Characteristic chromatogram of Wantongkang preparation as well as construction method and application of characteristic chromatogram

Abstract

The invention discloses a characteristic spectrum of a Wantongkang preparation as well as a construction method and application of the characteristic spectrum. (1) preparing a sample; (2) performing high performance liquid chromatography analysis; and (3) constructing a characteristic spectrum. According to the method, the specific chromatogram detection conditions are optimized, 11 components of the Wantongkang specific chromatogram are simultaneously determined by adopting a wavelength switching method in high performance liquid chromatography analysis, multi-component determination analysis of the specific chromatogram is realized, the detection period is greatly shortened, the analysis efficiency is improved, and the HPLC specific chromatogram analysis method is finally determined through methodological investigation. The method has the advantages of high specificity, high precision, high repeatability, high stability, capability of simultaneously quantifying 11 characteristic components and the like, is very suitable for controlling the overall quality of the Wantongkang, ensures the quality of a product and has a huge application prospect.

Description

Technical field:

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection and quality control, and specifically relates to a method for constructing a characteristic spectrum of a compound Chinese patent medicine Wantong Yankang preparation and its standard characteristic spectrum, as well as the application of the characteristic spectrum in the quality standard and quality control of Wantong Yankang medicine. Background technology:

[0002] Acute and chronic pharyngitis is a common disease, usually caused by viral and / or bacterial infection. Worldwide, there are approximately 615 million cases of pharyngitis each year in all age groups. So far, clinical treatment strategies for pharyngitis mainly include antibiotics, antiviral drugs and glucocorticoids to relieve pain, but there are problems with drug toxicity and drug resistance.

[0003] Wantong Yankang is a Chinese patent medicine composed of Radix Scrophulariae and Radix Sarcosae. Its tablets are included in the Chinese Pharmacopoeia (2020 Edition) Volume 1, and have the effects of dispelling wind and heat, detoxifying and reducing swelling. It is used for pharyngeal swelling, gingival swelling, sore and furuncle swelling and pain caused by exogenous wind-heat; acute and chronic pharyngitis, tonsillitis, gingivitis, sore and furuncle with the above symptoms. However, since Chinese patent medicines are composed of multiple medicinal materials, there are many deficiencies in the quality control of Wantong Yankang: (1) The Pharmacopoeia of the People's Republic of China only contains the method of determining the content of isofraquinone by high performance liquid chromatography under the item [Content Determination] of Wantong Yankang Tablets. However, Wantong Yankang is composed of two medicinal materials, Radix Scrophulariae and Radix Sarcosae (1:1). It is not enough to evaluate the quality and efficacy of the drug based solely on the content of isofraquinone in Radix Sarcosae. It is necessary to further improve the quality evaluation standards and establish more complete quality standards. In addition, the current standards for this drug issued by the State Food and Drug Administration also have deficiencies. There is no specific content determination method for rosmarinic acid, a chemical component of the prescription drug Radix Sarcosinae, and there is no content determination method for the chemical components of the prescription drug Radix Scrophulariae. The quality control level is low, which is not conducive to the monitoring of the production process and product quality. (2) There is a lack of characteristic fingerprint identification of the drug composition, and there is a lack of quality control and standards for its main active ingredients, which cannot meet the needs of the modernization of traditional Chinese medicine; it fails to accurately reflect the changes in production conditions and standard management. Therefore, it is urgent to construct the characteristic spectrum and quality control system of Wantong Yankang, comprehensively improve the quality standards and quality control level, and improve the monitoring of the production process and product quality. Summary of the invention:

[0004] The purpose of the present invention is to provide a method for constructing a characteristic spectrum of Wantong Yankang preparation and its characteristic spectrum, which can effectively control the quality of the medicine and monitor the production process to solve the problems raised in the background technology.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for constructing a standard characteristic spectrum of a Wantong Yankang preparation, the method comprising the following steps:

[0007] S1: Preparation of the test sample Wantong Yankang preparation solution, the reference sample solution and the negative control Scrophularia scabra and Sarcandra scutellariae solution;

[0008] S2: The test sample Wantong Yankang preparation solution, the reference substance solution and the negative control Scrophularia ningpoensis and Sarcandra suspensa solutions are respectively subjected to high performance liquid chromatography to obtain liquid chromatograms of the test sample Wantong Yankang preparation, the reference substance and the negative control Scrophularia ningpoensis and Sarcandra suspensa;

[0009] The reference substances in the reference solution include: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarascoside, apigenin-7-O glucuronic acid, rosmarinic acid, scrophulariaceae glycoside IA and scrophulariaceae glycoside IB;

[0010] The high performance liquid chromatography is carried out by using a high performance liquid chromatograph, C 18 Chromatographic column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution or 0.1% formic acid solution by volume, elution mode is gradient elution, and the gradient elution program is:

[0011] From 0 to 20 minutes, the volume fraction of the mobile phase A increases from 15% to 20%;

[0012] 20-35 minutes, the volume fraction of the mobile phase A increases from 20% to 30%;

[0013] 35-50 minutes, the volume fraction of the mobile phase A increases from 30% to 60%;

[0014] The retention time of the liquid phase spectrum of the test sample Wantong Yankang preparation solution is compared with the retention time of the liquid phase spectrum of the reference solution to obtain the characteristic spectrum of the Wantong Yankang preparation.

[0015] Preferably, the Wantong Yankang tablets or Wantong Yankang capsules.

[0016] Preferably, the HPLC conditions are: column temperature 30±5°C; flow rate 0.8-1.1 mL / min; detection wavelengths 264±2 nm and 324-343 nm.

[0017] The test sample Wan Tong Yan Kang preparation solution, the reference solution and the negative sample Scrophularia scabra and Sarcandra scutellariae solution

[0018] Preferably, the process of obtaining the test sample Wantong Yankang preparation solution is as follows: take 10 to 20 tablets / capsules of Wantong Yankang preparation, remove the coating or capsule shell, grind, accurately weigh 0.5 to 1.0 g, put in a stoppered conical bottle, accurately add 25 or 50 ml of 50 to 75% methanol, and weigh the weight. Ultrasonic treatment power 450 to 500 W, frequency 35 to 40 kHz, time 30 to 60 minutes, cool, weigh again, use 50 to 75% methanol to make up for the lost weight, shake well, filter, and take the filtrate to obtain;

[0019] The preparation process of the reference solution is: accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarascoside, apigenin-7-O-glucuronic acid, rosmarinic acid, scroscoside IA, and scroscoside IB reference substances, add 50-75% methanol to prepare a mixed reference solution containing 20-50 μg of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and caffeic acid, 50-100 μg of isofraquinone, 200-250 μg of verbascoside, rosmarinic acid, isovarascoside, and apigenin-7-O-glucuronic acid, and 100-800 μg of scroscoside IA and scroscoside IB per 1 ml;

[0020] The preparation process of negative Scrophularia ningpoensis and Radix Sarcosae is as follows: ① Preparation of Scrophularia ningpoensis solution: accurately weigh 0.8-1.0g of Scrophularia ningpoensis medicinal material powder, accurately add 25 or 50ml of 75-50% methanol solution, and weigh the weight; ultrasonic extraction for 30-60min with a power of 450-500W and a frequency of 35-40kHz, cool, make up the lost weight with 75-50% methanol, filter, and take the filtrate to obtain; Preparation of Radix Sarcosae solution: accurately weigh 0.8-1.0g of Radix Sarcosae medicinal material powder, accurately add 25 or 50ml of 75-50% methanol solution, and weigh the weight; ultrasonic extraction for 30-60min with a power of 450-500W and a frequency of 35-40kHz, cool, make up the lost weight with 75-50% methanol, filter, and take the filtrate to obtain.

[0021] Preferably, the characteristic spectrum of the Wantong Yankang preparation has a total of 11 chromatographic peaks, namely peak 1-neochlorogenic acid, peak 2-chlorogenic acid, peak 3-cryptochlorogenic acid, peak 4-caffeic acid, peak 5-isofracinidine, peak 6-verbascoside, peak 7-isoverbascoside, peak 8-apigenin-7-O-glucuronic acid, peak 9-rosmarinic acid, peak 10-scrophulariaceae glycoside IB, and peak 11-scrophulariaceae glycoside IA.

[0022] Preferably, the identification and attribution of the chromatographic peaks are mainly detected by UFLC-Q-TOF-MS / MS HPLC-MS, and the chromatographic conditions of HPLC-MS analysis are: the chromatographic column is C 18Chromatographic column; acetonitrile (A)-0.1% formic acid / acetic acid (B) as mobile phase, gradient elution; flow rate: 0.8~1.1ml / min; and use a diverter valve to adjust the flow rate entering the mass spectrometer to 0.2~0.3ml / min; column temperature 30℃, injection volume 5~10μl; mass spectrometry conditions ESI electrospray ion source, positive and negative ion modes, ion spray voltage: positive mode 5500V, negative mode -4500V; ion source parameters: spray gas 50psi; auxiliary heating gas 50psi; ion source temperature 500℃; curtain gas 35psi; collision gas pressure 10psi, primary mass spectrometry parent ion scanning range: m / z 100~2000, collision activation scan is 15.0, and dynamic background subtraction is turned on.

[0023] The present invention also provides the application of the characteristic spectrum constructed above in the evaluation and control of the product quality of Wantong Yankang preparation.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. For the first time, a determination method for the characteristic spectrum of Wantong Yankang preparations was constructed. According to the structural characteristics and physicochemical properties of the active ingredients contained in Wantong Yankang preparations, the analysis conditions such as the treatment method of the test sample, mobile phase, detection wavelength, elution procedure, column temperature, flow rate, etc. were screened and optimized, and a systematic methodological verification was completed. The obtained Wantong Yankang characteristic spectrum has high separation and good peak shape. Each characteristic chromatographic peak has achieved good baseline separation, good stability, and many characteristic peaks. It can comprehensively, accurately, and intuitively evaluate the quality of Wantong Yankang preparations, and is suitable for the control of the quality of Wantong Yankang preparations.

[0026] 2. The present invention adopts HPLC wavelength switching method to simultaneously identify 11 components of the characteristic spectrum and determine their contents in the Wantong Yankang preparation, thereby achieving comprehensive evaluation and control of the quality of the Wantong Yankang preparation.

[0027] 3. The characteristic spectrum of the Wantong Yankang preparation established by the present invention overcomes the characteristics of the single control index, low specificity and low quality standard level in the current standard. The characteristic spectrum of the Wantong Yankang preparation established has 11 chromatographic peaks, namely peak 1 (neochlorogenic acid), peak 2 (chlorogenic acid), peak 3 (cryptochlorogenic acid), peak 4 (caffeic acid), peak 5 (isofraxin), peak 6 (verbascoside), peak 7 (isoverbascoside), peak 8 (apigenin-7-O-glucuronic acid), peak 9 (rosmarinic acid), peak 10 (scrophulariaceae glycoside IB), peak 11 (scrophulariaceae glycoside IA). There are 6 characteristic components belonging to the medicinal material of Radix Sarcosinae, and 5 characteristic components belonging to the medicinal material of Radix Scrophulariae. The quality of the Wantong Yankang preparation can be comprehensively evaluated, and the quality of the finished product can be effectively guaranteed. At the same time, it can be used as the internal control standard of the enterprise for large-scale production and the evaluation basis for the consistency of the efficacy of Wantong Yankang tablets, providing a guarantee for the safe, effective, uniform and stable standardized production of the Wantong Yankang preparation. Description of the drawings:

[0028] Figure 1 The UV absorption of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarascoside, apigenin-7-O glucuronic acid, rosmarinic acid, scrophulariaceae glycoside IA and scrophulariaceae glycoside IB;

[0029] Figure 2 This is a comparison of the similarity of HPLC characteristic spectra of 10 batches of Wantong Yankang tablets, R: control characteristic spectrum; S1-S10: batch numbers 22052201, 22071901, 22101002, 22110902, 23010303, 23011802, 23020302, 22101802, 21102701, 22010701;

[0030] Figure 3 It is the common pattern of the characteristic spectrum of Wantong Yankang Tablets (reference characteristic spectrum);

[0031] Figure 4 This is the HPLC chart of the reference medicinal material of Sarcandra glabra (batch number 230413);

[0032] Figure 5 This is the HPLC chart of the reference medicinal material of Scrophularia tataricola (batch number 220830);

[0033] Figure 6 This is the HPLC characteristic spectrum of Wantong Yankang Tablets;

[0034] Figure 7This is the common peak attribution diagram of the characteristic spectrum of Wantong Yankang Tablets, A: mixed reference substance; B: Sarcandra scabra; C: Scrophularia odorata; D: Wantong Yankang Tablets, Peak 1: neochlorogenic acid, Peak 2: chlorogenic acid, Peak 3: cryptochlorogenic acid, Peak 4: caffeic acid, Peak 5: isofraquinone, Peak 6: verbascoside, Peak 7: isoverbascoside, Peak 8: apigenin-7-O-glucuronic acid, Peak 9: rosmarinic acid, Peak 10: Scrophularia odorata glycoside IB, Peak 11: Scrophularia odorata glycoside IA;

[0035] Figure 8 This is the characteristic spectrum of Wantong Yankang Capsule;

[0036] Fig. 9 This is the total ion current diagram of Wantong Yankang Tablets; Specific implementation method:

[0037] The present invention is further described below by way of examples, but is not intended to be limiting of the present invention.

[0038] 1. Experimental Preparation

[0039] 1. Instruments and test drugs

[0040] 1.1 Instrument

[0041] 1 / 100,000 electronic analytical balance (MS205DU, Mettler Toledo, Switzerland); ultrapure water device (Simplicity, Millipore, USA); CNC ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.); Ultimate 3000 high performance liquid chromatograph (Dionex, USA, LPG-3400SD quaternary pump, SRD-3600 degasser, WPS-3000SL autosampler, TCC3000-RS column oven, DAD detector, Chromeleon7.0 data processing software, instrument number: 418-6); UFLC-Q-TOF-MS / MS liquid-mass spectrometry system (UFLC XR ultra high performance liquid chromatograph, Shimadzu, Japan; Triple TOFTM 5600+, AB Sciex, USA).

[0042] Chromatographic column: 1.C 18 Column-Manufacturer: Welch Specification: UltimateXB-C18, 4.6*250, 5μm, SN: 60230803750

[0043] 2.C 18 Column-Manufacturer: ZORBAX Specification: Eclipse plus C18, 4.6*250, 5μm, SN: USUXA13448

[0044] 3.C 18Column-Manufacturer: Thermo Specification: Hypersil gold, 4.6*250, 5μm, SN: 20260745

[0045] 1.2 Drug testing

[0046] Experimental reagents:

[0047] Methanol (Manufacturer: Fisher; Batch No.: F23N5N202; Purity: HPLC)

[0048] Liquid chromatography reagents:

[0049] Formic acid (manufacturer: Sigma; batch number: BCBM4407; purity: LC-MS, ≥99%);

[0050] Acetonitrile (Manufacturer: Fisher; Batch No.: F23N5N202; Purity: HPLC)

[0051] 1.3 Samples

[0052] Wantong Yankang tablets (capsules), Sarcandra scabra and Scrophularia ningpoensis were provided by Guangxi Wantong Pharmaceutical Co., Ltd., as shown in Table 1; the reference substances are shown in Table 2.

[0053] Table 1 Wantong Yankang sample situation

[0054]

[0055] 1.4 Reference substances

[0056] Table 2 Reference material information

[0057]

[0058] Example 1

[0059] 1. Construction of the characteristic spectrum of Wantong Yankang tablets

[0060] 1.1 Preparation of test sample, reference sample and reference medicinal material solutions

[0061] Preparation of reference solution: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarascoside, apigenin-7-O-glucuronic acid, rosmarinic acid, scroscoside IA, and scroscoside IB reference substances, and add 50% methanol to prepare a mixed reference solution containing 20 μg of neochlorogenic acid, 20 μg of chlorogenic acid, 20 μg of cryptochlorogenic acid, 20 μg of caffeic acid, 50 μg of isofraquinone, 100 μg of verbascoside, 200 μg of isovarascoside, 200 μg of apigenin-7-O-glucuronic acid, 100 μg of rosmarinic acid, 800 μg of scroscoside IA, and 500 μg of scroscoside IB per 1 ml.

[0062] Preparation of test solution: Take 20 tablets of this product, remove the coating, accurately weigh, grind, take 0.6g (film-coated tablet), accurately weigh, put in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh the weight, ultrasonically treat (power 500W 45kHz) for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate.

[0063] Preparation of control medicinal material solutions: ① Preparation of the test solution of Radix Scrophulariae Radix: Accurately weigh 0.8g of Radix Scrophulariae Radix powder in the above table, accurately add 25mL of 60% methanol solution, and weigh the weight; ultrasonically extract for 1 hour (power 500W 45kHz), cool, make up the lost weight with 60% methanol, filter, and take the filtrate. ② Preparation of the test solution of Radix Sarcandrae Radix: Accurately weigh 1g of Radix Sarcandrae Radix powder in the above table, accurately add 25mL of 75% methanol solution, and weigh the weight; ultrasonically extract for 1 hour (power 500W 45kHz), cool, make up the lost weight with 75% methanol, filter, and take the filtrate.

[0064] Preparation of medicinal materials and test solution: Take 10 batches of Wantong Yankang tablets finished product and reference substance, Sarcandra scutellariae medicinal materials, and Scrophularia ningpoensis reference medicinal materials and prepare them according to the above-mentioned test solution preparation method to obtain corresponding test solution.

[0065] 1.2 Optimization and selection of chromatographic methods

[0066] Selection of mobile phase: In the 2020 edition of the Chinese Pharmacopoeia, the HPLC method for determining the content of isofraxidin in Radix Sarcodiformis and Radix Sarcodiformis tablets uses acetonitrile-0.1% phosphoric acid (20:80 v / v) as the mobile phase; the content of isofraxidin in Radix Sarcodiformis extract is determined using acetonitrile-0.1% formic acid (20:80 v / v) as the mobile phase. By comparing acetonitrile-0.1% phosphoric acid and acetonitrile-0.1% formic acid under the same elution gradient, different mobile phases and elution gradient conditions were compared and screened (as shown in Table 3 below).

[0067] Table 3 Optimization of mobile phase elution gradient conditions

[0068]

[0069] After repeated tests and comparisons, and considering that phosphoric acid solution is not suitable for full component detection of this product using UFLC-Q-TOF-MS / MS, acetonitrile-0.1% formic acid was finally selected as the mobile phase. The optimized chromatographic conditions for the determination of Wantong Yankang Tablets are determined as follows:

[0070] Table 4 Mobile phase elution gradient (acetonitrile-0.1% formic acid)

[0071]

[0072] Detection wavelength: Use the above elution procedure to detect the Wantong Yankang tablets sample, record the chromatogram, and observe the absorption of the chromatogram at the full wavelength of ultraviolet light: the maximum absorption wavelengths of 9 substances, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isoverbascoside, apigenin-7-O glucuronic acid and rosmarinic acid, are concentrated around 340nm, while the maximum absorption wavelength of scrophulariaceae IA and scrophulariaceae glycoside IB is 264nm. It can be seen that at 264nm and 340nm, as much component information as possible (such as Figure 1 Therefore, through analytical screening, 340nm and 264nm were selected as the detection wavelengths of the characteristic spectrum method.

[0073] Selection of chromatographic column: When other chromatographic conditions remained unchanged, three chromatographic columns from different manufacturers were investigated, namely Welch Altimate XB-C18 (4.6×250mm, 5μm), Agilent ZORBAX Eclipse Plus C18 (4.6×250mm, 5μm), and ThermoHypersil gold (4.6×250mm, 5μm). The peak shape of each chromatographic peak was good and basic separation could be achieved with a separation degree of more than 1.2. Therefore, the C18 column was selected for characteristic spectrum detection.

[0074] Determination of characteristic spectrum chromatographic conditions: Octadecylsilane bonded silica gel was used as filler (Welch Ultimate XB-C 18 , 4.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution according to Table 4; detection wavelengths at 340nm, 264nm; column temperature at 30℃; flow rate at 1mL / min. Detection time: 50 minutes, and switch the wavelength from 340nm to 264nm at 35 minutes; column temperature at 30℃; flow rate at 1mL / min.

[0075] 1.3 Methodological research

[0076] Methodological study on the optimized HPLC chromatographic conditions of Wantong Yankang Tablets

[0077] ①Specificity test

[0078] The blank solvent is 50% methanol solution, and the blank solvent chromatogram is obtained by injection. Wantong Yankang Tablets (batch number: 22101002) were operated according to the method under the test solution preparation item in Example 1, and analyzed under the same chromatographic conditions. The results showed that the identification of the 11 common chromatographic peaks was not interfered by factors such as solvents and excipients, and had good specificity.

[0079] ②Precision test

[0080] Weigh the same batch of Wantong Yankang tablets (batch number: 22101002) accurately, operate according to the method under the preparation of the test solution in Example 1, inject 6 times continuously on the high performance liquid chromatograph, and record the chromatogram. Among them, because the peak stability of scrophulariaceae glycoside IA is good, the separation is high, the peak area is large, and the transfer rate is high and the transferability tends to be stable during the production process, scrophulariaceae glycoside IA is selected as the reference peak, and the relative retention time of the reference peak and each peak is compared. However, since the position of the IA peak is later and belongs to scrophulariaceae, the test sample is composed of swollen joint herb and scrophulariaceae, so isofraquinone and rosmarinic acid belonging to scrophulariaceae are selected as reference peaks, and the two compound reference substances are widely available, easy to obtain, stable and high in separation. The results show that the RSD% of the relative retention value of each characteristic peak is less than 1% (see Table 5), indicating that the method has good precision and meets the requirements of the characteristic spectrum.

[0081] Table 5 Relative retention value results of the precision of each characteristic peak in the characteristic spectrum

[0082]

[0083]

[0084] ③Stability test

[0085] Accurately weigh the same batch of Wantong Yankang tablets (batch number: 22101002), and operate according to the method under the preparation of the test solution in Example 1. Samples were injected at 0, 12, 24, and 48 hours, and the chromatograms were recorded. The relative retention values ​​of each component in its characteristic spectrum (scrophulariaceae glycoside IA was the reference peak) were calculated. The results are shown in the following table (Table 6): The RSD values ​​of the relative retention values ​​of each characteristic peak were all less than 3.00%, indicating that the Wantong Yankang tablets test solution and the mixed reference solution had good stability within 48 hours.

[0086] Table 6 Relative retention value results of the stability of each characteristic peak in the characteristic spectrum

[0087]

[0088] ④Repeatability test

[0089] Accurately weigh 6 portions of the same batch of Wantong Yankang tablets (batch number: 22101002) in parallel, and operate according to the method under the preparation of the test solution in Example 1. Inject the samples separately according to the above-mentioned chromatographic conditions, record the chromatogram, and calculate the relative retention value of each component in the characteristic spectrum (scrophulariaceae glycoside IA is the reference peak). The results are shown in Table 7. The RSD values ​​of the relative retention values ​​of each characteristic peak are all less than 1.00%, indicating that the method has good repeatability.

[0090] Table 7 Relative retention value results of each characteristic peak repeatability in the characteristic spectrum

[0091]

[0092] 1.4 Common peak determination

[0093] The 10 batches of Wantong Yankang tablets were subjected to HPLC analysis according to the chromatographic conditions under the "Preparation of test solution" and the HPLC chromatograms were recorded. The chromatograms of the 10 batches of Wantong Yankang tablets were analyzed, and 11 chromatographic peaks were stably reproducible. Combined with the mixed reference substance, Sarcandra scutellariae, Scrophularia ningpoensis single medicinal materials and literature reports, the common peaks of the characteristic spectra were identified and attributed. The characteristic peaks attributed to Sarcandra scutellariae are peaks 1, 2, 3, 4, 5, and 6 (such as Figure 4 The characteristic peaks belonging to Scrophularia odorata are peaks 7, 8, 9, 10, and 11 (as shown in Figure 5 shown).

[0094] 1.5 Mass spectrometry confirms the common peak molecular structure of the characteristic spectrum of Wantong Yankang tablets

[0095] The UFLC-Q-TOF-MS / MS technology was used to detect the mixed reference solution, Wantong Yankang tablets test solution, and the medicinal materials of Sarcandra suspensa and Scrophularia ningpoensis. Combined with the molecular ion peaks and pyrolysis fragment information of mass spectrometry, the components corresponding to the chromatographic peaks were confirmed as follows: peak 1 (neochlorogenic acid), peak 2 (chlorogenic acid), peak 3 (cryptochlorogenic acid), peak 4 (caffeic acid), peak 5 (isofracinidine), peak 6 (verbascoside), peak 7 (isoverbascoside), peak 8 (apigenin-7-O-glucuronic acid), peak 9 (scrophularia ningpoensis glycoside IB), and peak 11 (scrophularia ningpoensis glycoside IA).

[0096] 1.5 Construction of feature maps

[0097] During the experiment, 10 batches of Wantong Yankang tablets were tested, with batch numbers S1-S10: 22052201, 22071901, 22101002, 22110902, 23010303, 23011802, 23020302, 22101802, 21102701, 22010701. Ten test solutions were prepared according to the method under "Preparation of test solution", and the chromatograms of 10 batches of Wantong Yankang tablets were obtained by determination according to the law. The similarity evaluation results are shown in Table 8. The similarity coefficients of the 10 batches of Wantong Yankang tablets are all greater than 0.970, indicating that the 10 batches of Wantong Yankang tablets have good correlation, proving that the production process of Wantong Yankang tablets is stable and the product uniformity is good.

[0098] Table 8 Similarity evaluation results of 10 batches of Wantong Yankang tablets

[0099]

[0100] The 2012 version of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" was used to perform pattern recognition on 10 batches of Wantong Yankang Tablets. The common pattern of the HPLC characteristic spectrum of Wantong Yankang Tablets was obtained by the average method ( Figure 3 ), establish a control feature map (such as Figure 3 The result is as shown Figure 2 As shown, from left to right, they are characteristic peaks 1 to 11; R is the reference characteristic spectrum; S1 to S10 are the characteristic spectra of batch numbers 22052201, 22071901, 22101002, 22110902, 23010303, 23011802, 23020302, 22101802, 21102701, and 22010701. The results show that the production process of Wantong Yankang is stable and the product uniformity is good.

[0101] Example 2

[0102] Wantong Yankang Capsules are another dosage form of Wantong Yankang preparations. There is no difference between Wantong Yankang Capsules and Wantong Yankang Tablets in terms of prescription composition, preparation process, etc. Both are composed of Scrophularia ningpoensis and Sarcandra chinensis, so the characteristic spectrum of Wantong Yankang Tablets can also be applied to Wantong Yankang Capsules.

[0103] 1Construction of characteristic spectrum of Wantongyankang capsule

[0104] 1.1 Preparation of reference solution: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarascoside, apigenin-7-O-glucuronic acid, rosmarinic acid, scroscoside IA, and scroscoside IB reference substances, and add 50% methanol to prepare a mixed reference solution containing 20 μg of neochlorogenic acid, 20 μg of chlorogenic acid, 20 μg of cryptochlorogenic acid, 20 μg of caffeic acid, 50 μg of isofraquinone, 100 μg of verbascoside, 200 μg of isovarascoside, 200 μg of apigenin-7-O-glucuronic acid, 100 μg of rosmarinic acid, 800 μg of scroscoside IA, and 500 μg of scroscoside IB per 1 ml.

[0105] 1.2 Preparation of test solution: Take the content of Wantong Yankang Capsules, about 0.6g, accurately weigh, place in a stoppered conical flask, accurately add 10ml of methanol, weigh the weight, ultrasonically treat (power 500w, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter through a 0.45μm filter membrane, and take the filtrate to obtain.

[0106] 1.3 Preparation of control medicinal material solutions: ① Preparation of the test solution of Radix Scrophulariae Radix: accurately weigh 0.8g of Radix Scrophulariae Radix powder, accurately add 25mL of 60% methanol solution, and weigh the weight; ultrasonic extraction for 1 hour (power 500W 45kHz), cool, make up the lost weight with 60% methanol, filter, and take the filtrate. ② Preparation of the test solution of Radix Sarcandrae Radix: accurately weigh 1g of Radix Sarcandrae Radix powder, accurately add 25mL of 75% methanol solution, and weigh the weight; ultrasonic extraction for 1 hour (power 500W 45kHz), cool, make up the lost weight with 75% methanol, filter, and take the filtrate.

[0107] 1.4 Preparation of medicinal materials and test solutions: Take 10 batches of Wantong Yankang capsule finished products and reference substances, Sarcandra scutellariae medicinal materials, and Scrophularia ningpoensis reference medicinal materials and prepare them according to the above-mentioned test solution preparation method to obtain the corresponding test solution.

[0108] 1.5 High performance liquid chromatography analysis: Chromatographic conditions: Welch UltimateXB-C column 18 , 4.6mm×250mm×5μm; acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution according to Table 1; detection wavelengths at 340nm, 264nm; column temperature at 30℃; flow rate at 1mL / min. Detection time: 50 minutes, and switch the wavelength from 340nm to 264nm at 35 minutes; column temperature at 30℃; flow rate at 1mL / min.

[0109] 1.6 Common peak determination:

[0110] The test solutions of Wantong Yankang Capsules from 10 batches were subjected to HPLC analysis according to the chromatographic conditions under "Preparation of test solution", and the HPLC chromatograms were recorded. The chromatograms of the 10 batches of Wantong Yankang Capsules were analyzed, and 11 chromatographic peaks were stably reproducible.

[0111] The mixed reference solution, Wantong Yankang capsule test solution, and Radix Sarcandrae and Radix Scrophulariae solutions were tested. The common peaks were identified and attributed by combining the analysis of the common peaks with the reference, reference medicinal materials, UV spectra and literature search. Finally, the characteristic spectrum of Wantong Yankang capsule was determined as follows: Figure 8 shown.

[0112] Example 3

[0113] Determination of content in Wantong Yankang tablets (capsules)

[0114] The present invention has investigated the characteristic spectrum methodology of Wantong Yankang preparations. The detection method has good stability, high precision, good repeatability, and can comprehensively and objectively evaluate the quality of Wantong Yankang. The above-mentioned chromatographic conditions can be used for content determination. Take 10 batches of Wantong Yankang tablets produced by Guangxi Wantong Pharmaceutical Co., Ltd.; 4 batches of Wantong Yankang capsules are prepared according to the method under "Preparation of test solution", and the reference substance is prepared according to the method under "Preparation of reference solution". The above-mentioned chromatographic conditions are used for content determination, and the contents of 11 characteristic peaks in 14 batches of Wantong Yankang tablets (capsules) are calculated. The results are shown in Tables 9 and 10, indicating that the characteristic spectrum of Wantong Yankang tablets can be applied to the content determination of characteristic peaks.

[0115] Table 9 Determination results of the content of each characteristic component in 10 batches of Wantong Yankang tablets

[0116]

[0117]

[0118] Table 10 Determination results of the content of each characteristic component in 4 batches of Wantong Yankang Capsules

[0119]

[0120]

[0121] Example 4

[0122] Confirmation of Mass Spectrum Peaks of Wantong Yankang Tablets (Capsules)

[0123] In the above investigation of the characteristic spectrum method of Wantong Yankang preparation, the common peaks of the characteristic spectrum were identified and attributed in combination with the mixed reference substance, Radix Sarcandrae and Radix Scrophulariae single herbs and literature reports (such as Figure 6 and 7 The test sample was prepared according to the method under "Preparation of test sample solution", and the reference sample was prepared according to the method under "Preparation of reference sample solution". The following mass spectrometry conditions were used for LC-MS detection and analysis. The results are shown in the figure. Fig. 9 shown.

[0124] LC-MS analysis: ① Chromatographic conditions: Welch UltimateXB-C 18 , 4.6mm×250mm×5μm; acetonitrile (A)-0.1% formic acid water (B) as mobile phase, gradient elution (as shown in the table below); flow rate: 1ml / min; and use a diverter valve to adjust the flow rate into the mass spectrometer to 0.3ml / min; column temperature 30℃, injection volume 10μl; elution program is as follows: mobile phase elution gradient

[0125]

[0126] ②Mass spectrometry conditions

[0127] ESI electrospray ion source, positive and negative ion modes, ion spray voltage: positive mode 5500V, negative mode -4500V; ion source parameters: spray gas 50psi; auxiliary heating gas 50psi; ion source temperature 500℃; curtain gas 35psi; collision gas pressure 10psi, primary mass spectrometry parent ion scanning range: m / z 100-2000. Collision activation scan was 15.0, and dynamic background subtraction was turned on.

[0128] The above experimental results show that the characteristic spectrum detection method provided by the present invention has good stability, high precision, good repeatability, and can comprehensively and objectively evaluate the quality of Wantong Yankang, which is of great significance for ensuring clinical efficacy.

[0129] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of Wantong Yankang preparation, characterized in that: The following steps are involved: S1: Preparation of the test sample Wantong Yankang preparation solution, the reference sample solution and the negative control Scrophularia scabra and Sarcandra scutellariae solution; S2: The test sample Wantong Yankang preparation solution, the reference substance solution and the negative bitter Scrophularia and Radix Sarcosae solutions are respectively subjected to high performance liquid chromatography to obtain liquid chromatograms of the test sample Wantong Yankang preparation, the reference substance and the negative bitter Scrophularia and Radix Sarcosae; The reference substances in the reference solution include: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarascoside, apigenin-7-O glucuronic acid, rosmarinic acid, scrophulariaceae glycoside IA and scrophulariaceae glycoside IB; The high performance liquid chromatography is carried out by using a high performance liquid chromatograph, C 18 Chromatographic column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution or 0.1% formic acid solution by volume, elution mode is gradient elution, and the gradient elution program is: From 0 to 20 minutes, the volume fraction of the mobile phase A increases from 15% to 20%; 20-35 minutes, the volume fraction of the mobile phase A increases from 20% to 30%; 35-50 minutes, the volume fraction of the mobile phase A increases from 30% to 60%; The retention time of the liquid phase spectrum of the test sample Wantong Yankang preparation solution is compared with the retention time of the liquid phase spectrum of the reference solution to obtain the characteristic spectrum of the Wantong Yankang preparation.

2. The construction method according to claim 1, characterized in that: The described ones are Wantong Yankang tablets or Wantong Yankang capsules.

3. The construction method according to claim 1, characterized in that: The HPLC conditions are as follows: column temperature 30±5°C; Flow rate: 0.8~1.1mL / min, detection wavelength: 264±2nm and 324~343nm.

4. The construction method according to claim 1, characterized in that: The preparation methods of the test sample Wantong Yankang preparation solution, the reference sample solution, and the negative bitter Scrophularia ningpoensis and Sarcandra scutellariae solutions are as follows: The process of obtaining the test sample Wantong Yankang preparation solution is as follows: take 10 to 20 tablets / capsules of Wantong Yankang preparation, remove the coating or capsule shell, grind finely, accurately weigh 0.5 to 1.0 g, put in a stoppered conical bottle, accurately add 25 or 50 ml of 50 to 75% methanol, and weigh the weight. Ultrasonic treatment power 450 to 500 W, frequency 35 to 40 kHz, time 30 to 60 minutes, cool, weigh again, use 50 to 75% methanol to make up for the lost weight, shake well, filter, and take the filtrate to obtain; The preparation process of the reference solution is: accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarascoside, apigenin-7-O-glucuronic acid, rosmarinic acid, scroscoside IA, and scroscoside IB reference substances, add 50-75% methanol to prepare a mixed reference solution containing 20-50 μg of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and caffeic acid, 50-100 μg of isofraquinone, 200-250 μg of verbascoside, rosmarinic acid, isovarascoside, and apigenin-7-O-glucuronic acid, and 100-800 μg of scroscoside IA and scroscoside IB per 1 ml; The preparation process of negative control Scrophularia ningpoensis and Sarcandra glabra solutions is as follows: ① Preparation of Scrophularia ningpoensis solution: accurately weigh 0.8-1.0g of Scrophularia ningpoensis medicinal material powder, accurately add 25 or 50ml of 75-50% methanol solution, and weigh the weight; ultrasonic extraction for 30-60min with a power of 450-500W and a frequency of 35-40kHz, cool, make up the lost weight with 75-50% methanol, filter, and take the filtrate to obtain; Preparation of Sarcandra glabra solution: accurately weigh 0.8-1.0g of Sarcandra glabra medicinal material powder, accurately add 25 or 50ml of 75-50% methanol solution, and weigh the weight; ultrasonic extraction for 30-60min with a power of 450-500W and a frequency of 35-40kHz, cool, make up the lost weight with 75-50% methanol, filter, and take the filtrate to obtain.

5. The construction method according to claim 1, characterized in that: The characteristic spectrum of the Wantong Yankang preparation has a total of 11 chromatographic peaks, namely peak 1-neochlorogenic acid, peak 2-chlorogenic acid, peak 3-cryptochlorogenic acid, peak 4-caffeic acid, peak 5-isofracinidine, peak 6-verbascoside, peak 7-isoverbascoside, peak 8-apigenin-7-O-glucuronic acid, peak 9-rosmarinic acid, peak 10-scrophulariaceae glycoside IB, and peak 11-scrophulariaceae glycoside IA.

6. The construction method according to claim 1, characterized in that The test solution and reference solution were analyzed by high performance liquid chromatography to obtain the high performance liquid chromatography fingerprint of Wantong Yankang; the main chromatographic peaks were attributed by UFLC-Q-TOF-MS / MS liquid chromatography-mass spectrometry technology. The chromatographic conditions for liquid chromatography-mass spectrometry analysis were: the chromatographic column was C 18 Chromatographic column; acetonitrile (A)-0.1% formic acid / acetic acid (B) as mobile phase, gradient elution; flow rate: 0.8~1.1ml / min; and use a diverter valve to adjust the flow rate entering the mass spectrometer to 0.2~0.3ml / min; column temperature 30℃, injection volume 5~10μl; mass spectrometry conditions ESI electrospray ion source, positive and negative ion modes, ion spray voltage: positive mode 5500V, negative mode -4500V; ion source parameters: spray gas 50psi; auxiliary heating gas 50psi; ion source temperature 500℃; curtain gas 35psi; collision gas pressure 10psi, primary mass spectrometry parent ion scanning range: m / z 100~2000, collision activation scan is 15.0, and dynamic background subtraction is turned on.

7. A characteristic spectrum prepared according to any one of the preparation methods of claims 1-6.

8. Application of the characteristic spectrum described in claim 7 in the evaluation and control of the quality of Wantong Yankang preparation products.

9. The use according to claim 8, characterized in that: The described ones are Wantong Yankang tablets or Wantong Yankang capsules.

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