CN114660194B - HPLC fingerprint construction method and detection method of Yakui Niu Hazhan wave prescription - Google Patents
HPLC fingerprint construction method and detection method of Yakui Niu Hazhan wave prescription Download PDFInfo
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Abstract
The invention discloses an HPLC fingerprint construction method and a detection method of a ya-together Niu Hazhan wave prescription. According to the invention, the HPLC fingerprint spectrum is combined with the content measurement of multiple indexes for the first time, and the quality of the Yakuan Niu Hazhan wave particles is better monitored and evaluated through the qualitative identification and the content measurement of the Yakuan Niu Hazhan wave particles in multiple batches, so that the method guides standardized production and has higher application value; provides research clues for the establishment of quality standards of the yaku Niu Hazhan wave particles with multiple components and multiple indexes, and lays a solid foundation for the deep development of the yaku Niu Hazhan wave particles. The fingerprint prepared by the method can not only perform qualitative and quantitative detection on the Yakuan Niu Hazhan wave granules, but also perform qualitative and quantitative detection on the Yakuan Niu Hazhan wave pills, extractum and other dosage forms.
Description
Technical Field
The invention belongs to the technical field of chemical detection, and particularly relates to an HPLC fingerprint construction method and a detection method of a Yakui Niu Hazhan wave prescription.
Background
The ya-in Niu Hazhan wave is a traditional meridian prescription of Dai medicine, is taken in Dai medicine meridian book "Feng Ha Yazhao Wan na" for more than 1300 years, is composed of herba Aristolochiae Mollissimae, herba Clerodendranthi Spicati, celastrus conical, coicis semen root and Glycyrrhrizae radix, has effects of clearing heat and promoting urination, and can be used for treating acute and chronic nephritis, nephropyelitis, urinary infection, kidney, ureter and vesical calculus. The Dai medical hospital of Xishuangbanna develops a medical institution preparation of "five-stranguria fossil capsule" (Dian medicine manufactured by Chinese character (Z) 20082254K) on the basis of the traditional meridian prescription for 20 years in clinic, and has definite curative effect and high safety in the aspect of treating urinary tract infection and calculi. Meanwhile, when the curative effect of the prescription preparation is researched by hospitals, the prescription preparation can reduce blood and urinary creatinine and urea content besides the calculus removing effect, and the prescription preparation is suggested to have better kidney function protecting effect. However, the Dai medicine is slow to develop at present, and the elegant Niu Hazhan wave prescription lacks modern controllable quality control indexes, so that the quality standard is required to be improved and perfected.
The quality detection research report of the Yakuan Niu Hazhan wave is less at present, the quality standard research of the Yakuan Niu Hazhan wave prescription is developed from two aspects of qualitative and quantitative by establishing an HPLC fingerprint spectrum and multi-component synchronous content determination method, the quality evaluation standard suggestion is provided, a clue is provided for the quality standard research of the Yakuan Niu Hazhan wave prescription, and a reference is provided for the explanation of the material basis of other Dai medical traditional classical name prescriptions.
Disclosure of Invention
The first aim of the invention is to provide a construction method of an HPLC fingerprint spectrum of a Yakuan Niu Hazhan wave prescription, and the second aim of the invention is to provide a detection method of a Yakuan Niu Hazhan wave prescription.
The first aim of the invention is realized in such a way that the construction method of the HPLC fingerprint of the ya-together Niu Hazhan wave prescription is realized according to the following steps:
1) Preparation of test solution: dissolving the Yazhu Niu Hazhan wave particle powder with an organic solvent, performing ultrasonic extraction, and filtering to obtain a sample solution A;
2) Preparation of a control solution:
6 main active ingredients of a Yakui Niu Hazhan wave prescription, namely danshensu, protocatechuic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B are used as reference substances, and 40-70% methanol is used as a solvent to prepare mixed reference substance solutions with different mass concentrations;
3) Construction of a fingerprint:
sucking the sample solution A and the mixed reference substance solution, and injecting into a liquid chromatograph for high performance liquid chromatography analysis to obtain the HPLC fingerprint of the Yazhi Niu Hazhan wave prescription; and (3) establishing a linear relation between the concentration or content of each substance in the mixed reference substance liquid and the peak area, and carrying out sample injection according to the chromatographic conditions in the step (3), and detecting the peak areas of 6 effective components of the to-be-detected yaku Niu Hazhan wave prescription, so as to obtain the content of each substance, namely the content of the corresponding main active component.
The conditions of the liquid chromatography are as follows: the diamondsil C18 chromatographic column has the following specification: 4.6X250 mm,5 μm; the mobile phase is acetonitrile-0.5% glacial acetic acid water solution; the flow rate is 0.6-1.0 mL-min -1 The sample injection amount is 15-30 mu L, the detection wavelength is 260-330 nm,the column temperature is 20-45 ℃;
gradient elution conditions were as follows: 0-7 min,5% A; 7-15 min, 5-10% acetonitrile; 15-30 min, 10-14% acetonitrile; 30-35 min, 14-18% acetonitrile; 35-55 min, 18-21% acetonitrile; 55-87 min, 21-32% acetonitrile; 87-90 min, 32-95% acetonitrile; 90-100 min,95% acetonitrile; 100-102 min,95% -5% acetonitrile; 102-110 min,5% acetonitrile.
The second purpose of the invention is realized in such a way that the detection method of the yaku Niu Hazhan wave prescription is realized according to the following steps:
1) Dissolving, extracting and filtering a to-be-detected Yakuan Niu Hazhan wave prescription sample to obtain a to-be-detected sample solution B, and injecting the to-be-detected sample solution B into a high performance liquid chromatograph for measurement under the liquid chromatography condition to obtain a map of the to-be-detected Yakuan Niu Hazhan wave prescription;
2) Comparing the spectrum of the to-be-detected Yakuan Niu Hazhan wave prescription with the relative retention time of each peak and/or the peak area of each peak in the constructed fingerprint spectrum to finish the qualitative and/or quantitative determination of the to-be-detected Yakuan Niu Hazhan wave prescription.
The beneficial effects of the invention are as follows:
1) According to the invention, the fingerprint spectrum conditions of the Yakuan Niu Hazhan wave particles are optimized, the chromatographic conditions and the extraction conditions of the Yakuan Niu Hazhan wave particles are determined, the HPLC fingerprint spectrum method of the Yakuan Niu Hazhan wave particles is established for the first time by combining methodology investigation, 32 common peaks in a plurality of batches of samples are calibrated at 280nm, 6 components, namely danshensu, protocatechuic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B are identified as characteristic peaks, and the qualitative detection of the Yakuan Niu Hazhan wave particles can be realized, and the stability and the reproducibility are good.
2) The invention utilizes the chromatographic separation condition of the fingerprint spectrum, and simultaneously realizes the synchronous quantitative analysis of 6 main active components in the yaku Niu Hazhan wave particles.
3) According to the invention, the HPLC fingerprint spectrum is combined with the content measurement of multiple indexes for the first time, and the quality of the Yakuan Niu Hazhan wave particles is better monitored and evaluated through the qualitative identification and the content measurement of the Yakuan Niu Hazhan wave particles in multiple batches, so that the method guides standardized production and has higher application value; provides research clues for the establishment of quality standards of the yaku Niu Hazhan wave particles with multiple components and multiple indexes, and lays a solid foundation for the deep development of the yaku Niu Hazhan wave particles.
4) The fingerprint prepared by the method can not only perform qualitative and quantitative detection on the Yakuan Niu Hazhan wave granules, but also perform qualitative and quantitative detection on the Yakuan Niu Hazhan wave pills, extractum and other dosage forms.
Drawings
FIG. 1 is a superimposed HPLC fingerprint (280 nm) of 11 batches of Yakuan Niu Hazhan wave particles;
FIG. 2 shows the HPLC fingerprint pattern (R, 280 nm) of the Yaku Niu Hazhan wave particles;
FIG. 3 is an HPLC chart (280 nm) of the mixed control (A) and Yakuan Niu Hazhan wave particles with the component drug taste (B);
FIG. 411 is a cluster analysis chart of a group of furnished Niu Hazhan wave particle samples (S1-S11);
FIG. 5 11 shows an OPLS-DA score for a batch of Niu Hazhan wave particle samples (S1-S11);
FIG. 6 11 is an OPLS-DA load diagram of a batch of furnished Niu Hazhan wave particle samples (S1-S11);
FIG. 711 shows projection graphs of the OPLS-DA variable importance of a batch of Niu Hazhan wave particle samples (S1-S11) (red: VIP >1; green: VIP < 1).
Detailed Description
The invention is described in further detail below with reference to the drawings and examples, but is not limited in any way to any changes or modifications made based on the teachings of the invention, which fall within the scope of the invention.
The invention discloses a construction method of an HPLC fingerprint spectrum of a Yakuan Niu Hazhan wave prescription, which is realized by the following steps:
1) Preparation of test solution: dissolving the Yazhu Niu Hazhan wave particle powder with an organic solvent, performing ultrasonic extraction, and filtering to obtain a sample solution A;
2) Preparation of a control solution:
6 main active ingredients of a Yakui Niu Hazhan wave prescription, namely danshensu, protocatechuic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B are used as reference substances, and 40-70% methanol is used as a solvent to prepare mixed reference substance solutions with different mass concentrations;
3) Construction of a fingerprint:
sucking the sample solution A and the mixed reference substance solution, and injecting into a liquid chromatograph for high performance liquid chromatography analysis to obtain the HPLC fingerprint of the Yazhi Niu Hazhan wave prescription; and (3) establishing a linear relation between the concentration or content of each substance in the mixed reference substance liquid and the peak area, and carrying out sample injection according to the chromatographic conditions in the step (3), and detecting the peak areas of 6 effective components of the to-be-detected yaku Niu Hazhan wave prescription, so as to obtain the content of each substance, namely the content of the corresponding main active component.
The conditions of the liquid chromatography are as follows: the diamondsil C18 chromatographic column has the following specification: 4.6X250 mm,5 μm; the mobile phase is acetonitrile-0.5% glacial acetic acid water solution; the flow rate is 0.6-1.0 mL-min -1 The sample injection amount is 15-30 mu L, the detection wavelength is 260-330 nm, and the column temperature is 20-45 ℃;
gradient elution conditions were as follows: 0-7 min,5% A; 7-15 min, 5-10% acetonitrile; 15-30 min, 10-14% acetonitrile; 30-35 min, 14-18% acetonitrile; 35-55 min, 18-21% acetonitrile; 55-87 min, 21-32% acetonitrile; 87-90 min, 32-95% acetonitrile; 90-100 min,95% acetonitrile; 100-102 min,95% -5% acetonitrile; 102-110 min,5% acetonitrile.
In the step 1, the organic solvent is 40-70% methanol, 2-4 g of yazhi Niu Hazhan wave particles are dissolved in 25-50 mL of methanol, ultrasonic extraction is carried out for 25-35 min, the mixture is cooled to room temperature and then weighed, the lost weight is complemented by methanol, and a 0.45 mu m filter membrane is used for filtering.
In the step 2, the concentration of salvianic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B in the reference substance solution is 38-42 mug.mL respectively -1 ，15~16μg•mL -1 ，3.20~3.25μg•mL -1 ，8.5~9.0μg•mL -1 ，36.5~37.0μg•mL -1 ，27.5~28.0μg•mL -1 。
The HPLC fingerprint of the Yakuan Niu Hazhan wave prescription has 32 specified total peaks at 280nm, uses No. 17 peak as a reference peak and uses No. 4, 5, 8, 17, 24 and 30 peaks which can represent main active ingredients in the prescription as characteristic peaks; wherein, peak 4 is danshensu, peak 5 is protocatechuic acid, peak 8 is protocatechuic aldehyde, peak 17 is p-coumaric acid, peak 24 is rosmarinic acid, and peak 30 is salvianolic acid B.
Comparing correlation peaks of the finger print of the Yakui Niu Hazhan wave particles and the single drug according to ultraviolet spectrum information and retention time, and determining source attribution of characteristic peaks, wherein chromatographic peaks 5, 8, 11, 14 and 18 are attributed to the calyx Seu fructus Physalis, chromatographic peaks 4, 7, 8, 15, 23, 24, 25, 26, 28, 29, 30 and 32 are attributed to the clerodendranthus spicatus, chromatographic peaks 12 and 14 are attributed to the celastrus orbiculatus, chromatographic peak 17 is attributed to the coix lacryma-jobi root, and chromatographic peaks 18 and 19 are attributed to the liquorice.
The Yakui Niu Hazhan wave prescription comprises granule, decoction, pill, tablet and extract.
The invention also provides a detection method of the yakui Niu Hazhan wave prescription, which is realized according to the following steps:
1) Dissolving, extracting and filtering a to-be-detected Yakuan Niu Hazhan wave prescription sample to obtain a to-be-detected sample solution B, and injecting the to-be-detected sample solution B into a high performance liquid chromatograph for measurement under the liquid chromatography condition to obtain a map of the to-be-detected Yakuan Niu Hazhan wave prescription;
2) Comparing the spectrum of the to-be-detected Yakuan Niu Hazhan wave prescription with the relative retention time of each peak and/or the peak area of each peak in the constructed fingerprint spectrum to finish the qualitative and/or quantitative determination of the to-be-detected Yakuan Niu Hazhan wave prescription.
When the measurement is carried out, the detection wavelength of 63-78 min is 330nm and the detection wavelength of the rest time is 280nm during the synchronous quantitative analysis of 6 main active components.
Example 1 preparation of HPLC finger print of Yakui Niu Hazhan wave granule
1. Experimental materials
Instrument: LC-20A high performance liquid chromatograph (DAD detector, shimadzu); KQ-300B ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); XS105DU analytical balance (meltrel-tolidox, switzerland).
Reagent: methanol (lot number: 10044118, national pharmaceutical systems chemical Co., ltd.), acetonitrile (lot number: 193502, siemens technology (China Co., ltd.), glacial acetic acid (lot number: 20190530, tianjin optical recovery technology development Co., ltd.), purified water (lot number: 3212TJ, waha purified water Co., ltd.).
Sample to be detected: yazhu Niu Hazhan wave granule 11 lot (S1-S11, lot number is 200514, 200610, 200612, 200613, 200614, 200710, 200711, 200712, 200713, 200714, 200810, 10 g/bag in order), and Botrytis cinerea (Botrytis cinerea of the family golden tiger)Aspidopterys obcordataVine stem), herba Clerodendranthi Spicati (Labiatae plant herba Clerodendranthi Spicati)Clerodendranthus spicatusIs prepared from herba Celastri Orientiae, and caulis Celastri Coni (Celastraceae plant Cone-sequence Celastrus orbiculatus)Celastrus paniculatusRoot of Coix lacryma-jobi L.var.jobi L.of GramineaeCoix lacryma-jobiRoot of Glycyrrhiza uralensis (Glycyrrhiza uralensis Fisch. Of Leguminosae)Glycyrrhiza uralensisThe dry roots and the rhizomes) and other medicinal materials are provided by Xishuangbanna version of medical industry Limited liability company, 5 medicinal materials with a formula are identified by a master pharmacist Zhao Yinggong of Dai medical hospital in the Dai medical department of the Xishuangbanna, and all meet the requirements of related standards. All the samples are crushed and pass through a No. 4 sieve for experimental study.
Standard substance: sodium danshensu (lot number: CHB180731, purity. Gtoreq.98%), protocatechuic acid (lot number: CHB180929, purity. Gtoreq.98%), protocatechuic aldehyde (lot number: CHB180928, purity. Gtoreq.98%), p-coumaric acid (lot number: CHB180223, purity. Gtoreq.98%), salvianolic acid B (lot number: CHB180108, purity. Gtoreq.98%) were all supplied by Chengkomar biotechnology Co., ltd, rosmarinic acid (lot number: PS012101, purity. Gtoreq.98%) was supplied by Cheng Doupu s biotechnology Co.
2. Test methods and results
1. Preparation of test solutions
A. Preparing a finished medicine sample solution: 2g of Yakuan Niu Hazhan wave granule S5 (batch number: 200614) powder is taken, precisely weighed, 25mL of 50% methanol is precisely added, weighed, ultrasonically extracted for 30min, cooled to room temperature, and then supplemented with a 0.45 μm filter membrane, and filtered, thus obtaining the sample solution A.
B. Single drug test solution preparation: taking proper amounts of the medicinal herbs of the dun bugle, the clerodendranthus spicatus, the celastrus orbiculatus, the coix seed, the liquorice and the like according to the prescription proportion, and preparing the solution B of the test sample of each single medicine according to the method.
2. Preparation of control solution
Precisely weighing the reference substances, and preparing into preparations containing salvianic acid A, protocatechuic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B with concentration of 40 μg.mL respectively with 50% methanol -1 ，15.6μg•mL -1 ，3.22μg•mL -1 ，8.8μg•mL -1 ，36.8μg•mL -1 ，27.8μg•mL -1 Is used as a reference solution.
3. Chromatographic conditions
Diamonsil C 18 The column (4.6X250 mm,5 μm) was subjected to gradient elution with acetonitrile (A) -0.5% glacial acetic acid in water (B) as mobile phase, and the gradient elution conditions were as follows: 0-7 min,5% A; 7-15 min, 5-10% of A; 15-30 min, 10-14% of A; 30-35 min, 14-18% of A; 35-55 min, 18-21% of A; 55-87 min, 21-32% of A; 87-90 min, 32-95% of A; 90-100 min,95% A; 100-102 min, 95-5%A; 102-110 min,5% A. The flow rate was 0.8 mL/min -1 The column temperature is 40 ℃, the sample injection amount is 20 mu L, and the detection wavelength is 280nm.
4. HPLC fingerprint establishment methodology investigation of Yakui Niu Hazhan wave particles
1) Precision test
Taking a sample solution A, continuously sampling for 6 times according to the chromatographic conditions, recording a chromatogram, taking a 17-number peak as a reference peak, and calculating the relative retention time and the RSD value of the relative peak area of each chromatogram peak. The result is a RSD value of <3% for the relative retention time of 32 peaks and <5% for the relative peak area. Indicating that the instrument has good precision.
2) Stability test
Taking a sample solution A, measuring at 0, 2, 4, 8, 10 and 24 hours according to the chromatographic conditions, taking 17 # peaks as reference peaks, calculating relative retention time and RSD value of relative peak area of each chromatographic peak, and obtaining RSD value <3% of relative retention time of 32 peaks and RSD value <5% of relative peak area. Indicating that the test solution is substantially stable for 24 hours.
3) Repeatability test
Taking a Yakuan Niu Hazhan wave particle sample S5, preparing 6 parts of sample solution (CFX-1-CFX-6) in parallel according to the method of the sample solution A, and calculating the relative retention time and the RSD value of the relative peak area of each chromatographic peak by taking 17 # peak as a reference peak according to the chromatographic condition, wherein the RSD value of the relative retention time of 32 peaks is less than 3% and the RSD value of the relative peak area is less than 5%. Indicating that the method has good repeatability.
5. Establishment of yakui Niu Hazhan wave particle fingerprint
1) 11 batches of samples (S1-S11) with the waves of the elegant furs Niu Hazhan are taken, a sample solution A is prepared, and HPLC analysis is carried out according to the chromatographic conditions. 11 batches of sample chromatograms are exported into a CDF format by using an Shimadzu workstation, then files are imported into a software 'traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2004A edition)', the Yaku Niu Hazhan wave particles S1 are used as reference fingerprints, the time window width is set to be 0.2min, multipoint correction is carried out, automatic matching is carried out, the Yaku Niu Hazhan wave particle HPLC fingerprint (figure 1) is obtained, and a reference fingerprint is generated.
2) And (3) taking the generated control spectrum as a reference spectrum (R), carrying out similarity evaluation on 11 batches of samples, and calculating the relative retention time and RSD value of the relative peak area of each chromatographic peak by taking the 17 # peak as the reference peak, wherein the fingerprint chromatogram is shown in figures 1 and 2. The results show that the similarity between S1-S11 batches of the furled Niu Hazhan wave particle samples and the control map is greater than 0.980, and the difference between 11 batches of samples is smaller, and the chemical components are basically consistent. The relative retention time RSD values of the 32 common peaks are all less than 1%, and the RSD of the relative peak areas is between 0% and 17.21%, so that the fingerprint spectrum of samples in different batches is totally unchanged, but the chromatographic peak heights or peak areas are changed to a certain extent, and the method can be used for qualitative identification of the elegant Niu Hazhan wave particles.
3) Chromatographic peak assignment
32 peaks were detected in total at 280nm and 6 peaks were identified by comparison with the control. Wherein peak 4 is salvianic acid, peak 5 is protocatechuic acid, peak 8 is protocatechuic aldehyde, peak 17 is p-coumaric acid, peak 24 is rosmarinic acid, and peak 30 is salvianolic acid B. The mixed control solution is shown in FIG. 3A.
4) The chromatographic peak of each single medicine is attributed to
Comparing correlation peaks of the yaku Niu Hazhan wave particles and the fingerprint of the single medicine according to the ultraviolet spectrum information and the retention time, and determining source attribution of characteristic peaks, wherein the result is shown in figure 3B. Wherein chromatographic peaks 5 (protocatechuic acid), 8 (protocatechuic aldehyde), 11, 14, 18 are from herba Aristolochiae Mollissimae, chromatographic peaks 4 (salvianic acid), 7, 8 (protocatechuic aldehyde), 15, 23, 24 (rosmarinic acid), 25, 26, 28, 29, 30 (salvianolic acid B), 32 are from herba Clerodendri Bungei, chromatographic peaks 12, 14 are from herba Centipedae, chromatographic peak 17 (p-coumaric acid) is from radix Coicis, and chromatographic peaks 18, 19 are from radix Glycyrrhizae. Wherein chromatographic peaks 1, 2, 3 and 31 are not shown in each single medicine, and can be new components generated after compatibility.
5) Chemometric analysis
The common peak area of 11 batches of the yaku Niu Hazhan wave particles was introduced into SIMCA 13.0 data analysis software to perform cluster analysis (HCA). From the results of the cluster analysis chart (fig. 4), 11 batches of samples can be classified into 2 types, wherein S6, S5, S3 and S4 can be clustered into one type; s1, S2, S8, S10, S9, S7, S11 can be grouped into a second class, indicating that there is some difference in the content of 32 components. This may be related to differences between the process of producing the yakui Niu Hazhan wave granules and the batches of medicinal materials.
Based on HCA, an orthogonal partial least squares discriminant analysis (OPLS-DA) was established, and the matrix diagram is shown in FIG. 5. Wherein index R 2 Y is 0.963, which can reflect the stability of the model; index Q 2 0.903, which may reflect the predictability of the model; both values are greater than 0.5, indicating good stability and predictability of the model.
Furthermore, an OPLS-DA load map (fig. 6) is built up to further predict the differences in the parameters, wherein the further the parameters on the load map are from the origin, the greater the weight representing the variation of the component. Meanwhile, components with larger component content differences among batches are screened by combining a variable importance projection (variabl importancein the projection, VIP) method, wherein a variable with a VIP value greater than 1 (figure 7) can be used as a difference marker. As can be seen from fig. 7, there were 14 chemical components in 11 samples with a large variation, and the inclusion of chromatographic peaks indicated that these 14 components could be used as differential markers for the quality of the yaku Niu Hazhan wave particles.
EXAMPLE 2 determination of the content of 6 active ingredients in Yazhui Niu Hazhan wave particles
1. Methodology investigation
1. Linear relationship investigation
Preparing mixed reference substance solution according to the item of 2.2, respectively injecting 2 mu L, 4 mu L,8 mu L, 10 mu L, 12 mu L, 16 mu L and 20 mu L, taking peak area as ordinate and reference substance injection amount as abscissa, performing linear regression, drawing a standard curve, fitting a linear equation, calculating a linear range and R 2 Values. The linear relation examination results are shown in Table 1. It can be seen that salvianic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B are respectively 8.0-80 μg/mL -1 ，3.2~32μg•mL -1 ，0.6~6.0μg•mL -1 ，1.8~18μg•mL -1 ，7.4~74μg•mL -1 ，5.6~56μg•mL -1 In good linear relation with peak area, R 2 Are all greater than 0.999.
Table 16 examination of the linear relationship of the ingredients
2. Precision test
Taking a Yakui Niu Hazhan wave particle sample S5, preparing a sample solution according to the method under the item "2.1", continuously sampling for 6 times according to the chromatographic condition under the item "2.3", and obtaining RSD values of peak areas of 6 components such as salvianic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, salvianolic acid B and the like which are less than 3%. Indicating that the instrument has good precision.
3. Stability test
Taking a Yakui Niu Hazhan wave particle sample S5, preparing a test solution according to the method under the item of 2.1, and carrying out sample injection analysis on the sample at 0, 2, 4, 6, 8, 10 and 24 hours according to the chromatographic condition under the item of 2.3, wherein the RSD values of peak areas of 6 components such as danshensu, protocatechuic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B in the test solution are all less than 3 percent. The test sample has good stability within 24 hours.
4. Repeatability test
Taking a Yakui Niu Hazhan wave particle sample S5, preparing 6 parts of test sample solutions in parallel according to the method under the item "2.1", and respectively injecting samples according to the chromatographic conditions under the item "2.3", wherein the content of salvianic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B in the test sample is 0.245mg/g,0.072mg/g,0.015mg/g,0.058mg/g,0.255mg/g and 0.188mg/g respectively. RSD was 0.31%,1.04%,0.00%,2.77%,0.20%,2.94%, respectively. Indicating that the method has good repeatability.
5. Sample recovery test
6 parts of each 1g of Yakui Niu Hazhan wave particle sample (S5) are taken, precisely weighed, and 6 parts of sample solutions are prepared in parallel according to the method under the item "2.1" by adding the same amount of salvianic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B reference substances into each part of sample, sampling is carried out according to the chromatographic condition under the item "2.3", the peak area is recorded, and the sample adding recovery rate is calculated, and is shown in Table 2. Experimental results show that the average sample recovery rate of the salvianic acid, the protocatechuic aldehyde, the p-coumaric acid, the rosmarinic acid and the salvianolic acid B is 100.66% -101.99%, and the RSD is less than 3%, which indicates that the sample recovery rate of the salvianic acid, the protocatechuic aldehyde, the p-coumaric acid, the rosmarinic acid and the salvianolic acid B is good.
TABLE 2 sample recovery test results
2. Content determination of 6 active ingredients
11 batches of samples (S1-S11) of the particles with the waves of the elegant furs Niu Hazhan are taken, a sample solution is prepared according to the method in the example 1, sample injection analysis is carried out according to a method, 280nm is selected as the detection wavelength for measuring the content of salvianic acid, protocatechuic aldehyde and p-coumaric acid, and 330nm is selected as the detection wavelength for rosmarinic acid and salvianolic acid B. The results of the content measurement of the ya together Niu Hazhan wave granules are shown in Table 3.
Table 3 determination of the content of yakui Niu Hazhan wave granules (mg/g, n=11)
Analysis of results:
as is clear from Table 1, the average content of rosmarinic acid in the Yakui Niu Hazhan granule is 0.257mg/g at the highest, and the content of the other components is 0.249mg/g of salvianolic acid, 0.183mg/g of salvianolic acid B, 0.073mg/g of protocatechuic acid, 0.057mg/g of p-coumaric acid and 0.014mg/g of protocatechuic aldehyde from high to low.
Wherein the content of rosmarinic acid is the highest, which accords with the previous research result; the content of salvianic acid A is 0.249mg/g and the content of salvianolic acid B is 0.183mg/g, and according to the measurement results of more than 10 batches of samples, the salvianic acid A can be incorporated into the quality control standard of the Yaku Niu Hazhan wave particles. In view of the low content of protocatechuic acid and p-coumaric acid, and the total content of organic acid components of rosmarinic acid and salvianolic acid B in the ya kou Niu Hazhan wave granule can reach 0.570mg/g, the total content of organic acid components can be considered as a quality control index.
Example 3
The fingerprint prepared in example 1 was used to detect yaku Niu Hazhan wave decoction:
preparation of test solution: 2g of yakui Niu Hazhan wave decoction freeze-dried powder is taken, precisely weighed, 30mL of 50% methanol is precisely added, weighing, ultrasonic extraction is carried out for 30min, the weight is complemented after cooling to room temperature, and a 0.45 mu m filter membrane is filtered, thus obtaining the sample solution A. The rest of the procedure is the same as in example 1.
The fingerprint result shows that compared with the control spectrum, 32 common peaks appear, and the similarity is more than 0.95. The results show that the fingerprint constructed in the embodiment 1 can also be used for detecting the yaku Niu Hazhan wave decoction.
Example 4
The detection of the yakui Niu Hazhan wave extract was carried out by using the fingerprint prepared in example 1:
preparation of test solution: 3g of Yakui Niu Hazhan wave extract is taken, precisely weighed, 25mL of 70% methanol is precisely added, weighed, ultrasonically extracted for 30min, cooled to room temperature, and then supplemented with a 0.45 mu m filter membrane, and filtered, thus obtaining the sample solution A. The rest of the procedure is the same as in example 1.
The fingerprint result shows that compared with the control spectrum, 32 common peaks appear, and the similarity is more than 0.95. The results show that the fingerprint constructed in the embodiment 1 can also be used for detecting the Yaku Niu Hazhan wave extract.
Example 5
The detection of the yaku Niu Hazhan waveplate agent was performed by using the fingerprint prepared in example 1:
preparation of test solution: 4g of Yain Niu Hazhan wave plate agent is taken, precisely weighed, 50mL of 60% methanol is precisely added, weighed, ultrasonically extracted for 35min, cooled to room temperature, and then the weight is complemented, and a 0.45 mu m filter membrane is filtered, thus obtaining the sample solution A. The rest of the procedure is the same as in example 1.
The fingerprint result shows that compared with the control spectrum, 32 common peaks appear, and the similarity is more than 0.95. The results show that the fingerprint constructed in example 1 can also be used for detecting the yaku Niu Hazhan wave tablet.
Example 6
Detection of ya kou Niu Hazhan Bolus with the fingerprint prepared in example 1:
preparation of test solution: 3g of Yakui Niu Hazhan wave pill is taken, precisely weighed, precisely added with 40mL of 50% methanol, weighed, ultrasonically extracted for 25min, cooled to room temperature, and then supplemented with a 0.45 mu m filter membrane, and filtered, thus obtaining the sample solution A. The rest of the procedure is the same as in example 1.
The fingerprint result shows that compared with the control spectrum, 32 common peaks appear, and the similarity is more than 0.95. The results show that the fingerprint constructed in the example 1 can also be used for detecting the ya kou Niu Hazhan wave pill.
Claims (1)
1. The detection method of the Yakuan Niu Hazhan wave prescription is characterized in that the Yakuan Niu Hazhan wave prescription comprises granules, decoction, pills, tablets or extractum, and the detection method is realized according to the following steps:
1) Sample solution preparation: dissolving 2-4 g of yazhi Niu Hazhan wave granule powder with 25-50 mL of 40-70% methanol, ultrasonically extracting for 25-35 min, cooling to room temperature, weighing, supplementing the lost weight with methanol, and filtering with a 0.45 μm filter membrane to obtain a sample solution A;
2) Preparing a reference substance solution: the preparation method comprises the steps of preparing mixed reference substance solutions with the quality concentration of 38-42 mug/mL, 15-16 mug/mL, 3.20-3.25 mug/mL, 8.5-9.0 mug/mL, 36.5-37.0 mug/mL and 27.5-28.0 mug/mL respectively by taking 6 main active ingredients of a Yazhi Niu Hazhan wave prescription, namely danshensu, protocatechuic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B as reference substances and 40-70% methanol as solvents;
3) Construction of a fingerprint: sucking the sample solution A and the mixed reference substance solution, and injecting into a liquid chromatograph for high performance liquid chromatography analysis to obtain the HPLC fingerprint of the Yakui Niu Hazhan wave prescription; the fingerprint spectrum designates 32 total peaks at 280nm, takes 17 # peak as a reference peak, and takes 4 # peaks, 5 # peaks, 8 # peaks, 17 # peaks, 24 # peaks and 30 # peaks which can respectively represent main active components of tanshinol, protocatechuic acid, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid and salvianolic acid B in the formula as characteristic peaks; the liquid chromatography conditions were: the diamondsil C18 chromatographic column has the following specification: 4.6X250 mm,5 μm; the mobile phase is acetonitrile-0.5% glacial acetic acid water solution; the flow rate is 0.6-1.0 mL-min -1 The sample injection amount is 15-30 mu L, the detection wavelength is 280nm, and the column temperature is 40 ℃; the gradient elution conditions were: 0-7 min,5% acetonitrile; 7-15 min, 5-10% acetonitrile; 15-30 min, 10-14% acetonitrile; 30-35 min, 14-18% acetonitrile; 35-55 min, 18-21% acetonitrile; 55-87 min, 21-32% acetonitrile; 87-90 min, 32-95% acetonitrile; 90-100 min,95% acetonitrile; 100-102 min,95% -5% acetonitrile; 102-110 min,5% acetonitrile;
establishing a linear relation between the concentration or content of each substance in the mixed reference substance liquid and the peak area, and carrying out sample injection according to the liquid chromatography condition in the step 3), and detecting the peak areas of 6 main active ingredients of the to-be-detected yakui Niu Hazhan wave prescription, so as to obtain the content of the corresponding main active ingredients;
4) Qualitative and quantitative determination of the yakui Niu Hazhan wave prescription: dissolving, extracting and filtering a to-be-detected elegant Niu Hazhan wave prescription sample according to the method of the step 1) to prepare a to-be-detected sample solution B, and injecting the to-be-detected sample solution B into a high performance liquid chromatograph for measurement according to the liquid chromatography condition of the step 3) to obtain a spectrum of the to-be-detected elegant Niu Hazhan wave prescription; comparing the spectrum of the to-be-detected Yakuan Niu Hazhan wave prescription with the relative retention time of each peak and the peak area of each peak in the fingerprint constructed in the step 3), and thus completing the qualitative and quantitative determination of the to-be-detected Yakuan Niu Hazhan wave prescription; in the measurement, if 6 main active ingredients are analyzed synchronously and quantitatively, the detection wavelength of 63-78 min is 330nm, and the detection wavelength of the rest time is 280nm.
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| WO2005088295A1 (en) * | 2004-03-17 | 2005-09-22 | Tianjin Tasly Pharmaceutical Co., Ltd. | A quality controlling method for the compound danshen dropping pills |
| CN105467059A (en) * | 2015-12-30 | 2016-04-06 | 云南理想药业有限公司 | Quality detecting method for traditional Chinese medicine composition for treating hematuresis |
| CN108445125A (en) * | 2018-05-30 | 2018-08-24 | 吉林大学 | The HPLC fingerprint atlas detection methods of pain-relieving and illness-eliminating capsule |
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|---|---|---|---|---|
| WO2005088295A1 (en) * | 2004-03-17 | 2005-09-22 | Tianjin Tasly Pharmaceutical Co., Ltd. | A quality controlling method for the compound danshen dropping pills |
| CN105467059A (en) * | 2015-12-30 | 2016-04-06 | 云南理想药业有限公司 | Quality detecting method for traditional Chinese medicine composition for treating hematuresis |
| CN108445125A (en) * | 2018-05-30 | 2018-08-24 | 吉林大学 | The HPLC fingerprint atlas detection methods of pain-relieving and illness-eliminating capsule |
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| 张兰等.傣肾宁化学成分的HPLC-Q-TOF-MS/MS分析.中国实验方剂学杂志.2021,第27卷(第13期),前言、第2.2-2.3节、第2.1.1节、表1. * |
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