A HPLC fingerprint detection method for throat tablets

Abstract

The invention discloses an HPLC fingerprint detection method for throat tablets. The throat tablets are prepared from ten Chinese medicinal materials, namely, Scrophularia ningpoensis, Radix Ophiopogonis, Periostracum Cicadae, Fructus Terminaliae, Platycodon grandiflorum, Radix Isatidis, Sterculia lychnophorae, Radix Rehmanniae, Fructus Cibotii and Radix Glycyrrhizae. The fingerprint established by the invention comprises 16 common characteristic peaks, which are attributed to the nine medicinal materials, namely, Scrophularia ningpoensis, Radix Ophiopogonis, Fructus Terminaliae, Platycodon grandiflorum, Radix Isatidis, Sterculia lychnophorae, Fructus Cibotii and Radix Glycyrrhizae. Therefore, the nine medicinal materials in the Chinese medicine can be identified. Meanwhile, three components therein are identified, namely, gallic acid, liquiritin and ammonium glycyrrhizinate. Therefore, the invention can control the quality of drugs more comprehensively, better solve the problems of drug adulteration and counterfeiting, and ensure the safety and effectiveness of clinical drugs.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine detection and analysis, and particularly relates to an HPLC fingerprint detection method for throat tablets. Background Art

[0002] The Chinese Pharmacopoeia (2020 Edition, Part I) contains a traditional Chinese medicine that can clear the throat, nourish yin and promote the production of body fluid, and detoxify and purge fire. The medicine is called "Jianmin Throat Tablets", which is an exclusive product of Jianmin Pharmaceutical Group Co., Ltd., with the approval number "National Medicine Standard Z42021798". The medicine is made of ten traditional Chinese medicinal materials: Scrophularia, Ophiopogon japonicus, Periostracum Cicadae, Terminalia chebula, Platycodon grandiflorum, Radix Isatidis, Sterculia lychnophora, Rehmannia glutinosa, Cicadae, and Licorice. The specific prescription and preparation method have been disclosed in the patent document CN1559508A. The traditional Chinese medicine has a good effect on sore throat, aphonia, and upper respiratory tract inflammation caused by excessive heat and damage to body fluid and excessive heat and toxicity.

[0003] In the existing national standards, the quality of the product is controlled by thin layer identification of menthol and citronella, and HPLC content determination of an organic acid component common to terminalia and citronella. First, thin layer identification and content determination require extraction of samples separately, and different instruments and equipment are required, which has a long detection cycle and a large amount of labor; second, only the three medicinal materials in the drug can be identified, which makes it difficult to fully reflect and monitor the quality of the drug, and it is impossible to fundamentally solve the problem of counterfeit and inferior drugs, which has great limitations.

[0004] Fingerprints are a comprehensive and quantifiable method for detecting Chinese medicinal materials and Chinese medicinal preparations. They can be used to identify authenticity and evaluate the uniformity of the quality of raw medicinal materials, semi-finished products and preparations. They are currently widely used in the Chinese medicine industry. Especially in the current situation where the effective ingredients of Chinese medicine compound preparations have not been fully elucidated, fingerprints can more comprehensively reflect the types and quantities of Chinese medicine chemical components, thereby achieving a comprehensive evaluation of the intrinsic quality of Chinese medicine and effective control of its overall material. They are currently the most effective means of quality control of Chinese medicine and its preparations. The detection methods of Chinese medicine fingerprints can be divided into chromatography, spectroscopy, and spectroscopy according to the principle. Chromatography mainly includes thin layer chromatography, high performance liquid chromatography, gas chromatography, etc. Among them, high performance liquid chromatography (HPLC) is simple, efficient, sensitive, and reproducible, and is the mainstream method for studying Chinese medicine fingerprints. Summary of the invention

[0005] In view of the shortcomings of the current throat lozenge quality detection methods and evaluation systems, the purpose of the present invention is to establish an HPLC fingerprint detection and analysis method for throat lozenges, aiming to monitor and evaluate the quality of drugs as a whole and ensure the safety and effectiveness of clinical use of drugs.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A HPLC fingerprint detection method for throat tablets, wherein the throat tablets are made of ten Chinese medicinal materials: Scrophularia ningpoensis, Radix Ophiopogonis, Periostracum Cicadae, Fructus Terminaliae Terminaliae, Platycodon grandiflorum, Radix Isatidis, Sterculia lychnophorae, Radix Rehmanniae, Fructus Cicadae, and Glycyrrhiza uralensis, and the detection method comprises the following steps:

[0008] (1) Ultrasonic extraction of the sample to be tested with methanol, filtering with a microporous filter membrane, and taking the filtrate to obtain a test solution;

[0009] (2) The sample solution is drawn and tested on a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the detection wavelength of the ultraviolet detector is set to 210-290 nm, and the stationary phase of the high performance liquid chromatograph is C 18 Chromatographic column, mobile phase A is acetonitrile, mobile phase B is acid solution, gradient elution is adopted, flow rate is 0.5-2.0ml / min, column temperature is 25-35℃, theoretical plate number is not less than 3000;

[0010] (3) Record the chromatogram of the test solution, and use the Chinese Pharmacopoeia Committee's Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to import data, perform multi-point correction, and match data on the chromatogram of the test solution to obtain a standard fingerprint of the throat tablets. Perform a similarity evaluation between the standard fingerprint and the fingerprint of the sample to be tested.

[0011] Preferably, the methanol is 70% methanol.

[0012] Preferably, the ultrasonic extraction has a power of 300 W, a frequency of 40 kHz, and a duration of 30 minutes.

[0013] Preferably, the chromatographic column is Agilent Eclipse XDB-C18; 250 mm×4.6 mm, 5 um.

[0014] Preferably, the detection wavelength of the ultraviolet detector is set to 275 nm.

[0015] Preferably, the mobile phase B is 0.5% glacial acetic acid.

[0016] Preferably, the procedure of the gradient elution is shown in Table 4.

[0017] Preferably, the column temperature is 25°C.

[0018] Preferably, the injection volume of the test solution is 10ul.

[0019] The standard fingerprint contains 16 common peaks, which are respectively attributed to nine medicinal materials: Scrophularia, Ophiopogon, Terminalia chebula, Platycodon, Isatis indigotica, Sterculia lychnophora, Rehmannia glutinosa, Cibotium barometz and Glycyrrhiza uralensis. Among them, Peak 1 belongs to Terminalia chebula and Cibotium barometz; Peaks 2, 6 and 9 belong to Scrophularia; Peaks 3 and 8 belong to Platycodon; Peak 4 belongs to Ophiopogon; Peak 5 belongs to Terminalia chebula; Peaks 7 and 12 belong to Rehmannia glutinosa; Peaks 10 and 16 belong to Glycyrrhiza uralensis; Peaks 11 and 13 belong to Isatis indigotica; Peak 14 belongs to Sterculia lychnophora; Peak 15 belongs to Cibotium barometz; and after identification by reference substances, Peak 1 is gallic acid, Peak 10 is glycyrrhizin, and Peak 16 is ammonium glycyrrhizinate.

[0020] Using the Chinese medicine chromatographic fingerprint similarity evaluation system (2004A version), the chromatogram of the sample to be tested was compared with the fitted standard liquid fingerprint to obtain the similarity. The samples with a similarity greater than 0.920 were judged to be qualified.

[0021] The percentages involved in the present invention are all volume percentages. For example, 70% methanol means that 100ml of methanol aqueous solution contains 70ml of methanol, and 0.5% glacial acetic acid means that 100ml of glacial acetic acid aqueous solution contains 0.5ml of glacial acetic acid.

[0022] The beneficial effects of the present invention are:

[0023] The present invention successfully establishes a fingerprint spectrum detection and analysis method for throat tablets. The established fingerprint spectrum includes 16 common characteristic peaks, which are attributed to nine medicinal materials, namely, Scrophularia ningpoensis, Ophiopogon japonicus, Terminalia chebula, Platycodon grandiflorum, Radix Isatidis, Sterculia lychnophora, Rehmannia glutinosa, Cibotium barometz and Licorice. Therefore, the nine medicinal materials in traditional Chinese medicine can be identified. At the same time, three components therein are also identified, namely, gallic acid, liquiritin and ammonium glycyrrhizinate. Therefore, the present invention can control the quality of drugs more comprehensively, better solve the problems of drug adulteration and counterfeiting, and ensure the safety and effectiveness of clinical drug use.

[0024] Compared with the existing detection methods for this product, the detection method of the present invention is simple and easy to operate, and can complete the detection through one extraction and sampling, shortening the detection cycle, reducing the instrument equipment and detection costs. The method also has the advantages of good stability, reproducibility, and high precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the liquid chromatogram prepared under the optimal conditions.

[0026] Figure 2 is a comparative chromatogram of the extraction solvents.

[0027] Figure 3 It is a comparative chromatogram at different wavelengths.

[0028] Figure 4This is the liquid chromatogram obtained under the mobile phase gradient elution condition (1).

[0029] Figure 5 This is the liquid chromatogram obtained under the mobile phase gradient elution condition (2).

[0030] Figure 6 This is the liquid chromatogram obtained under the mobile phase gradient elution condition (3).

[0031] Figure 7 Chromatogram overlay of repeatability test.

[0032] Figure 8 Overlay of chromatograms from the precision test.

[0033] Fig. 9 Chromatographic overlay of stability test.

[0034] Fig.10 Overlay of chromatograms from ten batches of samples.

[0035] Fig.11 Comparative identification diagram of standard fingerprint and chromatograms of each reference substance. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the embodiments.

[0037] Instruments and reagents: Shimadzu LC-2040C CN liquid chromatograph, including diode array detector, Labsolutions version workstation; Milli-Q Direct 8 ultrapure water instrument; Mettler ME203E, Mettler ME104E, Mettler TOLEDO XPE105 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.). Agilent Eclipse XDB-C18 chromatographic column (5μm, 250×4.6mm), the theoretical plate number calculated based on liquiritin should be no less than 3500;

[0038] Gallic acid reference substance (purity 91.5%, batch number 110831-201906), liquiritin reference substance (purity 95.0%, batch number 111610-201908), and ammonium glycyrrhizinate reference substance (purity 94.4%, batch number 110731-202122) were all purchased from China Food and Drug Inspection Institutes; acetonitrile was chromatographically pure, and methanol, ethanol, ethyl acetate, phosphoric acid, etc. were all analytically pure.

[0039] The throat tablet preparations are from Jianmin Pharmaceutical Group Co., Ltd., with batch numbers: 220611, 220590, 220570, 220550, 220430, 220310, 220389, 220269, 220249, 220139, denoted by S1-S10, and the formula and preparation method are as follows:

[0040] 50g of Scrophularia, 34g of Ophiopogon, 20g of Periostracum Cicadae, 34g of Terminalia Terminalia, 34g of Platycodon, 34g of Isatis Root, 2g of Sterculia Lycopodiella, 50g of Rehmannia, 10g of Cicadae, and 20g of Licorice.

[0041] Add water to the above ten ingredients and boil them three times, 2 hours each for the first and second times, and 1 hour for the third time, filter, combine the filtrates, and concentrate into a thick paste; add appropriate amounts of sucrose powder, starch and cocoa powder, mix well, granulate, dry, cool, spray with ethanol solution containing peppermint oil and menthol, add appropriate amount of orange powder, press into 1000 tablets, and film-coat.

[0042] Example 1 Exploration of detection conditions

[0043] 1. Investigation of sample extraction solvent

[0044] Four different extraction solvents were investigated: 70% methanol, ethanol, water-saturated n-butanol, and ethyl acetate.

[0045] Take the product (batch number: 220430) under the filling amount item, grind it, mix it, take about 1g, weigh it accurately, put it in a conical flask, weigh 4 portions in total, accurately add 70% methanol, ethanol, water-saturated n-butanol, and ethyl acetate 25ml respectively, plug it tightly, weigh it, treat it with ultrasound (power 300W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with the corresponding solvents, shake it well, filter it, and take the filtrate to obtain it.

[0046] The chromatographic conditions were as follows:

[0047] Chromatographic column: Agilent Eclipse XDB-C18 (250 mm × 4.6 mm, 5 um);

[0048] Mobile phase: acetonitrile and 0.5% glacial acetic acid;

[0049] Gradient elution program: see Table 1;

[0050] The flow rate is 1 ml / min;

[0051] The column temperature was 25 °C;

[0052] The injection volume was 10ul;

[0053] Detection wavelength: 275nm;

[0054] The number of theoretical plates is not less than 3000; the chromatographic recording time is 90 minutes.

[0055] By comparison, it can be seen (see Figure 2 ), under the same detection conditions, water-saturated n-butanol and ethyl acetate could not effectively extract the components in the sample; 70% methanol and ethanol could both extract a large number of components, and the overall effect of the chromatogram was basically the same, among which the peaks prepared by 70% methanol extraction had stronger responsiveness than ethanol, and the number of peaks was slightly more than ethanol, so 70% methanol was preferred as the extraction solvent.

[0056] 2. Wavelength selection

[0057] 220430 batches of throat lozenge samples were tested, and the chromatogram effects at different wavelengths such as 210nm, 230nm, 245nm, 260nm, 275nm, and 290nm were investigated respectively. 70% methanol was used as the extraction solvent, and other chromatographic conditions were the same as in Example 1.

[0058] Results Figure 3 As can be seen from the figure, when the wavelength is 210 and 230nm, the overall chromatogram baseline fluctuates greatly and the noise is large. When the wavelength is 245 and 260nm, the chromatographic peaks before 28min are generally small or missing. At 245nm, some chromatographic peaks after 40min have poor responsiveness. In comparison, the overall chromatogram effect is better when the wavelength is 275nm and 290nm, especially when the wavelength is 275nm, the number of peaks is more. Therefore, considering the overall situation, the preferred wavelength of 275nm is the optimal detection wavelength of the fingerprint spectrum.

[0059] 3. Optimization of mobile phase elution program

[0060] According to experimental exploration, the acetonitrile-0.5% acid solution system is the best mobile phase. The gradient elution program with acetonitrile (A): 0.5% glacial acetic acid (B) as the mobile phase system was optimized, and the following four gradient elution conditions were investigated:

[0061] Table 1 Gradient elution conditions 1

[0062] Time (min) A(％) B(％) 0～12 3～7 97～93 12～25 7～12 93～88 25～40 12～25 88～75 40～50 25～50 75～50 50～60 50～80 50～20 60～70 80 20 70～90 80～3 20～97

[0063] Table 2 Gradient elution conditions 2

[0064] Time (min) A(％) B(％) 0～18 3～9 97～91 18～27 9 91 27～48 9～15 91～85 48～55 15～40 85～60 55～60 40～80 60～20 60～70 80 20 70～90 80～3 20～97

[0065] Table 3 Gradient elution conditions 3

[0066] Time (min) A(％) B(％) 0～18 3～9 97～91 18～25 9 91 25～33 9～12 91～88 33～42 12～8 88～92 42～55 8～25 92～75 55～75 25～70 75～30 75～90 70～3 30～97

[0067] Table 4 Gradient elution conditions 4

[0068] Time (min) A(％) B(％) 0～18 3～9 97～91 18～25 9 91 25～33 9～12 91～88 33～42 12～8 88～92 42～52 8～19 92～81 52～60 19 81 60～80 19～60 81～40 80～90 60～3 40～97

[0069] Results: Figure 4 , 5 As shown in Figures 6 and 1, under gradient elution conditions (1) and (2), the chromatographic peaks are relatively concentrated and some main peaks are not separated; under gradient elution condition (3), the overall effect of the chromatogram is slightly better, but the number of peaks is still relatively small and some peaks are not completely separated; in comparison, under gradient elution condition (4), the main chromatographic peaks have a suitable distribution of peak time and good peak separation, so the gradient elution condition (4) is preferred.

[0070] The final test conditions are as follows:

[0071] Preparation of test solution: Take the sample to be tested, grind it, mix it, take about 1g, weigh it accurately, put it in a conical flask, accurately add 25ml of 70% methanol, plug it tightly, weigh it, and treat it with ultrasound (power 300W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it with a microporous filter membrane, and take the filtrate to obtain it.

[0072] The chromatographic conditions were as follows:

[0073] Chromatographic column: Agilent Eclipse XDB-C18 (250 mm × 4.6 mm, 5 um);

[0074] Mobile phase: acetonitrile and 0.5% glacial acetic acid;

[0075] Gradient elution program: see Table 4;

[0076] The flow rate is 1 ml / min;

[0077] The column temperature was 25 °C;

[0078] The injection volume was 10ul;

[0079] Detection wavelength: 275nm;

[0080] The number of theoretical plates is not less than 3000; the chromatographic recording time is 90 minutes.

[0081] The following methodological investigations were all carried out under the finalized detection conditions.

[0082] Example 2 Methodological Investigation

[0083] 1. Repeatability test

[0084] Take the same batch of throat tablets (batch number: 220430), prepare 6 test solutions according to the optimized conditions and measure them to examine the repeatability. Take peak 8 as the reference peak, calculate the RSD value of the relative retention time and relative peak area of ​​each common peak. The results show that the relative standard deviation of the relative retention time of each chromatographic peak is less than 1.00%, and the relative standard deviation of the relative peak area of ​​each chromatographic peak is less than 2.50%, and the repeatability is good. See Table 5-6 and Figure 7 .

[0085] Table 5 Relative retention time of each peak in the repeatability test of throat tablets

[0086] Peak 1 2 3 4 5 6 average value RSD 1 0.153 0.152 0.155 0.153 0.154 0.151 0.153 0.92％ 2 0.216 0.212 0.214 0.213 0.215 0.211 0.214 0.88％ 3 0.239 0.238 0.237 0.240 0.241 0.236 0.239 0.78％ 4 0.621 0.624 0.623 0.619 0.625 0.618 0.622 0.45％ 5 0.679 0.672 0.675 0.680 0.673 0.678 0.676 0.49％ 6 0.768 0.771 0.762 0.765 0.769 0.763 0.766 0.46％ 7 0.798 0.789 0.786 0.793 0.795 0.793 0.792 0.54％ 8(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 9 1.057 1.042 1.041 1.055 1.056 1.054 1.051 0.70％ 10 1.129 1.120 1.127 1.130 1.128 1.133 1.128 0.39％ 11 1.231 1.225 1.221 1.233 1.241 1.230 1.230 0.56％ 12 1.321 1.335 1.345 1.342 1.316 1.332 1.332 0.86％ 13 1.379 1.384 1.377 1.389 1.392 1.372 1.382 0.55％ 14 1.409 1.408 1.401 1.387 1.392 1.399 1.399 0.62％ 15 1.442 1.432 1.429 1.427 1.436 1.449 1.436 0.58％ 16 1.509 1.487 1.496 1.492 1.513 1.497 1.499 0.67％

[0087] Table 6 Relative peak area of ​​each peak in the repeatability test of throat tablets

[0088]

[0089]

[0090] 2. Precision test

[0091] Take the test solution of the same batch of throat tablets (batch number: 220430), repeat the injection for 6 times, and examine the precision. Take the chromatographic peak No. 8 as the reference peak, and calculate the RSD of the relative retention time and relative peak area of ​​each common peak. The results show that the relative standard deviation of the relative retention time of each common peak is less than 0.80%, and the relative standard deviation of the relative peak area is less than 2.00%, which shows good precision. See Table 7-8 and Figure 8 .

[0092] Table 7 Relative retention time of each peak in throat tablet precision test

[0093]

[0094]

[0095] Table 8 Relative peak area of ​​each peak in throat tablet precision test

[0096] Peak 1 2 3 4 5 6 average value RSD 1 2.263 2.251 2.231 2.247 2.241 2.276 2.252 0.71％ 2 0.259 0.252 0.257 0.247 0.251 0.248 0.252 1.90％ 3 0.215 0.217 0.226 0.218 0.215 0.219 0.218 1.87％ 4 0.240 0.237 0.238 0.244 0.238 0.233 0.238 1.52％ 5 0.190 0.184 0.185 0.188 0.184 0.187 0.186 1.30％ 6 1.185 1.171 1.176 1.192 1.189 1.177 1.182 0.70％ 7 0.247 0.250 0.248 0.254 0.252 0.251 0.250 1.03％ 8(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 9 0.991 1.005 0.998 1.002 0.999 1.006 1.000 0.55％ 10 0.678 0.665 0.661 0.679 0.667 0.673 0.671 1.09％ 11 0.156 0.148 0.153 0.150 0.149 0.151 0.151 1.94％ 12 0.187 0.185 0.189 0.181 0.191 0.185 0.186 1.88％ 13 0.143 0.146 0.149 0.144 0.143 0.146 0.145 1.60％ 14 0.877 0.879 0.871 0.885 0.883 0.876 0.879 0.57％ 15 0.825 0.831 0.842 0.833 0.842 0.838 0.835 0.81％ 16 0.122 0.123 0.120 0.122 0.119 0.124 0.122 1.53％

[0097] 3. Stability test

[0098] Take the test solution of the same batch of throat tablets (batch number: 220430), place it at room temperature, and inject it into the liquid chromatograph at 0h, 4h, 8h, 12h, and 24h respectively. Take the chromatographic peak No. 8 as the reference peak to examine the RSD of the relative retention time and relative peak area of ​​each common peak. The results show that the relative standard deviation of the relative retention time of each chromatographic peak is less than 1.00%, and the relative standard deviation of the relative peak area of ​​each chromatographic peak is less than 2.00%, and the stability is good. See Table 9-10 and Fig. 9 .

[0099] Table 9 Relative retention time of each peak in the throat tablet stability test

[0100]

[0101]

[0102] Table 10 Relative peak areas of each peak in the throat tablet stability test

[0103] Peak 0h 4h 8h 12h 24h average value RSD 1 2.267 2.244 2.227 2.255 2.262 2.251 0.71％ 2 0.261 0.263 0.268 0.259 0.261 0.262 1.31％ 3 0.218 0.219 0.225 0.220 0.217 0.220 1.42％ 4 0.241 0.239 0.242 0.247 0.239 0.242 1.36％ 5 0.191 0.187 0.186 0.182 0.183 0.186 1.92％ 6 1.183 1.175 1.173 1.199 1.178 1.182 0.88％ 7 0.249 0.252 0.253 0.249 0.253 0.251 0.82％ 8(S) 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 9 0.997 1.011 0.995 1.006 0.994 1.001 0.75％ 10 0.679 0.678 0.672 0.684 0.673 0.677 0.72％ 11 0.151 0.148 0.152 0.150 0.149 0.150 1.05％ 12 0.188 0.186 0.189 0.183 0.190 0.187 1.48％ 13 0.142 0.145 0.147 0.144 0.143 0.144 1.33％ 14 0.879 0.878 0.873 0.881 0.887 0.880 0.58％ 15 0.827 0.832 0.841 0.836 0.843 0.836 0.78％ 16 0.121 0.125 0.121 0.122 0.122 0.122 1.34％

[0104] Example 5 Establishment and Analysis of Fingerprint

[0105] 1. Establishment of standard fingerprint and determination of common peaks

[0106] According to the determined throat lozenge test solution preparation method and liquid chromatography detection conditions, 10 batches of throat lozenge samples were taken for detection and analysis. The chromatograms of the 10 batches of samples are shown in the figure. Fig.10 The main chromatographic peaks obtained by the determination all appeared within 90 minutes. The obtained fingerprints were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004A Edition)" software for processing. The median method was used, the time window width was set to 0.2, and the multi-point correction method was used to perform full peak matching on the chromatogram. The standard reference fingerprint was fitted and 16 common peaks were determined ( Fig.11 ), taking chromatographic peak No. 8 as the reference peak, the relative retention times of other common peaks in 10 batches of samples were calculated (Table 11). The average relative retention times (peak numbers) were 0.151(1), 0.213(2), 0.240(3), 0.621(4), 0.681(5), 0.768(6), 0.790(7), 1.000(8), 1.049(9), 1.129(10), 1.235(11), 1.332(12), 1.385(13), 1.407(14), 1.438(15), and 1.508(16), respectively. The relative deviations were all within 1.0%.

[0107] Table 11-1 Relative retention time of common peaks in 10 batches of throat lozenges

[0108]

[0109] Table 11-2 Relative retention time of common peaks in 10 batches of throat lozenge samples

[0110]

[0111]

[0112] 2. Identification of common peaks

[0113] Preparation of reference solution: Accurately weigh appropriate amounts of gallic acid, liquiritin, and ammonium glycyrrhizinate reference substances, and prepare each single reference solution of about 20 ug / ml with 70% methanol.

[0114] The chromatograms of the reference solutions of gallic acid, liquiritin and ammonium glycyrrhizinate were compared with the standard fingerprints of the samples, and the chromatographic peaks at the same retention time as the reference solutions were identified. Fig.11 The results showed that among the common peaks, peaks 1, 10, and 16 were identified as gallic acid, liquiritin, and ammonium glycyrrhizinate, respectively. Peak 8 was in the middle position, with a suitable peak size and good peak shape, so peak 8 was selected as the reference peak.

[0115] 3. Correlation between the fingerprints of medicinal materials and finished products

[0116] Preparation of medicinal material test solution: Extract each medicinal material in the prescription according to the granule preparation process of this product to obtain an extract, and assign the chromatographic peaks according to the standard fingerprint conditions. The results are shown in Table 12.

[0117] Table 12 Correlation between throat tablets and medicinal materials

[0118]

[0119]

[0120] The 16 common peaks were attributed to medicinal materials, and a total of nine medicinal materials in the prescription were identified, among which gallic acid No. 1 was attributed to Terminalia chebula and Cibotium barometz, peaks 2, 6, and 9 were attributed to Scrophularia ningpoensis, peaks 3 and 8 were attributed to Platycodon grandiflorum, peak 4 was attributed to Ophiopogon japonicus, peak 5 was attributed to Terminalia chebula, peaks 7 and 12 were attributed to Rehmannia glutinosa, glycyrrhizin No. 10 and ammonium glycyrrhizinate No. 16 were attributed to Glycyrrhiza uralensis, peaks 11 and 13 were attributed to Isatis indigotica, peak 14 was attributed to Sterculia lychnophora, and peak 15 was attributed to Cibotium barometz.

[0121] 4. Similarity evaluation and sample qualification determination

[0122] The color spectra of 10 batches of samples were compared with the standard reference fingerprint spectra, and similarity evaluation was performed according to the "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine" (2004A Edition). See Table 13. The similarities of the 10 batches of samples were 0.954, 0.989, 0.977, 0.971, 0.958, 0.935, 0.928, 0.948, 0.929, and 0.922, respectively. The similarities of the 10 batches were all above 0.900, indicating that the quality stability and uniformity of the samples were good. After comprehensive consideration, the samples with a similarity greater than 0.920 were selected as qualified products.

[0123] Table 13 Similarity evaluation of 10 batches of throat lozenge samples

[0124] batch number Similarity batch number Similarity 220611 0.954 220310 0.935 220590 0.989 220389 0.928 220570 0.977 220269 0.948 220550 0.971 220249 0.929 220430 0.958 220139 0.922

Claims

1. A HPLC fingerprint detection method for throat tablets, wherein the throat tablets are made of ten Chinese medicinal materials: Scrophularia ningpoensis, Radix Ophiopogonis, Periostracum Cicadae, Fructus Terminaliae Terminaliae, Platycodon grandiflorum, Radix Isatidis, Sterculia lychnophorae, Radix Rehmanniae, Fructus Cicadae, and Radix Glycyrrhizae, characterized in that: The detection method comprises the following steps: (1) The sample to be tested is ultrasonically extracted with 70% methanol, filtered with a microporous filter membrane, and the filtrate is taken to obtain a test solution; (2) The sample solution was drawn and tested on a high performance liquid chromatograph, wherein the high performance liquid chromatograph contained an ultraviolet detector, the detection wavelength of the ultraviolet detector was set to 275 nm, and the stationary phase of the high performance liquid chromatograph was C 18 Chromatographic column, mobile phase A is acetonitrile, mobile phase B is 0.5% glacial acetic acid, gradient elution is adopted, flow rate is 0.5-2.0ml / min, column temperature is 25-35°C, theoretical plate number is not less than 3000; (3) Record the chromatogram of the test solution, and use the Chinese Pharmacopoeia Committee's Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to import data, perform multi-point correction, and match data on the chromatogram of the test solution to obtain the standard fingerprint of the throat tablets. Perform similarity evaluation between the standard fingerprint and the fingerprint of the sample to be tested. The procedure of the gradient elution is as follows:

2. The HPLC fingerprint detection method of throat lozenges according to claim 1, characterized in that: The ultrasonic extraction has a power of 300 W, a frequency of 40 kHz, and a time of 30 minutes.

3. The HPLC fingerprint detection method of throat tablets as claimed in claim 1, characterized in that: The chromatographic column is Agilent Eclipse XDB-C18; 250 mm×4.6 mm, 5 um.

4. The HPLC fingerprint detection method of throat lozenges according to claim 1, characterized in that: The column temperature was 25°C.

5. The HPLC fingerprint detection method of throat tablets as claimed in claim 1, characterized in that: The injection volume of the test solution was 10ul.

6. The HPLC fingerprint detection method of throat lozenges according to claim 1, characterized in that: The standard fingerprint contains 16 common peaks, which are respectively attributed to nine medicinal materials: Scrophularia, Ophiopogon, Terminalia chebula, Platycodon, Isatis indigotica, Sterculia lychnophora, Rehmannia glutinosa, Cibotium barometz and Glycyrrhiza uralensis. Among them, Peak 1 belongs to Terminalia chebula and Cibotium barometz; Peaks 2, 6 and 9 belong to Scrophularia; Peaks 3 and 8 belong to Platycodon; Peak 4 belongs to Ophiopogon; Peak 5 belongs to Terminalia chebula; Peaks 7 and 12 belong to Rehmannia glutinosa; Peaks 10 and 16 belong to Glycyrrhiza uralensis; Peaks 11 and 13 belong to Isatis indigotica; Peak 14 belongs to Sterculia lychnophora; Peak 15 belongs to Cibotium barometz; and after identification by reference substances, Peak 1 is gallic acid, Peak 10 is glycyrrhizin, and Peak 16 is ammonium glycyrrhizinate.

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