CN115436497A - Centipede medicinal material quality detection method - Google Patents
Centipede medicinal material quality detection method Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicine analysis, and particularly discloses a quality detection method of centipede medicinal materials, which is based on a high performance liquid chromatography technology and adopts a one-label multi-detection analysis method, hypoxanthine is taken as an internal reference substance, and the contents of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in the centipede medicinal materials are detected. The established quality detection method has the advantages of simple and convenient operation, stability, reliability, strong specificity and good reproducibility, can comprehensively and effectively detect the component content of the centipede medicinal material, is favorable for stabilizing the product quality, and ensures the safety and effectiveness of clinical medication, thereby better meeting the requirements of medical treatment and market.
Description
Technical Field
The invention relates to a quality detection method of centipede medicinal materials, and belongs to the field of quality control of traditional Chinese medicinal materials.
Background
Centipede is originally recorded in the traditional Chinese medicine classic Shen nong Ben Cao Jing, has more than two thousand years of medicinal history in China and is an indispensable traditional Chinese medicinal material in clinic. The centipede medicinal materials are dry polypide of Scolopendra subspinipes mutilans L.Koch of centipede family specified in the 'Chinese pharmacopoeia' 2020 edition, and the main effects of calming endogenous wind and relieving spasm, dredging collaterals and relieving pain, and counteracting toxic substances and dissipating stagnation. Modern pharmacological research shows that centipede has pharmacological actions of analgesia, anti-inflammation, antibiosis, anti-tumor, anticonvulsant, anticoagulation fibrinolysis, immunity regulation, anticoagulation, blood pressure reduction, blood fat reduction and the like, and can be used for treating various surgical inflammations, malignant tumors, diabetes, epilepsy, rheumatoid arthritis, various nerve pains, vascular embolism diseases, pertussis, nerve headache, tuberculosis and other diseases in modern medicine. However, the effective substance basis of animal medicinal materials is not very clear, and the research on centipede medicinal materials is also difficult to avoid.
In the first part of the Chinese pharmacopoeia 2020 edition, only 5 items of characters, water, total ash, aflatoxin and extract are used for quality control of centipedes, and the content determination of specificity is lacked, so that the quality of the centipede medicinal material is difficult to effectively ensure, and the quality standard needs to be improved and perfected urgently. At present, the research on the quality control of centipede medicinal materials at home and abroad is less, and most of the prior art is to measure amino acid, histamine, nucleoside and purine components in centipedes, but the components widely exist in most animal and plant tissues, and the specificity of the components for the quality control is not strong.
Chinese patent CN105004809A discloses a quality control method of centipede medicinal materials, which uses 3, 8-dihydroxyquinoline in centipede as an index component and adopts high performance liquid chromatography or thin layer chromatography to control the quality of the centipede medicinal materials. Wherein the high performance liquid chromatography adopts methanol-10 mmol/L potassium dihydrogen phosphate solution =28:64 is a mobile phase, the flow rate is 0.6ml/min, the detection wavelength is 252nm, the column temperature is 35 ℃, and the specific quantitative detection of the 3, 8-dihydroxyquinoline can be realized. However, the detection method can realize accurate quantification of the single component of the 3, 8-dihydroxyquinoline, and cannot effectively separate other quinoline or purine components during simultaneous detection, so that accurate quantitative detection cannot be realized.
Chinese patent CN110357815A discloses quinoline compounds, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate, in two centipede medicinal materials; chinese patent CN111471012A discloses a centipede quinoline compound-3, 8-dihydroxy-4-methoxyquinoline. The research of a multi-means and multi-index quality control method is carried out on the centipede medicinal materials, and the quality standard system of the centipede medicinal materials is favorably perfected, so that the quality of the medicinal materials and the safety and effectiveness of clinical medication are guaranteed. The inventors tried to detect several of the above quinoline-based components and purine-based components at the same time, and the separation degree was not good, and accurate quantification could not be achieved.
Based on the above, the invention establishes a content detection method of 5 components in centipedes by high performance liquid chromatography, comprehensively evaluates and controls the quality of centipede medicinal materials, and has important significance for activity research of centipede pharmacodynamic substance basis, qualitative and quantitative analysis of active ingredients in animal medicaments and improvement of centipede medicinal material quality standard.
Disclosure of Invention
Aiming at the defects of the existing centipede medicinal material quality detection method, the invention aims to provide a new centipede medicinal material quality detection method. The inventor successfully establishes the HPLC method for simultaneously detecting the content of hypoxanthine (6-hydroxypurinine), xanthine (2, 6-dihydroxypurinine), 3-hydroxy-8-quinoline sulfate (jineol-8-sulfate), 3-hydroxy-4-methoxy-8-quinoline sulfate (3-hydroxy-4-methoxy quinolin-8-yl hydrogen sulfate) and 3, 8-dihydroxyquinoline (jineol) in centipede medicinal materials through long-time exploration and repeated tests, integrates the content detection of 5 components of the centipede medicinal materials, and provides a novel and more comprehensive and accurate centipede medicinal material detection method.
The method established by the invention has good applicability, takes hypoxanthine as an internal reference, determines relative correction factors of xanthine, 3-hydroxy-8-quinoline sulfate, 3-hydroxy-4-methoxy-8-quinoline sulfate and 3, 8-dihydroxyquinoline by a multi-point correction method, and establishes a content detection method of the 4 components by adopting a one-test multi-evaluation method; the method can be used for evaluating the quality of the centipede medicinal materials through methodology verification, is accurate and low in cost, can more comprehensively reflect the internal quality of the centipede medicinal materials, and is more reasonable in controlling the quality of the medicinal materials by the total content of 5 components.
The invention aims to provide a centipede medicinal material quality detection method, which adopts a one-standard multi-detection analysis method, takes hypoxanthine as an internal reference substance, and detects the contents of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in centipede medicinal materials by HPLC.
Preferably, the HPLC column is Agilent ZORBAX XDB C18, agilent ZORBAX SB C18 or YMC-Pack ODS-AQ; further preferred is YMC-Pack ODS-AQ.
Preferably, the HPLC method takes acetonitrile-ammonium phosphate solution as a mobile phase; further preferred is acetonitrile: 10-20 mmol/L ammonium phosphate solution =95:5 mobile phase a, acetonitrile: 10-20 mmol/L ammonium phosphate solution =25: mobile phase B is 75.
Preferably, the HPLC method employs a gradient of elution:
preferably, the elution flow rate is 0.6mL/min to 1.0mL/min; column temperature: 25-35 ℃; detection wavelength: 220-280nm; further preferably, the flow rate is 0.8mL/min; the column temperature is 30 ℃; the detection wavelength was 254nm.
Specifically, the one-standard multi-test method for detecting the content of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8 quinoline sulfate in the centipede medicinal materials comprises the following steps:
A. preparation of a test solution: taking a proper amount of centipede medicinal materials, crushing and sieving the medicinal materials to be used as a test sample for later use; and (3) taking a proper amount of sample powder, and adding the mixture into the sample powder according to a weight/volume ratio of 1: ultrasonic extracting with 20-50 times of methanol solution for 20-50min, cooling to room temperature, weighing, adding solvent, reducing weight loss, shaking, centrifuging, sucking supernatant, filtering, discarding the primary filtrate, and using the secondary filtrate as sample solution;
B. preparation of control solutions: preparing a single reference solution of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate, 3-hydroxy-4-methoxy-8-quinoline sulfate and a mixed reference solution of 5 components;
C. determination of relative retention time: and C, sucking the 5 single reference substance solutions and the 5 component mixed reference substance solutions in the step B, respectively injecting the solutions into HPLC (high performance liquid chromatography) for detection, recording the retention time of the 5 single reference substance solutions, simultaneously recording the retention time of each component in the 5 component mixed reference substance solutions, taking hypoxanthine as an internal reference substance, and performing detection by using a formula RRT = T k /T s Calculating the relative retention time of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfate, 3-hydroxy-4-methoxy-8-quinolinesulfate, relative to the internal reference hypoxanthine; t in the formula s Retention time for internal reference, T k The retention time of other 6 ingredients to be tested in the mixed reference solution;
D. determination of the relative correction factor: and D, sucking the mixed reference substance solution in the step B, injecting HPLC, detecting, recording peak areas of 5 components of the mixed reference substance solution, taking hypoxanthine as an internal reference substance, and calculating a formula f according to a relative correction factor k/s ＝(A s ×C k )/(A k ×C s ) Calculating relative correction factors f for xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfate, 3-hydroxy-4-methoxy-8-quinolinesulfate k/s (ii) a In the formula A s Is the peak area of the internal reference substance, C s Is the mass concentration of the internal reference substance, A k As peak area of other component, C k The mass concentration of other components;
E. detecting the content of multiple components: respectively sucking the test solution in the step A and the hypoxanthine reference solution in the step B, injecting HPLC, detecting, and performing HPLC according to formula C Sample (II) ＝(A Sample (II) ×C For is to )/A To pair Calculating the content of hypoxanthine in the test solution; according to the step C, the relative retention time RRT is used for characterizing 4 components of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in the test solution, and according to the step D, the relative correction factor f is used k/s Calculating the content of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in the test solution; in the formula A Sample (A) Is the peak area of the test article, C To pair Concentration of control, A For is to Peak area for control.
Preferably, the concentration of methanol used in step A is 40-60%, preferably 50%, and the filtering step uses an organic filter head of 0.22 μm.
Specifically, the preparation method of the test solution in the step a comprises the following steps:
taking a proper amount of centipede medicinal materials, grinding, and sieving with a 80-mesh sieve; as a test sample, for standby; sampling 2.0g of powder, accurately weighing, placing in a 50mL round-bottom flask, accurately adding 50mL of 50% methanol solution, sealing, weighing, refluxing for 30min, cooling to room temperature, weighing again, complementing the weight loss amount with 50% methanol solution, shaking up, centrifuging, sucking supernatant, passing through a 0.22 μm organic filter head, discarding the primary filtrate, and taking the subsequent filtrate as a test solution.
Preferably, the preparation method of the mixed control solution in the step B comprises the following steps:
precisely weighing appropriate amounts of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate, respectively dissolving with 50% methanol, and respectively preparing into reference stock solutions with concentrations of 0.1311mg/mL, 0.3810mg/mL, 0.2980mg/mL, 0.3316mg/mL and 0.2748mg/mL for later use;
diluting the stock solution of the reference substance into a solution containing hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate, 3-hydroxy-4-methoxy-8-quinoline sulfate 0.06555mg/mL, 0.03810mg/mL, 0.02980mg/mL, 0.04145mg/mL and 0.01099mg/mL in each 1mL, namely the mixed reference substance solution.
Preferably, the HPLC chromatographic conditions are: employing YMC-Pack ODS-AQ (250 mm. Times.4.6 mm,5 μm) column; and (2) mixing acetonitrile: 10-20 mmol/L ammonium phosphate solution =95:5 as mobile phase a, in acetonitrile: 10-20 mmol/L ammonium phosphate solution =25: and 75, carrying out linear gradient elution on the mobile phase B, wherein the elution procedure is as follows:
flow rate: 0.6mL/min-1.0mL/min; column temperature: 25-35 ℃; sample injection amount: 2-10 μ L; detection wavelength: 220-280nm.
Preferably, the flow rate is 0.8mL/min in the chromatographic conditions; the column temperature is 30 ℃; the sample amount was 5. Mu.L, and the detection wavelength was 254nm.
Preferably, the relative retention time of the xanthine, the 3, 8-dihydroxyquinoline, the 3-hydroxy-8-quinoline sulfate and the 3-hydroxy-4-methoxy-8-quinoline sulfate in the step C relative to the reference hypoxanthine is 1.22 +/-0.05, 3.51 +/-0.2, 2.66 +/-0.04 and 3.07 +/-0.05 respectively; further preferably, the relative retention times are 1.22, 3.52, 2.65, 3.04, respectively.
D, the relative correction factors of the xanthine, the 3, 8-dihydroxyquinoline, the 3-hydroxy-8-quinoline sulfate and the 3-hydroxy-4-methoxy-8-quinoline sulfate in the step D are respectively 1.68 +/-0.08, 0.76 +/-0.02, 9.09 +/-0.26 and 5.06 +/-0.35;
preferably, the relative correction factors are 1.67, 0.76, 9.01, 5.03, respectively.
Compared with the prior art, the invention has the following beneficial effects:
1) According to the invention, 5 components in the centipede medicinal materials are simultaneously detected by one test and multiple evaluations, and synchronous detection of a plurality of components can be realized by detecting the content of one component of hypoxanthine, so that the detection time is saved, the detection efficiency is improved, the problems of shortage of reference substances and high detection cost can be solved, the consumables are saved, the detection efficiency is improved, and the labor cost is saved.
2) The invention establishes a method for detecting the content of 5 components in centipede medicinal materials, adopts an HPLC method to simultaneously detect the content of 5 components of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in the centipede medicinal materials, and has the advantages of linearity, precision, repeatability, stability and recovery rate test results meeting the requirements, good applicability, capability of further effectively controlling the quality of the centipede medicinal materials and realization of comprehensively evaluating the preparation quality.
3) The invention adopts a one-label multi-detection method to simultaneously detect the content of 5 components of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in centipede medicinal materials, and the method has the advantages of high accuracy, good stability, high precision, excellent repeatability, high recovery rate and simple operation.
Drawings
FIG. 1 is a chromatogram of a centipede drug reference solution, wherein the reference marks in the drawing are as follows:
wherein 1 is hypoxanthine; 2 is xanthine; 3 is 3-hydroxy-8-quinoline sulfate; 4 is 3-hydroxy-4-methoxy-8 quinoline sulfate; 5 is 3, 8-dihydroxyquinoline.
FIG. 2 is a chromatogram of a test solution of Scolopendra materials, with the following scores:
1 is hypoxanthine; 2 is xanthine; 3 is 3-hydroxy-8-quinoline sulfate; 4 is 3-hydroxy-4-methoxy-8 quinoline sulfate; 5 is 3, 8-dihydroxyquinoline.
Detailed Description
The present invention will be further described below in the context of exemplary embodiments for a more complete understanding of its practice. Meanwhile, since the above examples are only preferred embodiments of the present invention, the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that various modifications and improvements can be made by those skilled in the art without departing from the principle and spirit of the invention, and these modifications and improvements should also fall into the scope of the invention.
The centipede medicinal materials can be obtained by commercial means.
Example 1 research on methodology for detecting content of effective components of centipede medicinal materials by one-label multi-measurement method
1 Instrument and reagent
1.1 reagents
Hypoxanthine (6-dihydroxypurinine, CAS:68-94-0, purity not less than 98%), xanthine (2, 6-dihydroxypurinine, CAS:69-89-6, purity not less than 98%), the above reference substances are provided by Shanghai-sourced leaf Biotechnology GmbH; 3-hydroxy-8-quinoline sulfate (jineol-8-sulfate, purity is more than or equal to 98%), 3-hydroxy-4-methoxy-8-quinoline sulfate (3-hydroxy-4-methoxy quinolin-8-yl hydrogen sulfate, purity is more than or equal to 97%), 3, 8-dihydroxy quinoline (jineol, purity is more than or equal to 98%), and all the above reference substances can be prepared by adopting the method in the prior art. Acetonitrile, methanol (chromatographically pure, semer feishell technologies ltd).
1.2 instruments
An Agilent 1100 hplc (including DAD detector, quaternary low pressure gradient pump, agilent open Lab chromatographic workstation, agilent technologies, inc.); one in ten thousand electronic analytical balance (TOLEDO LE204E, METTLER); one ten thousandth electronic analytical balance (01193-YP 601N, METTLER); a numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); an organic filter (0.22 μm pore size, asimer technologies ltd); pure water/ultrapure water systems.
1.3 test drugs
Scolopendra, available from Shandong-Kanpu pharmaceutical Co., ltd, is shown in Table 1;
TABLE 1 Centipede herb sample statistical table (n = 14)
2 methods and results
2.1 preparation of the solution
(1) Preparation of a test solution: taking a proper amount of centipede medicinal materials, grinding, and sieving by a 80-mesh sieve; as a test sample for standby; sampling 2.0g of powder, precisely weighing, placing in a 50mL round-bottom flask, precisely adding 50mL of 50% methanol solution, sealing, weighing, refluxing for 30min, cooling to room temperature, weighing again, complementing the weight loss by 50% methanol solution, shaking up, centrifuging, sucking supernatant, filtering with a 0.22 μm organic filter head, discarding primary filtrate, and taking subsequent filtrate as sample solution.
(2) Preparation of control solutions: precisely weighing appropriate amounts of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate, respectively dissolving with 50% methanol, and respectively preparing into reference stock solutions with concentrations of 0.1311mg/mL, 0.3810mg/mL, 0.2980mg/mL, 0.3316mg/mL and 0.2748mg/mL for later use; diluting the stock solution of the reference substance into a solution containing hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate, 3-hydroxy-4-methoxy-8-quinoline sulfate 0.06555mg/mL, 0.03810mg/mL, 0.02980mg/mL, 0.04145mg/mL and 0.01099mg/mL in each 1mL, namely the mixed reference substance solution.
2.2 chromatographic conditions
A chromatographic column: and (3) chromatographic column: YMC-PackODS-AQ (250 mm. Times.4.6 mm,5 μm); a mobile phase A: acetonitrile: 20mmol/L ammonium phosphate solution =95:5, mobile phase B: acetonitrile: 20mmol/L ammonium phosphate solution =25:75; flow rate: 0.8mL/min; solvent: methanol-ultrapure water (50; column temperature: 30 ℃; sample introduction amount: 5 mu L of the solution; detection wavelength: 254nm; gradient elution.
TABLE 2 elution gradient chart
2.3 recording chromatograms
Injecting the reference solution and the sample solution into a high performance liquid chromatography system, recording a chromatogram, and calculating the content of each component. The HPLC profile of the mixed control solution is shown in FIG. 1, and the HPLC profile of the test solution is shown in FIG. 2.
2.4 methodological considerations
2.4.1 Linear Range inspection
Diluting the reference stock solution into a series of concentrations, detecting peak areas according to a chromatographic method under 2.2, sequentially injecting hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate, calculating a regression equation (Y = aX + b) by taking the concentration (mg/mL) as an abscissa (X) and the peak area (A) as an ordinate (Y), and drawing a standard curve, wherein the linear regression equation of 5 active ingredients is shown in Table 3.
TABLE 3 Linear equation of active ingredients
The test result shows that 5 components of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxyl-8-quinoline sulfate have good linear relation in respective corresponding mass concentration ranges.
2.4.2 precision test
And taking the mixed reference substance solution, carrying out sample injection according to the chromatographic method under the item 2.2, carrying out continuous sample injection for 6 times, and recording the peak area. The average peak area RSD (%) of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate is obtained, and the results show that the RSD (%) of 4 and 5 components in the table is less than 2.0 percent, which shows that the chromatographic method is applicable to systems and the instrument is precise and stable.
Table 4 active ingredient precision test (n = 6)
2.4.3 repeatability tests
210601 batches of centipede medicinal material sample powder is precisely weighed for 6 times, the test solution is prepared according to the test solution preparation method under item 2.1, sample introduction is respectively carried out according to the chromatographic method under item 2.2, peak areas are recorded, and the content of the components is calculated, and the result is shown in table 5. The average value RSD (%) of the 6-time content of each component is less than 2.5%, which shows that the method for detecting the content of the active ingredients in the centipede medicinal materials has good repeatability.
Table 5 active ingredient repeatability tests (n = 6)
2.4.4 stability test
210601 batches of centipede medicinal material test samples are precisely weighed, the test solution is prepared according to the test solution preparation method under item 2.1, samples are respectively injected for 0h, 2h, 4h, 6h, 8h, 12h, 24h and 48h according to the chromatographic method under item 2.2, and the peak area is recorded. Obtaining the average peak areas of hypoxanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8 quinoline sulfate, and calculating the RSD (%), wherein the RSD (%) of 5 components is less than 2.0 percent, which shows that the centipede medicinal material sample solution is stable within 48 hours, and the results are shown in Table 6.
TABLE 6 stability test of active ingredients
2.4.5 accuracy test (sample recovery test)
Accurately weighing 6 parts of centipede medicinal material sample powder with known content, respectively and accurately adding reference substance stock solution containing equal amount of each component of reference substance, preparing sample solution according to the preparation method of test solution under item 2.1, injecting sample according to the chromatographic method under item 2.2, and obtaining results shown in Table 7. The results show that the average recovery rate RSD (%) of each component is less than 4.0%, which indicates that the accuracy of the two content detection methods is good.
Table 7 active recovery test (n = 6)
2.5 one-label multi-detection method for detecting content of 5 components in centipede medicinal material
2.5.1 calculation of relative correction factor
(1) Slope correction method: in the standard curve, X = (Y-b)/a = Y/a-b/a, the b value is usually caused by error, in a/b>At 100, b/a is usually negligible and can be calculated directly with X = Y/a, when the correction factor is calculated by the formula: f. of k/s ＝a s /a k The mass concentration calculation formula of the components to be measured is as follows: c k’ ＝(A k’ ×f k/s )/a s (a s : slope of reference, a k : slope of other control components). Calculating by slope correction method to obtain f of other components relative to hypoxanthine k/s F of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate, 3-hydroxy-4-methoxy-8-quinoline sulfate with respect to hypoxanthine k/s See table 8.
TABLE 8 slope correction method correction factor (f) k/s )
(2) Multi-point correction method: f calculated at a plurality of mass concentration points k/s Taking the average value as the quantitative f k/s . The correction factor calculation formula: f. of k/s ＝(A S ×C K )/(A k ×C s ). The mass concentration calculation formula of the components to be measured is as follows: c k’ ＝(f k/s ×C s ×A k’ )/A S (As: reference substance chromatographic Peak area, C S : reference mass concentration of substance, A K : peak area of chromatographic peak of other control component, C k : mass concentration of other control Components, A k : peak area of chromatographic peak of component to be measured). The results are shown in Table 9.
TABLE 9 correction factor (f) for multiple point correction method k/s ，n＝5)
2.5.2 calculation of relative Retention values
And (4) positioning by using relative retention time, namely determining by using retention time of chromatographic peaks and hypoxanthine chromatographic peaks of components to be detected. Relative retention time calculation formula: RRT = T k /T s (T s Retention time for reference, T k Retention time for other components to be tested), the results are shown in table 10.
TABLE 10 relative retention of multipoint correction method (RRT, n = 7)
Comparing the 3-test and multi-evaluation results with the detection results of the external standard method
3.1 one-test and multi-evaluation results are compared with the content detection results of the external standard method
Taking 14 batches of centipede medicinal material test samples to prepare a reference substance solution and a test sample solution according to the condition of 2.1, injecting samples according to the chromatographic condition of 2.2, respectively carrying out content detection on the samples by a one-standard multi-detection method and an external standard method, and comparing the results of the two detection methods, which are shown in a table 11. As can be seen from the table, the 5 active ingredient content RSD (%) values obtained by different calculation methods are all less than 3%, and the applicability is good.
TABLE 11 one-test-multiple evaluation results and external standard method content test results (n = 14)
3.2 multipoint corrected relative retention value is compared with the external standard actual retention time
The theoretical retention time of 14 centipede drug samples calculated by the multipoint correction factor is compared with the actual retention time of an external standard method, and the result has no significant difference, which is shown in table 12. As can be seen from the table, the RSD (%) values between the calculated retention value and the actual retention value of 5 active ingredients were all less than 1.0%, and the applicability was good.
Table 12 one-test-multiple evaluation results compared to external standard retention time (n = 14)
In conclusion, the research adopts the high performance liquid chromatography to establish a content detection method for detecting the content of the centipede medicinal material (QAMS), and the method establishes a liquid chromatography method for simultaneously detecting 5 components of the centipede medicinal material by investigating the aspects of an extraction method, chromatographic conditions, elution gradient and the like, and further detects the content of the centipede medicinal materialOne step takes hypoxanthine as an internal reference substance, determines relative correction factors of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxyl-8 quinoline sulfate by a multipoint correction method, and establishes a content detection method of the 4 components by adopting a one-standard multi-measurement method; relative correction factor (f) of these 4 components k/s ) 1.67, 9.01, 5.03, 0.76, respectively, and relative retention values (RRT) of 1.22, 2.65, 3.04, 3.52, respectively;
the invention adopts QAMS method to detect 14 batches of centipede medicinal materials, compares the content of 5 components obtained by QAMS method with the content detection value of External Standard Method (ESM), and RSD (%) is less than 1.0%, compares the calculated retention value obtained by QAMS method with the actual retention value obtained by ESM method, and RSD (%) is less than 1.0%, which shows that the method established by the invention is accurate, low in cost, stable and reliable, strong in specificity and good in reproducibility, can reflect the internal quality of centipede medicinal materials more comprehensively, and provides technical basis for further improving the quality standard of centipede medicinal materials.
Claims (10)
1. A centipede medicinal material quality detection method is characterized in that a one-standard multi-detection method is adopted, hypoxanthine is taken as an internal reference substance, and the contents of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in the centipede medicinal material are detected by an HPLC method.
2. The method of claim 1, wherein the HPLC column is Agilent ZORBAX XDB C18, agilent ZORBAX SB C18, or YMC-Pack ODS-AQ.
3. The method of claim 1, wherein the HPLC column is YMC-Pack ODS-AQ.
4. The method of claim 1, wherein the HPLC method uses acetonitrile-ammonium phosphate solution as the mobile phase.
5. The quality inspection method according to claim 4, wherein the acetonitrile: 10-20 mmol/L ammonium phosphate solution =95 as mobile phase a, acetonitrile: 10-20 mmol/L ammonium phosphate solution =25: mobile phase B is 75.
6. The method of claim 5, wherein the HPLC method uses the following gradient for elution:
7. the quality detection method of claim 6, wherein the elution stream is from 0.6mL/min to 1.0mL/min; column temperature: 25-35 ℃; detection wavelength: 220-280nm.
8. The quality inspection method according to claim 7, wherein the flow rate is 0.8mL/min; the column temperature is 30 ℃; the detection wavelength was 254nm.
9. The quality inspection method of any one of claims 1 to 8, comprising the steps of:
A. preparing a test solution: taking a proper amount of centipede medicinal materials, crushing and sieving the centipede medicinal materials to be used as a test sample for later use; and (3) taking a proper amount of sample powder, and adding the mixture into the sample powder according to a weight/volume ratio of 1: ultrasonic extracting with 20-50 times of methanol solution for 20-50min, cooling to room temperature, weighing, adding solvent, reducing weight loss, shaking, centrifuging, sucking supernatant, filtering, discarding the primary filtrate, and using the secondary filtrate as sample solution;
B. preparation of control solutions: preparing a single reference solution of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate, 3-hydroxy-4-methoxy-8-quinoline sulfate and a mixed reference solution of 5 components;
C. determination of relative retention time: sucking the 5 single reference substance solutions and the 5 component mixed reference substance solutions in the step B, respectively injecting into HPLC for detection, and recordingRecording the retention time of 5 single control solutions and the retention time of each component in 5 component mixed control solutions, taking hypoxanthine as an internal reference, and adopting the formula RRT = T k /T s Calculating the relative retention time of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfate, 3-hydroxy-4-methoxy-8-quinolinesulfate, relative to the internal reference hypoxanthine; in the formula T s Retention time for internal reference, T k The retention time of other 6 ingredients to be tested in the mixed reference solution;
D. determination of the relative correction factor: and C, sucking the mixed reference substance solution in the step B, injecting HPLC (high performance liquid chromatography), detecting, recording peak areas of 5 components of the mixed reference substance solution, taking hypoxanthine as an internal reference substance, and calculating a formula f according to a relative correction factor k/s ＝(A s ×C k )/(A k ×C s ) Calculating relative correction factors f for xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfate, 3-hydroxy-4-methoxy-8-quinolinesulfate k/s (ii) a In the formula A s Is the peak area of the internal reference substance, C s Is the mass concentration of the internal reference, A k As peak area of other component, C k The mass concentration of other components;
E. and (3) detecting the content of multiple components: respectively sucking the test solution obtained in step A and the hypoxanthine reference solution obtained in step B, injecting into HPLC, detecting, and analyzing according to formula C Sample (A) ＝(A Sample (A) ×C To pair )/A To pair Calculating the content of hypoxanthine in the test solution; according to the step C, the relative retention time RRT is used for characterizing 4 components of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in the test solution, and according to the step D, the relative correction factor f is used k/s Calculating the content of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in the test solution; in the formula A Sample (A) Is the peak area of the test article, C To pair Concentration of control, A To pair Peak area of control.
10. The detection method according to claim 9, wherein the test solution in step a and the control solution in step B are prepared with 40% to 60% methanol.
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| CN105004809A (en) * | 2015-07-13 | 2015-10-28 | 鲁南制药集团股份有限公司 | Centipede medicinal material quality control method |
| CN109490446A (en) * | 2018-12-31 | 2019-03-19 | 云南中医学院 | Method that is a kind of while measuring 5 kinds of flavone compounds in ground centipede medicinal material |
| CN110357815A (en) * | 2018-04-09 | 2019-10-22 | 鲁南制药集团股份有限公司 | Centipede quinolines and preparation method thereof, purposes |
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| CN105004809A (en) * | 2015-07-13 | 2015-10-28 | 鲁南制药集团股份有限公司 | Centipede medicinal material quality control method |
| CN110357815A (en) * | 2018-04-09 | 2019-10-22 | 鲁南制药集团股份有限公司 | Centipede quinolines and preparation method thereof, purposes |
| CN109490446A (en) * | 2018-12-31 | 2019-03-19 | 云南中医学院 | Method that is a kind of while measuring 5 kinds of flavone compounds in ground centipede medicinal material |
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