Construction Method of HPLC Control Fingerprint and Fingerprint of Sample to be Tested for Biyantong Spray

Abstract

The invention discloses a method for constructing an HPLC reference fingerprint spectrum and a fingerprint spectrum of a sample to be detected for Biyantong Spray, including: injecting 15 batches of samples of Biyantong Spray into a liquid chromatograph to measure the single-needle fingerprint spectrum, recording the fingerprint spectrum, and substituting it into a similarity software for fitting to obtain the reference fingerprint spectrum; injecting the test solution into the liquid chromatograph for determination and recording the chromatographic peaks to obtain the fingerprint spectrum of the sample to be detected; the adopted chromatographic conditions include: using octadecylsilane chemically bonded silica gel as the filler; using acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, and adopting a gradient elution program for elution. By optimizing the chromatographic conditions, different wavelengths are used at different time periods to adjust the proportion of the common characteristic peaks of the entire characteristic spectrum, effectively improving the quality of the fingerprint characteristic spectrum. The method of the invention has good specificity, and the characteristic peaks are not interfered by impurity peaks; it has good precision, repeatability and stability, is stable and reliable, and has good adaptability.

Description

Technical Field

[0001] The present invention relates to a method for constructing a fingerprint of traditional Chinese medicine preparations, and particularly to a method for constructing an HPLC reference fingerprint and a fingerprint of a sample to be detected for Biyantong Spray, belonging to the field of constructing a reference fingerprint and a fingerprint of a sample to be detected for Biyantong Spray. Background Art

[0002] China is the birthplace of traditional Chinese medicine, and the history of using traditional Chinese medicine has been thousands of years. However, due to the complexity of the formula compatibility of traditional Chinese medicine and the instability of medicinal materials, the quality control means of traditional Chinese medicine products are relatively lacking in scientificity, it is difficult to confirm the correlation between efficacy and quality, and it cannot be expressed by modern pharmaceutical science, resulting in a small share of traditional Chinese medicine in the international market transactions, seriously hindering the development process of traditional Chinese medicine. An effective means to solve this problem is to refer to the quality control methods of foreign botanical drugs and establish a fingerprint of traditional Chinese medicine commonly used in the international community.

[0003] The fingerprint of traditional Chinese medicine refers to a chromatogram or spectrogram obtained by using certain analysis means after appropriate treatment of certain traditional Chinese medicinal materials or traditional Chinese medicine preparations, which can mark their chemical characteristics. It can comprehensively reflect the types and quantities of chemical components contained in medicinal materials, effectively reflect the integrity and comprehensive effect of traditional Chinese medicine components, and has been widely used in aspects such as analysis and identification and quality control of traditional Chinese medicine due to its characteristics such as rapidity and accuracy.

[0004] Biyantong Spray is a commonly used pure traditional Chinese medicine preparation in clinical practice, without hormones, and atomizes traditional Chinese medicine to directly spray on the lesion. It is a nasal spray directly acting on the nose, specially used for symptoms such as acute and chronic rhinitis, allergic rhinitis, nasal congestion, nasal itching, sneezing, runny nose and nasal discomfort, and has good curative effects on rhinitis and sinusitis. The main components of Biyantong Spray are composed of Lonicera hypoglauca Miq., Flos Magnoliae Essential Oil, Borneol, Baicalin, and Ephedrine Hydrochloride, and has the functions of dispersing wind and clearing heat, and promoting lung qi and opening orifices.

[0005] So far, there are only two items, Baicalin and Ephedrine Hydrochloride, in the content determination method of the quality standard of Biyantong Spray, which cannot comprehensively reflect the quality of other components. As a traditional Chinese medicine preparation, this standard is difficult to comprehensively reflect the overall quality characteristics of the product and cannot control the quality of the product as a whole, and it is urgently needed to be improved. Summary of the Invention

[0006] The main object of the present invention is to provide a method for constructing an HPCL fingerprint of Biyantong Spray. The constructed fingerprint can comprehensively reflect the quality information of Biyantong Spray, and has advantages such as quickness, stability, high precision, and strong reproducibility, and can be used for the quality control of Biyantong Spray.

[0007] To achieve the above object, the present invention provides a method for constructing an HPLC fingerprint of Biyantong spray, including a method for constructing an HPLC reference fingerprint of Biyantong spray and a method for constructing an HPLC fingerprint of the sample to be detected of Biyantong spray. Among them, the method for constructing the HPLC reference fingerprint of Biyantong spray includes:

[0008] Inject 15 batches of samples of Biyantong spray into a liquid chromatograph to measure the single-needle fingerprint, record the fingerprint, substitute it into the similarity software, and fit to obtain the HPLC reference fingerprint of Biyantong spray;

[0009] The method for constructing the HPLC fingerprint of the sample to be detected of Biyantong spray includes:

[0010] (1) Preparation of the test solution: Dissolve Biyantong spray with methanol, dilute it, shake well, filter, and take the continuous filtrate to obtain it.

[0011] (2) Absorb the test solution and inject it into the liquid chromatograph, measure it, and record the chromatographic peaks within 100 minutes to obtain it.

[0012] Among them, the chromatographic conditions for measuring the HPLC reference fingerprint of Biyantong spray or the HPLC fingerprint of the sample to be detected of Biyantong spray by a liquid chromatograph include:

[0013] Using octadecylsilane-bonded silica gel as the filler; using acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table:

[0014]

[0015] The detection wavelength is: from 0 to 23 min, the detection wavelength is 215 nm; from 23 to 60 min, the detection wavelength is 326 nm; from 60 to 100 min, the detection wavelength is 215 nm.

[0016] As a preferred specific implementation of the present invention, the chromatographic conditions further include: the chromatographic column is selected from Waters Xbridge, Welch Ultimate XB C18 or Phenomenex Luna C18; the flow rate is 0.5 - 1.5 ml / min; the column temperature is 25 - 35 °C; the theoretical plate number calculated by the chlorogenic acid peak should be not less than 10000; more preferably, the chromatographic column is Phenomenex Luna C18; the flow rate is 1.0 ml / min; the column temperature is 30 °C.

[0017] As a preferred specific implementation of the present invention, the method for constructing the HPLC reference fingerprint of the Biyantong spray further includes: preparing a reference substance solution, sucking the reference substance solution and injecting it into a liquid chromatograph for determination, and recording the chromatographic peaks within 100 minutes; the role of the reference substance in the reference fingerprint is to confirm which specific compound some of the common peaks are.

[0018] As a preferred specific implementation of the present invention, the preparation of the reference substance solution includes: taking appropriate amounts of chlorogenic acid reference substance, neochlorogenic acid reference substance, cryptochlorogenic acid reference substance, isochlorogenic acid A reference substance, isochlorogenic acid B reference substance, isochlorogenic acid C reference substance, ephedrine hydrochloride reference substance, and baicalin reference substance, accurately weighing them, and making a mixed solution containing 100 μg of ephedrine hydrochloride, 800 μg of baicalin, and 5 μg of each of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in 1 ml with methanol, that is, obtaining it.

[0019] As a preferred specific implementation of the present invention, the preferred method for preparing the test sample solution is: measuring 1 ml of the Biyantong spray and placing it in a 25-ml volumetric flask, adding methanol to the scale and shaking well, filtering, and taking the subsequent filtrate, that is, obtaining it.

[0020] In order to determine the most suitable detection wavelength, the present invention conducted a screening test with a wavelength scanning range of 190 nm - 400 nm. The results showed that the characteristic chromatographic peaks (ephedrine hydrochloride, benzyl alcohol, and magnolia flower oil) of the Biyantong spray only had absorption at 215 nm in the ultraviolet range. However, at 215 nm, the absorption of the characteristic chromatographic peak of baicalin was too large. Therefore, the present invention preferably uses the following segmented detection wavelengths as the detection wavelengths for the fingerprint of the Biyantong spray: 0 - 23 min: 215 nm, 23 - 60 min: 326 nm; 60 min - 100 min: 215 nm.

[0021] The present invention compared dilute ethanol, ethanol, 50% methanol, and methanol as extraction solvents for preparing the test sample, and the results showed that when the extraction solvent was methanol, the baseline was relatively stable and the separation between each chromatographic peak was better. Therefore, the present invention preferably uses methanol as the extraction solvent when preparing the test sample solution.

[0022] In order to screen the most suitable chromatographic column, in the present invention, under the same gradient, the effects of chromatographic columns Waters Xbridge (4.6×250 mm, 5 μm), Welch Ultimate XB C18 (4.6×250 mm, 5 μm), and Phenomenex Luna C18 (4.6×250 mm, 5 μm) on the chromatogram were investigated respectively. The results showed that when using the Phenomenex Luna C18 (4.6×250 mm, 5 μm) column, the overall separation effect of the chromatographic peaks was the best. Therefore, the present invention preferably determines Phenomenex Luna C18 as the liquid chromatographic column for establishing the fingerprint of Biyantong Spray.

[0023] In order to determine the most suitable mobile phase system, in the present invention, the effects of acetonitrile - 0.2% phosphoric acid solution, acetonitrile - 0.1% formic acid solution, acetonitrile - 0.4% formic acid solution, acetonitrile - 0.4% glacial acetic acid solution, and methanol - 0.2% phosphoric acid solution as the mobile phase system on the chromatographic peaks were investigated respectively. The results showed that when the mobile phase system was acetonitrile - 0.2% phosphoric acid solution, the overall baseline was relatively stable, the resolution of the chromatographic peaks was better, the number of chromatographic peaks was larger, and the impurity peaks were fewer. Therefore, the present invention preferably uses acetonitrile - 0.2% phosphoric acid solution as the final mobile phase.

[0024] The present invention also investigated the effects of phosphoric acid solutions with different volume fractions on the chromatographic peaks. Acetonitrile - 0.1% phosphoric acid, acetonitrile - 0.2% phosphoric acid, and acetonitrile - 0.3% phosphoric acid were used as the mobile phase respectively, and other chromatographic conditions such as the elution gradient and column temperature were kept consistent. The results showed that the separation was better when the volume fraction of phosphoric acid was 0.2%. Therefore, the present invention preferably uses a phosphoric acid solution with a volume fraction of 0.2%.

[0025] The present invention also investigated the effects of different column temperatures (25 - 35°C) on the chromatographic peaks respectively. The results showed that when the column temperature was 30°C, the chromatographic peaks could achieve better separation. Therefore, the column temperature in the chromatographic conditions of the present invention is preferably 30°C.

[0026] Through a series of experimental comparisons, the present invention finally determined methanol as the solvent. Since baicalin is the main drug of Biyantong Spray and has a large absorption in the chromatogram, by optimizing the chromatographic conditions, different wavelengths were used at different time periods to adjust the proportion of the common characteristic peaks of the entire characteristic chromatogram, greatly improving the quality of the entire fingerprint chromatogram.

[0027] The fingerprint of Biyantong Spray constructed in the present invention has 14 characteristic peaks. By comparing with the reference substances, 9 of the characteristic peaks were finally identified based on the complete coincidence of retention time and UV characteristic fingerprint. They are Peak 1: Ephedrine Hydrochloride; Peak 2: Chlorogenic Acid Isomer; Peak 3: Chlorogenic Acid; Peak 4: Cryptochlorogenic Acid; Peak 5: Benzyl Alcohol; Peak 6: Isochlorogenic Acid B; Peak 7: Isochlorogenic Acid C; Peak 8: Isochlorogenic Acid A; Peak 9: Baicalin.

[0028] According to the prescription medicinal material data collected in the previous work, the ephedrine hydrochloride in the prescription contains the characteristic components of ephedrine hydrochloride, and the extract of Lonicera confusa DC contains the characteristic components of chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and chlorogenic acid isomer. The essential oil of Magnolia biondii Pamp., ephedrine hydrochloride, baicalin, and benzyl alcohol in the excipients also have their respective characteristic components. According to the pharmacodynamic components and main chemical properties of each medicinal flavor, the corresponding reference substance solutions were prepared for positioning and comparison, so that the constructed reference fingerprint can comprehensively reflect the quality information of Biyantong Spray, avoiding the disadvantages of single and one-sided existing quality control standards, and can be used for the comprehensive quality evaluation and control of Biyantong Spray.

[0029] The fingerprint of 15 batches of Biyantong Spray was determined in the present invention. The fingerprints of 15 batches of Biyantong Spray were imported into the Similarity Evaluation System for Traditional Chinese Medicine Fingerprints (2012 Edition), and matched with the mark peak to obtain the reference fingerprint by fitting, and a CDF format file was generated, which can provide the reference fingerprint for the determination of other Biyantong Spray fingerprints. After calculation, the similarity between the fingerprints of 15 batches of Biyantong Spray and the fitted reference fingerprint is above 0.99, indicating that the fingerprint quality of 15 batches of Biyantong Spray is highly consistent and the production process is stable and controllable.

[0030] The present invention uses the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints recommended by the Pharmacopoeia Commission of the People's Republic of China to identify the fingerprint, which is simple and fast to operate. The similarity results obtained in this way are used to evaluate the Biyantong Spray fingerprint, and the conclusion is relatively objective and accurate.

[0031] In summary, the present invention uses high performance liquid chromatography (HPLC) to compare the fingerprint spectra of 15 batches of Biyantong Spray, determines 14 common characteristic peaks, constructs the standard fingerprint spectrum of Biyantong Spray, and uses a mixed reference substance solution to identify these common peaks such as ephedrine hydrochloride, chlorogenic acid, baicalin, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C. The constructed reference fingerprint spectrum can comprehensively reflect the quality information of Biyantong Spray, has the advantages of being fast, stable, highly precise, and strong in reproducibility, avoids the disadvantages of the existing quality control standards being single and one-sided, and can be used for the comprehensive quality evaluation and control of Biyantong Spray. The newly established fingerprint spectrum is used to comprehensively compare the quality of 15 batches of Biyantong Spray samples, prove the quality consistency of the product, and the conclusion is relatively objective and accurate. Through the optimization of the chromatographic conditions, the fingerprint spectrum baseline of Biyantong Spray is more stable, the resolution of chromatographic peaks is better, the number of chromatographic peaks is more, the miscellaneous peaks are less, the peak emergence time is faster, and the information volume is large.

[0032] The present invention has carried out methodological verification on the established HPLC fingerprint spectrum method for Biyantong Spray. The verification results show that the HPLC fingerprint spectrum method established by the present invention has good specificity, and the characteristic peaks are not interfered by impurity peaks; the precision, repeatability, and stability all meet the requirements, indicating that the method of the present invention is stable and reliable and has good adaptability. Description of the Drawings

[0033] Figure 1 Are fingerprint spectra at different detection wavelengths.

[0034] Figure 2 Are fingerprint spectra at different detection wavelengths.

[0035] Figure 3 Are fingerprint spectra of different chromatographic columns.

[0036] Figure 4 Are fingerprint spectra of different types of mobile phases.

[0037] Figure 5 Are fingerprint spectra of mobile phases with different phosphoric acid concentrations.

[0038] Figure 6 Are fingerprint spectra at different chromatographic column temperatures.

[0039] Figure 7 Are fingerprint spectra of different extraction solvents.

[0040] Figure 8 Is the specific chromatogram of ephedrine hydrochloride.

[0041] Figure 9 Is the specific chromatogram of baicalin.

[0042] Figure 10 It is the specific chromatogram of the extract of Lonicera confusa DC..

[0043] Figure 11 It is the specific chromatogram of benzyl alcohol.

[0044] Figure 12 It is the specific chromatogram of Flos Magnoliae volatile oil.

[0045] Figure 13 Chromatogram of precision test.

[0046] Figure 14 It is the chromatogram of repeatability test.

[0047] Figure 15 It is the fingerprint of different operators.

[0048] Figure 16 It is the fingerprint of different liquid chromatographs.

[0049] Figure 17 It is the chromatogram of stability test.

[0050] Figure 18 It is the fingerprint of 15 batches of Biyantong Spray.

[0051] Figure 19 It is the reference fingerprint of Biyantong Spray; among the 14 common peaks, peak 1: ephedrine hydrochloride, peak 2: neochlorogenic acid, peak 3: chlorogenic acid, peak 4: cryptochlorogenic acid, peak 5(S): benzyl alcohol, peak 6: isochlorogenic acid B, peak 7: isochlorogenic acid A, peak 8: isochlorogenic acid C, peak 9: baicalin. Detailed implementation mode

[0052] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and forms of the present invention can be made without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0053] Experimental Example 1 Construction method and methodological verification of HPLC reference fingerprint and HPLC fingerprint of samples to be detected of Biyantong Spray

[0054] 1 Establishment of fingerprint chromatographic method

[0055] 1.1 Selection of detection wavelength

[0056] Method: Mobile phase A is acetonitrile and B is 0.2% phosphoric acid (V / V) solution. Linear gradient elution is performed according to the established procedure; the flow rate is 1 ml / min, the column temperature is 30 °C, PDA monitoring is carried out, and the wavelength scanning range is 190 nm - 400 nm. The results are Figure 1 and Figure 2 shown as

[0057] From Figure 1 and Figure 2 it can be seen that the characteristic chromatographic peaks (ephedrine hydrochloride, benzyl alcohol, magnolia flower oil) of Biyantong Spray only have absorption at 215 nm in ultraviolet light. However, at 215 nm, the absorption of the characteristic chromatographic peak of baicalin is too large. Therefore, 0 - 23 min: 215 nm, 23 - 60 min: 326 nm; 60 min - 100 min: 215 nm is selected as the detection wavelength for the fingerprint of Biyantong Spray by stages.

[0058] 1.2 Selection of chromatographic column

[0059] Under the same gradient, the effects on the chromatogram were investigated when the chromatographic columns were Waters Xbridge (4.6×250 mm, 5 μm), Welch Ultimate XB C18 (4.6×250 mm, 5 μm), and Phenomenex Luna C18 (4.6×250 mm, 5 μm) respectively. The results are as Figure 3 shown. From Figure 3 it can be seen that when using the Phenomenex Luna C18 (4.6×250 mm, 5 μm) column, the overall separation effect of the chromatographic peaks is the best. Therefore, Phenomenex Luna C18 is selected as the liquid chromatographic column for the fingerprint of Biyantong Spray.

[0060] 1.3 Selection of types of mobile phase

[0061] The effects on the chromatographic peaks were investigated when acetonitrile - 0.2% phosphoric acid solution by volume, acetonitrile - 0.1% formic acid solution by volume, acetonitrile - 0.4% formic acid solution by volume, acetonitrile - 0.4% glacial acetic acid solution by volume, methanol - 0.2% phosphoric acid solution by volume were used as the mobile phase systems respectively. The results are shown in Figure 4 .

[0062] The results show that when the mobile phase system is acetonitrile - 0.2% phosphoric acid solution by volume, the overall baseline is relatively stable, the resolution of the chromatographic peaks is better, the number of chromatographic peaks is larger, and there are fewer impurity peaks. Therefore, acetonitrile - 0.2% phosphoric acid solution by volume is selected as the final mobile phase.

[0063] 1.4 Selection of phosphoric acid concentration

[0064] Investigate the influence of glacial acetic acid solutions with different volume fractions on chromatographic peaks. Use acetonitrile - 0.1% phosphoric acid, acetonitrile - 0.2% phosphoric acid, and acetonitrile - 0.3% phosphoric acid, and keep other chromatographic conditions such as the elution gradient and column temperature consistent. The measurement results are as Figure 5 shown.

[0065] The results show that when the volume fraction of phosphoric acid is 0.2%, the separation is better. Therefore, a phosphoric acid solution with a volume fraction of 0.2% is selected.

[0066] 1.5 Selection of column temperature

[0067] Investigate the influence on the chromatogram peaks when the column temperature is 25°C, 30°C, and 35°C respectively. The test results are shown in Figure 6 . According to Figure 6 the results, when the column temperature is 30°C, the chromatographic peaks can achieve better separation. Therefore, a column temperature of 30°C is adopted.

[0068] 1.6 Determination of chromatographic conditions

[0069] Chromatographic column: Phenomenex Luna C18 (4.6×250mm, 5μm), A is acetonitrile, and B is 0.2% phosphoric acid solution. The detection wavelength is 215nm from 0 to 23min; 326nm from 23 to 60min; 215nm from 60min to 100min, and the column temperature is 30°C. Elute according to the following gradient.

[0070] Table 1 Elution gradient of fingerprint

[0071]

[0072] 2 Establishment of the preparation method of the test solution

[0073] Selection of extraction solvent: Accurately measure 1 ml of Biyantong Spray into a 25 - ml volumetric flask, add dilute ethanol, ethanol, 50% methanol, and methanol to the scale respectively, shake well, filter, and measure. The results are as Figure 7 shown. It can be found from the results that when the solvent is methanol, the baseline is relatively stable and the separation between chromatographic peaks is better. Therefore, methanol is selected as the solvent.

[0074] 3 Method validation

[0075] 3.1 Specificity test

[0076] Precisely pipette 10 μL of the corresponding negative control solution, reference substance solution, and test solution respectively, and inject them for measurement under the established chromatographic conditions. The results are as Figures 8 - 12 shown. There are no corresponding chromatographic peaks in the negative control solution at the characteristic peak positions of the reference medicinal material and the test solution. This indicates that there is no interference in the determination of the fingerprint of the test solution, and the method has good specificity.

[0077] 3.2 Precision Test

[0078] Take the test solution and continuously measure it 6 times under the proposed test conditions. Record the fingerprint chromatogram, compare it with the generated reference fingerprint chromatogram, calculate the similarity. The results show that the similarity is 1 in all cases, the RSD of the relative retention time of the common peaks is less than 1%, and the RSD of the relative peak area is less than 5%( Figure 13 ), indicating that the precision of the method is good.

[0079] Table 2 Ratio of Relative Retention Time of Main Chromatographic Peaks

[0080]

[0081] Table 3 Ratio of Relative Peak Area of Main Chromatographic Peaks

[0082]

[0083] Table 4 Results of Similarity Calculation

[0084]

[0085] 3.3 Repeatability Test

[0086] Experimenter A took the same batch of test samples (batch number: 20002, provided by Guoyao Group Dezhong (Foshan) Pharmaceutical Co., Ltd.), prepared 6 test solutions with reference to "Preparation of Test Solution", precisely pipetted 10 μL of the test solution, injected it into the liquid chromatograph, and carried out sample injection and determination with reference to "Chromatographic Conditions". Record the fingerprint chromatogram, compare it with the generated reference fingerprint chromatogram, calculate the similarity. The results show that the similarity is 1 in all cases, the RSD of the relative retention time of the common peaks is less than 2%, and the RSD of the relative peak area is less than 5%( Figure 14 ), indicating that the repeatability of the method is good.

[0087] Table 5 Ratio of Relative Retention Time of Main Chromatographic Peaks

[0088]

[0089]

[0090] Table 6 Ratio of Relative Peak Area of Main Chromatographic Peaks

[0091]

[0092] Table 7 Results of Similarity Calculation

[0093]

[0094] 3.4 Intermediate Precision Test

[0095] 3.4.1 Comparison among Different Operators

[0096] Experimenter B prepared 6 portions of the test sample solution (batch number: 20002, provided by Guoyao Group Dezhong (Foshan) Pharmaceutical Co., Ltd.) according to the requirements of "repetitive test", and determined it under the chromatographic conditions in the above "1 Establishment of Fingerprint Chromatographic Method". Record the fingerprint chromatogram, input it into the similarity software. Each fingerprint chromatogram should have 14 characteristic peaks, and calculate the similarity with the reference fingerprint chromatogram respectively. The results showed that under the preparation by different personnel, the similarity of each measured fingerprint chromatogram with the reference fingerprint chromatogram was 1( Figure 15 ), indicating that the intermediate precision of this method is good.

[0097] 3.4.2 Comparison of Different Liquid Chromatographs

[0098] Another same test sample solution was taken and tested under the specified chromatographic conditions on a Waters e2695 liquid chromatograph and a Thermo Ultimate 3000 liquid chromatograph respectively. The results are shown in Figure 16 .

[0099] The results showed that the fingerprint chromatograms obtained by the Waters e2695 liquid chromatograph, the Thermo Ultimate 3000 liquid chromatograph, and the liquid chromatograph were analyzed by the similarity evaluation system, and the similarities were 1 and 0.997 in sequence, indicating that there was no significant difference in the results of liquid chromatographs of different brands or models, and the intermediate precision of the method was good.

[0100] 3.5 Stability Test

[0101] Take one portion of the test sample solution in the intermediate precision item and measure it at 0, 2, 8, 12, 16, 24, and 32 hours respectively. Record the fingerprint chromatogram, input it into the similarity software. Each fingerprint chromatogram should have 14 characteristic peaks. The results showed ([[]] Figure 17 ), among the measurement results at different time periods, the similarity of each fingerprint chromatogram with the fingerprint chromatogram at 0 hour was 1, and the RSD of the retention time was less than 1%. It indicated that the measurement results of the sample were stable within 32 hours.

[0102] Table 8 Relative Retention Time Ratio of Main Chromatographic Peaks

[0103]

[0104] Table 9 Relative Peak Area Ratio of Main Chromatographic Peaks

[0105]

[0106] 4 Establishment of Reference Fingerprint Chromatogram

[0107] 4.1 Medicinal Material Sources of Main Chromatographic Peaks

[0108] Referring to "Preparation of Test Solution", prepare the test solutions of the eight crude drugs in the prescription respectively. Precisely pipette 10 μl each and inject them into the liquid chromatograph, and perform injection tests according to the "Chromatographic Conditions". The results show that among the nine common peaks in the standard fingerprint of Biyantong Spray, the clear attribution of each peak has been basically found in the fingerprint of the crude drugs in the prescription. Among them: Peak 1 is attributed to ephedrine hydrochloride; Peaks 2, 3, 4, 6, 7, and 8 are attributed to the extract of Lonicerae Japonicae Flos; Peak 5 is attributed to benzyl alcohol; Peak 9 is attributed to baicalin; Peaks 10, 11, 12, 13, and 14 are attributed to Flos Magnoliae Essential Oil.

[0109] 4.2 Fitting of Control Chromatogram

[0110] Use the established method to determine the single-needle fingerprint of 15 batches of Biyantong Spray samples, record the fingerprints, input them into the similarity software, and obtain Figure 18 , generate a control chromatogram, and calculate the similarity between the 15 batches of test products and the control fingerprint.

[0111] Table 10 Similarity of Fingerprints of 15 Batches of Biyantong Spray

[0112]

[0113] Table 10 shows the similarity of fingerprints of 15 batches of Biyantong Spray. Figure 18 They are the fingerprints of 15 batches of Biyantong Spray. Figure 19 It is the control fingerprint of Biyantong Spray. As can be seen from Table 10, the similarity of fingerprints of 15 batches of Biyantong Spray is all above 0.99.

Claims

1. A method for constructing an HPLC fingerprint of Biyantong spray, characterized in that, Including the construction method of the HPLC reference fingerprint of Biyantong Spray and the construction method of the HPLC fingerprint of the Biyantong Spray to be detected. Among them, the construction method of the HPLC reference fingerprint of Biyantong Spray includes: Inject 15 batches of samples of Biyantong Spray into the liquid chromatograph to measure the single-needle fingerprint, record the fingerprint, substitute it into the similarity software, and fit to obtain the HPLC reference fingerprint of Biyantong Spray; The construction method of the HPLC fingerprint of the Biyantong Spray to be detected includes: (1) Preparation of the test solution: Dissolve Biyantong Spray with methanol, dilute it, shake well, filter, and take the filtrate to obtain; (2) Absorb the test solution and inject it into the liquid chromatograph for determination, record the chromatographic peaks within 100 minutes to obtain; Among them, the chromatographic conditions for measuring the HPLC reference fingerprint of Biyantong Spray or the HPLC fingerprint of the Biyantong Spray to be detected by the liquid chromatograph include: Using octadecylsilane chemically bonded silica gel as the filler; using acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, and the gradient elution program is as follows: 0 - 100 minutes, mobile phase A 5→95%, mobile phase B 95→5%; The detection wavelength is: 0 - 23 min, the detection wavelength is 215 nm; 23 - 60 min, the detection wavelength is 326 nm; 60 - 100 min, the detection wavelength is 215 nm.

2. The construction method according to claim 1, characterized in that, The chromatographic conditions also include: The theoretical plate number calculated by the chlorogenic acid peak is not less than 10,000.

3. The construction method according to claim 1, characterized in that, The chromatographic conditions also include: The chromatographic column is selected from Waters Xbridge, Welch Ultimate XB C18 or Phenomenex Luna C18.

4. The construction method according to claim 1, characterized in that, The chromatographic conditions also include: The flow rate is 0.5 - 1.5 ml / min.

5. The construction method according to claim 4, characterized in that, The flow rate is 1.0 ml / min.

6. The construction method according to claim 1, characterized in that, The chromatographic conditions also include: The column temperature is 25 - 35 °C.

7. The construction method according to claim 6, characterized in that, The column temperature is 30 °C.

8. The construction method according to claim 1, characterized in that, The construction method of the HPLC reference fingerprint of Biyantong Spray also includes: Prepare the reference substance solution, absorb the reference substance solution and inject it into the liquid chromatograph for determination, and record the chromatographic peaks within 100 minutes.

9. The construction method according to claim 8, characterized in that, The preparation method of the reference substance solution is: Weigh the reference substances of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, ephedrine hydrochloride, and baicalin, add methanol to make a mixed solution containing 100 μg of ephedrine hydrochloride and 800 μg of baicalin per 1 ml, and containing 5 μg of each of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C to obtain.

10. The construction method according to claim 1, characterized in that, Bring the fingerprints of 15 batches of Biyantong Spray into the "Similarity Evaluation System for Traditional Chinese Medicine Fingerprints" 2012 version, match with the mark peak, and fit to obtain the reference fingerprint.

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