CN110057950B - Detection wavelength switching-based Pudilan antiphlogistic tablet HPLC fingerprint construction method - Google Patents
Detection wavelength switching-based Pudilan antiphlogistic tablet HPLC fingerprint construction method Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
Abstract
The invention provides a construction method of a Pudilan antiphlogistic tablet HPLC fingerprint spectrum based on detection wavelength switching, which comprises the steps of absorbing a Pudilan antiphlogistic tablet test solution, injecting the test solution into a liquid chromatograph for high performance liquid chromatography, carrying out multi-point correction and full spectrum peak matching, and generating a Pudilan antiphlogistic tablet standard fingerprint spectrum by adopting an average method; wherein the detection wavelength in the chromatographic condition is 0-10 min 323nm and 10-40 min 280 nm. The invention provides a Pudilan antiphlogistic tablet standard fingerprint, and carries out quality analysis and evaluation on samples on the market through similarity evaluation; the method is simple and convenient to operate, has high repeatability, precision and stability, and can comprehensively and accurately evaluate the process rationality and the product uniformity of the Pudilan anti-inflammatory tablet.
Description
Technical Field
The invention relates to a construction method of Pudilan antiphlogistic tablet HPLC fingerprint spectrum based on detection wavelength switching, and the Pudilan antiphlogistic tablet HPLC fingerprint spectrum obtained by the method, and belongs to the technical field of pharmaceutical analysis.
Background
The Pudilan anti-inflammatory tablet is composed of four traditional Chinese medicines of scutellaria baicalensis, dandelion, isatis root and corydalis bungeana, has the functions of clearing heat and removing toxicity, resisting inflammation and diminishing swelling, and is widely used for treating furuncle, pharyngitis, tonsillitis and the like. At present, a plurality of manufacturers of Pudilan antiphlogistic tablets are available, and the quality of products of all the manufacturers is uneven. The reason is that the quality standard is single and has great relation with the collection and carrying items of the existing quality standard. The detection of the effective components in the current standard of the Pudilan anti-inflammatory tablet only carries a target component, namely baicalin content determination. The method for quantitatively controlling only a single component has large one-sidedness, and is difficult to characterize the overall facial features of the complex components of the traditional Chinese medicine, so that the internal quality of the Pudilan anti-inflammatory tablet cannot be comprehensively and accurately controlled and evaluated.
The traditional Chinese medicine fingerprint spectrum refers to a spectrum which can mark chemical characteristics of traditional Chinese medicines and a traditional Chinese medicine prescription preparation obtained by adopting a certain analysis means after the traditional Chinese medicines and the traditional Chinese medicine prescription preparation are properly processed. The fingerprint spectrum emphasizes the front-back sequence and the mutual relation of all the characteristic peaks forming the fingerprint, has more scientificity and comprehensiveness compared with a quality control method of a single component, avoids the one-sidedness of the quality of the traditional Chinese medicine determined by measuring the single component, reduces the possibility of manual treatment for reaching the standard of the quality, and can more comprehensively reflect the internal quality of the preparation. Wherein, the High Performance Liquid Chromatography (HPLC) method is the most common method for establishing the traditional Chinese medicine fingerprint at present and has the characteristics of high analysis speed, wide application range, high sensitivity and the like. The method for constructing the traditional Chinese medicine fingerprint by using the high-efficiency liquid multi-wavelength switching technology has been widely applied in the field of quality control and evaluation of Chinese patent medicines in recent years.
At the present stage, the relation between the effective components and the drug effect of the Chinese patent medicine is mostly unclear, and the integrity and the fuzziness of the fingerprint spectrum of the Chinese patent medicine are just suitable for the characteristic. At present, the traditional Chinese medicine fingerprint spectrum technology becomes one of the most effective quality control modes for controlling the quality of traditional Chinese medicines internationally recognized, and has great significance for the modernization and internationalization of traditional Chinese medicines.
Disclosure of Invention
The invention aims to solve the problems that the existing standard detection item of the Pudilan anti-inflammatory tablet is single, and the quality control and evaluation have large area, and provides a wavelength switching technology-based Pudilan anti-inflammatory tablet HPLC fingerprint spectrum detection method, and a scientific and reliable technical means for the internal quality analysis and evaluation of the Pudilan anti-inflammatory tablet by comparing the fingerprint spectrum obtained by the method and combining the similarity evaluation of the fingerprint spectrum.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for constructing HPLC fingerprint of Pudilan anti-inflammatory tablet based on detection wavelength switching comprises sucking a sample solution of Pudilan anti-inflammatory tablet, injecting into a liquid chromatograph, and measuring to obtain fingerprint. Generating a Pudilan anti-inflammatory tablet comparison fingerprint spectrum by adopting an average method through multipoint correction and full spectrum peak matching; wherein the detection wavelength in the chromatographic condition is 0-10 min 323nm and 10-40 min 280 nm.
Preferably, the construction method comprises the following steps:
(1) preparation of a test solution: taking Pudilan anti-inflammatory tablets, removing coatings, grinding, placing into a conical flask with a plug, adding an extraction solvent, sealing the plug, weighing, heating and refluxing, taking out, cooling, weighing again, supplementing the weight loss with the extraction solvent, shaking up, filtering, and taking a subsequent filtrate to obtain the medicine;
(2) preparation of control solutions: precisely weighing the reference substances of monocaffeoyltartaric acid, baicalin, oroxylin A-7-O-beta-D-glucuronide, wogonoside, baicalein, wogonin and oroxylin A, and adding methanol to prepare a mixed reference solution containing monocaffeoyltartaric acid, baicalin, oroxylin A-7-O-beta-D-glucuronide, wogonoside, baicalein, wogonin and oroxylin A10.20, 210.38, 25.92, 41.37, 16.30, 8.74 and 2.29 μ g per 1 mL;
(3) chromatographic conditions are as follows: agilent Poroshell 120 SB-C using octadecylsilane chemically bonded silica as filler18A chromatographic column; performing gradient elution by using methanol as a mobile phase A, acetonitrile as a mobile phase B and 0.1% phosphoric acid solution as a mobile phase C; flow rate: 0.3 mL/min; detection wavelength: 323nm in 0-10 min and 280nm in 10-40 min; column temperature: 30 ℃;
(4) and (3) HPLC determination: respectively sucking 2 mu L of reference solution and 2 mu L of test solution, injecting into a high performance liquid chromatograph, recording the chromatogram of the reference solution and the sample, and obtaining the measured chromatogram as the sample fingerprint;
(5) the establishment of the Pudilan antiphlogistic tablet contrast fingerprint spectrum: taking 1 batch of Pudilan anti-inflammatory tablets from a plurality of different manufacturers, respectively preparing a sample solution according to the step (1), injecting a sample, recording a chromatogram, introducing software of 'a traditional Chinese medicine chromatogram fingerprint spectrum evaluation system' version 2012.130723, performing multi-point correction and full spectrum peak matching, and generating a Pudilan anti-inflammatory tablet comparison fingerprint spectrum by adopting an average method;
(6) evaluating the similarity of the sample fingerprint spectra: and (5) determining the fingerprint of each sample, taking the comparison fingerprint as a reference, and adopting the evaluation software in the step (5) to evaluate the similarity of the samples, wherein the similarity is more than 0.95.
Preferably, the chromatographic conditions of high performance liquid chromatography are as follows: agilent Poroshell 120 SB-C using octadecylsilane chemically bonded silica as filler18Is a chromatographic column; performing gradient elution by using methanol as a mobile phase A, acetonitrile as a mobile phase B and 0.1% phosphoric acid solution as a mobile phase C; flow rate: 0.3 ml/min; detection wavelength: 323nm in 0-10 min and 280nm in 10-40 min; column temperature: at 30 ℃.
Preferably, Poroshell 120 SB-C18The column size was 2.1mm X50 mm, 2.7. mu.m.
Preferably, the requirements for mobile phase gradient elution are:
0-5min, mobile phase A10 → 20%, mobile phase B5%, mobile phase C85 → 75%;
5-20min, mobile phase A20 → 25%, mobile phase B5 → 10%, mobile phase C75 → 65%;
20-30min, mobile phase A25 → 40%, mobile phase B10 → 15%, mobile phase C65 → 45%;
30-40min, mobile phase A40%, mobile phase B15% and mobile phase C45%.
Preferably, the wavelength switching time is selected to be 10 minutes before the baicalin peak retention time.
Preferably, the heating reflux method in the step (1) can effectively inhibit the activity of the enzyme, and the baicalein is completely inactivated at the reflux temperature of 85 ℃ and the heating reflux time is 2 hours.
Preferably, the sample extraction solvent in step (1) is 50% methanol.
Preferably, 7 components of the reference substance are identified by using the retention time of the chromatographic peak and the ultraviolet absorption spectrogram, and the peak 1 is judged to be monocaffeoyltartaric acid, the peak 5 is baicalin, the peak 9 is oroxylin A-7-O-beta-D-glucuronide, the peak 10 is wogonoside, the peak 12 is baicalein, the peak 13 is wogonin, and the peak 14 is oroxylin A.
Preferably, the similarity between the fingerprint of the sample and the fingerprint of the control in the step (5) is more than 0.95.
In order to confirm the effect of the invention, a large number of experiments are carried out, including detection wavelength selection, mobile phase optimization, sample extraction method and extraction solvent selection, fingerprint spectrum determination methodology verification and chromatographic peak identification in fingerprint spectrum, and the specific experiments are as follows:
(1) selection of detection wavelength: the method is characterized in that the flavone component in the scutellaria baicalensis and the phenolic acid component in the dandelion are detected by adopting a high performance liquid chromatography multi-wavelength switching technology, the maximum absorption wavelength of the flavone component in the scutellaria baicalensis is 272-284 nm, the maximum absorption wavelength of various phenolic acid components in the dandelion is 320-328 nm, the wavelength difference between the two is large, and the detection sensitivity of one component is inevitably reduced by adopting a single wavelength. Meanwhile, the difference between the structures and the polarities of the flavone and the phenolic acid components is large, so that the difference between the retention time is large, the phenolic acid component peak is in front, and the flavonoid component peak is behind, so that the optimal sensitivity of detecting each component can be obtained by wavelength switching, and the wavelength switching time is 10 minutes before the retention time of the baicalin peak.
The detection wavelength finally determined by the invention is as follows: 323nm in 0-10 min and 280nm in 10-40 min. The multi-component system is detected by adopting the multi-wavelength switching technology, so that the detection sensitivity of each component can be effectively improved, and the reliability of an analysis result is improved.
(2) Optimization of the mobile phase: the Pudilan anti-inflammatory tablet contains a plurality of effective components, wherein the structures of flavonoid components are relatively similar, a plurality of pairs of isomers exist, and the flavonoid components cannot be effectively separated by adopting isocratic elution, so that gradient elution is adopted, the phenolic acid and the flavonoid components are strongly retained in an acidic mobile phase, and a methanol-water-phosphoric acid system is firstly considered in the mobile phase. Through investigation of methanol-water-phosphoric acid systems with different proportions, wogonin and adjacent peaks are found to be difficult to realize baseline separation, and in order to achieve good separation of chromatographic peaks of various components, acetonitrile with a proper proportion is added into a mobile phase, so that separation among the chromatographic peaks can be obviously improved.
The final mobile phase gradient elution procedure identified by the present invention is table 1:
| time (min) | Mobile phase A (%) | Mobile phase B (%) | Mobile phase C (%) |
| 0～5 | 10→20 | 5 | 85→75 |
| 5～20 | 20→25 | 5→10 | 75→65 |
| 20～30 | 25→40 | 10→15 | 65→45 |
| 30～40 | 40 | 15 | 45 |
(3) Selection of an extraction method: in the determination of baicalin content currently carried by the Pudilan antiphlogistic tablet, the extraction method of the test sample comprises a reflux method and an ultrasonic method. The invention inspects the extraction efficiency and the solution stability of the two extraction methods, and finds that the extraction efficiency of the ultrasonic method is basically consistent with that of the reflux method, but the stability of the extraction solution has larger difference. The ultrasonic method is adopted, the contents of baicalin and wogonoside are gradually reduced along with the prolonging of ultrasonic time, and the contents of baicalein and wogonoside are gradually increased, which shows that in the ultrasonic process, baicalin components are hydrolyzed into aglycon under the action of baicalein-baicalinase, namely endogenous enzyme of scutellaria, and meanwhile, the test solution obtained by ultrasonic extraction still continues to undergo hydrolysis reaction in the process of placing at room temperature, so that the stability of the solution is poor. The heating reflux method can effectively inhibit the activity of the enzyme, and the baicalein is completely inactivated at the reflux temperature of 85 ℃. The influence of 3 different reflux times of 1 hour, 2 hours and 3 hours on the extraction result of each component is examined, the content of each component obtained by extracting for 2 hours under reflux is slightly more than 1 hour, and the condition that the reflux time for 1 hour is slightly insufficient is shown. When the reflux time is 3 hours, the content of each component is less than 2 hours, which shows that when the reflux time is more than 2 hours, the flavonoid component in the Pudilan anti-inflammatory tablet can be thermally degraded to a certain degree.
The method for extracting the test sample finally determined by the invention comprises the following steps: heated and refluxed for 2h at 85 ℃.
(4) Selection of an extraction solvent: in the currently accepted quality standard of the Pudilan antiphlogistic tablet, the baicalin content determination extraction solvent comprises methanol and ethanol with different concentrations. Considering the extraction effect of methanol and ethanol, under the same concentration condition, the chromatographic peak number obtained by methanol extraction is slightly more than that of ethanol, and the content of each component is slightly more than that of ethanol, so that a methanol solution is selected as an extraction solvent. Then, the extraction effects of 30-100% of 8 methanol solutions with different concentrations are examined, and the result shows that when the concentration of the methanol solution is less than 50%, the number of peaks in the chromatogram of the test sample is slightly reduced, and when the concentration of the methanol solution is more than 50%, the peak area is gradually reduced due to the gradual reduction of the solubility of phenolic acid and flavone components. Comprehensive analysis shows that the extraction solvent of the test sample is finally determined to be 50% methanol.
Methodology investigation
The sample for precision test is used for preparing a test sample solution according to the preparation method of the test sample, sample introduction is continuously carried out for 6 times according to the chromatographic conditions, a chromatogram is recorded, the similarity of the sample solution is evaluated according to a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.130723 edition) provided by the State pharmacopoeia Committee, full spectrum peak matching is adopted, the similarity is calculated according to the cosine of an included angle, and the similarity is 1.000. The instrument precision is good, and the technical requirements of the fingerprint spectrum are met.
The method comprises the steps of preparing 6 parts of test solution from samples in parallel in a repeatability test, measuring according to the chromatographic conditions, recording a chromatogram, evaluating the similarity according to a similarity evaluation system (2012.130723 version) of a traditional Chinese medicine chromatogram fingerprint provided by the State pharmacopoeia Committee, adopting full spectrum peak matching, and calculating the similarity according to the cosine of an included angle, wherein the similarity is 1.000. The method has good repeatability and meets the technical requirements of the fingerprint.
Stability test samples are prepared into test sample solution, the test sample solution is respectively measured for 0, 2, 4, 8, 12 and 24 hours according to the chromatographic conditions, chromatogram is recorded, the similarity of the test sample solution is evaluated according to a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 edition) provided by the State pharmacopoeia Committee, full spectrum peak matching is adopted, the similarity is calculated according to the cosine of an included angle, and the similarity is 1.000. The test solution is stable within 24 h.
(6) Fingerprint spectrum chromatography identification: the Pudilan antiphlogistic tablet fingerprint spectrum has 14 common peaks, the common peaks are matched with the ultraviolet spectrogram of a reference substance by DAD full scanning, and 7 common peaks are identified by combining the retention time of chromatographic peaks, namely the No. 1 peak: mono-caftaric acid; peak No. 5: baicalin; peak No. 9: oroxylin A-7-O-beta-D-glucuronide; peak No. 10: wogonoside; peak No. 12: baicalein; peak No. 13: wogonin; peak No. 14: oroxylin A.
The invention has the beneficial effects that: the invention provides a construction method of Pudilan antiphlogistic tablet fingerprint, provides a Pudilan antiphlogistic tablet contrast fingerprint, and carries out quality analysis on a sample on the market through similarity evaluation. The method is simple, convenient and practical, has high repeatability, precision and stability, and is suitable for the production process control and the overall quality evaluation of the Pudilan anti-inflammatory tablet.
The invention has the following advantages:
(1) by adopting a wavelength switching method, the maximum absorption of each effective component in the Pudilan anti-inflammatory tablet can be simultaneously met, and the detection sensitivity of each component is improved;
(2) extracting a sample by adopting 50% methanol, so that the peak number of each water-soluble and fat-soluble component obtained by HPLC is the largest, and the composition information of chemical components in the Pudilan anti-inflammatory tablet can be more comprehensively reflected;
(3) the reflux heating mode is adopted for extraction, so that the phenomenon that the baicalin component is converted into the baicalein component by hydrolase contained in the scutellaria baicalensis at normal temperature is avoided, and the stability of the test solution is obviously improved;
(4) the fingerprint spectrum established by the invention determines 14 common peaks, and combines with a reference substance to perform chemical identification, so that 7 chromatographic peaks are identified, the characteristics of the product can be expressed, and the product has good specificity.
Drawings
FIG. 1 is HPLC chromatogram of Pudilan antiphlogistic tablet.
FIG. 2 is a chromatogram of a mixed control solution, in which peak 1 is caftaric acid; peak 2 is baicalin; peak 3 is oroxylin A-7-O-beta-D-glucuronide; peak 4 is wogonoside; peak 5 is baicalein; peak 6 is wogonin; peak 7 is oroxylin A.
FIG. 3 is the overlay of fingerprint spectrum of Pudilan antiphlogistic tablet.
FIG. 4 is a comparison fingerprint of Pudilan antiphlogistic tablet with baicalin as reference peak (S).
FIG. 5 is a UV absorption spectrum of a Pudilan anti-inflammatory tablet monocaffeoyl tartaric acid control.
FIG. 6 is a UV absorption spectrum of a sample of Pudilan anti-inflammatory tablet monocaffeoyl tartaric acid.
FIG. 7 is a UV absorption spectrum of a baicalin control sample of Pudilan anti-inflammatory tablet.
FIG. 8 is a UV absorption spectrum of a sample of baicalin of Pudilan antiphlogistic tablet.
FIG. 9 is a UV absorption spectrum of a control sample of oroxylin A-7-O-beta-D-glucuronide of Pudilan anti-inflammatory tablet.
FIG. 10 is a UV absorption spectrum of a sample of oroxylin A-7-O-beta-D-glucuronide of Pudilan anti-inflammatory tablet.
FIG. 11 is a graph of UV absorption spectrum of a control of the Pudilan anti-inflammatory tablet, wogonin.
FIG. 12 is a graph of the ultraviolet absorption spectrum of a sample of wogonoside of the Pudilan anti-inflammatory tablet.
FIG. 13 is a UV absorption spectrum of a control of Pudilan anti-inflammatory tablet baicalein.
FIG. 14 is a UV absorption spectrum of a sample of Pudilan antiphlogistic tablet scutellarin.
FIG. 15 is the ultraviolet absorption spectrum of a wogonin control of Pudilan anti-inflammatory tablet.
FIG. 16 is a UV absorption spectrum of a sample wogonin of the Pudilan anti-inflammatory tablet.
FIG. 17 is a UV absorption spectrum of a control of oroxylin A of Pudilan anti-inflammatory tablet.
FIG. 18 is a UV absorption spectrum of a sample of Pudilan antiphlogistic oroxylin A.
FIG. 19 is a HPLC chart comparing the manufacturer with the other manufacturers with a similarity of less than 0.95.
Detailed Description
The invention is further explained below with reference to specific embodiments and the attached drawings:
the instruments and reagents used in the present invention are as follows:
instruments and reagents:
agilent 1260 liquid chromatograph (Agilent, Inc. of Agilent, USA, with DAD detector); XSE205DU electronic balance (mettler-toledo instruments ltd); HWS-11-8 electric heating constant temperature water bath (Shanghai Bingbo practice Co., Ltd.) medical equipment factory.
The monocaffeoyltartaric acid reference substance (batch number P30JTF 169909, purity ≥ 98%) is from Shanghai leaf Biotech limited; the reference oroxylin A (lot No. 3945, purity greater than or equal to 98%) is from Shanghai Shidan De Biotechnology Co., Ltd; oroxylin A-7-O-beta-D-glucuronide reference substance (batch number CFS201801, purity more than or equal to 98%) is from Wuhantian plant biotechnology limited; 4 controls of baicalin (batch No. 111752) -201703 with a purity of 93.5%), wogonin (batch No. 112002-201702 with a purity of 98.5%), baicalein (batch No. 111595-201306 with a purity of 97.8%), and wogonin (batch No. 111514-201304) were obtained from the institute of food and drug assay.
The samples were analyzed with methanol and acetonitrile both in chromatographic purity (Merck, Germany); phosphoric acid is analytically pure (chemical reagents of national drug group, ltd); the water is ultrapure water.
Example 1 determination method of Pudilan antiphlogistic tablet fingerprint
Method and results
Preparation of a test solution: taking 10 tablets of Pudilan anti-inflammatory tablet, removing the coating, grinding, taking about 0.3g, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of 50% methanol, sealing the plug, weighing, heating and refluxing at 85 ℃ for 2h, taking out, cooling, weighing again, complementing the weight loss by 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the medicine.
Preparation of control solutions: taking appropriate amount of monocaffeoyl tartaric acid, baicalin, oroxylin A-7-O-beta-D-glucuronide, wogonoside, baicalein, wogonin, oroxylin A reference substances, precisely weighing, and adding methanol to obtain mixed reference substance solution containing monocaffeoyl tartaric acid, baicalin, oroxylin A-7-O-beta-D-glucuronide, wogonoside, baicalein, wogonin, oroxylin A10.202, 210.375, 25.921, 41.370, 16.305, 8.736, and 2.289 μ g per 1 mL.
Chromatographic conditions are as follows: the chromatographic column is Agilent Poroshell 120 SB-C18(2.1X 50mm, 2.7 μm); gradient elution is carried out according to the table 1 by taking methanol as a mobile phase A, acetonitrile as a mobile phase B and 0.1% phosphoric acid solution as a mobile phase C; flow rate: 0.3 mL/min; detection wavelength: 323nm in 0-10 min and 280nm in 10-40 min; column temperature: at 30 ℃.
TABLE 1 mobile phase gradient elution Table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Mobile phase C (%) |
| 0～5 | 10→20 | 5 | 85→75 |
| 5～20 | 20→25 | 5→10 | 75→65 |
| 20～30 | 25→40 | 10→15 | 65→45 |
| 30～40 | 40 | 15 | 45 |
Fingerprint spectrum determination: precisely sucking 2 μ l of each of the mixed reference solution and the sample solution, injecting into a high performance liquid chromatograph, operating according to the above chromatographic conditions, and recording HPLC chromatogram, namely the fingerprint of the Pudilan anti-inflammatory tablet, as shown in figure 1 and figure 2.
Example 2 construction of Pudilan antiphlogistic tablet by comparison with fingerprint
1. Sample information
Selecting 1 batch of 12 Pudilan anti-inflammatory tablet samples of different manufacturers, wherein the batch comprises the following steps: anhui Jiren pharmaceutical industry Co Ltd (batch No. 2171208), Gansu Minhai pharmaceutical industry Co Ltd (batch No. 171110), Guangdong Xinbao pharmaceutical industry science and technology Co Ltd (batch No. 20180120), Harbin City Longsheng Bei drug engineering Co Ltd (batch No. 20171112), Hunan Sheng pharmacy Co Ltd (batch No. 170616), Jilin dao Jun drug industry Co Ltd (batch No. 20180103), Jinzhou Tian drug industry Co Ltd (batch No. 170903), Shandong Kong pharmacy Co Ltd (batch No. 171203), Shandong Xian He drug industry Co Ltd (batch No. 70940 BO), Wuhan Shuanglong drug industry Co Ltd (batch No. 170401), Yunnan Bai Yao drug group Co Ltd (batch No. ZLB), and Yunnan Longfa drug manufacturing Co Ltd (batch No. 171131).
2. Method and results
2.1 creation of control fingerprint
Accurately weighing 0.3g of the 12 batches of samples, respectively preparing a sample solution according to the sample preparation method, injecting samples according to the chromatographic conditions, measuring the fingerprints of the 12 batches of samples, introducing the samples into 2012.130723 version software of a traditional Chinese medicine chromatographic fingerprint evaluation system recommended by the State pharmacopoeia Committee by taking the fingerprint of the A factory (batch number 2171208) as a reference, performing multi-point correction and full spectrum peak matching, and obtaining 12 batches of Pudilan antiphlogistic tablet fingerprint superposition maps and a reference fingerprint (R) by adopting an average method and a time window of 0.1 s.
2.2 determining common fingerprint peaks and performing finger-print analysis on the Pudilan anti-inflammatory tablet, selecting 14 common peaks with relatively large peak areas and good separation degree, calculating area percentage, and finally determining that the 14 common peaks are the common fingerprint peaks of the Pudilan anti-inflammatory tablet, wherein the retention time of each common fingerprint peak is 2.713min for the No. 1 peak, 13.782min for the No. 2 peak, 16.025min for the No. 3 peak, 22.210min for the No. 4 peak, 23.246min for the No. 5 peak (S), 26.225min for the No. 6 peak, 26.985min for the No. 7 peak, 27.603min for the No. 8 peak, 28.072min for the No. 9 peak, 28.647min for the No. 10 peak, 30.123min for the No. 11 peak, 30.668min for the No. 12 peak, 34.288min for the No. 13 peak and 35.391min for the No. 14 peak.
The components in the Pudilan antiphlogistic tablet are analyzed by DAD full scan, and 7 components are identified by combining the retention time of chromatographic peaks and ultraviolet absorption spectrograms, wherein the peak 1 is monocaffeoyltartaric acid (shown in figures 5 and 6), the peak 5 is baicalin (shown in figures 7 and 8), the peak 9 is oroxylin A-7-O-beta-D-glucuronide (shown in figures 9 and 10), the peak 10 is wogonoside (shown in figures 11 and 12), the peak 12 is baicalein (shown in figures 13 and 14), the peak 13 is wogonin (shown in figures 15 and 16), and the peak 14 is oroxylin A (shown in figures 17 and 18).
2.4 selection of reference (S)
Referring to fig. 3 and 4, the reference substance of the fingerprint is usually selected from index components which are easily obtained, stable in property and high in content, and is mainly used for determining the technical parameters of the fingerprint. The No. 5 peak is baicalin which is an index component of the Pudilan anti-inflammatory tablet, has moderate retention time, the maximum peak area and good separation degree from adjacent peaks, and therefore the No. 5 peak (baical skullcap root peak) is used as a reference peak. The relative retention time and the relative peak area of the common peak of the 12 samples were calculated with the retention time and the peak area of the reference peak as 1, respectively, and the results are shown in tables 2 and 3.
TABLE 2, 12 samples sharing relative retention time of fingerprint peaks
Table 3 and 12 samples sharing relative peak area of fingerprint peak
Example 3 evaluation of similarity of fingerprint of Pudilan antiphlogistic tablet
1. Sample information
Anhui Ji pharmaceutical industry Co Ltd (2171208, 2180214), Gansu Minhai pharmaceutical industry Co Ltd (171110, 171129, 171136, 180124, 180127, 180309), Guangdong Xinbao pharmaceutical industry science and technology Co Ltd (20180120, 20180122), Longsheng North pharmaceutical engineering Co Ltd of Harbin city (20171112, 20171206, 20171233, 20180102, 20180108, 20180204, 20180304), Hunan Fangsheng pharmaceutical industry Co Ltd (170616, 171224, 180305, 180328), Jilin daojun pharmaceutical industry Co Ltd (20180103, 20180109), the pharmaceutical companies of york Hongyan (170903, 171201, 171215, 180207, 180212), Shandong Kongfu pharmaceutical company GmbH (171203, 180101), Shandong Xianhe river pharmaceutical company GmbH (70940BO, 70940B6), Wuhan Shuanglong pharmaceutical company GmbH (170401, 170414, 171006, 180122, 180208), Yunnan Baiyao group GmbH (ZLB1720, ZDD1801, ZBB1806, ZBB1809, ZBB1814, ZBB1828), Yunnan Longfa pharmaceutical company GmbH (171131, 171227), and 45 samples.
2. Method and results
The sample solutions of 45 batches were prepared according to the above-mentioned sample preparation method, and the samples were measured under the above-mentioned chromatographic conditions, and the HPLC chromatogram was recorded, as shown in FIG. 19. The obtained chromatographic data is led into software of 2012.130723 version of Chinese medicine chromatographic fingerprint evaluation system, the time window is 0.1s, the similarity of 45 batches of samples is calculated by taking the contrast fingerprint as reference, and the result is shown in Table 4. The calculation result shows that the similarity of 43 samples is more than 0.95, but the similarity of 2 samples is only 0.923 and 0.901 which is lower than 0.95, and the 2 samples are produced by the same manufacturer. Analyzing the HPLC fingerprint of the sample, and finding that the contents of baicalin and wogonin in the 2 batches of samples are obviously lower than those of other samples, the contents of aglycon-baicalein and wogonin corresponding to the two samples are obviously increased, and the contents of all the components are integrally obviously lower than those of other samples. The problem of the quality of the medicinal materials or the preparation process of the factory is shown, the baicalin component in the scutellaria baicalensis is seriously hydrolyzed, and the quality of the medicinal materials is poor. Therefore, the quality of the Pudilan anti-inflammatory tablet medicinal material and the quality of the production process can be quickly analyzed and judged by combining the similarity evaluation of the sample fingerprint with the HPLC fingerprint.
Table 4, 45 samples fingerprint similarity calculation results
Claims (7)
1. A construction method of Pudilan antiphlogistic tablet HPLC fingerprint based on detection wavelength switching is characterized in that a sample solution of the Pudilan antiphlogistic tablet is absorbed and injected into a liquid chromatograph for high performance liquid chromatography analysis, and a Pudilan antiphlogistic tablet contrast fingerprint is generated by adopting an average method through multi-point correction and full spectrum peak matching; and (3) combining the comparison fingerprint spectrum and the ultraviolet spectrogram of the comparison product, identifying components corresponding to 7 common peaks in the comparison fingerprint spectrum: the peaks with retention time of 2.713min, 23.246min, 28.072min, 28.647min, 30.668min, 34.288min and 35.391min are respectively monocaffeyltartaric acid, baicalin, oroxylin A-7-O-beta-D-glucuronide, wogonin, baicalein, wogonin and oroxylin A;
the chromatographic conditions of the high performance liquid chromatography are as follows: agilent Poroshell 120 SB-C18 using octadecylsilane chemically bonded silica as filler is used as chromatographic column, and the specification of the chromatographic column is 2.1mm multiplied by 50mm and 2.7 μm; the detection wavelength is 0-10 min 323nm and 10-40 min 280 nm; methanol is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, and 0.1 percent phosphoric acid solution is taken as a mobile phase C, gradient elution is carried out, and the elution procedure is as follows:
0-5min, mobile phase A10 ￫ 20, mobile phase B5 and mobile phase C85 ￫ 75%;
5-20min, mobile phase A20 ￫ 25%, mobile phase B5 ￫ 10%, and mobile phase C75 ￫ 65%;
20-30min, mobile phase A25 ￫ 40%, mobile phase B10 ￫ 15%, and mobile phase C65 ￫ 45%;
30-40min, mobile phase A40%, mobile phase B15% and mobile phase C45%.
2. The construction method of HPLC fingerprint spectrum of Pudilan antiphlogistic tablet based on detection of wavelength switching as claimed in claim 1, characterized in that the construction method comprises the following steps:
(1) preparation of a test solution: taking Pudilan anti-inflammatory tablets, removing coatings, grinding, placing into a conical flask with a plug, adding an extraction solvent, sealing the plug, weighing, heating and refluxing, taking out, cooling, weighing again, supplementing the weight loss with the extraction solvent, shaking up, filtering, and taking a subsequent filtrate to obtain the medicine;
(2) preparation of control solutions: preparing a mixed reference solution from caftaric acid, baicalin, oroxylin A-7-O-beta-D-glucuronide, wogonoside, baicalein, wogonin, oroxylin A with methanol;
(3) and (3) HPLC determination: respectively sucking 2 mu L of reference solution and 2 mu L of test solution, injecting into a high performance liquid chromatograph, recording the chromatogram of the reference solution and the sample, and obtaining the measured chromatogram as the sample fingerprint;
(4) the establishment of the Pudilan antiphlogistic tablet contrast fingerprint spectrum: taking 1 batch of Pudilan anti-inflammatory tablets of a plurality of different enterprises, respectively preparing a test solution according to the test preparation method in the step (1), injecting a sample, measuring a fingerprint of the sample, introducing the sample into software of 2012.130723 edition of a Chinese medicine chromatography fingerprint evaluation system of the national pharmacopoeia committee, performing multipoint correction and full spectrum peak matching, and generating a Pudilan anti-inflammatory tablet comparison fingerprint by adopting an average method;
(5) evaluating the similarity of the sample fingerprint spectra: and (4) determining the fingerprint of each sample, and evaluating the similarity of the samples by using the comparison fingerprint as a reference and adopting the evaluation software in the step (4).
3. The method for constructing the HPLC fingerprint spectrum of the Pudilan antiphlogistic tablet based on the detection of the wavelength switching as claimed in claim 1 or 2, wherein the chromatographic conditions of the high performance liquid chromatography are as follows: flow rate: 0.3 ml/min; column temperature: at 30 ℃.
4. The method for constructing the HPLC fingerprint spectrum of Pudilan antiphlogistic tablet based on the detection of the wavelength switching as claimed in claim 1 or 2, wherein the wavelength switching time is selected to be 10 minutes before the retention time of baicalin peak.
5. The construction method of the HPLC fingerprint of the Pudilan antiphlogistic tablet based on the detection of the wavelength switching according to claim 2, wherein the heating reflux method in the step (1) can effectively inhibit the activity of the enzyme, the baicalein is completely inactivated at the reflux temperature of 85 ℃, and the heating reflux time is 2 h.
6. The method for constructing HPLC fingerprint of Pudilan antiphlogistic tablet based on detection wavelength switching as claimed in claim 2, wherein the sample extraction solvent in step (1) is 50% methanol.
7. The method for constructing the HPLC fingerprint of the Pudilan antiphlogistic tablet based on the detection of the wavelength switching as claimed in claim 2, wherein the similarity between the sample fingerprint and the comparison fingerprint in the step (5) is greater than 0.95.
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