CN115436497B - 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, adopts a one-standard-multi-measurement analysis method, uses hypoxanthine as an internal reference, and detects 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. The established quality detection method is simple and convenient to operate, stable and reliable, strong in specificity and good in reproducibility, can comprehensively and effectively detect the component content of the centipede medicinal material, is beneficial to stabilizing the product quality, ensures the safety and effectiveness of clinical medication, and further better meets the needs 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
The centipede is recorded in the classic Shennong Ben Cao Jing of Chinese traditional medicine at the earliest, has more than two thousand years of medical history in China, and is an indispensable traditional Chinese medicinal material in clinic. The centipede medicinal material is Scolopendra subspinipes mutilans L.Koch dried body of centipede, which is a centipede family animal, and has the main effects of calming endogenous wind, relieving spasm, dredging collaterals, relieving pain, counteracting toxic substances and resolving hard mass. Modern pharmacological researches have shown that centipedes have pharmacological effects of relieving pain, resisting inflammation, resisting bacteria, resisting tumor, resisting convulsion, resisting fibrinolysis, regulating immunity, resisting blood coagulation, reducing blood pressure and blood lipid, 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 the problem.
In the quality control of centipedes in the first part of Chinese pharmacopoeia 2020 edition, only 5 items of properties, moisture, total ash content, aflatoxin and extract are included, the content measurement of specificity is lacking, the quality of centipede medicinal materials is difficult to effectively guarantee, and the quality standard of the centipede medicinal materials needs to be improved and perfected. At present, the quality control research of centipede medicinal materials at home and abroad is less, in the prior art, amino acids, histidines, nucleosides and purines in centipedes are mostly measured, but the components are widely existed in most animal and plant tissues and are not strong in specificity for quality control.
Chinese patent CN105004809A discloses a quality control method of centipede medicinal materials, wherein 3, 8-dihydroxyquinoline in centipede is used as an index component, and high performance liquid chromatography or thin layer chromatography is adopted to control the quality of the centipede medicinal materials. Wherein high performance liquid chromatography uses 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, and the detection is carried out at the column temperature of 35 ℃, so that the specific quantitative detection of the 3, 8-dihydroxyquinoline can be realized. However, the detection method can accurately quantify the single component of 3, 8-dihydroxyquinoline, and can not effectively separate other quinoline or purine components during simultaneous detection, and can not accurately quantify the detection.
Chinese patent CN110357815a discloses quinoline compounds, 3-hydroxy-8-quinoline sulfate, 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 the multi-means and multi-index quality control method is carried out on the centipede medicinal material, which is beneficial to perfecting the quality standard system of the centipede medicinal material, thereby ensuring the quality of the medicinal material and the safety and effectiveness of clinical medication. The inventors tried to detect the above-mentioned several quinoline components and purine components simultaneously, and the degree of separation was poor, and accurate quantification was not possible.
Based on the method, the content detection method of 5 components in the centipedes is established through the high performance liquid chromatography, the quality of the centipede medicinal materials is comprehensively evaluated and controlled, and the method has important significance for activity research of the centipede medicinal material foundation, qualitative and quantitative analysis of the effective components in the animal medicaments and improvement of the quality standard of the centipede medicinal materials.
Disclosure of Invention
Aiming at the defects of the existing centipede medicinal material quality detection method, the invention aims to provide a novel centipede medicinal material quality detection method. The inventor successfully establishes the method for simultaneously detecting the content of hypoxanthine (6-hydroxy-purine), xanthine (2, 6-dihydroxy-purine), 3-hydroxy-8-quinoline sulfate (jineol-8-sulfate), 3-hydroxy-4-methoxy-8-quinoline sulfate (3-hydroxy-4-method-quinone-8-yl hydrogen sulfate) and 3, 8-dihydroxyquinoline (jineol) in centipede medicinal materials by using an HPLC method through long-time exploration and repeated experiments, combines the content detection of 5 components of the centipede medicinal materials into one, and provides a novel method for detecting the centipede medicinal materials more comprehensively and accurately.
The method has good applicability, uses 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 multipoint correction method, and establishes a content detection method of the 4 components by adopting a one-measurement-multiple-evaluation method; the method provided by the invention can be used for evaluating the quality of centipede medicinal materials through methodological verification, is accurate and low in cost, can more comprehensively reflect the internal quality of centipede medicinal materials, and is more reasonable in controlling the quality of medicinal materials by the total content of 5 components.
The invention aims to provide a centipede medicinal material quality detection method, which adopts an analysis method of one standard and multiple tests, takes hypoxanthine as an internal reference substance, and detects the content 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 chromatographic column is Agilent ZORBAX XDB C, agilent ZORBAX SB C18 or YMC-Pack ODS-AQ; YMC-Pack ODS-AQ is more preferable.
Preferably, the HPLC method uses acetonitrile-ammonium phosphate solution as mobile phase; acetonitrile is further preferred: 10-20 mmol/L ammonium phosphate solution = 95:5 is mobile phase a, acetonitrile: 10-20 mmol/L ammonium phosphate solution = 25:75 is mobile phase B.
Preferably, the HPLC method uses the following gradient elution:
preferably, the elution flow rate is 0.6mL/min-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-measurement 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 centipede medicinal materials comprises the following steps:
A. preparation of test solution: taking a proper amount of centipede medicinal materials, crushing and sieving the centipede medicinal materials to serve as a sample for standby; the powder of the sample is proper, and the ratio of the added feed liquid is 1: performing ultrasonic extraction with 20-50 times of methanol solution for 20-50min, cooling to room temperature, weighing, adding solvent to reduce weight, shaking, centrifuging, collecting supernatant, filtering, and discarding primary filtrate to give filtrate as sample solution;
B. preparation of a control solution: preparing single reference substance solutions of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate and mixed reference substance solutions 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 the 5 single reference substance solutions and the 5 component mixed reference substance solutions into HPLC 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 RRT=T according to the formula k /T s Calculating the relative retention time of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfonate, 3-hydroxy-4-methoxy-8-quinolinesulfonate relative to the reference hypoxanthine; t in the formula s Retention time for internal reference, T k Retention time for other 6 components to be tested in the mixed reference solution;
D. determination of the relative correction factor: sucking the mixed reference substance solution in the step B, injecting into HPLC, detecting, recording peak areas of 5 components of the mixed reference substance solution, taking hypoxanthine as an internal reference substance, and calculating formula f according to relative correction factors k/s ＝(A s ×C k )/(A k ×C s ) Calculating relative correction factor f of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfonate, 3-hydroxy-4-methoxy-8-quinolinesulfonate k/s The method comprises the steps of carrying out a first treatment on the surface of the In the formula A s Peak area of internal reference substance, C s A is the concentration of the reference substance k Peak area for other components, C k Is the mass concentration of other components;
E. and (3) detecting the content of multiple components: respectively sucking the sample solution in step A and the hypoxanthine reference substance solution in step B, injecting into HPLC, detecting, and determining according to formula C Sample ＝(A Sample ×C For a pair of )/A For a pair of Calculating the content of hypoxanthine in the sample solution; the 4 components of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in the test sample solution are qualitatively determined according to the relative retention time RRT in the step C, and the relative correction factor f is determined according to the step D k/s Calculating the content of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in a sample solution; in which A Sample C as the peak area of the sample For a pair of As the concentration of the reference substance, A For a pair of The peak area of the control.
Preferably, the methanol concentration used in step A is 40-60%, preferably 50%, and the filtration step uses an organic filter head of 0.22 μm.
Specifically, the preparation method of the sample solution in the step A comprises the following steps:
grinding appropriate amount of Scolopendra, and sieving with 80 mesh sieve; as a sample for standby; 2.0g of sample powder is precisely weighed, placed in a 50mL round bottom flask, 50mL of 50% methanol solution is precisely added, the mixture is sealed, weighed, refluxed for 30min, cooled to room temperature, re-weighed, the weight loss is compensated by 50% methanol solution, shaken uniformly, centrifuged, supernatant is sucked, and the supernatant is filtered by a 0.22 mu m organic filter head, and the primary filtrate is discarded to be used as a sample solution.
Preferably, the preparation method of the mixed reference substance 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 preparing 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;
and (3) diluting the reference substance stock solution with a proper volume to obtain 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 of the reference substance stock solution, thereby obtaining a mixed reference substance solution.
Preferably, the HPLC chromatographic conditions are: a YMC-Pack ODS-AQ (250 mm. Times.4.6 mm,5 μm) column was used; acetonitrile: 10-20 mmol/L ammonium phosphate solution = 95:5 is mobile phase a, acetonitrile: 10-20 mmol/L ammonium phosphate solution = 25:75 is mobile phase B subjected to a linear gradient elution with the following procedure:
flow rate: 0.6mL/min-1.0mL/min; column temperature: 25-35 ℃; sample injection amount: 2-10 mu L; detection wavelength: 220-280nm.
Preferably, in the chromatographic conditions, the flow rate is 0.8mL/min; the column temperature is 30 ℃; the sample injection amount is 5 mu L, and the detection wavelength is 254nm.
Preferably, the relative retention times of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfonate, 3-hydroxy-4-methoxy-8-quinolinesulfonate relative to the reference hypoxanthine in step C are 1.22±0.05, 3.51±0.2, 2.66±0.04, 3.07±0.05, respectively; further preferably, the relative retention times are 1.22, 3.52, 2.65, 3.04, respectively.
The relative correction factors of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 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 material are detected simultaneously by one measurement and multiple evaluation, and the 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 a reference substance and high detection cost can be relieved, the material consumption is saved, the detection efficiency is improved, and the labor cost is saved.
2) The invention establishes a content detection method for 5 components in centipede medicinal materials, adopts an HPLC method to simultaneously detect the contents 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 the established method has the advantages of meeting the requirements of linearity, precision, repeatability, stability and recovery rate test results, having good applicability, further effectively controlling the quality of the centipede medicinal materials, and realizing comprehensive evaluation of the quality of the preparation.
3) The method adopts a one-standard-multiple-measurement method to simultaneously detect the contents 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 has the advantages of high accuracy, good stability, high precision, excellent repeatability, high recovery rate and simple operation.
Drawings
FIG. 1 is a centipede drug control solution chromatogram, labeled as follows:
wherein 1 is hypoxanthine; 2 is xanthine; 3 is 3-hydroxy-8-quinolinesulfonate; 4 is 3-hydroxy-4-methoxy-8 quinoline sulfate; 5 is 3, 8-dihydroxyquinoline.
FIG. 2 is a chromatogram of a sample solution of Scolopendra, labeled as follows:
1 is hypoxanthine; 2 is xanthine; 3 is 3-hydroxy-8-quinolinesulfonate; 4 is 3-hydroxy-4-methoxy-8 quinoline sulfate; 5 is 3, 8-dihydroxyquinoline.
Detailed Description
The invention will be further described by way of exemplary embodiments for a more complete understanding of the practice of the invention. Meanwhile, since the above examples are only preferred embodiments of the present invention, the description thereof is more specific and detailed, but should not be construed as limiting the scope of the present invention. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the principles and concepts of the present invention, which are also intended to be within the scope of the present invention.
The centipede medicinal materials can be obtained through commercial paths.
Example 1 method for detecting content of drug effect component of centipede drug by one-standard-multiple measurement method
1 instrument and reagent
1.1 reagents
Hypoxanthine (CAS: 68-94-0, purity not less than 98%), xanthine (2, 6-dihydroxypuree, CAS:69-89-6, purity not less than 98%), the above reference substances were supplied by Shanghai Yuan leaf Biotechnology Co., ltd; 3-hydroxy-8-quinoline sulfate (jineol-8-sulfate, purity greater than or equal to 98%), 3-hydroxy-4-methoxy-8-quinoline sulfate (3-hydroxy-4-methoxy-8-yl hydrogen sulfate, purity greater than or equal to 97%), 3, 8-dihydroxyquinoline (jineol, purity greater than or equal to 98%), all of which can be prepared by the prior art methods. Acetonitrile, methanol (chromatographic purity, zemoer feishi technologies limited).
1.2 instruments
Agilent 1100 high performance liquid chromatograph (including DAD detector, quaternary low pressure gradient pump, agilent open Lab chromatographic workstation, agilent technologies); a ten-thousandth electronic analytical balance (toldo LE204E, METTLER); one ten-thousandth electronic analytical balance (01193-YP 601N, METTLER); numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); organic filter head (0.22 μm pore size, elsamer technologies); pure water/ultrapure water systems.
1.3 test drug
Centipede is provided by Lunan thick Pu pharmaceutical Co., ltd., see Table 1;
table 1 centipede herb sample statistics (n=14)
2 methods and results
2.1 preparation of solutions
(1) Preparation of test solution: grinding appropriate amount of Scolopendra, and sieving with 80 mesh sieve; as a sample for standby; 2.0g of sample powder is precisely weighed, placed in a 50mL round bottom flask, 50mL of 50% methanol solution is precisely added, the mixture is sealed, weighed, refluxed for 30min, cooled to room temperature, re-weighed, the weight loss is compensated by 50% methanol solution, shaken uniformly, centrifuged, supernatant is sucked, and the supernatant is filtered by a 0.22 mu m organic filter head, and the primary filtrate is discarded to be used as a sample solution.
(2) Preparation of a control solution: 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 preparing 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; and (3) diluting the reference substance stock solution with a proper volume to obtain 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 of the reference substance stock solution, thereby obtaining a mixed reference substance solution.
2.2 chromatographic conditions
Chromatographic column: chromatographic column: YMC-PackODS-AQ (250 mm. Times.4.6 mm,5 μm); 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 (50:50); column temperature: 30 ℃; sample injection amount: 5. Mu.L; detection wavelength: 254nm; gradient elution.
TABLE 2 elution gradient table
2.3 recording of chromatograms
Injecting the reference substance 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 reference solution is shown in FIG. 1, and the HPLC profile of the test solution is shown in FIG. 2.
2.4 methodology investigation
2.4.1 Linear Range investigation
Diluting a control stock solution into a series of concentrations, detecting peak areas according to a chromatographic method under 2.2 items, sequentially injecting hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate, calculating 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 35 active ingredient linear equation
The test results show that 5 components of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate have good linear relation in the respective corresponding mass concentration ranges.
2.4.2 precision test
Taking mixed reference substance solution, sampling according to the chromatographic method under 2.2 items, continuously sampling for 6 times, and recording peak area. The average peak areas RSD (%) of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate are obtained, the results are shown in Table 4, and the RSD (%) of 5 components are less than 2.0%, which shows that the system of the chromatographic method is applicable and the instrument is precise and stable.
Table 45 precision test of active ingredients (n=6)
2.4.3 repeatability test
210601 batches of centipede medicinal material sample powder are precisely weighed for 6 times, the sample solution is prepared according to the sample solution preparation method under the condition of 2.1, sample injection is respectively carried out according to the chromatographic method under the condition of 2.2, the peak area is recorded, the component content is calculated, and the result is shown in Table 5. The average value RSD (%) of 6 times of content of each component is less than 2.5%, which shows that the method for detecting the content of the active ingredients of the centipede medicinal materials has good repeatability.
Table 55 active ingredient repeatability test (n=6)
2.4.4 stability test
Precisely weighing 210601 batches of centipede medicinal materials to be tested, preparing a test solution according to the test solution preparation method under 2.1, respectively sampling at 0h, 2h, 4h, 6h, 8h, 12h, 24h and 48h according to the chromatographic method under 2.2, and recording peak areas. The average peak areas of hypoxanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate are obtained, RSD (%) is calculated, and RSD (%) of 5 components is less than 2.0%, which shows that centipede medicinal materials are stable in 48 hours when used as sample solutions, and the results are shown in Table 6.
Table 65 stability test of active ingredients
2.4.5 accuracy test (sample recovery test)
6 parts of centipede medicinal material sample powder with known content are precisely weighed, reference substance stock solutions containing the reference substances with equal amounts of components are accurately added respectively, a sample solution is prepared according to the method for preparing the sample solution under the condition of 2.1, and sample injection is carried out according to the chromatographic method under the condition of 2.2, and the obtained results are 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 75 active ingredient recovery test (n=6)
2.5 one-standard multi-measurement method for detecting content of 5 components in centipede medicinal material
2.5.1 calculation of relative correction factors
(1) Slope correction method: in the standard curve, the values of x= (Y-b)/a=y/a-b/a, b are usually caused by errors, at a/b>At 100, b/a is usually negligible and can be directly calculated with x=y/a, where the correction factor is calculated by the formula: f (f) k/s ＝a s /a k The mass concentration calculation formula of the component to be measured: c (C) k’ ＝(A k’ ×f k/s )/a s (a s : slope of reference, a k : other control component slopes). Calculating by slope correction method to obtain f of other components relative to hypoxanthine k/s Xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfonate, 3-hydroxy-4-methoxy-8-quinolinesulfonate relative to hypoxanthine f k/s See table 8.
Table 8 slope correction method correction factor (f) k/s )
(2) Multipoint correction method: calculated f with a plurality of mass concentration points k/s Taking average value as f for quantification k/s . Correction factor calculation formula: f (f) k/s ＝(A S ×C K )/(A k ×C s ). The mass concentration calculation formula of the component to be measured: c (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 : other control component chromatographic peak area, C k : other control component Mass concentration, A k : chromatographic peak area of the component to be measured). The results are shown in Table 9.
TABLE 9 Multi-Point correction method correction factors (f k/s ，n＝5)
2.5.2 calculation of relative Retention value
The relative retention times are used for localization, i.e. the retention times of the chromatographic peak of the respective component to be tested and the chromatographic peak of hypoxanthine. Relative retention time calculation formula: rrt=t k /T s (T s For reference retention time, T k Retention time for other components to be tested) and the results are shown in table 10.
Table 10 relative retention value by multipoint correction method (RRT, n=7)
3 comparing the multiple evaluation results with the detection results of the external standard method
3.1 comparing the multiple evaluation results with the content detection results of the external standard method
Taking 14 batches of centipede medicinal materials for sample preparation, preparing a reference substance solution and a sample solution according to the conditions of 2.1, sampling according to the conditions of 2.2 chromatography, detecting the content of the sample solution by a one-standard multi-test method and an external standard method respectively, and comparing the results of the two detection methods, wherein the results are shown in Table 11. As can be seen from the table, the RSD (%) values of the 5 active ingredients obtained by different calculation methods are all less than 3%, and the applicability is good.
Table 11 one measurement of multiple evaluation results and content detection result by external standard method (n=14)
3.2 comparison of the multipoint corrected relative Retention value with the external reference actual Retention time
The theoretical retention time of 14 batches of centipede medicinal materials tested products calculated by the multipoint correction factors is compared with the actual retention time of an external standard method, and the results have no significant difference, as shown in Table 12. As can be seen from the table, the RSD (%) values between the calculated retention values and the actual retention values of the 5 active ingredients are all less than 1.0%, and the applicability is good.
Table 12 comparison of one-test multiple evaluation results with external standard retention time (n=14)
In summary, the content detection method of centipede medicine material one-standard-multiple measurement (QAMS) is established by adopting the high performance liquid chromatography, the method establishes the liquid chromatography method for simultaneously detecting 5 components of the centipede medicine material by examining the aspects of an extraction method, chromatographic conditions, elution gradient and the like, further uses hypoxanthine as an internal reference, determines relative correction factors of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8 quinoline sulfate by adopting a multipoint correction method, and establishes the content detection method of the 4 components by adopting a one-standard-multiple measurement method; the relative correction factor (f) of the 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;
according to the invention, 14 batches of centipede medicinal materials are detected by adopting a QAMS method, the content of 5 components obtained by the QAMS method is compared with the content detection value of an External Standard Method (ESM), RSD (%) is less than 1.0%, and the calculated retention value obtained by the QAMS method is compared with the actual retention value of the ESM method, so that RSD (%) is less than 1.0%, and the established method is accurate, low in cost, stable and reliable, strong in specificity and good in reproducibility, can reflect the internal quality of the centipede medicinal materials more comprehensively, and provides technical basis for further improving the quality standard of the centipede medicinal materials.
Claims (6)
1. The centipede medicinal material quality detection method is characterized in that a one-standard multi-measurement method is adopted, hypoxanthine is used as an internal reference substance, and 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 material is detected by an HPLC method;
the HPLC chromatographic column is Agilent ZORBAX XDB C, agilent ZORBAX SB C or YMC
-Pack ODS-AQ；
The mobile phase of the HPLC method is acetonitrile: 10-20 mmol/L ammonium phosphate solution = 95:5 mobile phase a, acetonitrile: 10-20 mmol/L ammonium phosphate solution = 25:75 is mobile phase B;
the HPLC method uses the following gradient for elution:
。
2. the method for detecting mass according to claim 1, wherein the HPLC column is YMC-
Pack ODS-AQ。
3. The mass detection method according to claim 1, wherein the elution flow rate is 0.6mL/min to 1.0 mL-
min; column temperature: 25-35 ℃; detection wavelength: 220-280nm.
4. A mass detection method according to claim 3, wherein the flow rate is 0.8mL/min; the column temperature is 30 ℃; the detection wavelength was 254nm.
5. The quality inspection method according to any one of claims 1 to 4, comprising the steps of:
A. preparation of test solution: taking a proper amount of centipede medicinal materials, crushing and sieving the centipede medicinal materials to serve as a sample for standby; the powder of the sample is proper, and the ratio of the added feed liquid is 1: performing ultrasonic extraction with 20-50 times of methanol solution for 20-50min, cooling to room temperature, weighing, adding solvent to reduce weight, shaking, centrifuging, collecting supernatant, filtering, and discarding primary filtrate to give filtrate as sample solution;
B. preparation of a control solution: preparing single reference substance solutions of hypoxanthine, xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate and mixed reference substance solutions 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 the 5 single reference substance solutions and the 5 component mixed reference substance solutions into HPLC 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 RRT=T according to the formula k /T s Calculating the relative retention time of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfonate, 3-hydroxy-4-methoxy-8-quinolinesulfonate relative to the reference hypoxanthine; t in the formula s Retention time for internal reference, T k Retention time for other 6 components to be tested in the mixed reference solution;
D. determination of the relative correction factor: sucking the mixed reference substance solution in the step B, injecting into HPLC, detecting, recording peak areas of 5 components of the mixed reference substance solution, taking hypoxanthine as an internal reference substance, and calculating formula f according to relative correction factors k/s =（A s ×C k ）/(A k ×C s ) Calculating relative correction factor f of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinolinesulfonate, 3-hydroxy-4-methoxy-8-quinolinesulfonate k/s The method comprises the steps of carrying out a first treatment on the surface of the In the formula A s Peak area of internal reference substance, C s A is the concentration of the reference substance k Peak area for other components, C k Is the mass concentration of other components;
E. and (3) detecting the content of multiple components: sucking the sample solution in step A and the hypoxanthine reference solution in step B, injecting into HPLC, and detecting according toFormula C Sample =（A Sample ×C For a pair of ）/A For a pair of Calculating the content of hypoxanthine in the sample solution; the 4 components of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in the test sample solution are qualitatively determined according to the relative retention time RRT in the step C, and the relative correction factor f is determined according to the step D k/s Calculating the content of xanthine, 3, 8-dihydroxyquinoline, 3-hydroxy-8-quinoline sulfate and 3-hydroxy-4-methoxy-8-quinoline sulfate in a sample solution; in which A Sample C as the peak area of the sample For a pair of As the concentration of the reference substance, A For a pair of The peak area of the control.
6. The method according to claim 5, wherein the sample solution in step A and the reference solution in step B are prepared with 40% -60% methanol as the solvent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Non-Patent Citations (1)
| Title |
|---|
| 刘武占 ; 范建伟 ; 李艳芳 ; 苏瑞强 ; 张贵民 ; .蜈蚣药材中3,8-二羟基喹啉的定性定量分析方法研究.药物分析杂志.2017,(第04期),第57-61页. * |
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