A fingerprint detection method for children's hot cough oral solution

Abstract

The present invention provides a method for detecting the fingerprint of oral liquid for children's hot cough. On the basis of the existing standards, the similarity evaluation of drugs is added to make the quality control indicators of drugs more comprehensive; the fingerprint detection method of the present invention has good precision, stability and reproducibility. The detection method of the present invention includes the preparation of reference solution; the preparation of test solution; chromatographic conditions: using octadecyl bonded silica gel chromatographic column mobile phase A as water, mobile phase B as acetonitrile, and gradient elution; chromatographic peak determination: accurately aspirate the above solution, inject it into the chromatograph, and use the traditional Chinese medicine chromatographic fingerprint similarity evaluation system to perform data analysis.

Description

Technical Field

[0001] The invention relates to a fingerprint spectrum detection method for oral liquid for children's hot cough, and belongs to the field of inspection and analysis of Chinese patent medicines. Background Art

[0002] Children's hot cough oral liquid is made from ten kinds of herbs extracted from honey ephedra, gypsum, bitter almond, forsythia, rhubarb, trichosanthes, mulberry bark, patrinia, safflower, and licorice. It has the effects of clearing heat and promoting lung function, resolving phlegm and relieving cough. It is used for cough caused by phlegm-heat blocking the lung, yellow phlegm or phlegm sound in the middle of the cough, fever, sore throat, thirst, dry stool, and acute bronchitis in children with the above symptoms.

[0003] The current legal standard YBZ00452012 only has the commonly used identification and content. In order to further explore the indicator ingredients of Children's Hot Cough Oral Liquid, the fingerprint spectrum method is now used to control its similarity, making the quality control of the drug more comprehensive. Summary of the invention

[0004] The purpose of the present invention is to provide a fingerprint detection method for children's hot cough oral liquid, which adopts high performance liquid chromatography to effectively separate samples, fits more than 10 batches of samples to control fingerprints, and uses the Chinese medicine evaluation system (2012 version) for evaluation, with a similarity requirement greater than 0.90; further improves the control standard of children's hot cough oral liquid to ensure the quality stability and uniformity of the children's hot cough oral liquid.

[0005] The detection method of the present invention comprises the following steps:

[0006] (a) Preparation of reference solution: Take an appropriate amount of forsythiaside reference substance, add methanol to make a solution, and filter to obtain the solution.

[0007] (b) Preparation of test solution: Accurately pipette the sample into a volumetric flask, dilute to the mark with methanol, shake well, and filter.

[0008] (c) Chromatographic conditions: an octadecyl bonded silica gel column with mobile phase A being water and mobile phase B being acetonitrile, with gradient elution at a flow rate of 0.8 to 1.2 ml / min; column temperature being 15 to 35°C; detection wavelength being 197 nm; and the plate number being not less than 5000 based on forsythiaside.

[0009] (d) Chromatographic peak determination: Accurately pipette 10 μl of each of the above solutions and inject into the chromatograph. Use the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) to perform data analysis. The similarity should not be less than 0.90.

[0010] As a preferred embodiment of the present invention, in the preparation of the reference solution in step (a), the concentration of forsythin is 0.04-1 mg / mL.

[0011] As a preferred embodiment of the present invention, in the preparation of the test solution in step (b), take the test solution under the filling volume difference item, accurately pipette 1 ml into a 10 ml volumetric flask, add 50% methanol to dilute to the scale, shake well, and filter to obtain.

[0012] As a preferred embodiment of the present invention, in the chromatographic conditions of step (c), the mobile phase A of the octadecyl bonded silica gel chromatographic column is water, the mobile phase B is acetonitrile, and linear gradient elution is performed at a flow rate of 1.0 ml / min; the column temperature is 30° C.; the detection wavelength is 197 nm; the plate number is not less than 5000 based on forsythiaside, and the linear gradient elution conditions used for the mobile phase are:

[0013] 0-5min, the volume ratio of mobile phase A is 100%, and the volume ratio of mobile phase B is 0%;

[0014] 5-10min, the volume ratio of mobile phase A is: 100%→95%, the volume ratio of mobile phase B is 0%→5%;

[0015] 10-30min, the volume ratio of mobile phase A is: 95%→90%, the volume ratio of mobile phase B is 5%→10%;

[0016] 30-32min, the volume ratio of mobile phase A is: 90%→85%, the volume ratio of mobile phase B is 10%→15%;

[0017] 32-75min, the volume ratio of mobile phase A is 85%, and the volume ratio of mobile phase B is 15%;

[0018] 75-80min, the volume ratio of mobile phase A is: 85%→70%, the volume ratio of mobile phase B is 15%→30%;

[0019] 80-85min, the volume ratio of mobile phase A is: 70%→60%, the volume ratio of mobile phase B is 30%→40%;

[0020] 85-90min, the volume ratio of mobile phase A is: 60%→40%, the volume ratio of mobile phase B is 40%→60%;

[0021] 90-95min, the volume ratio of mobile phase A is: 40%→100%, the volume ratio of mobile phase B is 60%→0%;

[0022] 95-100min, the volume ratio of mobile phase A is 100%, and the volume ratio of mobile phase B is 0%.

[0023] Most preferably, the detection method of the present invention comprises the following steps:

[0024] The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler, water as mobile phase A, acetonitrile as mobile phase B, gradient elution, flow rate 1.0 ml / min; column temperature 25°C; detection wavelength 197 nm; theoretical plate number calculated based on forsythiaside shall not be less than 5000.

[0025]

[0026] Preparation of reference solution: Take an appropriate amount of forsythiaside reference substance, add methanol to make a 0.2 mg / ml solution, and filter to obtain the solution.

[0027] Preparation of test solution: Take the test solution under the item of volume difference, accurately pipette 1 ml into a 10 ml volumetric flask, add 50% methanol to dilute to the scale, shake well, filter, and obtain.

[0028] Determination method: Accurately pipette 10 μl of each of the above solutions, inject into the chromatograph, and use the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) to perform data analysis. The similarity should not be lower than 0.90.

[0029] The detection method of the present invention has the following beneficial effects:

[0030] 1. The present invention can effectively evaluate the stability and uniformity of drug batches;

[0031] 2. The high performance liquid phase method established by the present invention has high precision (RSD is 0.5-4.5%), stability (RSD is 0.2-3.2%), and repeatability (RSD is 1.1-4.4%). The experimental results show that the test method has the advantages of accurate measurement, simple operation, and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 - Scanning for UV wavelengths

[0033] Figure 2 - is the liquid phase chromatogram of the forsythiaside reference substance solution;

[0034] Figure 3 - is the liquid chromatogram of the test product;

[0035] Figure 4 - is a superimposed liquid chromatogram;

[0036] Figure 5 -For reference fingerprints; DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0038] In the present invention, the test sample processing method, reference sample concentration, mobile phase ratio, precision, repeatability, stability, confirmation of reference materials, and generation of reference fingerprints were investigated. Pediatric hot cough oral solution was provided by Jiangxi Jimin Kexin Pharmaceutical Co., Ltd., with methodological validation batch numbers: 190902, 190903, 190904, 190905, 190906, 190907.

[0039] Embodiment 1

[0040] 1. Wavelength selection: Accurately pipette 1 ml into a 50 ml volumetric flask, dilute to scale with water, shake well, take the solution into the UV detector, scan at 190-400 nm, and the result is that the maximum absorption value is at λ = 197 nm. Therefore, 197 nm is selected as the detection wavelength (see attached Figure 1 ).

[0041] 2. Selection of mobile phase: Since the detection wavelength is 197 nm, acetonitrile-water is selected as the mobile phase to avoid the terminal absorption of methanol.

[0042] 3. Selection of mobile phase ratio: After examining 6 gradients, the above linear gradient elution was finally selected.

[0043] 4. Solvent selection: Water, 50% methanol, and methanol were used as the investigation conditions. The results showed that the chromatographic peaks extracted with 50% methanol as the solvent were richer. Therefore, 50% methanol was selected as the solvent.

[0044] 5. Methodological validation:

[0045] 5.1、Instrument precision test Take Xiaoer Reke oral solution (batch number: 190902) and conduct the test according to the main text. Inject the sample 6 times continuously and record the retention time and peak area of ​​each common peak. The results show that the retention time RSD (n=6) of each common peak is less than 0.4%, and the common peak area RSD (n=6) is less than 4.5%, indicating that the instrument precision of this method is good and meets the technical requirements of fingerprint spectrum. See Table 1 and 2.

[0046] Table 1 Precision test - retention time

[0047]

[0048] Table 2 Precision test - peak area

[0049]

[0050]

[0051] 5.2 Repeatability test Take Xiaoer Reke oral solution (batch number: 190902) and conduct the test according to the main text. Take 6 samples respectively and record the retention time and peak area of ​​each common peak. The results show that the RSD of the retention time of each common peak (n=6) is less than 0.5%, and the RSD of the common peak area (n=6) is less than 4.4%, indicating that the repeatability of this method is good and meets the technical requirements of fingerprint spectrum. See Tables 3 and 4.

[0052] Table 3 Repeatability test - retention time

[0053]

[0054] Table 4 Repeatability test-peak area

[0055]

[0056] 5.3 Stability Test Take the solution of the precision test sample and inject it into the chromatograph at 0, 4, 8, 12 and 24 hours, record the retention time and peak area of ​​each common peak, and the results show that the RSD of the retention time of each common peak (n=5) is less than 0.5%, and the RSD of the common peak area (n=5) is less than 3.5%, indicating that the stability of the test solution is good within 24 hours, which meets the technical requirements of the fingerprint spectrum. See Tables 5 and 6.

[0057] Table 5 Stability-Retention Time

[0058]

[0059] Table 6 Stability-Peak Area

[0060]

[0061] 5.4. Establishment of reference map:

[0062] Thirteen batches of samples of pediatric hot cough oral solution produced by Jiangxi Jimin Kexin Pharmaceutical Co., Ltd. in 2018-2019 were tested according to the text. As a result, 5 batches showed small or missing common peaks. Therefore, 8 batches were selected as the superposition of the control spectrum (see Appendix Figure 4 ), the reference spectrum generation parameters are time window 1min, median method. Generate the reference fingerprint spectrum (see attached Figure 5 ).

[0063] 5.5. Confirmation of reference objects:

[0064] The index components of the 10 medicinal materials in the prescription were identified and it was found that the retention time of the forsythin reference substance was consistent with that of peak 9, and the UV spectrum was also consistent. Therefore, peak 9 was confirmed to be forsythin. Figure 2 , 3 .

[0065] 5.6. Sample determination:

[0066] Three batches of samples were tested, and the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) was used to pour the sample and reference spectra into the data analysis, and the similarity results were all greater than 0.90. See Table 7.

[0067] Table 7 Sample similarity determination

[0068]

[0069]

[0070] Example 2: Screening of chromatographic conditions - selection of mobile phase,

[0071] The maximum absorption wavelength of the test solution is 197nm, and the chromatographic peaks at 197nm are relatively abundant. Considering that methanol has terminal absorption below 200nm, water (A)-acetonitrile (B) was selected as the mobile phase; after the following 6 gradients were investigated, the peak separation of gradient 6 was >1.5. Finally, the above linear gradient elution was selected.

[0072] Gradient 1:

[0073]

[0074] Gradient 2

[0075]

[0076] Gradient 3

[0077]

[0078]

[0079] Gradient 4

[0080]

[0081] Gradient 5

[0082]

[0083] Gradient 6

[0084]

[0085]

[0086] Embodiment 3, the detection method of the present invention

[0087] (a) Preparation of reference solution: Take an appropriate amount of forsythiaside reference substance, add methanol to make a solution, and filter to obtain a solution with a concentration of 0.1 mg / mL forsythiaside.

[0088] (b) Preparation of test solution: Accurately pipette the sample into a 5 ml volumetric flask, dilute to the mark with 50% methanol, shake well, and filter.

[0089] (c) Chromatographic conditions: using an octadecyl bonded silica gel column with water as mobile phase A and acetonitrile as mobile phase B, gradient elution, flow rate 0.8 ml / min; column temperature 25°C; detection wavelength 197 nm; the plate number calculated based on forsythiaside should not be less than 5000;

[0090] (d) Chromatographic peak determination: Accurately pipette 10 μl of each of the above solutions and inject into the chromatograph. Use the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) to perform data analysis. The similarity should not be less than 0.90.

[0091] The linear gradient elution conditions used in the mobile phase are:

[0092] 0-5min, the volume ratio of mobile phase A is 100%, and the volume ratio of mobile phase B is 0%;

[0093] 5-10min, the volume ratio of mobile phase A is: 100%→95%, the volume ratio of mobile phase B is 0%→5%;

[0094] 10-30min, the volume ratio of mobile phase A is: 95%→90%, the volume ratio of mobile phase B is 5%→10%;

[0095] 30-32min, the volume ratio of mobile phase A is: 90%→85%, the volume ratio of mobile phase B is 10%→15%;

[0096] 32-75min, the volume ratio of mobile phase A is 85%, and the volume ratio of mobile phase B is 15%;

[0097] 75-80min, the volume ratio of mobile phase A is: 85%→70%, the volume ratio of mobile phase B is 15%→30%;

[0098] 80-85min, the volume ratio of mobile phase A is: 70%→60%, the volume ratio of mobile phase B is 30%→40%;

[0099] 85-90min, the volume ratio of mobile phase A is: 60%→40%, the volume ratio of mobile phase B is 40%→60%;

[0100] 90-95min, the volume ratio of mobile phase A is: 40%→100%, the volume ratio of mobile phase B is 60%→0%;

[0101] 95-100min, the volume ratio of mobile phase A is 100%, and the volume ratio of mobile phase B is 0%.

[0102] Example 4: Formula and preparation method of oral liquid for children with hot cough

[0103] Prescription: Ephedra (honey-fried) 60g, Gypsum 200g, Bitter Apricot 120g, Forsythia suspensa 200g, Rhubarb 40g, Trichosanthes 120g, Morus alba bark 120g, Patrinia 200g, Safflower 120g, Licorice (honey-fried) 60g

[0104] Preparation method: add ten times the amount of water to the above ten ingredients, decoct three times, 1 hour each time, filter, combine the filtrate, concentrate under reduced pressure to a relative density of 1.10-1.20 (60°C), add ethanol to make the alcohol content reach 65%, stir well, let stand for 24 hours, take the supernatant, and recover the ethanol until there is no alcohol taste. Add 400ml of water, refrigerate, filter, boil, add 150g of sucrose, stir well, add water to adjust the total amount to 1000ml, stir well, let stand, filter, pot, sterilize, and you are done.

Claims

1. A method for detecting fingerprints of oral liquid for treating pediatric cough. It is characterized in that The detection method comprises the following steps: (a) Preparation of reference solution: Take an appropriate amount of forsythiaside reference substance, add methanol to make a solution, and filter to obtain a solution with a concentration of 0.1 mg / mL forsythiaside. (b) Preparation of test solution: Accurately pipette the sample into a 5 ml volumetric flask, dilute to the mark with 50% methanol, shake well, and filter. (c) Chromatographic conditions: using an octadecyl bonded silica gel column, mobile phase A is water, mobile phase B is acetonitrile, gradient elution, flow rate 0.8 ml / min; column temperature 25°C; detection wavelength 197 nm; plate number calculated based on forsythiaside is not less than 5000; (d) Chromatographic peak determination: Accurately pipette 10 μl of each of the above solutions, inject into the chromatograph, and use the Chinese medicine chromatographic fingerprint similarity evaluation system to perform data analysis, and the similarity is not less than 0.90; The linear gradient elution conditions used in the mobile phase are: 0-5min, the volume ratio of mobile phase A is 100%, and the volume ratio of mobile phase B is 0%; 5-10min, the volume ratio of mobile phase A is: 100%→95%, the volume ratio of mobile phase B is 0%→5%; 10-30min, the volume ratio of mobile phase A is: 95%→90%, the volume ratio of mobile phase B is 5%→10%; 30-32min, the volume ratio of mobile phase A is: 90%→85%, the volume ratio of mobile phase B is 10%→15%; 32-75min, the volume ratio of mobile phase A is 85%, and the volume ratio of mobile phase B is 15%; 75-80min, the volume ratio of mobile phase A is: 85%→70%, the volume ratio of mobile phase B is 15%→30%; 80-85min, the volume ratio of mobile phase A is: 70%→60%, the volume ratio of mobile phase B is 30%→40%; 85-90min, the volume ratio of mobile phase A is: 60%→40%, the volume ratio of mobile phase B is 40%→60%; 90-95min, the volume ratio of mobile phase A is: 40%→100%, the volume ratio of mobile phase B is 60%→0%; 95-100min, the volume ratio of mobile phase A is 100%, and the volume ratio of mobile phase B is 0%.

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