Characteristic chromatogram of platelet-increasing capsule, construction method of characteristic chromatogram and quality detection method of platelet-increasing capsule

Abstract

The invention relates to a platelet-increasing capsule characteristic chromatogram, a construction method thereof and a quality detection method of the platelet-increasing capsule, and belongs to the technical field of traditional Chinese medicine detection.In the construction method of the platelet-increasing capsule characteristic chromatogram, the platelet-increasing capsule characteristic chromatogram is established by controlling liquid chromatography conditions, mobile phase composition and elution procedures; seven common characteristic peaks are obtained. Chromatographic peaks of the specific chromatogram are completely presented, the uniformity is good, the separation degree of the characteristic chromatographic peaks is good, the types and the number of chemical components contained in the platelet-increasing capsule are comprehensively reflected, overall description and evaluation of the quality of the platelet-increasing capsule are achieved, and the effectiveness and the safety of the platelet-increasing capsule are guaranteed. The specific chromatogram detection method has the advantages of simplicity and convenience in operation, stability, reliability, high precision, good reproducibility, easiness in mastering and the like.

Description

Technical Field

[0001] The invention relates to a platelet-raising capsule characteristic spectrum and a construction method thereof and a platelet-raising capsule quality detection method, belonging to the technical field of traditional Chinese medicine detection. Technical Background

[0002] Platelet-raising capsules are a compound preparation of traditional Chinese medicine made from five Chinese medicinal herbs: Indigo, Forsythia, Agrimoniae, Paeonia suffruticosa, and Licorice. They have the effects of clearing away heat and detoxifying, cooling blood and stopping bleeding, dispersing blood stasis and removing spots. They are mainly used for idiopathic thrombocytopenic purpura. The quality standard currently implemented for platelet-raising capsules is WS-10028 (ZD-0028) -2002-2012Z-2017. The original standard included the methods for determining the contents of Indigo and Paeonia suffruticosa, but there was no control method for characteristic spectra or fingerprint spectra. The main literature reports on the quality inspection of platelet-raising capsules include "Determination of the content of indigo in platelet-raising capsules by high performance liquid chromatography", "Simultaneous determination of the contents of paeonol and indigo in platelet-raising capsules by HPLC", "Determination of the content of forsythiaside in platelet-raising capsules by reversed-phase high performance liquid chromatography", and "Determination of the content of indigo in platelet-raising capsules by RP-HPLC". Content"; The above literature mainly studies the content determination method of platelet-raising capsules. At present, there is no relevant research on the establishment of fingerprint or characteristic spectrum methods, resulting in incomplete quality inspection of platelet-raising capsules. In order to better control the consistency of product quality, referring to the "Technical Guidance for the Study of Characteristic Spectra of Traditional Chinese Medicine Preparations" promulgated by the Drug Review Center of the State Drug Administration on February 27, 2024, the current version of the "Chinese Pharmacopoeia" and the "National Drug Standard Work Manual" and other relevant requirements, the characteristic spectrum method of platelet-raising capsules is now developed and verified to ensure the scientificity and rationality of the characteristic spectrum method. Establishing a comprehensive and quantifiable comprehensive ingredient quality monitoring method plays an important role in improving the quality stability and consistency of platelet-raising capsules. Summary of the invention

[0003] The purpose of the present invention is to solve the problems existing in the prior art. By providing a platelet-raising capsule characteristic spectrum and a construction method thereof and a platelet-raising capsule quality detection method, the quality of the platelet-raising capsule can be evaluated as a whole, thereby ensuring that the product quality is controllable and stable.

[0004] Therefore, the present invention discloses a method for constructing a characteristic spectrum of a platelet-raising capsule, comprising the following steps:

[0005] (1) Chromatographic conditions and system suitability: Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, 0.05% phosphoric acid solution was used as the mobile phase B, and gradient elution was performed as specified in the following table. The flow rate was 1.0 ml / min, the column temperature was 30°C, the detection wavelength was 230 nm, and the theoretical plate number calculated based on the forsythiaside peak should be no less than 5000;

[0006]

[0007]

[0008] (2) Blank solvent: N,N-dimethylformamide;

[0009] (3) Preparation of test solution: Take the contents of this product, grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of N,N-dimethylformamide, stopper it tightly, weigh it, treat it with ultrasound for 30 minutes, cool it, weigh it accurately, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and obtain it;

[0010] (4) Preparation of reference solution: Take an appropriate amount of forsythiaside reference substance and add N,N-dimethylformamide to prepare a solution containing 0.1 mg forsythiaside per 1 ml;

[0011] (5) Determination method: Accurately pipette 10 μl of blank solvent, reference solution and test solution respectively, inject into liquid chromatograph, measure and record the chromatogram to obtain the characteristic spectrum of platelet-raising capsule.

[0012] Preferably, the method further includes the construction of a control characteristic map of platelet-raising capsules, and the characteristic maps obtained by testing multiple batches of platelet-raising capsule test samples are generated by using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 edition of the State Pharmacopoeia Committee's "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System".

[0013] Preferably, after generating the control characteristic spectrum of platelet-raising capsules using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) of the State Pharmacopoeia Committee, the method further includes the step of marking common characteristic peaks.

[0014] Preferably, the characteristic spectrum of the platelet-raising capsule includes 7 characteristic peaks, and the common characteristic peaks are: Peak 1 is a common characteristic peak, Peak 2 is a common characteristic peak, Peak 3 is forsythiaside, Peak 4 is paeonol, Peak 5 is a common characteristic peak, Peak 6 is indigo, and Peak 7 is indigotin.

[0015] Preferably, the characteristic spectrum of the platelet-raising capsule includes 7 common characteristic peaks, wherein peak 3 is the reference peak S of forsythin; the relative retention time of each characteristic peak and the S peak is within the range of ±8% of the specified value; the specified values ​​are: 0.37 (peak 1), 0.42 (peak 2), 1.00 (peak 3), 1.33 (peak 4), 1.37 (peak 5), 1.93 (peak 6), 2.03 (peak 7);

[0016] Preferably, the chromatographic column used in step (1) of the method for constructing the characteristic spectrum of the platelet-raising capsule is selected from GL Inert Sustain AQ C 18 The column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm.

[0017] Preferably, the conditions of the ultrasonic treatment used in step (3) of the method for constructing the characteristic spectrum of the platelet-raising capsule are: power 300 W, frequency 40 KHz.

[0018] The present invention also provides a characteristic spectrum of a platelet-raising capsule, which is obtained by any of the above-mentioned construction methods.

[0019] The present invention also provides a control characteristic spectrum of a platelet-raising capsule, selected from any one of the following (1)-(3): (1) it has 7 common characteristic peaks, and the retention times are 26.470 min, 30.330 min, 71.643 min, 95.440 min, 98.547 min, 138.337 min, and 145.577 min, respectively;

[0020] (2) It has 7 common characteristic peaks, of which Peak 3 is the reference peak S of forsythin; the relative retention time of each characteristic peak and the S peak is within ± 8% of the specified value; the specified values ​​are: 0.37 (peak 1), 0.42 (peak 2), 1.00 (peak 3), 1.33 (peak 4), 1.37 (peak 5), 1.93 (peak 6), and 2.03 (peak 7);

[0021] (3) Using multiple batches of platelet-raising capsules, the characteristic spectra obtained according to the construction method described above are used to prepare a control characteristic spectrum by using the average value or median method.

[0022] The present invention measures 15 batches of platelet-raising capsules and determines 7 common characteristic peaks. The selection of the common characteristic peaks can be performed by using fingerprint similarity evaluation software of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 edition formulated by the State Pharmacopoeia Committee.

[0023] The present invention also discloses a method for constructing any of the above-mentioned platelet-raising capsule characteristic maps or a use of the above-mentioned platelet-raising capsule control characteristic map in the quality detection of platelet-raising capsule products.

[0024] The present invention also provides a quality detection method for platelet-raising capsules, comprising the step of comparing a characteristic spectrum of a platelet-raising capsule product to be tested with a platelet-raising capsule control characteristic spectrum; the characteristic spectrum of the platelet-raising capsule product to be tested is obtained using the platelet-raising capsule product to be tested according to any of the construction methods described above.

[0025] If the similarity between the characteristic spectrum of the platelet-raising capsule product to be tested and the characteristic spectrum of the platelet-raising capsule control is not less than 0.90, the quality is qualified; if it is less than 0.90, it is unqualified. Specifically, the similarity is obtained by using the fingerprint similarity evaluation software of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 edition formulated by the State Pharmacopoeia Committee.

[0026] Preferably, the platelet-raising capsule product and the reference substance are tested in the same manner as any of the above-mentioned methods for constructing the platelet-raising capsule characteristic spectrum to obtain corresponding characteristic spectra, and the characteristic spectrum of the platelet-raising capsule product to be tested is compared with the platelet-raising capsule control characteristic spectrum and the reference substance characteristic spectrum to evaluate their quality.

[0027] The beneficial effects of the present invention are as follows:

[0028] 1. Analyze the representative ingredients of each medicinal flavor in Shengxuexiaoban Capsule. Among them, Qingdai has the effects of clearing away heat and detoxifying, cooling blood and removing spots, purging fire and calming convulsions. It can be used to treat febrile toxic spots, blood-heat vomiting and epistaxis, chest pain and hemoptysis, mouth sores, mumps, throat paralysis, infantile convulsions, etc. The main ingredients are indigo carmine and indigo. The original quality standard also controlled the content of indigo carmine; Forsythia suspensa is a holy medicine for sores. It has the effects of clearing heat and detoxifying, removing swelling and dispersing nodules, and evacuating wind-heat. It can be used to treat carbuncle, arthralgia, breast carbuncle, erysipelas, wind-heat cold, the initial stage of febrile disease, warm and heat entering the camp, high fever and thirst, coma and spots, hot stranguria and pain, and can also regulate blood pressure and treat thrombocytopenic hemorrhagic purpura. The main quality control ingredient is forsythia glycoside; Paeonia suffruticosa has the effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis, and can be used to treat Heat enters the blood, warm poison causes spots, vomiting blood and epistaxis, hot at night and cool in the morning, no sweat and bone steaming, amenorrhea and dysmenorrhea, injuries and pains from falls, carbuncles and sores, etc. It also has a certain protective effect on the cardiovascular system, and its main quality control ingredient is paeonol; Herba Agrimoniae has the effects of astringing and stopping bleeding, stopping symptoms, stopping dysentery, detoxifying, and replenishing deficiency. It can be used for hemoptysis, vomiting blood, metrorrhagia, dysentery, swelling and sores, itchy vaginal discharge, and fatigue. Its main active ingredient is Herba Agrimoniae phenols; Licorice has the effects of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving emergency and relieving pain, and harmonizing various medicines. It is used for spleen and stomach weakness, fatigue, palpitations and shortness of breath, cough with sputum, abdominal and limb cramps and pain, carbuncles and sores, etc. It also has the effect of relieving the toxicity and potency of drugs. Its main ingredient is glycyrrhizic acid, a terpene ingredient. In summary, indigo and indirubin in Indigo, forsythin in Forsythia, agrimonol B in Agrimoniae, paeonol in Paeonia suffruticosa and glycyrrhizic acid in Glycyrrhiza uralensis were selected as indicator components, and the same liquid phase method was used for positioning analysis, and a characteristic spectrum method was established to strengthen the overall quality control of Shengxuexiaoban Capsules.

[0029] 2. The method for constructing the characteristic spectrum of the platelet-raising capsule described in the present invention overcomes the defect that the quality of the related dosage form cannot be fully detected from multiple angles due to the interference caused by the complex chemical composition of the platelet-raising capsule; the present invention obtains 7 common characteristic peaks and realizes effective separation of the 7 common characteristic peaks with good peak shape and no interference, thereby realizing the quality detection of the effectiveness of the platelet-raising capsule from multiple angles and further realizing the quality control of the platelet-raising capsule, and the quality detection method is simple to operate, has good reproducibility and stability, reliable recovery rate, low detection cost and high detection efficiency after methodological verification, and the application of this method plays a vital role in the quality stability of the platelet-raising capsule product.

[0030] 3. A set of rapid, comprehensive and specific quality testing methods for platelet-raising capsules has been established.

[0031] 4. The characteristic spectrum can conduct all-round quality control of platelet-raising capsules from multiple angles, providing more powerful guarantees for the authenticity identification and large-scale production of platelet-raising capsules.

[0032] 5. The characteristic spectrum / fingerprint test results of each batch of platelet-raising capsules show that corresponding characteristic peaks are detected in different batches of platelet-raising capsules, and the similarity of fingerprint spectrum is greater than 0.90, indicating that the product quality is uniform and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below in conjunction with the accompanying drawings:

[0034] Figure 1 It is the experimental example of the present invention 1 liter platelet capsule chromatographic conditions investigation - method development 1 - gradient 1 reference solution UV wavelength scanning diagram (190 ~ 400nm);

[0035] Figure 2 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 1 - gradient 1 test solution - 3D scanning diagram (190-400nm);

[0036] Figure 3 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 1 - gradient 1 test sample solution chromatogram overlay;

[0037] Figure 4 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 1 - gradient 1 chromatogram overlay (230nm);

[0038] Figure 5 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 2 - gradient 2 test solution 1 (N, N-dimethylformamide) 3D scanning diagram (190-400nm);

[0039] Figure 6 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 2 - gradient 2 test solution 2 (methanol) 3D scan (190-400nm);

[0040] Figure 7 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 2 - gradient 2 chromatogram overlay (230nm);

[0041] Figure 8 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 3 - gradient 3 chromatogram overlay (230nm);

[0042] Fig. 9 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 4 - gradient 4 chromatogram overlay (230nm);

[0043] Fig.10It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 5 - gradient 4 chromatogram overlay (230nm);

[0044] Fig.11 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 6 - gradient 5 chromatogram overlay (230nm);

[0045] Fig.12 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 7 - gradient 6 chromatogram overlay (230nm);

[0046] Fig.13 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 8 - gradient 7 full wavelength UV scan;

[0047] Fig.14 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 8 - gradient 7 chromatogram overlay (230nm);

[0048] Fig.15 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 9 - gradient 8 chromatogram overlay (230nm);

[0049] Fig.16 It is the present invention experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 10 - gradient 9 chromatogram overlay (230nm);

[0050] Fig.17 It is the present invention Experimental Example 1 Liter Platelet Capsule Chromatographic Conditions Investigation - Method Development 11 - Gradient 10 Chromatographic Overlay (230nm);

[0051] Fig.18 It is the present invention Experimental Example 1 Liter Platelet Capsule Chromatographic Conditions Investigation - Method Development 12 - Gradient 11 Chromatographic Overlay (230nm);

[0052] Fig.19 It is the present invention Experimental Example 1 Liter Platelet Capsule Chromatographic Conditions Investigation - Method Development 13 - Gradient 12 Chromatographic Overlay (230nm);

[0053] Fig. 20 It is the present invention Experimental Example 1 Liter Platelet Capsule Chromatographic Conditions Investigation - Method Development 14 - Gradient 13 Chromatographic Overlay (230nm);

[0054] Fig.21 It is the present invention Experimental Example 1 Liter Platelet Capsule Chromatographic Conditions Investigation - Method Development 15 - Gradient 14 Chromatographic Overlay (230nm);

[0055] Fig. 22It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 16 - gradient 15 chromatogram overlay (230nm);

[0056] Fig.23 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - method development 17 - gradient 16 chromatogram overlay (230nm);

[0057] Fig.24 It is the present invention Experimental Example 1 Liter Platelet Capsule Chromatographic Conditions Investigation - Method Development 18 - Gradient 17 Chromatographic Overlay (230nm);

[0058] Fig.25 It is the chromatographic condition investigation of the 1-liter platelet capsule of the experimental example of the present invention - ultrasonic time investigation of the preparation of the test solution - full wavelength UV scanning diagram;

[0059] Fig.26 It is the present invention's experimental example 1 liter platelet capsule chromatographic conditions investigation - test solution preparation ultrasound time investigation - chromatogram overlay (230nm);

[0060] Fig. 27 It is the chromatographic condition investigation of the 1-liter platelet capsule of the present invention's experimental example - the peak attribution overlay of the control medicinal material;

[0061] Fig.28 It is the HPLC characteristic spectrum methodological verification of the 1-liter platelet capsule of the experimental example of the present invention - the specificity chromatogram overlay;

[0062] Fig.29 This is the peak matching diagram of the HPLC characteristic spectrum of the samples of 15 batches of finished product detection of 1 liter platelet capsules in the experimental example of the present invention;

[0063] Fig.30 This is the HPLC standard characteristic spectrum of 1 liter platelet capsule established in the experimental example of the present invention - HPLC overlap diagram of 15 batches of samples;

[0064] Fig.31 This is the establishment of the HPLC standard characteristic spectrum of the 1-liter platelet capsule in the experimental example of the present invention - the reference characteristic spectrum. DETAILED DESCRIPTION

[0065] The following examples are provided for further understanding of the present invention, but are not intended to limit the best mode of implementation and do not limit the content and protection scope of the present invention.

[0066] The experimental reference substances, samples, reagents, and instruments used in the embodiments of the present invention are shown in Tables 1-4.

[0067] Table 1 Test reference substances

[0068] name batch number source Indigo Reference 110716-202313 China Food and Drug Inspection Institute Agrimonia phenol B reference substance 111896-202003 China Food and Drug Inspection Institute Indigo Red Reference Substance 110717-202106 China Food and Drug Inspection Institute Paeonol reference substance 110708-202309 China Food and Drug Inspection Institute Forsythiaside Reference 110821-202318 China Food and Drug Inspection Institute Licorice glycoside reference substance 111610-202209 China Food and Drug Inspection Institute Ammonium Glycyrrhizinate Reference Substance 110731-202122 China Food and Drug Inspection Institute

[0069] Table 2 Test samples

[0070]

[0071]

[0072] Table 3 Test reagents

[0073] name batch number Grade (Purity) Manufacturer (Source) Phosphoric acid 20220710 HPLC Tianjin Komiou Chemical Reagent Co., Ltd. Phosphoric acid 20230407 HPLC Tianjin Komiou Chemical Reagent Co., Ltd. Methanol F23NBF201 HPLC Fisher Acetonitrile F23N2E201 HPLC Fisher N,N-Dimethylformamide 20075264 HPLC TEDIA Anhydrous ethanol 23095140 HPLC TEDIA water 20240401 / Wahaha water 20240517 / Wahaha

[0074] Table 4 Test equipment

[0075]

[0076] Example 1

[0077] This embodiment provides a method for constructing a characteristic map of a platelet-raising capsule, comprising the following steps:

[0078] (1) Chromatographic conditions and system suitability: Octadecylsilane bonded silica gel was used as filler (GLInertSustainAQ C 18 , 4.6mm*250mm, 5μm); acetonitrile was used as mobile phase A, 0.05% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in the following table, the flow rate was 1.0ml / min, the column temperature was 30℃, and the detection wavelength was 230nm. The theoretical plate number calculated based on the forsythiaside peak should not be less than 5000;

[0079] Table 5 Gradient elution program

[0080]

[0081] (2) Blank solvent: N,N-dimethylformamide;

[0082] (3) Preparation of test solution: Take the contents of this product, grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of N,N-dimethylformamide, stopper it tightly, weigh it, treat it with ultrasound (power 300W, frequency 40kHz) for 30 minutes, let it cool, weigh it accurately, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and obtain it;

[0083] (4) Preparation of reference solution: Take an appropriate amount of forsythiaside reference substance and add N,N-dimethylformamide to prepare a solution containing 0.1 mg forsythiaside per 1 ml;

[0084] (5) Determination method: Accurately pipette 10 μl of blank solvent, reference solution and test solution respectively, inject into liquid chromatograph, measure and record the chromatogram to obtain the characteristic spectrum of platelet-raising capsule.

[0085] An embodiment of the present invention also provides a quality detection method for platelet-raising capsules, including a characteristic spectrum of a platelet-raising capsule to be tested obtained by a method for constructing a characteristic spectrum of a platelet-raising capsule, and also including a step of comparing the characteristic spectrum of the platelet-raising capsule to be tested with a control characteristic spectrum of the platelet-raising capsule, wherein the characteristic spectrum of the platelet-raising capsule to be tested includes 7 common characteristic peaks, and the retention times are 26.470 min, 30.330 min, 71.643 min, 95.440 min, 98.547 min, 138.337 min, and 145.577 min, respectively; and the quality is judged to be qualified.

[0086] Experimental Example 1

[0087] 1. Investigation of chromatographic conditions

[0088] 1.1 Method development 1- Gradient 1

[0089] Referring to the chromatographic conditions under the content determination item under the licorice herbal medicine item in the 2020 edition of the "Chinese Pharmacopoeia", acetonitrile-0.05% phosphoric acid solution was used as the mobile phase, and the elution gradient was preliminarily formulated. The preparation method of the test solution was prepared according to its quality standard WS-10028 (ZD-0028) -2002-2012Z-2017. The method was developed and the chromatographic conditions are shown in Table 6.

[0090] Table 6 HPLC-DAD assay - Gradient 1

[0091]

[0092] (1) Preparation of reference stock solution

[0093] Stock solution of paeonol reference substance: Accurately weigh about 10 mg of paeonol reference substance, place it in a 25 ml volumetric flask, add N,N-dimethylformamide to dissolve and dilute to the scale, shake well, and obtain.

[0094] Stock solution of indigo red reference substance: Accurately weigh about 10 mg of indigo red reference substance, place it in a 20 ml volumetric flask, add N, N-dimethylformamide to dissolve and dilute to the scale, shake well, and obtain.

[0095] Ammonium glycyrrhizinate reference substance stock solution: Accurately weigh about 10 mg of ammonium glycyrrhizinate reference substance, place it in a 10 ml volumetric flask, add N,N-dimethylformamide to dissolve and dilute to the scale, shake well, and obtain.

[0096] (2) Preparation of reference solution

[0097] Paeonol reference solution: Accurately measure 5 ml of paeonol reference stock solution, place it in a 20 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0098] Indigo red reference solution: Accurately measure 3 ml of indigo red reference stock solution, place it in a 50 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0099] Ammonium glycyrrhizinate reference solution: Accurately measure 2 ml of ammonium glycyrrhizinate reference stock solution, place it in a 10 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0100] (3) Preparation of test solution: Grind the contents of 24 tablets of this product (batch number: 2401024) into powder, take out about 0.5 g, weigh accurately, place in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper tightly, weigh accurately, treat with ultrasound (300 W, 40 kHz) for 30 minutes, let cool, weigh accurately again, make up the lost weight with N,N-dimethylformamide, shake well, filter, and take the filtrate to obtain the solution.

[0101] Accurately pipette 10 μl of blank solvent (N,N-dimethylformamide), the reference solution and the test solution respectively, inject into the liquid chromatograph, use the DAD detector to perform full wavelength scan at 190-400 nm, record the chromatogram, and the results are shown in Figure 1 , the test solution 190 ~ 400nm absorption 3D scan, the results are shown in Figure 2 .

[0102] The results showed that in the UV wavelength scanning diagram (190-400nm) of each reference solution, paeonol had maximum absorption at 212nm, 228nm, and 275nm, indirubin at 206nm, 240nm, and 290nm, and glycyrrhizic acid at 252nm; in the 3D scanning diagram of the test solution, at the end of the ultraviolet absorption at 210-240nm, the absorption value was large, and more components could be detected. Therefore, combined with the maximum absorption wavelength of each reference solution, the chromatograms of the test solution at wavelengths of 230nm, 252nm, 275nm, and 290nm were extracted for comparative analysis. The results are shown in Figure 2. Figure 3 .

[0103] The results showed that at a wavelength of 230nm, the overall response was large, the number of chromatographic peaks detected was large, and glycyrrhizic acid, paeonol, and indirubin could all be detected; at a wavelength of 252nm, glycyrrhizic acid had a large absorption, but the number of other chromatographic peaks was small; at a wavelength of 275nm, paeonol had a large absorption, the number of other chromatographic peaks was also small, and glycyrrhizic acid did not peak; at a wavelength of 290nm, indirubin had a large absorption, but the overall detectable chromatographic peaks were significantly reduced, and glycyrrhizic acid did not peak. In order to ensure that the known peak glycyrrhizic acid could be detected, the wavelength of 230nm was selected for method development.

[0104] Extract each chromatographic peak at an absorption wavelength of 230nm for analysis, and the overlap diagram is shown in Figure 4 .

[0105] The results showed that in the chromatogram of the reference solution, paeonol, indirubin and glycyrrhizic acid all had good peak shapes, with a peak matching degree of 1000, indicating good peak purity; in the chromatogram of the test solution, paeonol, indirubin and glycyrrhizic acid could all be detected with good peak shapes, among which the peak matching degrees of paeonol and indirubin were both greater than 990, indicating good peak purity; the peak matching degree of glycyrrhizic acid was 979, less than 990, which did not meet the requirements; the overall number of chromatographic peaks detected in the test solution from 5 to 30 minutes was large, and the baseline was not stable, and the separation was poor, so the acetonitrile rise rate from 5 to 30 minutes was reduced, and the gradient method was adjusted to continue the investigation. At the same time, methanol was added as the extraction solvent to investigate the preparation method of the test solution.

[0106] 1.2 Method Development 2- Gradient 2

[0107] According to the above test results, the elution gradient was adjusted and the method development was continued. The chromatographic conditions are shown in Table 7.

[0108] Table 7 HPLC-DAD assay - Gradient 2

[0109]

[0110]

[0111] The preparation of the reference solution is the same as that of (2) reference solution under “1.1 Method Development 1- Gradient 1”.

[0112] The preparation of test solution 1 is the same as that of the test solution under “1.1 Method Development 1- Gradient 1”.

[0113] Preparation of test solution 2: Take 0.5004 g of the fine powder under the preparation of the test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of methanol, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0114] Accurately pipette 10 μl of blank solvent (N,N-dimethylformamide, methanol), the reference solution and the test solution, inject them into the liquid chromatograph, use the DAD detector to scan the full wavelength at 190-400 nm, record the chromatogram, and the results are shown in Figures 5-6 .

[0115] Extract the chromatogram of the test solution at 230nm wavelength for comparative analysis. The results are shown in Figure 7 .

[0116] The results showed that in the chromatograms of each reference solution, the peak shapes of paeonol, indirubin and glycyrrhizic acid were all good, and the peak matching degrees were all 1000, indicating good peak purity. In the chromatogram of test solution 1, the peak matching degree of paeonol was 1000, indicating good peak purity; the peak shape of indirubin was good, but the peak matching degree of 986 was less than 990, which did not meet the requirements; the peak shape of glycyrrhizic acid was bifurcated, and the peak matching degree of 989 was less than 990, which did not meet the requirements; the separation and baseline of the two test solutions were still poor at 20 to 40 minutes, so the overall gradient elution time was extended and the gradient method was adjusted to continue the investigation; the overall response of the chromatographic peak of the test solution extraction solvent N, N-dimethylformamide was significantly greater than that of methanol, and more chromatographic peaks were detected, so N, N-dimethylformamide was continued to be used as the extraction solvent for subsequent investigation.

[0117] 1.3 Method Development 3- Gradient 3

[0118] According to the above test results, the elution gradient was adjusted and the method development was continued. The chromatographic conditions are shown in Table 8.

[0119] Table 8 HPLC-DAD assay - Gradient 3

[0120]

[0121] Preparation of test solution: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0122] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide) and the above test solution respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, the results are shown in Figure 8 .

[0123] The results showed that in the chromatogram of the test solution, the overall separation of the chromatographic peaks was significantly improved, but the baseline from 0 to 30 minutes was still poor, so the acetonitrile increase rate from 10 to 50 minutes was reduced, and the acetonitrile increase rate from 60 to 110 minutes was slightly accelerated, and the gradient method was adjusted to continue the investigation.

[0124] 1.4 Method Development 4- Gradient 4

[0125] According to the above test results, the elution gradient was adjusted and the method development was continued. The chromatographic conditions are shown in Table 9.

[0126] Table 9 HPLC-DAD Assay - Gradient 4

[0127]

[0128]

[0129] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), reference solution under "1.1 Method Development 1-Gradient 1" and test solution under "1.3 Method Development 3-Gradient 3" respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, and the results are shown in the table. Fig. 9 .

[0130] The results showed that in the chromatogram of the reference solution, paeonol, indirubin and glycyrrhizic acid all had good peak shapes, with a peak matching degree of 1000, indicating good peak purity; in the chromatogram of the test solution, the paeonol peak matching degree was 1000, indicating good peak purity; the peak matching degrees of indirubin and glycyrrhizic acid were also greater than 990, and the peak purity met the requirements; the separation and baseline of the test solution were significantly improved in 20 to 40 minutes, and the chromatographic peaks were basically effectively separated in 50 to 110 minutes, but the overall baseline was slightly poor, and there were many small impurity peaks, so it was considered to replace the chromatographic column for investigation.

[0131] 1.5 Method Development 5- Gradient 4

[0132] The column used is GL InertSustain AQ C 18 (4.6mm*250mm, 5μm) chromatographic column, the elution method remains unchanged, and the chromatographic condition parameters are the same as Table 9.

[0133] Preparation of test solution: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0134] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide) and the above test solution respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm for comparative analysis, the results are shown in Fig.10 .

[0135] The results showed that in the chromatogram of the test solution, the overall separation of the chromatographic peaks was significantly improved, but the baseline interference was large from 10 to 50 minutes, there were many small impurity peaks, and the peak shape and peak matching were poor. Therefore, the elution was accelerated by increasing the proportion of acetonitrile from 10 to 50 minutes, and the subsequent elution time was extended to optimize the separation of the chromatographic peaks from 50 to 110 minutes.

[0136] 1.6 Method Development 6- Gradient 5

[0137] According to the above test results, the chromatographic conditions were optimized, see Table 10.

[0138] Table 10 HPLC-DAD determination - gradient 5

[0139]

[0140] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), reference solution under "1.1 Method Development 1-Gradient 1" and test solution under "1.5 Method Development 5-Gradient 4" respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, and the results are shown in the table. Fig.11 .

[0141] The results showed that in the chromatogram of the reference solution, paeonol, indirubin and glycyrrhizic acid all had good peak shapes, and the peak matching degrees were greater than 990, indicating good peak purity; in the chromatogram of the test solution, the peak matching degrees of paeonol and glycyrrhizic acid were both 1000, indicating good peak purity; the peak matching degree of indirubin was 999 greater than 990, and the peak purity met the requirements; the peaks of the test solution were too dense in 10-50 minutes, and the separation and baseline were significantly deteriorated. From 50 to 130 minutes, the separation effect of each chromatographic peak was good, and the peak matching degrees were all greater than 990, which met the requirements, but the number of peaks was small. In order to detect more chromatographic peaks that met the requirements, the elution gradient was further adjusted to reduce the acetonitrile increase rate from 10 to 60 minutes for investigation.

[0142] 1.7 Method Development 7- Gradient 6

[0143] According to the above test results, the chromatographic conditions were optimized, see Table 11.

[0144] Table 11 HPLC-DAD assay - Gradient 6

[0145]

[0146]

[0147] Preparation of test solution: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0148] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide) and the above test solution respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, the results are shown in Fig.12 .

[0149] The results showed that in the chromatogram of the test solution, the separation effect of the chromatographic peaks was significantly enhanced from 10 to 60 minutes, and basically effective separation could be achieved, but some small impurity peaks still had poor peak shapes and affected the integration of other chromatographic peaks. The baseline was also unstable from 60 to 120 minutes, and the separation effect of some chromatographic peaks was poor, with peak wrapping. Therefore, the elution gradient was further adjusted to reduce the acetonitrile increase rate from 10 to 90 minutes, and the elution time was extended for investigation.

[0150] 1.8 Method development 8- Gradient 7

[0151] According to the above test results, the chromatographic conditions were optimized, see Table 12.

[0152] Table 12 HPLC-DAD assay - Gradient 7

[0153]

[0154]

[0155] (1) Preparation of reference stock solution

[0156] Stock solution of Forsythiaside reference substance: Accurately weigh about 10 mg of Forsythiaside reference substance, place it in a 10 ml volumetric flask, add N,N-dimethylformamide to dissolve and dilute to the scale, shake well, and obtain.

[0157] Licorice reference substance stock solution: Accurately weigh about 10 mg of licorice reference substance, place it in a 20 ml volumetric flask, add N, N-dimethylformamide to dissolve and dilute to the scale, shake well, and obtain.

[0158] Preparation of Agrimonia phenol B reference substance stock solution: Accurately weigh about 10 mg of Agrimonia phenol B reference substance, place it in a 10 ml volumetric flask, add N, N-dimethylformamide to dissolve and dilute to the scale, shake well, and obtain.

[0159] Indigo reference substance stock solution: Accurately weigh about 10 mg of indigo reference substance, place it in a 25 ml volumetric flask, add N,N-dimethylformamide to dissolve and dilute to the scale, shake well, and obtain.

[0160] (2) Preparation of reference solution

[0161] Forsythiaside reference solution: Accurately measure 2 ml of Forsythiaside reference stock solution, place it in a 10 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0162] Licorice glycoside reference solution: Accurately measure 2 ml of licorice glycoside reference stock solution, place it in a 50 ml volumetric flask, add N, N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0163] Agrimonia phenol B reference substance solution: Accurately measure 2 ml of Agrimonia phenol B reference substance stock solution, place it in a 10 ml volumetric flask, add N, N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0164] Indigo reference solution: Accurately measure 5 ml of indigo reference stock solution, place it in a 20 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0165] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), reference solution under "1.1 Method Development 1- Gradient 1", the above reference solution and test solution under "1.7 Method Development 7- Gradient 6" respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, and the results are shown in the table. Fig.13 .

[0166] Extract the chromatogram at 230nm wavelength for comparative analysis. The results are shown in Fig.14 .

[0167] The results showed that in the chromatogram of the reference solution, paeonol, indirubin, glycyrrhizic acid, forsythiaside and liquiritin all had good peak shapes, with a peak matching degree of 1000, indicating good peak purity, and herbicide B was not detected. The indigo peak matching degree was 999, which met the requirements, but the peak shape was poor and the tailing was serious; in the chromatogram of the test solution, paeonol, indirubin and glycyrrhizic acid peak matching degrees were all greater than 990, indicating good peak purity, and forsythiaside and indigo peak matching degrees were both less than 990, which did not meet the requirements. The ultraviolet absorption wavelength of the peak corresponding to liquiritin in the test sample was extracted, and the results showed that the absorption values ​​were inconsistent, so the chromatographic peak in the test sample was not liquiritin, and liquiritin and herbicide B were not detected; overall, the chromatographic peaks were basically able to achieve effective separation, but there were many small impurity peaks from 70 to 100 minutes, which affected the integration, so the acetonitrile increase rate was reduced and the investigation continued.

[0168] 1.9 Method Development 9- Gradient 8

[0169] According to the above test results, the chromatographic conditions were adjusted, see Table 13.

[0170] Table 13 HPLC-DAD assay - gradient 8

[0171]

[0172] Preparation of test solution: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0173] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide) and the above test solution respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, the results are shown in Fig.15 .

[0174] The results showed that in the chromatogram of the test solution, the separation effect of the chromatographic peaks was significantly enhanced from 45 to 90 minutes, but there were still some small impurity peaks with poor peak shape and affected the integration of other chromatographic peaks, so the elution gradient was further adjusted for investigation.

[0175] 1.10 Method Development 10- Gradient 9

[0176] According to the above test results, the chromatographic conditions were adjusted, see Table 14.

[0177] Table 14 HPLC-DAD assay - gradient 9

[0178]

[0179] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), ammonium glycyrrhizinate reference solution under "1.1 Method Development 1-Gradient 1", forsythiaside and indigo reference solution under "1.8 Method Development 8-Gradient 7", and test solution under "1.9 Method Development 9-Gradient 8", respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, and the results are shown in the table. Fig.16 .

[0180] The results showed that in the chromatogram of the reference solution, glycyrrhizic acid and forsythin had good peak shapes and a peak matching degree of 1000, indicating that the peak purity was good, and the indigo peak matching degree was 1000, which met the requirements, but the peak shape was poor and the tailing was serious; in the chromatogram of the test solution, the glycyrrhizic acid, forsythin and indigo peak matching degrees were all greater than 990, indicating that the peak purity was good, but forsythin had obvious peak wrapping and the integration was not accurate enough; overall, the chromatographic peaks were basically able to achieve effective separation, but the peaks were too dense in 20 to 40 minutes, and the baseline was not stable enough, affecting the integration, so the acetonitrile increase rate was reduced. Since there were more chromatographic peaks with high polarity eluted from 0 to 10 minutes, the initial gradient acetonitrile ratio was increased and the investigation continued.

[0181] 1.11 Method Development 11- Gradient 10

[0182] According to the above test results, the chromatographic conditions were adjusted, see Table 15.

[0183] Table 15 HPLC-DAD assay - gradient 10

[0184]

[0185] Preparation of test solution: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0186] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide) and the above test solution respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, the results are shown in Fig.17 .

[0187] The results showed that in the chromatogram of the test solution, the separation effect of each chromatographic peak was improved at 30 to 50 minutes. Some chromatographic peaks with larger peak areas were greatly interfered by other chromatographic peaks with smaller peak areas, and the peak matching degree did not meet the requirements. Therefore, the acetonitrile increase rate was further reduced from 30 to 50 minutes, and the elution gradient was adjusted for investigation.

[0188] 1.12 Method Development 12- Gradient 11

[0189] According to the above test results, the chromatographic conditions were adjusted, see Table 16.

[0190] Table 16 HPLC-DAD assay - gradient 11

[0191]

[0192] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), ammonium glycyrrhizinate reference solution under "1.1 Method Development 1-Gradient 1", forsythiaside and indigo reference solution under "1.8 Method Development 8-Gradient 7", and test solution under "1.11 Method Development 11-Gradient 10", inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, and the results are shown in the table. Fig.18 .

[0193] The results showed that in the chromatogram of the reference solution, glycyrrhizic acid and forsythin had good peak shapes and a peak matching degree of 1000, indicating good peak purity. The indigo peak matching degree was 1000, which met the requirements, but the peak shape was poor and the tailing was serious. In the chromatogram of the test solution, the indigo and forsythin peak matching degrees were both greater than 990, indicating good peak purity. However, forsythin had obvious peak inclusion, and the integration was not accurate enough. The glycyrrhizic acid peak matching degree of 621 was less than 990, which did not meet the peak purity requirements. The overall separation of the chromatographic peaks was significantly better. In order to improve the peak shape of forsythin and enable it to be effectively separated from the included chromatographic peak, the 90-110 minutes were adjusted to isocratic elution for further investigation.

[0194] 1.13 Method Development 13- Gradient 12

[0195] According to the above test results, the chromatographic conditions were adjusted, see Table 17.

[0196] Table 17 HPLC-DAD assay - Gradient 12

[0197]

[0198] Preparation of test solution: Take about 0.50 of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0199] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide) and the above test solution respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, the results are shown in Fig.19 .

[0200] The results showed that in the chromatogram of the test solution, the overall separation of the chromatographic peaks was good, but there were more substances with higher polarity from 0 to 30 minutes. In order to elute them quickly, the acetonitrile ratio of the initial gradient was increased and the gradient elution time was shortened to continue the investigation.

[0201] 1.14 Method Development 14- Gradient 13

[0202] According to the above test results, the chromatographic conditions were adjusted, see Table 18.

[0203] Table 18 HPLC-DAD assay - gradient 13

[0204]

[0205]

[0206] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), paeonol, indirubin, and ammonium glycyrrhizinate reference solution under "1.1 Method Development 1-Gradient 1", forsythiaside, indigo, and agrimonol B reference solution under "1.8 Method Development 8-Gradient 7", and test solution under "1.13 Method Development 13-Gradient 12", inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, and the results are shown in the table. Fig. 20 .

[0207] The results showed that in the chromatogram of the reference solution, indirubin, paeonol, glycyrrhizic acid, and forsythin all had good peak shapes, and the peak matching degree was 1000, indicating good peak purity. The indigo peak matching degree was 1000, which met the requirements, but the peak shape was poor, the tailing was serious, and agrimonol B was not detected; in the chromatogram of the test solution, the peak matching degrees of indigo (peak 10), indirubin (peak 11), paeonol (peak 7), glycyrrhizic acid (peak 9), and forsythin (peak 4) were all greater than 990, indicating good peak purity, but forsythin (peak 4) had obvious peak inclusion, and the integration was not accurate enough; the separation effect of unknown peaks 2 and 3 was poor, and the peak 2 matching degree was 988 less than 990, which did not meet the requirements. The separation effect of the remaining chromatographic peaks was good, and the peak purity was greater than 990, which met the requirements. In order to reduce baseline interference and achieve effective separation of peaks 2 and 3, the initial gradient elution time was extended and the acetonitrile increase rate was reduced from 20 to 40 minutes, and the investigation was continued.

[0208] 1.15 Method Development 15- Gradient 14

[0209] According to the above test results, the chromatographic conditions were adjusted, see Table 19.

[0210] Table 19 HPLC-DAD assay - gradient 14

[0211]

[0212]

[0213] Preparation of test solution: Take 0.5041 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0214] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide) and the above test solution respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm for comparative analysis, the results are shown in Fig.21 .

[0215] The results showed that in the chromatogram of the test solution, unknown peaks 2 and 3 were effectively separated, and the matching degrees were greater than 990, which met the requirements. The separation effects of the other chromatographic peaks were also good, but the matching degree of peak 5 was 968, less than 990, which did not meet the requirements. In order to reduce baseline interference and make the separation effect of peaks 6 and 7 better, the acetonitrile increase rate was reduced from 60 to 90 minutes, and the investigation was continued.

[0216] 1.16 Method Development 16- Gradient 15

[0217] According to the above test results, the chromatographic conditions were adjusted, see Table 20.

[0218] Table 20 HPLC-DAD determination - gradient 15

[0219]

[0220]

[0221] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), ammonium glycyrrhizinate reference solution under "1.1 Method Development 1-Gradient 1", forsythiaside and indigo reference solution under "1.8 Method Development 8-Gradient 7", and test solution under "1.15 Method Development 15-Gradient 14", respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, and the results are shown in the table. Fig. 22 .

[0222] The results showed that in the chromatogram of the test solution, the peak shapes of the 11 chromatographic peaks were good, and the peak matching degrees were all greater than 990, indicating that the peak purity was good, but the retention times of peaks 1, 2, and 3 were too close to the impurity peaks of 0 to 20 minutes, and the baseline of 40 to 65 minutes was poor, and peak 4 had a peak wrapping phenomenon, which affected the accuracy of the integration. In order to reduce interference, the time of the initial elution ratio was extended and the elution rate of 40 to 60 minutes was reduced, and the investigation continued.

[0223] 1.17 Method Development 17- Gradient 16

[0224] According to the above test results, the chromatographic conditions were adjusted, see Table 21.

[0225] Table 21 HPLC-DAD assay - gradient 16

[0226]

[0227]

[0228] Preparation of test solution: Take 0.5029 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0229] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide) and the above test solution respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, the results are shown in Fig.23 .

[0230] The results showed that 11 chromatographic peaks were detected in the chromatogram of the test solution. The peak shape and separation of each chromatographic peak were good, and the peak matching degree was greater than 990, indicating that the peak purity was good, but there was a small packet peak after peak 10, which may affect the integration. In order to reduce interference, the acetonitrile increase rate was slightly reduced from 130 to 150 minutes for investigation and optimization.

[0231] 1.18 Method Development 18 - Gradient 17

[0232] According to the above test results, the chromatographic conditions were adjusted, see Table 22.

[0233] Table 22 HPLC-DAD assay - gradient 17

[0234]

[0235]

[0236] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), ammonium glycyrrhizinate reference solution under "1.1 Method Development 1-Gradient 1", forsythiaside and indigo reference solution under "1.8 Method Development 8-Gradient 7", and test solution under "1.17 Method Development 17-Gradient 16", inject into liquid chromatograph, measure with DAD detector, record chromatogram, extract chromatogram at 230 nm wavelength for comparative analysis, and the results are shown in the table. Fig.24 .

[0237] The results showed that in the chromatogram of the reference solution, the peak matching degrees of forsythiaside, glycyrrhizic acid and indigo were all 1000, indicating good peak purity; in the chromatogram of the test solution, a total of 11 chromatographic peaks were detected, and the peak shape and separation of each chromatographic peak were good, and the peak matching degrees were all greater than 990, indicating good peak purity, indicating that the gradient method is suitable for the determination of the characteristic spectrum of this product, and the preparation method of the test solution was further investigated and optimized.

[0238] 2. Ultrasonication time for preparation of test solution

[0239] The test solution was extracted by ultrasonic method, and different ultrasonic times were set for investigation. Other chromatographic conditions were the same as Table 22.

[0240] Preparation of test solution 1: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 20 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0241] Preparation of test solution 2: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0242] Preparation of test solution 3: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 40 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0243] Preparation of test solution 4: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 50 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0244] Preparation of test solution 5: Take about 0.5 g of the fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 60 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0245] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), paeonol, indirubin, ammonium glycyrrhizinate reference solution under "1.1 Method Development 1-Gradient 1", forsythiaside, indigo reference solution under "1.8 Method Development 8-Gradient 7", and the above test solution 1-5, respectively, inject into liquid chromatograph, measure with DAD detector, record chromatogram, and the results are shown in the table. Fig.25 .

[0246] Extract the chromatogram at 230nm wavelength for comparative analysis. The results are shown in Fig.26 The peak areas of each chromatographic peak are shown in Table 23.

[0247] Table 23 Peak area results of test solution

[0248] name Test solution 1 Test solution 2 Test solution 3 Test solution 4 Test solution 5 Unknown Peak 1 31.5708 31.9240 31.6587 32.4907 31.8848 Unknown Peak 2 33.4417 33.7757 33.5259 34.2309 33.6629 Unknown Peak 3 39.9239 40.2381 39.1083 40.1068 38.5079 Forsythiaside 49.7917 50.1312 49.9811 50.8813 49.9999 Unknown Peak 5 8.8093 9.0143 8.7930 9.0091 8.8204 Paeonol 249.6740 252.2068 250.2383 255.2238 251.4805 Unknown Peak 7 21.3589 21.5588 21.4701 21.5965 21.1612 Unknown Peak 8 8.7535 8.8782 8.8922 9.0366 8.8337 Glycyrrhizic acid 3.8206 3.7314 3.6717 3.7795 3.6359 Indigo 28.7232 28.2458 28.5899 28.6521 27.3865 Indigo 14.1034 14.2235 14.3601 14.5414 14.2875

[0249] The results showed that in the chromatogram of the reference solution, the peak matching of the five reference solutions was 1000, indicating good peak purity; in the chromatograms of the five test solutions, 11 chromatographic peaks were detected, and five known peaks were identified, namely, forsythin (peak 4), paeonol (peak 6), glycyrrhizic acid (peak 9), indigo (peak 10) and indirubin (peak 11). The peak shape and separation of each chromatographic peak were good, and the peak matching was greater than 990, indicating good peak purity. There was no significant difference in the peak area of ​​each chromatographic peak of the five test solutions, indicating that the ultrasonic time had no effect on the preparation of the test solution, so the moderate ultrasonic time of 30 minutes was selected for the peak attribution determination and methodological verification of the unknown peaks.

[0250] 3. Peak attribution

[0251] In order to attribute the 11 chromatographic peaks marked in the test solution, five medicinal materials, namely, Indigo Naturalis, Forsythia suspensa, Herba Agrimoniae, Cortex Moutan and Glycyrrhizae, were taken for determination, and the preparation was carried out according to the quality standard of Shengxuexiaoban Capsules WS-10028 (ZD-0028) -2002-2012Z-2017 and the method specified under "2 Ultrasonic time investigation for preparation of test solution".

[0252] Preparation of Indigo Naturalis Medicinal Material Solution: Take about 0.19 g of ground Indigo Naturalis medicinal material (equivalent to the amount of Indigo Naturalis medicinal material in 0.5 g of Shengxuexiaoban Capsule), accurately weigh it, put it in a 20 ml volumetric flask, accurately add an appropriate amount of N, N-dimethylformamide, seal it, weigh it, and treat it with ultrasound (300 W, 40 kHz) for 30 minutes. Take it out, let it cool, dilute it to the scale with N, N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain the solution.

[0253] Preparation of Forsythia suspensa solution: Take about 56g (0.11 times the prescribed amount) of crushed Forsythia suspensa medicinal material, add 336ml (6 times the amount) of water and soak for 4 hours, extract volatile oil by steam distillation for 6 hours, collect 1.20ml of volatile oil and store it in another container, and use the decoction for later use. Add 280ml (5 times the amount) of water to the residue and boil it twice, each time for 1.5 hours, filter, combine the three decoctions, mix well, measure the volume to 748ml, measure 7.5ml of the filtrate (equivalent to the amount of Forsythia suspensa in 0.5g of Shengxuexiaoban Capsule), put it in an evaporating dish, evaporate it to dryness in a water bath, add an appropriate amount of N,N-dimethylformamide to the residue to dissolve it, and transfer it to a 20ml volumetric flask, add 20μl of volatile oil, and dilute to the scale with N,N-dimethylformamide, shake well, filter, and take the filtrate.

[0254] Preparation of Herba Agrimoniae decoction pieces solution: Take about 5.6 g of Herba Agrimoniae (0.011 times the prescribed amount), weigh it accurately, place it in a stoppered conical flask, add 28 ml of 80% ethanol (5 times the amount), extract twice, each time for 2 hours, filter, combine the two ethanol extracts, mix well, measure a volume of 40 ml, measure 4 ml of the filtrate (equivalent to the amount of Herba Agrimoniae decoction pieces in 0.5 g of Shengxuexiaoban Capsule), place it in an evaporating dish, evaporate to dryness in a water bath, add an appropriate amount of N, N-dimethylformamide to the residue to dissolve it, and transfer it to a 20 ml volumetric flask, dilute to the scale with N, N-dimethylformamide, shake well, filter, and take the filtrate to obtain.

[0255] Preparation of peony bark slice solution: Take about 9.2g of crushed peony bark medicinal material, weigh it accurately, put it in a stoppered conical flask, add 46ml (5 times the amount) of 80% ethanol, extract twice, each time for 2 hours, filter, combine the two ethanol extracts, mix well, measure the volume to 73ml, measure 7.3ml of the filtrate (equivalent to the amount of peony bark slice in 0.5g of platelet-raising capsule), put it in an evaporating dish, evaporate to dryness in a water bath, add an appropriate amount of N,N-dimethylformamide to the residue to dissolve it, and transfer it to a 20ml volumetric flask, dilute to the scale with N,N-dimethylformamide, shake well, filter, and take the filtrate.

[0256] Preparation of licorice slice solution: Take about 28g of licorice material (0.11 times the prescribed amount), add 140ml of water (5 times the amount), decoct twice, each time for 1.5 hours, filter, combine the two decoctions, mix well, measure a volume of 222ml, measure 2.2ml of the filtrate (equivalent to the amount of licorice slices in 0.5g of Shengxuexiaoban Capsule), place in an evaporating dish, evaporate to dryness in a water bath, add an appropriate amount of N,N-dimethylformamide to the residue to dissolve it, and transfer it to a 20ml volumetric flask, dilute to the scale with N,N-dimethylformamide, shake well, filter, and take the filtrate.

[0257] Preparation of test solution: Take 0.5053 g of fine powder under the preparation of test solution in "1.1 Method Development 1-Gradient 1", accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and take the filtrate to obtain it.

[0258] Accurately pipette 10 μl of blank solvent (N, N-dimethylformamide), each of the above medicinal material solutions and the test solution, inject them into the liquid chromatograph, measure them with the DAD detector, record the chromatogram, and the results are shown in the table. Fig. 27 .

[0259] The results showed that among the 11 characteristic peaks marked in the chromatogram of the test sample, peaks 1, 4, 5, and 8 belonged to Forsythia suspensa; peak 9 belonged to Licorice; peaks 2, 3, 6, and 7 belonged to Paeonia suffruticosa; and peaks 10 and 11 belonged to Indigo naturalis. All chromatographic peaks were confirmed to belong to each medicinal flavor, among which no corresponding chromatographic peaks were detected in Agrimonia scabra, which was related to the main components contained in the medicinal material itself. The main components of Agrimonia scabra were flavonoids and phloroglucinol. Among them, Agrimonia phenol B was positioned and analyzed during the method development, and Agrimonia phenol B was not detected. The analysis results can be found in the conclusion under "1.14 Method Development 14-Gradient 13". Due to the characteristics of the ingredients and the extraction process, it is not controlled for the time being. In summary, the established characteristic spectrum method effectively locates the product and is suitable for overall control of product quality.

[0260] 4. HPLC characteristic spectrum method validation

[0261] 4.1 Exclusivity

[0262] Chromatographic conditions and system suitability tests were performed using octadecylsilane bonded silica gel as filler (GLInertSustain AQ C 18 , 4.6mm*250mm, 5μm); acetonitrile as mobile phase A, 0.05% phosphoric acid as mobile phase B; flow rate of 1.0ml / min, column temperature of 30°C, detection wavelength of 230nm, theoretical plate number calculated based on forsythiaside peak should not be less than 5000. The elution gradient is shown in Table 24.

[0263] Table 24 Elution gradient

[0264]

[0265] Preparation of Forsythiaside Reference Stock Solution Accurately weigh about 10 mg of Forsythiaside Reference, place in a 20 ml volumetric flask, add an appropriate amount of N,N-dimethylformamide to dissolve, dilute to the scale, shake well, and obtain.

[0266] Preparation of the stock solution of paeonol reference substance: Accurately weigh about 10 mg of paeonol reference substance, place it in a 20 ml volumetric flask, add an appropriate amount of N,N-dimethylformamide to dissolve it, dilute to the scale, shake well, and obtain.

[0267] Preparation of ammonium glycyrrhizinate reference stock solution: Accurately weigh about 10 mg of ammonium glycyrrhizinate reference, place in a 20 ml volumetric flask, add an appropriate amount of N,N-dimethylformamide to dissolve, dilute to the scale, shake well, and obtain the stock solution.

[0268] Preparation of indigo reference stock solution: Accurately weigh about 10 mg of indigo reference, place in a 20 ml volumetric flask, add an appropriate amount of N,N-dimethylformamide to dissolve, dilute to the scale, shake well, and obtain.

[0269] Preparation of Indigo Red Reference Stock Solution Accurately weigh about 10 mg of Indigo Red Reference, place in a 25 ml volumetric flask, add an appropriate amount of N,N-dimethylformamide to dissolve, dilute to the scale, shake well, and obtain.

[0270] Preparation of Forsythiaside Reference Solution Accurately measure 5 ml of Forsythiaside Reference Stock Solution, place in a 25 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0271] Preparation of paeonol reference solution: Accurately measure 5 ml of paeonol reference stock solution, place in a 25 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0272] Preparation of ammonium glycyrrhizinate reference solution: Accurately measure 5 ml of ammonium glycyrrhizinate reference stock solution, place it in a 25 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0273] Preparation of indigo reference solution: Accurately measure 5 ml of indigo reference stock solution, place it in a 25 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0274] Preparation of Indigo Red Reference Solution: Accurately measure 2 ml of Indigo Red reference stock solution, place in a 25 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0275] Preparation of mixed reference solution: Accurately measure 5 ml each of forsythiaside, paeonol, ammonium glycyrrhizate, indigo reference stock solution, and 2 ml of indirubin reference stock solution, place in a 25 ml volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain.

[0276] Preparation of test solution: Take the contents of this product (batch number: 2401024), grind it into powder, take out about 0.5 g, weigh it accurately, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it tightly, weigh it, treat it with ultrasound (power 300 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with N,N-dimethylformamide, shake it well, filter it, and obtain it.

[0277] Determination method: Precisely aspirate 10 μl of blank solvent (N,N-dimethylformamide), the above-mentioned reference solution, mixed reference solution and test solution, inject into liquid chromatograph, and determine. The results are shown in the table. Fig.28 .

[0278] The standard stipulates that the blank solvent should not interfere with the determination of the characteristic spectrum.

[0279] The results showed that 11 characteristic peaks were detected, and the blank solvent had no interference in the determination of the characteristic spectrum of Shengxuexiaoban Capsules.

[0280] 4.2 Instrument precision

[0281] Accurately pipette 10 μl of the mixed reference solution under “4.1 Specificity” and inject it into the liquid chromatograph. Measure three times continuously. The results are shown in Tables 25 and 26.

[0282] The standard stipulates: the retention time RSD of each control peak is ≤1.0% (n=3); the peak area RSD is ≤3.0% (n=3).

[0283] Table 25 Instrument precision test results - retention time

[0284] Serial number Forsythiaside Paeonol Glycyrrhizic acid Indigo Indigo Mixed reference solution 1 71.623 95.427 125.273 138.257 145.577 Mixed reference solution 2 71.643 95.380 125.310 138.273 145.597 Mixed reference solution 3 71.630 95.347 125.297 138.270 145.600 RSD(%) 0.01 0.04 0.01 0.01 0.01

[0285] Table 26 Instrument precision test results - peak area

[0286] Serial number Forsythiaside Paeonol Glycyrrhizic acid Indigo Indigo Mixed reference solution 1 31.8992 57.0733 6.7584 17.4922 19.6183 Mixed reference solution 2 31.9263 57.1145 6.7648 17.3972 19.6358 Mixed reference solution 3 32.0127 57.2016 6.7677 16.7378 19.6868 RSD(%) 0.19 0.11 0.07 2.39 0.18

[0287] The results showed that after three consecutive injections of the mixed reference solution, the retention time RSD of each reference peak was less than 1.0%; the peak area RSD was less than 3.0%, indicating that the instrument precision of this method was good.

[0288] 4.3 Repeatability

[0289] Take the contents of this product (batch number: 2401024), grind it into powder, take about 0.5g, prepare 6 portions in parallel according to the preparation method of the test solution under "4.1 Specificity", accurately pipette 10μl of each test solution, inject it into the liquid chromatograph, and measure it. Take forsythin as the S peak, and calculate the relative retention time and relative peak area of ​​each main chromatographic peak and the S peak. The results are shown in Table 27 and Table 28.

[0290] Note: Repeatability 1 is the same as the sample solution spectrum under “4.1 Specificity”.

[0291] The standard stipulates that the relative retention time RSD of each main chromatographic peak of 6 test solution is ≤2.0% (n=6), and the relative peak area RSD is ≤10.0% (n=6).

[0292] Table 27 Repeatability test results - relative retention time

[0293]

[0294]

[0295] Table 28 Repeatability test results - relative peak area

[0296]

[0297] The results showed that the relative retention time RSD of each main chromatographic peak of 6 test sample solutions was less than 2.0%, and the relative peak area RSD was less than 10.0%, indicating that the method had good repeatability.

[0298] 4.4 Intermediate precision

[0299] The effects of different dates, different analysts, and different instruments on precision were investigated.

[0300] Take the contents of this product (batch number: 2401024), grind it into powder, take about 0.5g, prepare 6 portions in parallel according to the preparation method of the test solution under "4.1 Specificity", accurately pipette 10μl of each test solution, inject it into the liquid chromatograph, and measure it. Take forsythin as the S peak, and calculate the relative retention time and relative peak area of ​​each main chromatographic peak and the S peak. The results are shown in Table 29 and Table 30.

[0301] The standard stipulates: 6 samples of the test solution relative retention time RSD ≤ 2.0%, relative peak area RSD ≤ 10.0% (n = 6). And under the repeatability item, a total of 12 samples of the test solution relative retention time RSD ≤ 3.0% (n = 12).

[0302] Table 29 Intermediate precision test results - relative retention time

[0303]

[0304]

[0305] Table 30 Intermediate precision test results - relative peak area

[0306]

[0307] The results showed that the intermediate precision of the method was good, with the RSD of the relative retention time of the 6 test solutions less than 2.0%, and the RSD of the relative peak area less than 10.0%. The RSD of the relative retention time of the main chromatographic peaks of the 12 test solutions under the repeatability item was less than 3.0%.

[0308] 4.5 Durability

[0309] 4.5.1 Test solution stability test

[0310] Take the test solution under "4.1 Specificity", place it at room temperature, and accurately draw 10μl at 0h, 8h, 19h, 27h, 36h, and 50h, inject it into the liquid chromatograph, and measure it. Take forsythiaside as the S peak, and calculate the relative retention time and relative peak area of ​​each main chromatographic peak and the S peak. The results are shown in Table 31 and Table 32.

[0311] The standard stipulates: The test solution is placed at room temperature for a period of time, and the relative retention time of each major chromatographic peak of the test solution is required to be RD≤2.0% compared with the result of 0h measurement, and the relative peak area RD≤10.0%.

[0312] Table 31 Stability test results - relative retention time

[0313]

[0314]

[0315] Table 32 Stability test results - relative peak area

[0316]

[0317]

[0318] The results showed that when the test solution was placed at room temperature for 50 hours, the relative retention time of each main chromatographic peak was less than 2.0% RD compared with the 0h detection result, and the relative peak area RD was less than 10.0%, indicating that the test solution had good stability when placed at room temperature for 50 hours.

[0319] 4.5.2 Chromatographic Conditions Robustness

[0320] The impact of slight changes in chromatographic conditions on the proposed method was investigated. The experimental conditions are shown in Table 33.

[0321] Table 33 Durability test conditions

[0322]

[0323] The standard stipulates: Taking the average relative retention time under "4.3 Repeatability" as the standard condition, the test solution should detect 11 corresponding chromatographic peaks under each condition. The relative retention time of each chromatographic peak should be within ±8% of the standard condition, and the relative peak area of ​​each chromatographic peak should be within ±15% of the standard condition. The results are shown in Tables 34 and 35.

[0324] Table 34 Durability test results - relative retention time

[0325]

[0326]

[0327] Table 35 Durability test results - relative peak area

[0328]

[0329]

[0330] The results showed that 11 main chromatographic peaks were detected under each condition, and the results measured by changing the column temperature and flow rate were all within the range of ±8% of the relative retention time measured under standard conditions; after replacing the chromatographic column of the same brand and model, the relative peak area and relative retention time of each main chromatographic peak were compared with the results measured under standard conditions, and the difference was not large, which met the needs of product quality comparison; after replacing the chromatographic column of the same brand but different models, except for the model GL Inertsil ODS-3, the relative retention time of each main chromatographic peak was compared with the relative retention time measured under standard conditions, and the results were quite different, but the relative peak area of ​​some chromatographic peaks (peak 2, peak 5, peak 6, peak 9) of the model GL Inertsil ODS-3 was compared with the results measured under standard conditions, and the difference was also large. In order to better control the product quality, the chromatographic column model needs to be fixed, which is GL InertSustain AQ C 18 (4.6mm*250mm, 5μm).

[0331] 5. Testing of 15 batches of finished products of platelet-raising capsules

[0332] Referring to the characteristic spectrum methodology validation method, 15 batches of samples of Shengxuexiaoplatelet Capsules were measured. The peak corresponding to the forsythin reference peak in the chromatogram was taken as the S peak. The relative retention time of each characteristic peak and the S peak was calculated. The calculation results are shown in Table 36. The peak area and peak matching results are shown in Tables 37 and 38.

[0333] Table 36 Test results of 15 batches of samples - relative retention time

[0334]

[0335] Table 37 Test results of 15 batches of samples - Peak area

[0336]

[0337] Table 38 15 batches of sample test results - peak matching

[0338]

[0339]

[0340] The results showed that 11 characteristic peaks corresponding to the control characteristic spectrum were present in the chromatograms of the 15 batches of samples; however, in the chromatogram of the raw platelet capsule (batch number: 2310207), the peak area of ​​the unknown peak 3 (peak 3) was only 4.3429, which was much lower than the peak area of ​​the same chromatogram of other batches, and the peak matching degree was less than 990, indicating that the chromatographic peak was impure; in the chromatogram of the raw platelet capsule (batch number: 2305079, 2305086), the peak matching degree of the unknown peak 4 (peak 5) was less than 990; and the raw platelet capsule ( In the chromatogram of the batch number: 2304054), the peak matching degree of unknown peak 6 (peak 8) is less than 990, indicating that the chromatographic peak is impure; in the chromatogram of platelet-producing capsules (batch numbers: 2207108, 2303036, 2304054, 2304064, 2304065, 2305079, 2309175, 2310207, 2311219, 2311220), the peak matching degree of glycyrrhizic acid (peak 9) is less than 990 due to the low peak area, indicating that the chromatographic peak is impure. Therefore, the chromatographic peaks that do not meet the peak purity requirements after removing the common peaks of the 15 batches of finished products are unknown peak 3 (peak 3), unknown peak 4 (peak 5), unknown peak 6 (peak 8), and glycyrrhizic acid (peak 9).

[0341] The chromatograms of 15 batches of Shengxuexiaoban Capsules samples were analyzed in the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) to generate a reference spectrum and match it with it. The peak matching diagram is shown in Fig.29 . The similarity between the characteristic spectrum of each sample and the generated control spectrum was calculated, and the results are shown in Table 39.

[0342] Table 39 Similarity comparison results

[0343] batch number 2207108 2303036 2304054 2304058 2304064 Similarity 0.996 0.999 0.996 1.000 0.998 batch number 2304065 2305074 2305079 2305086 2308157 Similarity 0.999 0.999 0.997 0.995 0.992 batch number 2309175 2310207 2311219 2311220 2401024 Similarity 0.991 0.994 0.999 0.998 0.993

[0344] The results showed that the similarities between the 15 batches of samples and the control spectra were all greater than 0.90. Therefore, from the overall spectral peak information, it can be seen that the types and quantities of chemical components contained in each test sample are basically the same.

[0345] 6. Establishment of HPLC standard characteristic spectrum

[0346] According to the test results under "5. Testing of 15 batches of finished products of platelet capsules", a standard characteristic spectrum was established, and the similarity evaluation system of Chinese medicine chromatographic fingerprint spectrum (2012 version) was used to generate a reference spectrum. Fig.30 , Fig.31 .

[0347] The results showed that the chromatographic peaks with good stability and appropriate response values ​​in the characteristic spectra of 15 batches of samples were selected as common peaks. A total of 7 common peaks were calibrated. By comparing the retention times of the peaks in the chromatograms of the test samples and the mixed reference samples, 4 of them can be identified, namely forsythin (peak 3), paeonol (peak 4), indigo (peak 6), and indirubin (peak 7). Among them, the peak 3 has a moderate elution time, a moderate peak area, a good peak shape, and good separation, so peak 3 is selected as the reference peak (S peak). The average relative retention time of 15 samples is taken as the specified value, and the specified values ​​are: 0.37 (peak 1), 0.42 (peak 2), 1.00 (peak 3), 1.33 (peak 4), 1.37 (peak 5), 1.93 (peak 6), and 2.03 (peak 7). Based on the results of the previous methodological validation investigation, the relative retention time is tentatively determined to be within ±8% of the specified value.

[0348] in conclusion:

[0349] The characteristic spectrum / fingerprint of platelet-raising capsules developed by this method has certain specificity, and the method has good durability and reproducibility. It can provide a basis for quality control of platelet-raising capsules, and be used to evaluate the uniformity, excellence and stability of preparation quality, and compare the consistency of product quality before and after changes.

[0350] The characteristic spectrum / fingerprint test results of each batch of platelet-raising capsules showed that corresponding characteristic peaks were detected in different batches of platelet-raising capsules, and the fingerprint spectrum similarity was greater than 0.90, indicating that the product quality was uniform and stable.

[0351] The characteristic spectrum method of platelet-raising capsules was developed and verified. The established method is scientific, feasible, and advanced. The verification results are scientific, reasonable, and accurate, and can meet the multi-component and overall quality control of the product.

Claims

1. A characteristic spectrum of a platelet-raising capsule and a method for constructing the same, characterized in that: The following steps are involved: (1) Chromatographic conditions and system suitability: Octadecylsilane bonded silica gel was used as filler; Use acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, perform gradient elution as specified in the following table, flow rate is 1.0ml / min, column temperature is 30℃, detection wavelength is 230nm, and the theoretical plate number calculated based on the forsythiaside peak should be no less than 5000; (2) Blank solvent: N,N-dimethylformamide; (3) Preparation of test solution: Take the contents of this product, grind it into powder, take about 0.5 g, weigh it accurately, put it in a stoppered conical flask, accurately add 20 ml of N,N-dimethylformamide, stopper it tightly, weigh it accurately, treat it with ultrasound for 30 minutes, let it cool, Weigh the weight, make up the lost weight with N,N-dimethylformamide, shake well, filter and obtain; (4) Preparation of reference solution: Take an appropriate amount of forsythiaside reference substance and add N,N-dimethylformamide to prepare a solution containing 0.1 mg forsythiaside per 1 ml; (5) Determination method: Accurately pipette 10 μl of blank solvent, reference solution and test solution respectively, inject into liquid chromatograph, measure and record the chromatogram to obtain the characteristic spectrum of platelet-raising capsule.

2. The method for constructing the characteristic spectrum of the platelet-raising capsule according to claim 1, characterized in that: The chromatographic column used in step (1) is selected from GL Inert Sustain AQ C 18 The column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm.

3. The method for constructing the characteristic spectrum of the platelet-raising capsule according to claim 1, characterized in that: The conditions of the ultrasonic treatment used in step (3) are: power 300W, frequency 40KHz.

4. A characteristic spectrum of a platelet-raising capsule, characterized in that: Obtained by the construction method described in any one of claims 1-3.

5. A control characteristic spectrum of platelet-raising capsule, characterized in that: Select any one of the following (1)-(3): (1) It has 7 common characteristic peaks, and the retention times are 26.470min, 30.330min, 71.643min, 95.440min, 98.547min, 138.337min, 145.577min; (2) It has 7 common characteristic peaks, of which Peak 3 is the reference peak S of forsythin; the relative retention time of each characteristic peak and the S peak is within ± 8% of the specified value; the specified values ​​are: 0.37 (peak 1), 0.42 (peak 2), 1.00 (peak 3), 1.33 (peak 4), 1.37 (peak 5), 1.93 (peak 6), and 2.03 (peak 7); (3) Using multiple batches of platelet-raising capsules and characteristic spectra obtained by the construction method described in any one of claims 1 to 3, a control characteristic spectra is prepared by using the average value or median method.

6. The method for constructing the platelet-raising capsule characteristic spectrum according to any one of claims 1 to 3 or the use of the platelet-raising capsule control characteristic spectrum according to claim 5 in the quality inspection of the platelet-raising capsule product.

7. A quality detection method for platelet-raising capsules, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the platelet-raising capsule product to be tested with the platelet-raising capsule control characteristic spectrum; the characteristic spectrum of the platelet-raising capsule product to be tested is obtained by using the platelet-raising capsule product to be tested according to the construction method described in any one of claims 1 to 5, and the platelet-raising capsule control characteristic spectrum is the platelet-raising capsule control characteristic spectrum described in claim 5.

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