A method for constructing a fingerprint spectrum of Xiao'er Zhenbei Powder and application thereof

Abstract

The present application relates to a kind of pediatric jianbei powder fingerprint spectrum construction method and its application, belong to the technical field of drug detection.The present application uses agarwood tetrol, taurine cholate, cholic acid and hyodeoxycholic acid as control, using methanol to dissolve test sample and control, with acetonitrile-carbonic acid aqueous solution as the mobile phase of high performance liquid chromatography, the chromatographic peak measured is good.The construction method of the present application has good precision, good stability, good repeatability and high accuracy;In the fingerprint spectrum obtained using the construction method, the number of chromatographic peaks is more, sensitive, baseline smooth, noise small, response acute, response value large, and good separation degree.The construction method of the present application and the obtained fingerprint spectrum and standard fingerprint spectrum can be used for quality analysis of pediatric jianbei powder before and after irradiation sterilization, and other quality detection of pediatric jianbei powder, to provide a more effective means for ensuring the safety, effectiveness and stability of pediatric jianbei powder product.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug detection, and particularly relates to a construction method of a fingerprint spectrum of Xiaoerzhenbeisan and application thereof. BACKGROUND

[0002] Xiaoerzhenbeisan has the effects of clearing heat, eliminating inflammation, relieving cough and reducing sputum, and is used for treating infantile tracheitis, bronchitis and asthmatic bronchitis. The prescription of Xiaoerzhenbeisan is as follows: ox gall 50 g, pearl 50 g, Chuanbeimu 300 g, Tianzhu Huang 200 g, Chenxiang 100 g, Dannaixing 50 g, calcined borax 50 g and Bingpian 10 g. The preparation method is as follows: the above eight kinds of ingredients are water-flying or finely pulverized into the finest powder, and are co-mixed with Chuanbeimu and the like to form a fine powder, and then are sieved and uniformly mixed to obtain Xiaoerzhenbeisan.

[0003] Xiaoerzhenbeisan is a traditional Chinese medicine preparation containing non-sterile raw medicinal powder, and the raw materials and production and storage process of Xiaoerzhenbeisan are extremely easy to be contaminated by microorganisms, so that sterilization treatment is needed in the process of drug preparation. Xiaoerzhenbeisan is sterilized by cobalt 60 irradiation. At present, there are some literatures reporting the influence of cobalt 60 irradiation sterilization on chemical components and content in traditional Chinese medicines, but whether cobalt 60 irradiation sterilization has an influence on the chemical components of Xiaoerzhenbeisan has not been reported.

[0004] The fingerprint spectrum of traditional Chinese medicines has the characteristics of whole, macroscopic and fuzzy analysis, and the fingerprint spectrum analysis can make the whole characteristics of various chemical components in traditional Chinese medicines visualized, so that the quality problems that cannot be found by conventional inspection can be exposed. The fingerprint spectrum is an important modern quality control method of traditional Chinese medicines from the perspective of “whole components”, and is an important comprehensive analysis method of traditional Chinese medicines with multiple components and multiple targets. At present, there is a lack of systematic qualitative and quantitative research on Xiaoerzhenbeisan.

[0005] Xiaoerzhenbeisan is a traditional Chinese medicine compound preparation, which contains complex chemical components. The prior art "Quality Standard Improvement Research of Xiaoerzhenbeisan" (Wang A, Cheng G, Pu J, et al. Quality Standard Improvement Research of Xiaoerzhenbeisan. Anhui Medicine, 2022, 26(6): 1094-1098.) Although the detection method of microscopic identification, thin layer chromatography and bilirubin content determination of Xiaoerzhenbeisan is newly established, but this method cannot fully reflect the influence of irradiation on the components contained in Xiaoerzhenbeisan; The prior art "Thin Layer Identification and Borneol Content Determination Method of Xiaoerzhenbeisan" (Chen Z, Huang Y. Thin Layer Identification and Borneol Content Determination Method of Xiaoerzhenbeisan. China Modern Drug Application, 2011, 5(6): 1-3.) Although the detection method of thin layer chromatography and borneol content determination of Xiaoerzhenbeisan is newly established, but the method used cannot fully reflect the influence of irradiation on the components contained in Xiaoerzhenbeisan. The above prior art cannot comprehensively and effectively judge whether the Co60 irradiation sterilization has an influence on the chemical components of Xiaoerzhenbeisan; It also cannot reflect the internal overall quality of Xiaoerzhenbeisan, and has certain limitations for ensuring the product quality consistency. Secondly, the quantitative detection of single component cannot comprehensively evaluate the chemical component change of Xiaoerzhenbeisan before and after irradiation sterilization, and it is difficult to fully and effectively reflect the influence of irradiation sterilization on the quality.

[0006] Therefore, it is necessary to establish the fingerprint of Xiaoerzhenbeisan, to qualitatively analyze the chemical components in Xiaoerzhenbeisan, and to evaluate the influence of Co60 irradiation on the chemical component change and quality of Xiaoerzhenbeisan by comparing the similarity of the fingerprint before and after irradiation. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of the prior art and provide a construction method of Xiaoerzhenbeisan fingerprint and its application. The construction method of the present application has good precision, repeatability and stability, and high accuracy. The construction method of the present application can be used for systematic qualitative research on the chemical components of Xiaoerzhenbeisan, and the influence of irradiation sterilization on the quality of Xiaoerzhenbeisan can be evaluated by comparing the similarity of the fingerprint before and after irradiation sterilization, to ensure the safety, effectiveness and stability of the product.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is:

[0009] In the first aspect, the present application provides a construction method of Xiaoerzhenbeisan fingerprint, comprising the following steps:

[0010] S1, preparation of test solution

[0011] Dissolve Xiaoerzhenbeisan sample in methanol and ultrasonic extraction, filter, and the filtrate is the test solution;

[0012] S2, preparation of control solution

[0013] The control substance agarotetrol, sodium taurocholate, cholic acid and hyodeoxycholic acid are mixed and dissolved in methanol to obtain a control substance solution;

[0014] S3, determining by high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD)

[0015] The test substance solution and the control substance solution are injected into a liquid chromatograph, and determined by combining an evaporative light scattering detector to obtain a test substance chromatogram and a control substance chromatogram respectively, the test substance chromatogram is identified and calibrated to obtain a fingerprint spectrum of the Xiaoerzhenbeisan; the chromatographic conditions of the liquid chromatograph are as follows: a C18 chromatographic column is used; acetonitrile is used as a mobile phase A and a formic acid aqueous solution is used as a mobile phase B; an elution program is as follows: 0-10 min, the volume percentage of the mobile phase A is gradually changed from 80% to 60%; 10-33 min, the volume percentage of the mobile phase A is gradually changed from 60% to 50%; 33-40 min, the volume percentage of the mobile phase A is gradually changed from 50% to 40%.

[0016] In the present application, agarotetrol, sodium taurocholate, cholic acid and hyodeoxycholic acid are used as control substances, methanol is used to dissolve the test substance and the control substance, and acetonitrile-formic acid aqueous solution is used as the mobile phase of high performance liquid chromatography, the determined chromatographic peaks are good, all the control substance chromatographic peaks can be separated, and the separation effect is good. In the chromatogram obtained by the construction method of the present application, the number of peaks is large, the sensitivity is high, the baseline is stable, the noise is small, the response is sensitive, the response value is large, and the separation degree is good. In addition, the test of the test substance solution by the blank solvent does not exist interference, and the specificity is good. The construction method of the present application has good precision, good stability, good repeatability and high accuracy.

[0017] The test substance chromatogram is compared with the control substance chromatogram, the characteristic peaks in the test substance chromatogram are identified according to the known characteristic peaks in the control substance solution chromatogram through the relative retention time. The common peaks of the chromatograms of different batches of test substances are calibrated by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The identification of the common peaks can be completed by comparing with the chromatogram of the control substance solution.

[0018] The fingerprint spectrum of the Xiaoerzhenbeisan obtained by the construction method of the present application includes 13 common peaks, wherein, taking the No. 7 peak as a reference, the relative retention times of the peaks are as follows:

[0019] Peak number Relative retention time (min) 1 6.157 2 6.841 3 12.108 4 14.73 5 16.885 6 18.631 7 20.68 8 22.069 9 23.724 10 28.279 11 29.723 12 35.063 13 36.928

[0020] After comparison and identification, the above No. 1 peak is agarotetrol, the No. 3 peak is sodium taurocholate, the No. 7 peak is cholic acid, and the No. 9 peak is hyodeoxycholic acid.

[0021] As a preferred embodiment of the construction method of the present application, in the step S1, the mass-volume ratio of the Xiaoerzhenbeisan to methanol is 1g:(10-30)mL.

[0022] In a preferred embodiment of the construction method of the present invention, in step S1, the mass-volume ratio of the pediatric granules to methanol is 1g:25mL.

[0023] In a preferred embodiment of the construction method described in this invention, in step S1, the methanol is analytical grade methanol.

[0024] In a preferred embodiment of the construction method of the present invention, in step S1, the ultrasound time is 10 to 40 minutes to achieve full extraction of the active ingredients.

[0025] In a preferred embodiment of the construction method of the present invention, in step S1, the ultrasound duration is 30 minutes; the ultrasound power is 600W and the ultrasound frequency is 40KHz.

[0026] In a preferred embodiment of the construction method described in this invention, the number of test samples used in step S1 is not particularly limited, and a suitable number of test samples can be easily selected; the number of test samples can be 5 to 30, or 10 to 20, etc. In step S1, the particle size of the Xiaozhenbei powder sample is not particularly limited, but preferably it is a particle size that can pass through a No. 3 or No. 4 sieve specified in the Chinese Pharmacopoeia.

[0027] In a preferred embodiment of the construction method of the present invention, in step S2, the concentration of linalool in the reference solution is 20-20.5 μg / mL, the concentration of sodium taurocholate is 50-50.5 μg / mL, the concentration of cholic acid is 150-150.5 μg / mL, and the concentration of porcine deoxycholic acid is 150-150.5 μg / mL.

[0028] In a preferred embodiment of the construction method of the present invention, in step S2, the concentration of linalool in the reference solution is 20 μg / mL, the concentration of sodium taurocholate is 50 μg / mL, the concentration of cholic acid is 150 μg / mL, and the concentration of porcine deoxycholic acid is 150 μg / mL.

[0029] The concentration of each reference standard in the reference solution of the present invention is not particularly limited, and the preferred concentration of reference standard content ratio is substantially equivalent to the content ratio of the corresponding component in the test solution.

[0030] In a preferred embodiment of the construction method described in this invention, in step S3, the C18 chromatographic column is an Agilent ZORBAX SB-C18 with dimensions of 250×4.6mm and 5μm.

[0031] In a preferred embodiment of the construction method of the present invention, in step S3, the mobile phase B is an aqueous solution of formic acid with a volume percentage of 0.1% to 0.2%.

[0032] The construction method of this invention uses a 0.1%–0.2% (v:v) formic acid aqueous solution as mobile phase B. The resulting fingerprint chromatogram exhibits good peak separation, well-defined peak shapes, no tailing, and high response values, providing a chromatogram covering 13 common peaks with a wider common peak range. However, below this range, such as using a 0.05% (v:v) formic acid aqueous solution as mobile phase B, some common peaks become undetectable, and the common peak range narrows.

[0033] In a preferred embodiment of the construction method of the present invention, in step S3, the mobile phase B is a 0.2% (v / v) aqueous solution of formic acid.

[0034] Using a 0.2% (v:v) formic acid aqueous solution as mobile phase B, the fingerprint chromatographic peaks were well separated, with good peak shape and no tailing phenomenon. The response value was better than that using a 0.1% (v:v) formic acid aqueous solution as mobile phase B, and even better using a 0.2% (v:v) formic acid aqueous solution as mobile phase B.

[0035] In a preferred embodiment of the construction method of the present invention, in step S3, the column temperature of the chromatographic column is 25-35℃.

[0036] In a preferred embodiment of the construction method of the present invention, in step S3, the column temperature of the chromatographic column is 30°C.

[0037] In a preferred embodiment of the construction method described in this invention, in step S3, the column flow rate under the chromatographic conditions is 1 mL / min. A column flow rate of 1 mL / min results in better flow separation and shorter elution time, compared to 0.8 mL / min or 1.2 mL / min, demonstrating superior separation performance.

[0038] In a preferred embodiment of the construction method of the present invention, in step S3, the injection volume of the test solution or reference solution is 5–10 μL. In this embodiment of the present invention, the injection volume is 10 μL.

[0039] In a preferred embodiment of the construction method described in this invention, in step S3, the drift tube temperature of the evaporative light scattering detector is 95–105°C, and the carrier gas volumetric flow rate is 2.1–2.9 L / min.

[0040] In a preferred embodiment of the construction method described in this invention, in step S3, the drift tube temperature of the evaporative light scattering detector is 105°C, and the carrier gas volumetric flow rate is 2.6 L / min.

[0041] In the construction method described in this invention, when the drift tube temperature of the liquid chromatograph is 95–105°C and the carrier gas flow rate is 2.1–2.9 L / min, the measured fingerprint chromatograms can all contain 13 common peaks, which is applicable to the detection of fingerprint chromatograms of Xiaor Zhenbei San (a traditional Chinese medicine). When the drift tube temperature is 105°C and the carrier gas flow rate is 2.6 L / min, the detected fingerprint chromatogram peaks are even better.

[0042] Secondly, the present invention provides the application of the fingerprint spectrum constructed by the construction method in detecting the quality of Xiaor Zhenbei San product.

[0043] As a preferred embodiment of the application described in this invention, the fingerprint spectrum constructed by the construction method is used in detecting the quality of Xiaor Zhenbeisan products after irradiation sterilization.

[0044] The fingerprint spectrum of Xiaoer Zhenbeisan before and after irradiation sterilization was detected using the construction method described above. A standard fingerprint spectrum was generated from the fingerprint spectrum. The similarity was obtained by comparing the fingerprint spectrum of the sample to be tested with the standard fingerprint spectrum. The product quality of the Xiaoer Zhenbeisan sample to be tested could be judged based on the similarity.

[0045] Thirdly, this invention provides a method for quality testing of children's Zhenbei powder, comprising the following steps:

[0046] (1) Using the construction method described above, a fingerprint spectrum of the test sample is obtained, and a standard fingerprint spectrum is generated using one of the fingerprint spectra of the test sample as a reference spectrum.

[0047] (2) The fingerprint spectrum of the pediatric Zhenbei powder to be tested is obtained by the construction method described above;

[0048] (3) Compare the fingerprint spectrum of the pediatric Zhenbei powder to be tested obtained in step (2) with the standard fingerprint spectrum obtained in step (1) and calculate the similarity. If the similarity is greater than 0.9, it means that the pediatric Zhenbei powder to be tested is of qualified quality.

[0049] There are no particular limitations on the method for generating standard fingerprint patterns; conventional methods in this field can be used.

[0050] As a preferred embodiment of the quality detection method of the present invention, in step (1), the fingerprint spectrum of the test sample is imported into the similarity evaluation system of chromatographic fingerprint spectrum of traditional Chinese medicine, and a standard fingerprint spectrum is generated by using one of the fingerprint spectra of the test sample as a reference spectrum and the median method and multi-point correction method.

[0051] As a preferred embodiment of the quality testing method of the present invention, in step (3), the comparison method is as follows: the fingerprint spectrum of the pediatric Zhenbei powder to be tested obtained in step (2) is compared with the standard fingerprint spectrum obtained in step (1) by importing it into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system.

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0053] This invention provides a method for constructing a fingerprint spectrum for pediatric Zhenbei powder. This method exhibits good precision, stability, repeatability, and accuracy. The fingerprint spectrum obtained using this method shows a large number of chromatographic peaks, high sensitivity, stable baseline, low noise, sharp response, large response value, and good resolution. The construction method and the obtained fingerprint spectrum and standard fingerprint spectrum of this invention can be used for quality testing of pediatric Zhenbei powder, such as quality analysis before and after irradiation sterilization, providing a more effective means to ensure the safety, efficacy, and stability of the product. Attached Figure Description

[0054] Figure 1 This is the chromatogram of the reference solution in Example 1 of the present invention;

[0055] Figure 2 This is the chromatogram of the test solution in Example 1 of the present invention;

[0056] Figure 3 The fingerprint spectra of samples S1-S6 in Example 2 of the present invention and the generated control fingerprint spectra (R);

[0057] Figure 4 The fingerprint spectrum results of precision, repeatability and stability detection in Example 3 of the present invention;

[0058] Figure 5 The fingerprint patterns obtained using different mobile phases in Example 4 of this invention;

[0059] Figure 6 This is the fingerprint spectrum obtained using different carrier gas volume flow rates in Embodiment 5 of the present invention;

[0060] Figure 7 The fingerprint spectrum obtained using different drift tube temperatures in Embodiment 5 of the present invention;

[0061] Figure 8 This is the fingerprint spectrum obtained in Example 6 of the present invention using different mass-to-volume ratios of pediatric Zhenbei powder samples and methanol;

[0062] Figure 9 The fingerprint spectrum obtained using different column flow rates in Example 7 of this invention. Detailed Implementation

[0063] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0064] The instruments used in the following embodiments include:

[0065] Agilent 1260II high performance liquid chromatograph (quaternary pump, autosampler, column oven); Alltech 6100 evaporative light scattering detector (ELSD); KQ-600DE digital control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); METTLER TOLEDO - ME204 and METTLER TOLEDO - MX5 electronic analytical balances; Pure water machine (Milli-Q Biocel - D24UV).

[0066] The reagents and test drugs used in the following examples are as follows:

[0067] Xiao'er Zhenbei Powder was provided by Guangzhou Baiyunshan Jingxiutang Pharmaceutical Co., Ltd., with batch numbers U02001, U02002, and U02003 respectively; the reference substance of costunolide was provided by the National Institutes for Food and Drug Control, with batch number 111980 - 201904; the reference substance of sodium taurocholate was provided by the National Institutes for Food and Drug Control, with batch number 110815 - 201911; the reference substance of deoxycholic acid was provided by the National Institutes for Food and Drug Control, with batch number 110724 - 201808; the reference substance of hyodeoxycholic acid was provided by the National Institutes for Food and Drug Control, with batch number 100087 - 201411; acetonitrile was chromatographically pure, produced by LABSCIENCE; water was ultrapure water; other reagents were analytically pure.

[0068] Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0069] Method for constructing the fingerprint of Xiao'er Zhenbei Powder in Example 1

[0070] The method for constructing the fingerprint of Xiao'er Zhenbei Powder includes the following steps:

[0071] 1. Preparation of the test solution

[0072] Precisely weigh about 1.0 g of the powder of Xiao'er Zhenbei Powder (batch number U02001), place it in a stoppered conical flask, precisely add 25 mL of methanol, weigh it, ultrasonically treat it for 30 min (ultrasonic power 600 W, ultrasonic frequency 40 KHz), let it cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0073] 2. Preparation of the reference solution

[0074] Take appropriate amounts of the reference substances of costunolide, sodium taurocholate, cholic acid, and hyodeoxycholic acid, precisely weigh them, and make a mixed solution with methanol with the concentration of costunolide being 20 μg / mL, sodium taurocholate being 50 μg / mL, cholic acid being 150 μg / mL, and hyodeoxycholic acid being 150 μg / mL to obtain the reference solution.

[0075] 3. Chromatographic conditions and evaporative light scattering detector detection conditions: Chromatographic column: C18 column with octadecylsilane-bonded silica gel as the packing material. 18 Column (Agilent ZORBAX SB-C18, dimensions 250mm × 4.6mm, 5μm);

[0076] Mobile phase: Acetonitrile was used as mobile phase A, and 0.2 (v:v)% formic acid aqueous solution was used as mobile phase B;

[0077] Gradient elution, the specific procedure for gradient elution is as follows:

[0078] 0-10 min, the volume ratio of phase A: phase B is 80:20-60:40;

[0079] 10-33 min, the volume ratio of phase A: phase B is 60:40-50:50;

[0080] 33-40 min, the volume ratio of phase A: phase B is 50:50-40:60;

[0081] The column flow rate was 1 mL / min; the column temperature was 30℃.

[0082] Evaporative light scattering detector: drift tube temperature 105℃; carrier gas volumetric flow rate 2.6L / min.

[0083] 4. Measurement

[0084] Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine their chromatograms. The chromatogram of the reference solution is shown below. Figure 1 As shown; the chromatogram of the test solution is as follows. Figure 2 As shown in the figure. The results show that the chromatographic peaks are well separated, with good peak shapes and no tailing phenomenon, indicating that the chromatographic system constructed by the method in Example 1 has good adaptability.

[0085] The chromatogram of the test solution is compared with that of the reference solution. Based on the known characteristic peaks in the chromatogram of the reference solution, the corresponding characteristic peaks in the chromatogram of the test solution are identified by relative retention time. The index components in the chromatogram of the test solution are assigned and located, thereby converting the chromatogram of the test solution into a fingerprint chromatogram of Xiaor Zhenbei San for application.

[0086] Example 2: Application of fingerprint spectrum of pediatric Zhenbei powder

[0087] Using the construction method of the fingerprint spectrum of Xiao'er Zhenbei Powder in Example 1, a total of 6 samples of Xiao'er Zhenbei Powder before and after irradiation in 3 batches with batch numbers U02001, U02002, and U02003 were measured, and the fingerprint spectra of the corresponding test samples were obtained respectively; the 6 samples were numbered S1 to S6, where S1 to S3 were the samples of batches U02001, U02002, and U02003 before cobalt-60 irradiation in sequence, and S4 to S6 were the samples of batches U02001, U02002, and U02003 after cobalt-60 irradiation in sequence.

[0088] The fingerprint spectrum data of the obtained test samples were imported into the software of "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2004A version) issued by the Pharmacopoeia Commission of the People's Republic of China to calibrate the common peaks; the fingerprint spectrum data of sample S1 were set as the reference spectrum, the time width was 0.1 min, the median method was selected to generate the control spectrum, and the multi-point calibration method was used to establish the control fingerprint spectrum, that is, the standard fingerprint spectrum.

[0089] The fingerprint spectra of samples S1 - S6 and the generated control fingerprint spectrum (R) are as Figure 3 shown. The results show that a total of 13 chromatographic peaks were calibrated as the common peaks of the fingerprint spectrum of Xiao'er Zhenbei Powder. The relative retention times and relative peak areas of the 13 common fingerprint peaks are as follows:

[0090] Peak 1, the relative retention time is 0.298, and the relative peak area is 0.255;

[0091] Peak 2, the relative retention time is 0.331, and the relative peak area is 0.066;

[0092] Peak 3, the relative retention time is 0.585, and the relative peak area is 0.859;

[0093] Peak 4, the relative retention time is 0.712, and the relative peak area is 0.163;

[0094] Peak 5, the relative retention time is 0.816, and the relative peak area is 0.060;

[0095] Peak 6, the relative retention time is 0.901, and the relative peak area is 0.018;

[0096] Peak 7, the relative retention time is 1.000, and the relative peak area is 1.000;

[0097] Peak 8, the relative retention time is 1.067, and the relative peak area is 0.012;

[0098] Peak 9, the relative retention time is 1.147, and the relative peak area is 1.067;

[0099] Peak 10 has a relative retention time of 1.367 and a relative peak area of ​​0.039.

[0100] Peak 11 has a relative retention time of 1.437 and a relative peak area of ​​0.020.

[0101] Peak 12 has a relative retention time of 1.696 and a relative peak area of ​​0.021.

[0102] Peak 13 has a relative retention time of 1.786 and a relative peak area of ​​0.011.

[0103] The aforementioned reference fingerprint chromatogram was compared with the chromatogram of the reference solution. Based on the known characteristic peaks in the chromatogram of the reference solution, four characteristic peaks in the reference fingerprint chromatogram of Xiaor Zhenbei San were identified by relative retention time. Peak 1 is agaricol, peak 3 is sodium taurocholate, peak 7 is cholic acid, and peak 9 is deoxycholic acid. Peak 7 (cholic acid) eluted at 20.68 min, with a relatively large peak area and good stability, indicating good chromatographic peak resolution. Therefore, peak 7 was selected as the reference peak (S peak).

[0104] Based on the recorded fingerprint spectra of samples S1-S6 and the generated control fingerprint spectra (R), the relative retention time and relative peak area of ​​each chromatographic peak are calculated, and the similarity of the fingerprint spectra is compared.

[0105] The relative retention times and RSD values ​​of the common peaks of samples S1-S6 are shown in Table 1 below. The relative peak areas and RSD values ​​of the common peaks of samples S1-S6 are shown in Table 2 below. The similarity between the fingerprint spectra of samples S1-S6 and the control fingerprint spectra (R) is shown in Table 3 below.

[0106] Table 1

[0107]

[0108] Table 2

[0109]

[0110] Table 3

[0111]

[0112] The results in Table 1-3 show that the relative retention time (RSD) values ​​of each common peak are between 0.00% and 0.14%, indicating that the elution time of the common peaks is stable; the relative peak area (RSD) values ​​of each common peak are between 2.4% and 14.7%, indicating that there are certain differences in the content of each component in different batches of Xiaor Zhenbei San samples; the similarity between the fingerprint spectra of each sample and the control fingerprint spectra is 1.000, which is extremely high.

[0113] The above results indicate that the types of components in the samples remained basically unchanged before and after irradiation, and the irradiation had little effect on the overall chemical composition of Xiaor Zhenbeisan. The overall consistency of Xiaor Zhenbeisan samples from different batches was good.

[0114] Example 3 Methodological Validation

[0115] 1. Precision

[0116] One sample of Xiaor Zhenbei San from batch U02001 was injected six times consecutively according to the fingerprint chromatogram construction method for Xiaor Zhenbei San in Example 1 above, and the chromatograms were recorded. Using peak 7 (cholic acid) as the reference peak, the relative retention times of each common peak and the relative peak areas of the main peaks (accounting for more than 5% of the total peak area) were calculated. The relative retention times and RSD results of the precision test are shown in Table 4 below; the relative peak areas and RSD results of the precision test are shown in Table 5 below.

[0117] Table 4

[0118]

[0119] Table 5

[0120]

[0121] The results in Tables 4 and 5 show that the RSD values ​​of the relative retention times of the common peaks in the test solution are between 0.00% and 0.18%, and the RSD values ​​of the relative peak areas of the main peaks (accounting for more than 5% of the total peak area) are between 0.73% and 0.88%, all less than 5%, indicating that the construction method has good precision.

[0122] 2. Repeatability

[0123] Six samples of Xiaor Zhenbei San (a traditional Chinese medicine) from batch U02001 were taken and injected six times consecutively according to the fingerprint chromatogram construction method for Xiaor Zhenbei San in Example 1 above. The chromatograms were recorded. Using peak 7 (cholic acid) as the reference peak, the relative retention time of each common peak and the relative peak area of ​​the main peak (accounting for more than 5% of the total peak area) were calculated. The relative retention times and RSD results of the repeatability test are shown in Table 6 below; the relative peak areas and RSD results of the repeatability test are shown in Table 7 below.

[0124] Table 6

[0125]

[0126] Table 7

[0127]

[0128] The results in Tables 6 and 7 show that the RSD values ​​of the relative retention times of the common peaks in the test solution are between 0.00% and 0.37%, and the RSD values ​​of the relative peak areas of the main peaks (accounting for more than 5% of the total peak area) are between 0.54% and 2.69%, all less than 5%, indicating that the construction method has good repeatability and high accuracy.

[0129] 3. Stability

[0130] Six samples of Xiaor Zhenbei San (a traditional Chinese medicine) from batch U02001 were taken. Following the fingerprint chromatogram construction method for Xiaor Zhenbei San in Example 1 above, the test solutions were prepared and incubated at room temperature for 0h, 2h, 4h, 8h, 12h, and 24h, respectively, before injection, and the chromatograms were recorded. Using peak 7 (cholic acid) as the reference peak, the relative retention times of each common peak and the relative peak areas of the major peaks (accounting for more than 5% of the total peak area) were calculated. The relative retention times and RSD results of the stability test are shown in Table 8 below; the relative peak areas and RSD results of the stability test are shown in Table 9 below.

[0131] Table 8

[0132]

[0133] Table 9

[0134]

[0135] The results in Tables 8 and 9 show that the RSD values ​​of the relative retention times of the common peaks in the test solution are between 0.02% and 0.59%, and the RSD values ​​of the relative retention areas of the main peaks (accounting for more than 5% of the total peak area) are between 0.43% and 4.26%, all less than 5%, indicating that the Xiaoer Zhenbei San test solution of the present invention has good stability after being placed at room temperature for 24 hours.

[0136] The fingerprint results of precision, repeatability and stability testing are as follows: Figure 4 As shown.

[0137] Example 4: The Influence of Flow Relatives on Fingerprint Detection

[0138] The fingerprint spectrum of pediatric Zhenbei San was constructed according to the construction method of Example 1. The mobile phase of Example 1 was replaced with the following three different mobile phases and tested respectively to study the effect of the mobile phase on the fingerprint spectrum of pediatric Zhenbei San.

[0139] Mobile phase 1: 0.1% glacial acetic acid-acetonitrile, with acetonitrile as mobile phase A and 0.1 (v:v)% glacial acetic acid aqueous solution as mobile phase B;

[0140] Mobile phase 2: 0.1% formic acid-acetonitrile, with acetonitrile as mobile phase A and 0.1 (v:v)% formic acid aqueous solution as mobile phase B;

[0141] Mobile phase 3: 0.05% formic acid-acetonitrile, with acetonitrile as mobile phase A and 0.05 (v:v)% formic acid aqueous solution as mobile phase B.

[0142] Fingerprint patterns obtained using different mobile phases, such as Figure 5 As shown, 0.2% formic acid-acetonitrile is the mobile phase of Example 1. The results show that when using 0.1% glacial acetic acid-acetonitrile as the mobile phase, the response values ​​of each chromatographic peak in the obtained fingerprint spectrum are significantly lower, and the peak shape of sodium taurocholate is poor. When using 0.1% formic acid-acetonitrile and 0.2% formic acid-acetonitrile as the mobile phase, the obtained fingerprint spectrum shows no tailing of the chromatographic peaks and good peak shapes. When using 0.1% formic acid-acetonitrile as the mobile phase, the response value of the chromatographic peak is slightly reduced. However, when using 0.05% formic acid-acetonitrile as the mobile phase, some chromatographic peaks disappear in the obtained fingerprint spectrum, meaning that some chromatographic peaks cannot be detected. The above results indicate that the mobile phase has a significant impact on the chromatographic peaks of the fingerprint chromatogram of Xiaor Zhenbei San. Acetonitrile and a 0.1%–0.2% formic acid aqueous solution are more suitable as the mobile phase, resulting in better chromatographic peak separation, good peak shape, no tailing phenomenon, and high response value. Among them, acetonitrile and a 0.2% formic acid aqueous solution are the best mobile phases.

[0143] Example 5: The Influence of Evaporative Light Scattering Detector Conditions on Fingerprint Spectra

[0144] 1. The effect of carrier gas volumetric flow rate on fingerprint patterns

[0145] The fingerprint spectrum of Xiaor Zhenbeisan was established according to the construction method of Example 1. The carrier gas volumetric flow rate of Example 1 was changed to 2.1 L / min, 2.3 L / min and 2.9 L / min respectively to test the effect of carrier gas volumetric flow rate on the fingerprint spectrum of Xiaor Zhenbeisan.

[0146] Fingerprint patterns obtained using different carrier gas volumetric flow rates, such as Figure 6 As shown, the results indicate that the peak height of the chromatographic peaks decreases with increasing carrier gas flow rate. Within the range of 2.1 to 2.9 L / min, all 13 common peaks can be detected, and the range of carrier gas flow rate is applicable from 2.1 to 2.9 L / min.

[0147] 2. The effect of drift tube temperature on fingerprint patterns

[0148] The fingerprint spectrum of Xiao'er Zhenbeisan was established according to the construction method of Example 1. The drift tube temperature of Example 1 was changed to 95℃ and 100℃ respectively to study the effect of drift tube temperature on the fingerprint spectrum of Xiao'er Zhenbeisan.

[0149] Fingerprint patterns obtained using different drift tube temperatures, such as Figure 7As shown, the results indicate that the peak height of the chromatographic peaks increases with the increase of the drift tube temperature. Within the range of 95℃ to 105℃ of carrier gas volumetric flow rate, all 13 common peaks can be detected. The drift tube temperature range of 95℃ to 105℃ is applicable.

[0150] Example 6: Effect of the mass-volume ratio of pediatric Zhenbei San sample to methanol on the fingerprint spectrum. The fingerprint spectrum of pediatric Zhenbei San was established according to the construction method of Example 1. The mass-volume ratio of pediatric Zhenbei San sample to methanol in Example 1 was changed to a mass-volume ratio of 1 and a mass-volume ratio of 1 respectively to be tested to study the effect of the mass-volume ratio of pediatric Zhenbei San sample to methanol on the fingerprint spectrum of pediatric Zhenbei San.

[0151] Mass-volume ratio 1: 1.0g of Xiaor Zhenbei Powder, added to 10mL of methanol (1g:10mL);

[0152] Mass-volume ratio 2: 1.0g of Xiaor Zhenbei Powder, added to 30mL of methanol (1g:30mL);

[0153] Fingerprints obtained using different mass-to-volume ratios of pediatric granules and methanol are as follows: Figure 8 As shown, the results indicate that the peak height of the chromatographic peaks decreases with the increase of the methanol volume ratio. Within the range of 1g:10mL to 1g:30mL of the mass-volume ratio of Xiaoer Zhenbei San to methanol, all 13 common peaks can be detected, meaning that Xiaoer Zhenbei San is applicable within the range of 1g:10mL to 1g:30mL of the mass-volume ratio of methanol.

[0154] Example 7: Effect of column flow rate on fingerprint detection

[0155] The fingerprint chromatogram of Xiaor Zhenbei San was constructed according to the construction method in Example 1. The column flow rate of Example 1 was changed to 0.8 mL / min and 1.2 mL / min respectively to study the effect of different column flow rates on the fingerprint chromatogram of Xiaor Zhenbei San.

[0156] Fingerprints obtained using different column flow rates, such as Figure 9 As shown in the figure, the results indicate that when the column flow rate is 0.8 mL / min, the number of total peaks decreases to 12; when the column flow rate is 1.2 mL / min, the number of total peaks decreases to 11. These results demonstrate that the column flow rate has a significant impact on the chromatographic peaks of the fingerprint chromatogram of Xiaor Zhenbei San (a traditional Chinese medicine), and that 1.0 mL / min is more suitable as the column flow rate for this method.

[0157] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of pediatric Zhenbei powder, characterized in that, Includes the following steps: S1. Preparation of the test solution Dissolve the pediatric Zhenbei powder sample in methanol and extract it by ultrasonication. Filter the solution and the filtrate is the test solution. S2. Preparation of reference solution The reference standards linalool, sodium taurocholate, cholic acid, and deoxycholic acid were mixed and dissolved in methanol to obtain the reference solution. S3, Determination by high performance liquid chromatography-evaporative light scattering detection method The test solution and the reference solution were injected into the liquid chromatograph and measured with an evaporative light scattering detector to obtain the chromatograms of the test solution and the reference solution, respectively. The chromatogram of the test solution was identified and calibrated to obtain the fingerprint chromatogram of Xiaor Zhenbei San. The chromatographic conditions of the liquid chromatograph are as follows: a C18 column is used; acetonitrile is used as mobile phase A, and formic acid aqueous solution is used as mobile phase B; the elution program is as follows: 0–10 min, the volume percentage of mobile phase A gradually changes from 80% to 60%; 10–33 min, the volume percentage of mobile phase A gradually changes from 60% to 50%. Over 33–40 minutes, the volume percentage of mobile phase A gradually changed from 50% to 40%. In step S3, the C18 chromatographic column has dimensions of 250 × 4.6 mm and 5 μm. In step S3, the drift tube temperature of the evaporative light scattering detector is 95–105°C, and the carrier gas volumetric flow rate is 2.1–2.9 L / min.

2. The construction method as described in claim 1, characterized in that, In step S1, the mass-volume ratio of the pediatric granules to methanol is 1 g: (10~30) mL.

3. The construction method as described in claim 2, characterized in that, In step S1, the mass-volume ratio of the pediatric powder to methanol is 1 g: 25 mL.

4. The construction method as described in claim 1, characterized in that, In step S2, the concentrations of agaric tetraol, sodium taurocholate, cholic acid, and porcine deoxycholic acid in the reference solution are 20-20.5 μg / mL, 50-50.5 μg / mL, 150-150.5 μg / mL, and 150-150.5 μg / mL, respectively.

5. The construction method as described in claim 1, characterized in that, In step S3, the C18 column is an Agilent ZORBAX SB-C18 column.

6. The construction method as described in claim 1, characterized in that, In step S3, the mobile phase B is an aqueous solution of formic acid with a volume percentage of 0.1% to 0.2%.

7. The construction method as described in claim 1, characterized in that, In step S3, the column flow rate under the chromatographic conditions is 1 mL / min.

8. The application of the fingerprint spectrum constructed by the construction method according to any one of claims 1 to 7 in the detection of the quality of Xiaor Zhenbei San product.

9. A method for quality testing of pediatric Zhenbei powder, characterized in that, Includes the following steps: (1) Obtain the fingerprint spectrum of the test sample using the construction method described in any one of claims 1 to 7, and generate a standard fingerprint spectrum using one of the fingerprint spectra of the test sample as a reference spectrum; (2) Obtain the fingerprint spectrum of the pediatric Zhenbei powder to be tested using the construction method described in any one of claims 1 to 7; (3) Compare the fingerprint spectrum of the pediatric Zhenbei powder to be tested obtained in step (2) with the standard fingerprint spectrum obtained in step (1) and calculate the similarity. If the similarity is greater than 0.9, it means that the pediatric Zhenbei powder to be tested is of qualified quality.

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