Construction method of fingerprint spectrum of Qutanling oral liquid and standard fingerprint spectrum thereof

Abstract

The present application belongs to the technical field of medicine quality control, and particularly relates to a construction method of a Qutanling oral liquid fingerprint spectrum and a standard fingerprint spectrum thereof, and comprises the following steps: 1) preparation of a test sample solution; 2) preparation of a reference sample solution of a single index component; and 3) establishment of the fingerprint spectrum: the reference sample solution and the test sample solution are respectively injected into a high performance liquid chromatograph, and detection is carried out according to high performance liquid chromatography conditions to obtain the fingerprint spectrum of the Qutanling oral liquid. The present application adopts high performance liquid chromatography, and the total number of common peaks in the standard fingerprint spectrum of the Qutanling oral liquid established by the present application is 9, 3 characteristic components, i.e. protocatechuic acid, p-hydroxybenzoic acid and guaiacol, are identified, and the internal components of the Qutanling oral liquid are effectively characterized, and the content of the quality standard is supplemented.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug quality control, and particularly relates to a construction method of a Qutanchenling oral liquid fingerprint and a standard fingerprint thereof. BACKGROUND

[0002] The Qutanchenling oral liquid is prepared from fresh bamboo juice and houttuynia cordata as main materials, with sucrose and sodium benzoate as auxiliary materials, has the function of clearing lung and reducing phlegm, and is used for treating cough, excessive phlegm and wheezing caused by phlegm-heat obstructing lung, and is also used for treating the above symptoms of acute and chronic bronchitis. However, there are few quality control methods for the drug preparation prepared from fresh bamboo juice and houttuynia cordata as main materials. The patent with the application number CN200610200242.0 discloses a cough-relieving and phlegm-reducing traditional Chinese medicine syrup, a preparation method and a quality control method, including appearance, thin layer chromatography and liquid chromatography. However, the method lacks scientific qualitative and quantitative control indexes of characteristic components, only establishes the chromatography of guaiacol, and fails to control the quality of the Qutanchenling oral liquid as a whole. The quality standard of the Qutanchenling oral liquid in the 2020 edition of the Chinese Pharmacopoeia is also relatively simple, and only the chromatography of guaiacol is established. The above methods only rely on a certain effective component and ignore other effective components, which are not comprehensive and scientific, and are not conducive to the comprehensive control of the quality of the drug.

[0003] The fingerprint is a comprehensive and quantifiable identification means, can systematically, integrally and exclusively represent the internal characteristics of traditional Chinese medicines, can be used for evaluating the quality of traditional Chinese medicinal materials, traditional Chinese medicinal preparations and semi-finished products, and is an effective means for the overall quality control of traditional Chinese medicines. Although the existing technology also discloses the detection research of the fingerprint UPLC of fresh bamboo juice and houttuynia cordata, the method only studies the raw drug and is not representative, and cannot comprehensively reveal the internal quality of the Qutanchenling oral liquid.

[0004] Therefore, the construction of the high-performance liquid chromatography fingerprint of the Qutanchenling oral liquid and the establishment of the effective chemical components through the analysis of the fingerprint are beneficial to providing a reference for the subsequent quantitative detection of the effective components, thereby being beneficial to guaranteeing the safety and effectiveness of the quality of the Qutanchenling oral liquid. SUMMARY

[0005] The application proposes the construction method of the Qutanchenling oral liquid fingerprint and the standard fingerprint thereof in view of the deficiencies of the prior art.

[0006] Specifically, the method is implemented by the following technical scheme:

[0007] A construction method of a Qutanchenling oral liquid fingerprint, comprising the following steps:

[0008] 1) Preparation of a test sample solution: filter multiple batches of Qutanchenling oral liquid through a 0.45 mu m filter membrane, and take the filtrate, namely the prepared test sample solution;

[0009] 2) Preparation of the reference solution of the single index component: 2 mg of the reference of the index component was weighed, dissolved in 50 ml of methanol, filtered through a 0.45 μm filter membrane, and the filtrate was taken to obtain the reference of the single index component; the index component is protocatechuic acid, p-hydroxybenzoic acid, guaiacol;

[0010] 3) Establishment of the fingerprint spectrum: 10 μL of the reference solution of the single index component and the sample solution were injected into the high performance liquid chromatograph, respectively, and detected according to the high performance liquid chromatography conditions; the spectrum and data of the sample solution and the reference solution within 48 min were recorded, respectively, and the obtained spectrum and data were introduced into the traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system for analysis to establish the fingerprint spectrum of the Qutanling oral liquid.

[0011] The detection conditions of the high performance liquid chromatography are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as the mobile phase A, and 0.1%-0.3% phosphoric acid solution is used as the mobile phase B for gradient elution, the detection wavelength is 210-350 nm, and the column temperature is 25-35℃.

[0012] Preferably, the detection conditions of the high performance liquid chromatography are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as the mobile phase A, and 0.3% phosphoric acid solution is used as the mobile phase B for gradient elution, the detection wavelength is 254 nm, and the column temperature is 25℃.

[0013] The specification of the chromatographic column is 250 mm x 4.6 mm, and the particle size is 5 μm.

[0014] The gradient elution is as follows: 0 min→9 min→15 min→15.01 min→24 min→35 min→48 min→48.01 min→50 min→60 min, the flow rate is 0.6 ml / min→0.6 ml / min→0.6 ml / min→1.0 ml / min→1.0 ml / min→1.0 ml / min→1.0 ml / min→0.6 ml / min→0.6 ml / min→0.6 ml / min, the percentage of the mobile phase A is 4%→4%→6%→6%→8%→14%→16%→16%→4%→4%, the percentage of the mobile phase is a volume percentage, and the sum of the volume percentages of the mobile phase A and the mobile phase B is 100%.

[0015] The present application provides a standard fingerprint spectrum of the Qutanling oral liquid, which is obtained by the construction method according to the present application.

[0016] The standard fingerprint spectrum comprises 9 common peaks, numbered from left to right, the 4th peak is protocatechuic acid, the 6th peak is p-hydroxybenzoic acid, and the 9th peak is guaiacol, and the 6th peak is used as a reference peak, and the relative retention time of the other 8 common peaks and the relative standard deviation of each common peak are all less than or equal to 3%.

[0017] The application also provides application of the standard fingerprint spectrum of the Qutanchenling oral liquid in quality detection of the Qutanchenling oral liquid.

[0018] The quality detection refers to evaluation of consistency of different batches of the Qutanchenling oral liquid and identification of authenticity of the Qutanchenling oral liquid.

[0019] In the above application, the quality detection is that the Qutanchenling oral liquid to be detected is prepared into a test sample solution according to the test sample solution preparation method, and then injected into a high performance liquid chromatograph for detection according to the above liquid chromatography conditions to obtain a fingerprint spectrum of the test sample solution, and then the fingerprint spectrum of the test sample is compared with the standard fingerprint spectrum.

[0020] The formula of the Qutanchenling oral liquid is as follows: fresh bamboo juice 450ml, fishy grass 180g, sucrose 125g, sodium benzoate 1.4g, and the pH value is adjusted to 4.2-5.3.

[0021] Beneficial effects:

[0022] The Qutanchenling oral liquid liquid fingerprint spectrum established by the application has a total of 9 common peaks, 3 characteristic components of protocatechuic acid, p-hydroxybenzoic acid and guaiacol are identified, the internal components of the Qutanchenling oral liquid are effectively represented, and the key chemical components related to the quality of the Qutanchenling oral liquid are effectively identified, the content of the quality standard of the Qutanchenling oral liquid is supplemented, the quality of the drug preparation is beneficial to be comprehensively controlled, and the safety and effectiveness of clinical use are ensured.

[0023] The Qutanchenling oral liquid fingerprint spectrum obtained by the method has many peaks and good peak shape, is easy to identify, has high similarity, is accurate and reliable, and can comprehensively reflect the chemical information in the Qutanchenling oral liquid, thereby being beneficial to reflect the quality stability between batches of the drug.

[0024] The method has the characteristics of high precision, good stability and good repeatability, and is simple, fast, accurate and stable, which is helpful to improve the scientificity and comprehensiveness of the quality control of the Qutanchenling oral liquid. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 : chromatograms of different wavelengths;

[0026] Figure 2 : chromatograms of different chromatographic columns;

[0027] Figure 3: chromatograms at different column temperatures;

[0028] Figure 4 : chromatograms at different mobile phase systems;

[0029] Figure 5 : chromatograms at different phosphoric acid concentrations;

[0030] Figure 6 : chromatogram using mobile phase elution gradient 1;

[0031] Figure 7 : chromatogram using mobile phase elution gradient 2;

[0032] Figure 8 : chromatogram using mobile phase elution gradient 3;

[0033] Figure 9 : chromatogram after collection time confirmation;

[0034] Figure 10 : chromatogram of selected reference;

[0035] Figure 11 : chromatogram of blank test;

[0036] Figure 12 : chromatogram of different batches of sample fingerprint;

[0037] Figure 13 : control fingerprint of Qutanling;

[0038] Figure 14 Attribution of single medicine flavor of Qutanling fingerprint;

[0039] Figure 15 Attribution of excipient of Qutanling fingerprint;

[0040] Figure 16 Identification results of chromatographic peak components of Qutanling fingerprint. DETAILED DESCRIPTION

[0041] The specific embodiments of the present application are further described in detail below, but the present application is not limited to these embodiments, and any improvement or replacement in the basic spirit of the present embodiments still belongs to the scope of protection claimed by the present application.

[0042] The following examples commonly use the following instruments and materials:

[0043] Instruments:

[0044] Agilent 1260 high performance liquid chromatograph, XSR205DU electronic balance (one hundredth of a million, Mettler Toledo Technology Co., Ltd.), ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), chromatographic column: Waters C18 (4.6 x 250 mm, 5 μm).

[0045] Reagents:

[0046] Vahaha purified water, acetonitrile (chromatographically pure, American Tiandi Co., Ltd., batch number: 22095174), glacial acetic acid (analytically pure, National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number: 20220304), phosphoric acid (chromatographically pure, Aladdin Reagent Co., Ltd., batch number: K22291043), formic acid (analytically pure, National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number: 20220712), methanol (analytically pure, Nanjing Chemical Reagent Co., Ltd., batch number: 211013595K), reference substance: p-hydroxybenzoic acid (for fingerprint, batch number: 2169, Shanghai Shidande Standard Technology Service Co., Ltd.), cryptogreen acid (for fingerprint, batch number: 3208, Shanghai Shidande Standard Technology Service Co., Ltd.), p-coumaric acid (for fingerprint, batch number: 5555, Shanghai Shidande Standard Technology Service Co., Ltd.), syringaldehyde (for fingerprint, batch number: 13286, Shanghai Shidande Standard Technology Service Co., Ltd.), 2,6-dimethoxyphenol (for fingerprint, batch number: 6224, Shanghai Shidande Standard Technology Service Co., Ltd.), tyrosine (for fingerprint, batch number: 140609-201914, China Institute for Drug Control), salicylic acid (for fingerprint, batch number: 100106-202106, China Institute for Drug Control), guaiacol (for fingerprint, batch number: 111510-202205, China Institute for Drug Control), 5-hydroxymethylfurfural (for fingerprint, batch number: 111626-202215, China Institute for Drug Control), protocatechuic acid (for fingerprint, batch number: 110809-201906, China Institute for Drug Control), furfural (for fingerprint, batch number: 111630-200301, China Institute for Drug Control), vanillin (for fingerprint, batch number: 100491-201902, China Institute for Drug Control), sodium benzoate (for fingerprint, batch number: 100433-202103, China Institute for Drug Control), chlorogenic acid (for fingerprint, batch number: 110753-202119, China Institute for Drug Control).

[0047] Test sample:

[0048] Shanghai Jingan Pharmaceutical production, batch number for 220128-2, 220308-1, 221123-2, 220121-1, 220207-1, 220704-1, 220706-2, 220228-1, 221218-1, 220707-2, 221122-1, 221226-1, 230110-2, 221220-2, 211112-1.

[0049] Establishment of standard fingerprint spectrum of Qutanling oral liquid

[0050] 1. Selection of detection wavelength

[0051] 1.1 Preparation of test solution: Take multiple batches of Qutanling oral liquid and filter through a 0.45 μm filter membrane. Take the filtrate to obtain the test solution;

[0052] 1.2 Chromatographic conditions: octadecylsilane-bonded silica gel as the filler (chromatographic column: Waters Xbridge C18, 250 mm x 4.6 mm, 5 μm); flow rate of 1.0 ml / min, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution, column temperature of 35°C;

[0053] The gradient elution is shown in Table 1:

[0054] Table 1

[0055]

[0056] 1.3 Determination method: precisely take 10 μl of the test solution and inject it into the high performance liquid chromatograph for detection, and investigate the chromatogram under different detection wavelengths (210 nm, 230 nm, 254 nm, 280 nm, 310 nm, 325 nm, 350 nm).

[0057] 1.4 Results: it can be seen from Figure 1 that compared with the chromatograms under other wavelengths, the fingerprint spectrum at 254 nm has a smooth baseline, beautiful peak shape, more key information, and can more fully reflect the chemical composition of the product, so 254 nm is selected as the detection wavelength.

[0058] 2. Selection of chromatographic column

[0059] 2.1 Preparation of test solution is the same as 1.1.

[0060] 2.2 Chromatographic conditions: flow rate of 1.0 ml / min, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution, detection wavelength of 254 nm, column temperature of 35°C; the gradient elution is the same as 1.2.

[0061] 2.3 Determination method: 10 μl of the test sample solution was precisely taken and injected into the high performance liquid chromatograph for detection, and the chromatograms under different chromatographic columns were investigated. The chromatographic column selection is shown in Table 2.

[0062] Table 2 Brand and model of chromatographic column to be investigated

[0063] Brand Model Specification Waters XBridge C18 250 x 4.6 mm 5 μm Kromasil 100-5-C18 250 x 4.6 mm 5 μm Welch Ulitimate XB 250 x 4.6 mm 5 μm

[0064] 2.4 Results: From the above results, it can be seen that the separation effect and peak shape of Waters Xbridge C18 (250 x 4.6 mm, 5 μm) chromatographic column are better than those of other models, and therefore, Waters Xbridge C18 (250 x 4.6 mm, 5 μm) chromatographic column is preferentially selected. Figure 2

[0065] 3. Selection of column temperature

[0066] 3.1 Preparation of test sample solution is the same as 1.1.

[0067] 3.2 Chromatographic conditions: octadecylsilane-bonded silica gel is used as the filler (chromatographic column: Waters Xbridge C18, 250 mm x 4.6 mm, 5 μm); the flow rate is 1.0 ml / min; acetonitrile is used as mobile phase A and 0.1% phosphoric acid solution is used as mobile phase B for gradient elution; the detection wavelength is 254 nm; and the gradient elution is the same as 1.2.

[0068] 3.3 Determination method: 10 μl of the test sample solution was precisely taken and injected into the high performance liquid chromatograph for detection, and the chromatograms under different column temperatures (25°C, 30°C and 35°C) were investigated.

[0069] 3.4 Results: From the above results, it can be seen that the peak shape and separation effect of each chromatographic peak are better at 25°C, and therefore, 25°C is the optimal detection column temperature. Figure 3

[0070] 4. Selection of mobile phase system

[0071] 4.1 Preparation of test sample solution is the same as 1.1.

[0072] 4.2 Chromatographic conditions: octadecylsilane-bonded silica gel is used as the filler (chromatographic column: Waters Xbridge C18, 250 mm x 4.6 mm, 5 μm); the initial flow rate is 0.6 ml / min; the detection wavelength is 254 nm; and the column temperature is 25°C.

[0073] ​​​​4.3.1 Determination method: 10 μl of the test solution was precisely taken and injected into the high performance liquid chromatograph for detection. The chromatograms of different mobile phase systems (acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% acetic acid, acetonitrile-0.1% formic acid) were investigated. Gradient elution was performed according to the corresponding mobile phase system. The gradient elution program was referred to Table 1.

[0074] 4.4.1 Results: From the above results, it can be seen that, compared with the two systems of acetonitrile-0.1% acetic acid and acetonitrile-0.1% formic acid, the chromatogram information is relatively more complete, the peak shape of each chromatographic peak is better, and the separation effect is better. Figure 4

[0075] 4.3.2 Determination method: Under the above conditions, the chromatograms of different concentrations (0.1%, 0.2%, 0.3%) of phosphoric acid solution were investigated.

[0076] 4.4.2 Results: From the above results, it can be seen that the influence of phosphoric acid concentration on the separation effect of chromatographic peaks mainly reflects in the first 10 minutes of the chromatogram. Compared with the other two concentrations, 0.3% phosphoric acid solution can make the separation effect of the chromatographic peaks of large polar substances better. Therefore, the system of acetonitrile-0.3% phosphoric acid solution was selected as the mobile phase. Figure 5

[0077] 5. Selection of elution gradient

[0078] 5.1 Preparation of the test solution was the same as 1.1.

[0079] 5.2 Chromatographic conditions: octadecylsilane-bonded silica gel was used as the filler (chromatographic column: Waters Xbridge C18, 250 mm x 4.6 mm, 5 μm); acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B; the detection wavelength was 254 nm; and the column temperature was 35°C.

[0080] 5.3 Determination method: 10 μl of the test solution was precisely taken and injected into the high performance liquid chromatograph for detection. The chromatograms of different elution gradients were investigated. The elution gradients were selected from the following:

[0081] Table 3 Elution gradient 1 of the mobile phase

[0082]

[0083] Table 4 Elution gradient 2 of the mobile phase

[0084]

[0085] Table 5 Elution gradient 3 of the mobile phase

[0086]

[0087] ​​

[0088] 5.4 Results: From the results shown in Table 4, it can be seen that the test solution of Jiafei Ling was separated well by using elution gradient 3 with varying flow rate. Figure 6-8

[0089] 6. Confirmation of the collection time

[0090] 6.1 Preparation of the test solution The same as 1.1.

[0091] 6.2 Chromatographic conditions: octadecylsilyl bonded silica as the filler (chromatographic column: Waters Symmetry® C18, 250 mm x 4.6 mm, 5 μm); initial flow rate of 0.6 ml / min, acetonitrile as mobile phase A and 0.3% phosphoric acid solution as mobile phase B, detection wavelength of 254 nm, gradient elution according to Table 6; column temperature of 25°C.

[0092] Table 6

[0093]

[0094] 6.3 Assay: 10 μl of the test solution was precisely pipetted and injected into the high performance liquid chromatograph for detection.

[0095] 6.4 Results: From the results shown in Table 4, it can be seen that the test solution of Jiafei Ling was separated well by using elution gradient 3 with varying flow rate. Figure 9

[0096] 7. Selection of the reference

[0097] 7.1 Preparation of the test solution The same as 1.1.

[0098] 7.2 Preparation of the reference solution:

[0099] 7.2.1 Preparation of the guaiacol reference solution: 2 mg of guaiacol reference was weighed into a 50 ml volumetric flask, dissolved in methanol and filtered through a 0.45 μm filter membrane. The filtrate was collected.

[0100] 7.2.2 Preparation of the p-hydroxybenzoic acid reference solution: 2 mg of p-hydroxybenzoic acid reference was weighed into a 50 ml volumetric flask, dissolved in methanol and filtered through a 0.45 μm filter membrane. The filtrate was collected.

[0101] 7.2.3 Preparation of the protocatechuic acid reference solution: 2 mg of protocatechuic acid reference was weighed into a 50 ml volumetric flask, dissolved in methanol and filtered through a 0.45 μm filter membrane. The filtrate was collected.

[0102] ​​​7.2.4 Preparation of cryptochlorogenic acid control solution: Weigh 2 mg of cryptochlorogenic acid control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0103] 7.2.5 Preparation of p-coumaric acid control solution: Weigh 2 mg of p-coumaric acid control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0104] 7.2.6 Preparation of syringaldehyde control solution: Weigh 2 mg of syringaldehyde control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0105] 7.2.7 Preparation of 2,6-dimethoxyphenol control solution: Weigh 2 mg of 2,6-dimethoxyphenol control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0106] 7.2.8 Preparation of tyrosine control solution: Weigh 2 mg of tyrosine control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0107] 7.2.9 Preparation of salicylic acid control solution: Weigh 2 mg of salicylic acid control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0108] 7.2.10 Preparation of 5-hydroxymethylfurfural control solution: Weigh 2 mg of 5-hydroxymethylfurfural control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0109] 7.2.11 Preparation of furfural control solution: Weigh 2 mg of furfural control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0110] 7.2.12 Preparation of vanillin control solution: Weigh 2 mg of vanillin control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0111] 7.2.13 Preparation of sodium benzoate control solution: Weigh 2 mg of sodium benzoate control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0112] 7.2.14 Preparation of chlorogenic acid control solution: Weigh 2 mg of chlorogenic acid control, add 50 ml of methanol to dissolve, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0113] 7.3 Chromatographic conditions refer to 6.2.

[0114] 7.4 Determination method: 10 μl of the test sample solution and the single component control sample solution described in 7.2 were precisely pipetted and injected into the high performance liquid chromatograph for detection.

[0115] 7.5 Results: From the results of the determination of the test sample solution and the single component control sample solution, it can be seen that the chemical components of each medicinal ingredient in Qutanling oral liquid were searched and preliminarily identified, and combined with the response value, separation degree, peak type, peak purity and stability of each main chromatographic peak, p-hydroxybenzoic acid (peak No. 6) was finally selected as the reference substance (S) of the standard fingerprint chromatogram of Qutanling oral liquid. Figure 10

[0116] Example 2

[0117] A method for constructing a fingerprint chromatogram of Qutanling oral liquid, comprising the following steps:

[0118] 1) Preparation of the test sample solution: Qutanling oral liquid was filtered through a 0.45 μm filter membrane, and the filtrate was obtained;

[0119] 2) Preparation of the guaiacol control sample solution: 2 mg of guaiacol control sample was weighed, dissolved in 50 ml of methanol, filtered through a 0.45 μm filter membrane, and the filtrate was obtained;

[0120] 3) Preparation of the p-hydroxybenzoic acid control sample solution: 2 mg of p-hydroxybenzoic acid control sample was weighed, dissolved in 50 ml of methanol, filtered through a 0.45 μm filter membrane, and the filtrate was obtained;

[0121] 4) Preparation of the protocatechuic acid control sample solution: 2 mg of protocatechuic acid control sample was weighed, dissolved in 50 ml of methanol, filtered through a 0.45 μm filter membrane, and the filtrate was obtained;

[0122] 5) Establishment of the fingerprint chromatogram: 10 μL of the control sample solution and the test sample solution were respectively injected into the high performance liquid chromatograph, and detected according to the high performance liquid chromatography conditions. The chromatogram and data of the test sample solution and the control sample solution within 48 min were respectively recorded, and the obtained chromatogram and data were introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system for analysis, so as to establish the standard fingerprint chromatogram of Qutanling oral liquid.

[0123] The detection conditions of the high performance liquid chromatograph were as follows: octadecylsilane-bonded silica gel was used as the filler (chromatographic column: Waters C18, 250 mm x 4.6 mm, 5 μm), acetonitrile was used as the mobile phase A, and 0.3% phosphoric acid solution was used as the mobile phase B, gradient elution was carried out according to the gradient elution program in Table 7, the detection wavelength was 254 nm, and the column temperature was 25 °C.

[0124] Table 7

[0125] ​​

[0126]

[0127] Example 3 Fingerprint method validation

[0128] 1. Blank test

[0129] 1.1 Chromatographic condition: same as that of experimental example 2.

[0130] 1.2 Measurement: take pure water as sample directly, record chromatogram for 48 minutes.

[0131] 1.3 Result: from the chromatogram, it can be seen that there is no residue and interference in the system. Figure 11

[0132] 2. Precision test

[0133] 2.1 Chromatographic condition: same as that of experimental example 2.

[0134] 2.2 Test sample solution: same as example 2.

[0135] 2.3 Measurement: continuously sample for 6 times, take peak No. 6 as reference peak, calculate relative retention time and relative peak area of each main chromatographic peak.

[0136] 2.4 Result: RSD of relative retention time of 9 common peaks is less than 3%, RSD of relative peak area is less than 5%, which indicates good precision of the instrument (see tables 8-9).

[0137] Table 8 Relative retention time of precision test of instrument

[0138]

[0139]

[0140] Table 9 Relative peak area of precision test of instrument

[0141]

[0142] 3. Reproducibility test

[0143] 3.1 Chromatographic condition: same as that of experimental example 2.

[0144] 3.2 Test sample solution: same as example 2, take 6 test sample solutions of the same batch.

[0145] 3.3 Measurement: use high performance liquid chromatograph for detection, take peak No. 6 as reference peak, calculate relative retention time and relative peak area of each main chromatographic peak.

[0146] ​3.4 Results: The RSDs of relative retention time of 9 common peaks were all less than 3%, and the RSDs of relative peak area were all less than 5%, which indicated that the method was reproducible well (see Tables 10-11).

[0147] Table 10 Relative retention time of reproducibility test

[0148]

[0149]

[0150] Table 11 Relative peak area of reproducibility test

[0151]

[0152] 4. Stability test

[0153] 4.1 Chromatographic condition: same as that of Example 2.

[0154] 4.2 Preparation of test solution: the same batch was used, and the test solution was prepared according to the method of Example 2.

[0155] 4.3 Determination: the high performance liquid chromatograph was used to determine the relative retention time and relative peak area of each main chromatographic peak at 0, 2, 4, 8, 12, 18, 24, 36 and 48 h after preparation of the test solution, respectively, with No. 6 peak as the reference peak.

[0156] 4.4 Results: the RSDs of relative retention time of 9 common peaks were all less than 3% within 36 h, and the RSDs of relative peak area were all less than 5%, which indicated that the solution was stable well within 36 h at room temperature (see Tables 12-13).

[0157] Table 12 Relative retention time of stability test

[0158]

[0159] Table 13 Relative peak area of stability test

[0160]

[0161] 5. Establishment of common peaks

[0162] The samples of 15 batches of Qutanling oral liquid produced by Shanghai Jingan Pharmaceutical Co., Ltd. were detected by the above established fingerprint determination method, and the sample batch numbers were 220128-2, 220308-1, 221123-2, 220121-1, 220207-1, 220704-1, 220706-2, 220228-1, 221218-1, 220707-2, 221122-1, 221226-1, 230110-2, 221220-2, and 211112-1.

[0163] 9 common chromatographic peaks were established Figure 12 , and the control fingerprint Figure 13 was generated by fitting using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 version) of the State Pharmacopoeia Commission.

[0164] 6. Chromatographic peak attribution and identification

[0165] 6.1 Chromatographic peak attribution

[0166] 6.1.1 Drug attribution

[0167] The construction method of Reference Example 2 was used for fingerprint detection to explore the specific drug attribution of the 9 common peaks in the fingerprint. The corresponding single drug extract was prepared according to the prescription process, and an appropriate amount of the single drug extract to be tested was taken. The detection method of Reference Example 2 was used for fingerprint detection.

[0168] The results are shown in Figure 14 . After comparison, all common peaks in the sample can be attributed to the corresponding drug, and the distilled liquid of Houttuynia cordata and fresh bamboo juice have their corresponding exclusive chromatographic peaks, indicating that the fingerprint can display the information of all drugs more completely.

[0169] 6.1.2 Auxiliary material attribution

[0170] The auxiliary materials were weighed according to the prescription, and the detection method of Reference Example 2 was used for fingerprint detection, and the results are shown in Figure 15 . After comparison, there is no corresponding chromatographic peak in the common peaks of the fingerprint at the same position as the auxiliary material chromatographic peak, i.e. there is no auxiliary material chromatographic peak in the sample fingerprint.

[0171] 6.2 Chromatographic peak identification

[0172] According to the attribution results, the chromatographic peaks were simply identified. The main drugs were subjected to chemical component retrieval, combined with the ultraviolet absorption curve of each chromatographic peak, and compared with the reference substance. Finally, 3 compounds were identified, and the results are shown in Figure 16 and Table 14.

[0173] Table 14 Summary of the identification of chromatographic peaks in the fingerprint of Qutianling

[0174]

[0175]

Claims

1. A method for constructing a fingerprint of Qutanling oral liquid, characterized in that: The steps include: 1) Preparation of test solution: Filter multiple batches of Qutanling oral solution through a 0.45 μm filter membrane and collect the filtrate; 2) Preparation of a single-component reference solution: Weigh 2 mg of the reference solution and dissolve it in 50 ml of methanol. Filter through a 0.45 μm filter membrane and collect the filtrate to obtain the single-component reference solution. The reference components are protocatechuic acid, p-hydroxybenzoic acid, and guaiacol. 3) Establishing a fingerprint: 10 μL of each of a single indicator component reference solution and a test solution were injected into a high-performance liquid chromatograph and tested according to high-performance liquid chromatography conditions. The spectra and data of the test solution and the reference solution were recorded within 48 minutes. The resulting spectra and data were imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for analysis to establish a fingerprint of Qutanling oral liquid. The fingerprint contained nine common peaks, numbered from left to right: peak 4 for protocatechuic acid, peak 6 for p-hydroxybenzoic acid, and peak 9 for guaiacol. The detection conditions of the high performance liquid chromatography are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, 0.1%-0.3% phosphoric acid solution is used as mobile phase B, gradient elution is performed, the detection wavelength is 254 nm, and the column temperature is 25-35°C; The gradient elution is: 。 2. The method for constructing a fingerprint of Qutanling oral liquid according to claim 1, characterized in that: The detection conditions of the high performance liquid chromatography are as follows: the chromatographic column uses octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, 0.3% phosphoric acid solution as the mobile phase B, gradient elution is performed, and the column temperature is 25°C.

3. A method for constructing a fingerprint of Qutanling oral liquid as described in claim 1 or 2, characterized in that: The specifications of the chromatographic column are 250 mm×4.6 mm, and the particle size is 5 μm.

4. Application of the method for constructing a fingerprint of Qutanling oral liquid as described in any one of claims 1 to 3 in the quality detection of Qutanling oral liquid.

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