into 10ml volumetric flasks, and fixing volume to scale mark with methanol solution to obtain reference 1-reference 6 mixed reference substance solution. The measurement was performed under the same chromatographic conditions. Linear regression was performed with the concentration of the control solution on the X-axis and the chromatographic peak area on the Y-axis, and the results of linear relationship examination are shown in table 5.
Table 5 5 regression equation, linear range, correlation coefficient of the components
2. Precision test
10 μl of the mixed control solution (control-3) was precisely aspirated, and samples were continuously fed 6 times to calculate RSD of 5 component peak areas, respectively. The results show that the peak areas RSD of the dehydroprocalcitonin, the dehydroisopycaintin, the dehydrocavidine, the palmatine hydrochloride and the berberine hydrochloride are respectively 0.24%, 0.21%, 0.25%, 0.27% and 0.35%, and are respectively less than 3%, which shows that the instrument precision is good, and the details are shown in Table 6.
3. Stability test
Taking the same sample solution (medicinal materials 210601, total alkali 200701 and capsule 210301), respectively carrying out sample injection analysis when the sample solution is placed for 0, 2, 4, 6, 8, 10, 12 and 24 hours, measuring the peak areas of all the substances to be detected, and calculating the peak areas RSD of dehydroprocaine, dehydrobacitracin, palmatine hydrochloride and berberine hydrochloride to be 0.27%, 0.34%, 0.29%, 0.32% and 0.42% (medicinal materials 210601); 0.22%, 0.31%, 1.06%, 0.29% (total base 200701); 0.74%, 0.69%, 0.74%, 1.12% (capsule 210301), all less than 3%, indicating that the test solutions were stable for 24 hours, as detailed in Table 6.
4. Repeatability test
6 parts of the same batch of corydalis saxicola bunting medicinal material (210601), corydalis saxicola bunting total alkali (200701) and corydalis saxicola bunting capsule (210301) are respectively taken, a sample solution is prepared by the same method, and the measurement is carried out under the same chromatographic condition. The content RSD of the dehydroprocalcitonin, dehydroisoparaffinovidine, dehydrocavidine, palmatine hydrochloride and berberine hydrochloride is measured to be 0.78%, 1.16%, 0.70%, 0.40% and 0.65% (medicinal material 210601) respectively; 0.42%, 0.51%, 0.41%, 0.30%, 0.60% (total base 200701); 0.38%, 0.54%, 0.43%, 0.28%, 0.38% (capsule 210301), all less than 3%, indicate good reproducibility of the process, as detailed in Table 6.
Table 65 precision, stability, repeatability test of the ingredients
5. Sample addition recovery test
9 parts of sample powder of corydalis saxicola bunting medicinal material (210601), corydalis saxicola bunting total alkali (200701) and corydalis saxicola bunting total alkali capsule (210301) with known contents are taken, about 0.1g of medicinal material, about 12.5mg of total alkali and about 35mg of capsule are precisely weighed, reference substances are respectively added according to 80%, 100% and 120% of 3 levels with known contents (0.8, 1.0 and 1.2ml of the mixed standard solution of the medicinal material sample-adding recovery test, 0.8, 1.0 and 1.2ml of the mixed standard solution of the total alkali sample-adding recovery test, 0.8, 1.0 and 1.2ml of the mixed standard solution of the capsule sample-adding recovery test, 3 parts of sample are added for each level, sample solution is prepared by the same method, sample-adding measurement is performed, and sample-adding recovery is calculated, and the sample-adding recovery rate is shown in tables 7-9.
TABLE 7 results of sample recovery test of medicinal materials
TABLE 8 Total alkali sample recovery test results
TABLE 9 Capsule sample recovery test results
6. Determination of relative correction factors
The mixed control solutions were taken for control 1-control 6, 10. Mu.L for each concentration. With dehydrogenation card dimension Ding Wei internal standard, according to formula f k/m ＝f k /f m ＝W k ×A m /W m ×A k (wherein A k For reference peak area, W k For reference concentration, A m Peak area for other component m, W m For the concentration of the other component m), correction factors of dehydrocavidine on dehydromethylcavidine, dehydroisoparaffinovidine, palmatine hydrochloride and berberine hydrochloride are calculated, and in order to confirm the positions of chromatographic peaks of other 4 components when only dehydrocavidine is adopted as a reference substance, the relative retention time between the chromatographic peaks of other 4 components to be detected and the chromatographic peak of dehydrocavidine Ding Sepu under different conditions is compared. At t=t k /T m (wherein T k For reference retention time, T m Retention time for the other component m) and the results are shown in tables 10 to 11.
Table 10 correction factors
TABLE 11 relative retention time
7 relative correction factor (f k/m ) Durability evaluation
7.1 different high Performance liquid chromatographs and column pairs f k/m Is to accurately absorb the mixed reference substance-3 solution, and to measure the same chromatographic conditions, and to examine different high performance liquid phase meters (Shimadzu LC-40D, agilent 1260 II, waters e-2695) and different chromatographic columns (ZORBAX SB-C18, waters)CSH TM C18, shimadzu Shim-pack GIST) pair f k/m The effect of (c) and the result shows f k/m The durability was good when using different instruments, different chromatographic columns, as detailed in tables 12-13.
Table 12 different pairs of hplc f k/m Influence of (2)
TABLE 13 different column pairs f k/m Influence of (2)
7.2 different flow Rate and column temperature vs f k/m Influence of the value
Precisely sucking the mixed reference substance-3 solution, measuring under the same chromatographic condition, and respectively observing f at different flow rates (0.8, 1.0, 1.2 mL/min) and different column temperatures (30deg.C, 35deg.C, 40deg.C) k/m The effect of (c) and the result shows f k/m Durability at different flow rates and different column temperaturesThe properties are good and detailed in tables 14 to 15.
TABLE 14 different flow Rate vs f k/m Influence of (2)
Table 15 different column temperatures vs f k/m Influence of (2)
8 chromatographic peak positioning of the component to be measured
The chromatographic peaks of the components to be detected are positioned by adopting the relative retention time, namely the ratio of the retention time between each component to be detected and dehydrocavidine, and the reproducibility of the relative retention value on different brands of high performance liquid chromatographs and different types of chromatographic columns and the reproducibility of the relative retention value under different flow rates and different column temperatures are examined, and the results show that the reproducibility is good, and are shown in tables 16-19 in detail.
TABLE 16 influence of different HPLC on t
TABLE 17 influence of different chromatographic columns on t
TABLE 18 influence of different flow rates on t
TABLE 19 influence of different column temperatures on t
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (6)
1. The method for detecting the one-measurement-multiple-evaluation content of five chemical components in corydalis saxicola bunting is characterized by comprising the following steps of:
s1: preparing reference substance solution
Dissolving the first product Ding Duizhao, the second product Ding Duizhao, the third product Ding Duizhao, the palmatine hydrochloride reference and the berberine hydrochloride reference with methanol to obtain reference solution;
s2: preparing test solution
The sample is selected from corydalis saxicola bunting medicinal materials, corydalis saxicola bunting total alkaloids and corydalis saxicola bunting total alkaloids capsules;
when the sample is corydalis saxicola bunting, adding 75% ethanol into the corydalis saxicola bunting, performing ultrasonic treatment, filtering with microporous membrane, and collecting subsequent filtrate;
when the sample is corydalis saxicola bunting total alkali, adding 75% ethanol into the corydalis saxicola bunting total alkali, performing ultrasonic treatment, filtering with a microporous filter membrane, and collecting the subsequent filtrate;
when the sample is the corydalis saxicola bunting total alkali capsule, adding 1% methanol hydrochloride into the content of the corydalis saxicola bunting total alkali capsule, performing ultrasonic treatment, filtering by a microporous filter membrane, and taking a subsequent filtrate to obtain the corydalis saxicola bunting total alkali capsule;
s3: high performance liquid chromatography detection
Precisely sucking the reference substance solution and the sample solution respectively, injecting into a liquid chromatograph, and calculating the content by adopting an external standard method and a QAMS method; the chromatographic conditions are as follows:
chromatographic column: octadecylsilane chemically bonded silica is used as a filler;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.01mol/L potassium dihydrogen phosphate solution, and the gradient elution is carried out;
detection wavelength: 345nm;
column temperature: 30 ℃;
flow rate: 0.8-1.2mL/min, preferably, flow rate: 1mL/min;
sample injection amount: 10uL;
the theoretical plate number is not lower than 3000 according to dehydrogenation Kav Ding Feng;
the gradient elution conditions were:
2. the method according to claim 1, wherein the concentration of dehydroprocalcitonin, dehydroisoparaffinavidine, dehydrocavidine, palmatine hydrochloride, berberine hydrochloride in the control solution of S1 is 100ug/mL, 40ug/mL, 160ug/mL, 100ug/mL, 20ug/mL.
3. The method according to claim 1, wherein the ultrasonic wave of S2 is a power of 500W and a frequency of 40KHz for 15 minutes.
4. The method according to claim 1, wherein when the sample is corydalis saxicola bunting in S2, the concentration of the sample solution is 4mg/mL based on the corydalis saxicola bunting.
5. The method according to claim 1, wherein when the sample is total alkaloid in S2, the concentration of the sample solution based on total alkaloid in S2 is 0.5mg/mL.
6. The method according to claim 1, wherein when the test substance is a total alkaloids capsule in S2, the concentration of the test substance solution based on the total alkaloids capsule content is 1.4mg/mL.
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