Construction method and application of fingerprint spectrum of Longqing preparation

Abstract

The present invention provides a method for constructing a fingerprint of a Longqing preparation and its application, and relates to the technical field of drug analysis. The construction method includes establishing a high performance liquid chromatography fingerprint of the Longqing preparation at four detection wavelengths of 230±2nm, 274±2nm, 327±2nm and 345±2nm, using acetonitrile as mobile phase A, formic acid aqueous solution as mobile phase B, and a gradient elution program. The fingerprint obtained by the method for constructing the fingerprint of the Longqing preparation provided by the present invention can more comprehensively reflect the compound information of the Longqing preparation, and each main chromatographic peak can achieve basic separation. The construction method provided by the present invention is verified, and its precision, repeatability and stability experimental results meet the requirements.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for constructing a fingerprint spectrum of a Longqing preparation and an application thereof. Background Art

[0002] The prescription of Longqing Tablets (National Medicine Standard No. Z10920030) is composed of Alisma, Plantago, Patrinia, Honeysuckle, Peony Root, Hedyotis Diffusa, Red Peony, Agrimonia, Coptis and Phellodendron, which has the effects of clearing away heat and detoxifying, cooling blood and relieving stranguria. It is mainly used for stranguria caused by damp heat in the lower part of the body, with symptoms such as frequent urination, urgency, pain, low back pain, and lower abdominal distension. Longqing Tablets have antibacterial, anti-inflammatory, analgesic and anti-prostatic hyperplasia pharmacological effects. Clinical studies have shown that it has a good therapeutic effect on chronic prostatitis, prostatic hyperplasia, urinary tract infection and other diseases. However, the research on the quality evaluation and quality control methods of Longqing Tablets is still very weak. In addition to the general inspection of tablets, the current quality standard only stipulates the microscopic identification of Alisma, thin layer identification of Coptis, Peony Root, Red Peony and the determination of berberine hydrochloride content. Relying solely on finished product quality standards cannot fully reflect the intrinsic quality of the product, nor can it timely detect fluctuations and changes in product quality. It is difficult to truly and effectively control the quality of traditional Chinese medicine. There are great limitations and it is not conducive to the production process control and quality risk prevention and control of enterprises. Therefore, it is urgent to carry out relevant research to evaluate the intrinsic quality of Longqing Tablets and improve the quality evaluation and control level of Longqing Tablets.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The first purpose of the present invention is to provide a method for constructing a fingerprint of a Longqing preparation to alleviate the technical problem of lacking a method for quality evaluation and quality control of a Longqing preparation.

[0005] The second object of the present invention is to provide the application of the method for constructing the fingerprint of the Longqing preparation in evaluating the Longqing preparation.

[0006] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0007] According to one aspect of the present invention, the present invention provides a method for constructing a fingerprint of a Longqing preparation, comprising analyzing a sample to be tested by high performance liquid chromatography to obtain a fingerprint of the Longqing preparation.

[0008] The Longqing preparation of the present invention refers to a pharmaceutical preparation whose prescription is composed of Rhizoma Alismatis, Semen Plantaginis, Herba Patriniae, Flos Lonicerae, Cortex Moutan, Herba Hedyotis Diffusae, Radix Paeoniae Rubra, Herba Agrimoniae, Rhizoma Coptidis and Cortex Phellodendri. The present invention does not limit the dosage form of the pharmaceutical preparation, for example, it can be but not limited to any dosage form acceptable in the art such as tablets, granules, powders, capsules or pills. The Longqing preparation is preferably Longqing tablets or Longqing capsules.

[0009] The Longqing preparation is constructed using high performance liquid chromatography, including obtaining high performance liquid chromatography fingerprints under at least four wavelength conditions, specifically including a fingerprint with a detection wavelength of 230±2nm, a fingerprint with a detection wavelength of 274±2nm, a fingerprint with a detection wavelength of 327±2nm, and a fingerprint with a detection wavelength of 345±2nm.

[0010] In some preferred embodiments, the fingerprint of the Longqing preparation consists of a fingerprint with a detection wavelength of 230 nm, a fingerprint with a detection wavelength of 274 nm, a fingerprint with a detection wavelength of 327 nm, and a fingerprint with a detection wavelength of 345 nm.

[0011] The samples were analyzed using the following HPLC conditions:

[0012] Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is formic acid aqueous solution. Longqing preparation contains a large amount of alkaloids, and adding an appropriate amount of acid to the mobile phase can improve separation.

[0013] In some preferred embodiments, the mobile phase B is a 0.1% to 0.2% formic acid solution, calculated by volume percentage, for example, it can be but not limited to 0.1%, 0.12%, 0.15%, 0.18% or 0.2%, preferably a 0.1% formic acid solution.

[0014] The gradient elution procedure is as follows, based on volume percentage:

[0015] 0 min, mobile phase A 5±2%, mobile phase B 95±2%; 50 min, mobile phase A 45±5%, mobile phase B 55±5%; 60 min, mobile phase A 100%, mobile phase B 0%.

[0016] In some preferred embodiments, the gradient elution procedure is as follows, based on volume percentage:

[0017] 0 min, mobile phase A 5±1%, mobile phase B 95±1%; 50 min, mobile phase A 45±1%, mobile phase B 55±1%; 60 min, mobile phase A 100%, mobile phase B 0%;

[0018] In some more preferred embodiments, the gradient elution procedure is as follows, by volume percentage:

[0019] 0 min, mobile phase A 5%, mobile phase B 95%; 50 min, mobile phase A 45%, mobile phase B 55%; 60 min, mobile phase A 100%, mobile phase B 0%.

[0020] In some preferred embodiments, the chromatographic column uses octadecylsilane bonded silica gel as a filler, and the chromatographic column model is preferably as follows: Aglient Extend C18 250 mm×4.6 mm, 5 μm.

[0021] In some optional embodiments, the sample to be tested is a Longqing preparation, and the Longqing preparation is prepared into a test solution for liquid chromatography analysis according to the following method: the Longqing preparation is made into a powder, extracted with an extraction solvent, and the weight loss is supplemented with the extraction solvent, and then the filtrate is taken as the test solution; wherein the extraction solvent is preferably an ethanol aqueous solution. In terms of volume percentage, the extraction solvent is preferably a 30% to 60% ethanol aqueous solution, for example, it can be but not limited to 30%, 40%, 50% or 60%, preferably a 60% ethanol aqueous solution. The extraction method can be selected from ultrasonic treatment or heating reflux, and the simpler ultrasonic treatment method is preferred.

[0022] In some optional embodiments, the sample to be tested also includes a reference solution, which includes chlorogenic acid, paeoniflorin, jatrorrhizine hydrochloride, coptisine hydrochloride, palmatine hydrochloride, berberine hydrochloride and paeonol, respectively. The concentration range of each reference substance can be selected as follows. The reference solution is prepared according to the following concentration range to obtain a more successful chromatogram: chlorogenic acid 0.1800-0.2100 mg / mL; paeoniflorin 0.4000-0.5050 mg / mL; jatrorrhizine hydrochloride 0.2200-0.2500 mg / mL; coptisine hydrochloride 0.1900-0.2100 mg / mL; palmatine hydrochloride 0.3000-0.3600 mg / mL; berberine hydrochloride 0.3700-0.4500 mg / mL; and paeonol 0.4500-0.4700 mg / mL.

[0023] The preferred concentrations of the reference solutions are independently: chlorogenic acid 0.2016 mg / mL; paeoniflorin 0.5020 mg / mL; jatrorrhizine hydrochloride 0.2495 mg / mL; coptisine hydrochloride 0.2060 mg / mL; palmatine hydrochloride 0.3594 mg / mL; berberine hydrochloride 0.4400 mg / mL; and paeonol 0.4672 mg / mL.

[0024] The solvent of each of the above-mentioned reference substance solutions is preferably methanol.

[0025] In some preferred embodiments, the construction method includes analyzing the sample to be tested by high performance liquid chromatography, and the sample to be tested includes a test solution and a reference solution.

[0026] The test solution is prepared as follows: the Longqing preparation is made into powder, a 60% ethanol aqueous solution is added, ultrasonic extraction is performed, a 60% ethanol aqueous solution is used to make up for the weight loss, and then the filtrate is taken as the test solution;

[0027] The reference solution includes: a chlorogenic acid reference solution with a concentration of 0.2016 mg / mL, a paeoniflorin reference solution with a concentration of 0.5020 mg / mL, a jatrorrhizine hydrochloride reference solution with a concentration of 0.2495 mg / mL, a coptisine hydrochloride reference solution with a concentration of 0.2060 mg / mL, a palmatine hydrochloride reference solution with a concentration of 0.3594 mg / mL, a berberine hydrochloride reference solution with a concentration of 0.4400 mg / mL, and a paeonol reference solution with a concentration of 0.4672 mg / mL; the reference solution uses methanol as a solvent; the analysis conditions of the high performance liquid chromatography are:

[0028] Mobile phase A was acetonitrile, and mobile phase B was 0.1% formic acid solution;

[0029] The gradient elution procedure is as follows, based on volume percentage:

[0030] 0 min, mobile phase A 5%, mobile phase B 95%;

[0031] 50 min, mobile phase A 45%, mobile phase B 55%;

[0032] 60min, mobile phase A 100%, mobile phase B 0%; the chromatographic column is Aglient Extend C18 250mm×4.6mm, 5μm; the detection wavelengths are 230nm, 274nm, 327nm and 345nm respectively.

[0033] In some optional embodiments, the construction method includes using high performance liquid chromatography to measure the test solution and the reference solution, confirming the main chromatographic peaks in the high performance liquid chromatography spectrum of the test solution based on the chromatographic peaks in the high performance liquid chromatography spectrum of the reference solution and determining the reference peak, and obtaining the relative retention time of the main chromatographic peak relative to the reference peak.

[0034] In some optional embodiments, the construction method further comprises providing fingerprints of different batches of Longqing preparations, calibrating common peaks and synthesizing a control fingerprint.

[0035] In some optional embodiments, the control fingerprint spectrum is composed of a control fingerprint spectrum with a detection wavelength of 230nm, a control fingerprint spectrum with a detection wavelength of 274nm, a control fingerprint spectrum with a detection wavelength of 327nm, and a control fingerprint spectrum with a detection wavelength of 345nm; in the control fingerprint spectrum, the berberine hydrochloride peak is used as the reference peak, and the relative retention time RSD of the common peak is not greater than 1%. The characteristics of the control fingerprint spectrum are as follows:

[0036] The control fingerprint with a detection wavelength of 230 nm contains 19 common peaks, peak 16 is the reference peak, and the average relative retention times of the common peaks are as follows: peak 1: 0.345; peak 2: 0.353; peak 3: 0.370; peak 4: 0.499; peak 5: 0.511; peak 6: 0.559; peak 7: 0.644; peak 8: 0.655; peak 9: 0.720; peak 10: 0.734; peak 11: 0.855; peak 12: 0.864; peak 13: 0.873; peak 14: 0.885; peak 15: 0.979; peak 16: 1.000; peak 17: 1.060; peak 18: 1.234; peak 19: 1.513;

[0037] The control fingerprint with a detection wavelength of 274 nm contains 16 common peaks, peak 15 is the reference peak, and the average relative retention times of the common peaks are as follows: peak 1: 0.124; peak 2: 0.251; peak 3: 0.345; peak 4: 0.371; peak 5: 0.499; peak 6: 0.553; peak 7: 0.560; peak 8: 0.644; peak 9: 0.655; peak 10: 0.855; peak 11: 0.864; peak 12: 0.873; peak 13: 0.885; peak 14: 0.979; peak 15: 1.000; peak 16: 1.234;

[0038] The control fingerprint with a detection wavelength of 327 nm contains 18 common peaks, peak 17 is the reference peak, and the average relative retention times of the common peaks are as follows: peak 1: 0.251; peak 2: 0.323; peak 3: 0.345; peak 4: 0.371; peak 5: 0.405; peak 6: 0.499; peak 7: 0.644; peak 8: 0.683; peak 9: 0.711; peak 10: 0.720; peak 11: 0.757; peak 12: 0.855; peak 13: 0.864; peak 14: 0.873; peak 15: 0.885; peak 16: 0.979; peak 17: 1.000; peak 18: 1.234;

[0039] The control fingerprint with a detection wavelength of 345 nm contains 16 common peaks, peak 15 is the reference peak, and the average relative retention times of the common peaks are as follows: peak 1: 0.251; peak 2: 0.323; peak 3: 0.345; peak 4: 0.371; peak 5: 0.499; peak 6: 0.644; peak 7: 0.683; peak 8: 0.710; peak 9: 0.720; peak 10: 0.855; peak 11: 0.864; peak 12: 0.873; peak 13: 0.885; peak 14: 0.979; peak 15: 1.000; peak 16: 1.234.

[0040] According to another aspect of the present invention, the present invention also provides the application of the method for constructing the fingerprint of the above-mentioned Longqing preparation in the evaluation of the Longqing preparation, preferably applied to the evaluation of Longqing tablets or Longqing capsules. It has been determined that the above-mentioned fingerprint is more effective in evaluating Longqing tablets.

[0041] In some optional embodiments, the Longqing preparation is evaluated in the following manner: the fingerprint of the Longqing preparation to be evaluated is compared with the reference fingerprint, and the similarity between the fingerprint of the Longqing preparation to be evaluated and the reference fingerprint is calculated.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The method for constructing the fingerprint of the Longqing preparation provided by the present invention adopts liquid chromatography, optimizes the detection wavelength and chromatographic conditions, and establishes a high-performance liquid chromatography fingerprint of the Longqing preparation at four detection wavelengths of 230±2nm, 274±2nm, 327±2nm and 345±2nm. Since the detection wavelengths of the index components of each medicinal material are quite different, it is easy to miss the information characteristics of the sample by using a single wavelength detection, while multi-wavelength detection is more effective in displaying the subtle differences of the samples. Combining the detection wavelengths of the index components of the prescription drugs and the chromatogram information at different detection wavelengths, it can be found that 230±2nm, 274±2nm, 327±2nm, and 345±2nm are used as the detection wavelengths. The information of the compounds on the chromatogram is relatively rich, the baseline is stable, and it has good representativeness within a certain wavelength range around.

[0044] The detection wavelength and chromatographic conditions of the present invention are used to construct the fingerprint spectrum, and the relative retention time of the Longqing preparation test sample has good reproducibility, and the RSD of the relative retention time of each common peak is less than 1.0%. The fingerprint spectrum obtained by the construction method provided by the present invention can more comprehensively reflect the compound information of the Longqing preparation, and each main chromatographic peak can achieve basic separation. The construction method provided by the present invention is verified, and its precision, repeatability and stability experimental results meet the requirements.

[0045] The fingerprint spectrum prepared by the method for constructing the fingerprint spectrum of the Longqing preparation provided by the present invention can comprehensively evaluate the intrinsic quality of the Longqing preparation, establish a feasible quality control method for the Longqing preparation, improve the internal quality control level of the product, and further provide a practical basis for the comprehensive evaluation, quality control and quality risk assessment of traditional Chinese medicine quality and explore a possible idea. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1a This is a high performance liquid chromatography three-dimensional ultraviolet (210-400nm) graph of Longqing Tablets (batch number 150120) of Example 1;

[0048] Figures 1b to 1k The detection wavelength of the Longqing Tablet (batch number 150120) of Example 1 was investigated by high performance liquid chromatography. Figures 1b to 1k The detection wavelengths are 210nm, 230nm, 254nm, 274nm, 300nm, 327nm, 345nm, 360nm, 380nm and 400nm;

[0049] Figure 2a to 2d This is the HPLC chromatogram for investigating the fingerprint chromatographic conditions of Longqing Tablets (Batch No. 150120). Figure 2a to 2d The detection wavelengths are 230nm, 274nm, 327nm and 345nm;

[0050] Figure 2e This is the HPLC chromatogram of Longqing Tablets (Batch No. 150120) of Example 1, with detection wavelengths of 230 nm, 274 nm, 327 nm and 345 nm;

[0051] Figure 3a~3d The HPLC chromatogram of the extraction solvent of the Longqing Tablet (batch number 150120) of Example 3 was investigated. Figure 3a~3d The detection wavelengths are 230nm, 274nm, 327nm and 345nm;

[0052] Figures 4a to 4d The HPLC chromatogram of the extraction method of Longqing Tablets (Batch No. 150120) of Example 4 is shown below. Figures 4a to 4d The detection wavelengths are 230nm, 274nm, 327nm and 345nm;

[0053] Figure 5a This is the HPLC chromatogram of the berberine hydrochloride reference substance at a detection wavelength of 345 nm in Example 5;

[0054] Figure 5b It is the HPLC chromatogram of Longqing Tablets (Batch No. 150120) with detection wavelength of 345 nm in Example 5;

[0055] Figure 5cThis is a full wavelength (210-400nm) scan of the chromatographic peak of the berberine hydrochloride reference substance in Example 5;

[0056] Figure 5d It is a full wavelength (210-400nm) scanning diagram of the corresponding position chromatographic peak (Rt=34.231) in the chromatogram of Longqing Tablets (Batch No. 150120) and berberine hydrochloride in Example 5;

[0057] Figures 5e to 5k is the HPLC chromatogram of the reference substance in Example 5, Figures 5e to 5k The order is chlorogenic acid reference substance (327nm), paeoniflorin reference substance (230nm), jatrorrhizine hydrochloride (345nm), coptisine hydrochloride (345nm), palmatine hydrochloride (345nm), berberine hydrochloride (345nm) and paeonol reference substance (274nm);

[0058] Figure 5l This is the full wavelength (210-400nm) scanning diagram of the chlorogenic acid reference substance in Example 5;

[0059] Figure 5m It is a full wavelength (210-400nm) scanning diagram of the chromatographic peak (Rt=11.711) at the corresponding position in the chromatogram of Longqing Tablets (Batch No. 150120) and chlorogenic acid in Example 5;

[0060] Figure 5n This is the full wavelength (210-400nm) scan of the paeoniflorin reference substance in Example 5;

[0061] Figure 5o It is a full wavelength (210-400nm) scanning diagram of the corresponding position chromatographic peak (Rt=17.370) in the chromatogram of Longqing Tablets (Batch No. 150120) and paeoniflorin in Example 5;

[0062] Figure 5p This is the full wavelength (210-400nm) scanning diagram of the jatrorrhizine hydrochloride reference substance in Example 5;

[0063] Figure 5q It is a full wavelength (210-400nm) scanning diagram of the corresponding position chromatographic peak (Rt=29.914) in the chromatogram of Longqing Tablets (Batch No. 150120) and Jatrorrhizine Hydrochloride in Example 5;

[0064] Figure 5r It is the full wavelength (210-400nm) scanning diagram of the coptisine hydrochloride reference substance in Example 5;

[0065] Figure 5s It is a full wavelength (210-400nm) scanning diagram of the corresponding position chromatographic peak (Rt=30.301) in the chromatogram of Longqing Tablets (batch number 150120) and Coptisine Hydrochloride in Example 5;

[0066] Figure 5t This is the full wavelength (210-400nm) scan of the bamipine hydrochloride reference substance in Example 5;

[0067] Figure 5u It is a full wavelength (210-400nm) scanning diagram of the corresponding position chromatographic peak (Rt=33.543) in the chromatogram of Longqing Tablets (Batch No. 150120) and Bamatin Hydrochloride in Example 5;

[0068] Figure 5v This is the full wavelength (210-400nm) scanning diagram of the paeonol reference substance in Example 5;

[0069] Figure 5w It is a full wavelength (210-400nm) scanning diagram of the corresponding position chromatographic peak (Rt=42.260) in the chromatogram of Longqing Tablets (Batch No. 150120) and Paeonol in Example 5;

[0070] Figure 5x The HPLC chromatogram for the chromatographic peak confirmation of Longqing Tablets (Batch No. 150120) in Example 5;

[0071] Figure 6a~6d This is the HPLC chromatogram of Longqing Tablets (Batch No. 150015) and the prescribed medicinal materials in Example 6. Figure 6a~6d The detection wavelengths are 230nm, 274nm, 327nm and 345nm;

[0072] Figures 7a to 7d This is the HPLC chromatogram of the fingerprint of 13 batches of Longqing Tablets samples in Example 7. Figures 7a to 7d The detection wavelengths are 230nm, 274nm, 327nm and 345nm;

[0073] Figures 8a to 8d This is the HPLC control fingerprint of Longqing Tablets in Example 9, Figures 8a to 8d The detection wavelengths are 230nm, 274nm, 327nm and 345nm;

[0074] Figures 9a to 9d The fingerprints of Longqing Capsule and Longqing Tablet in Example 10 are compared. Figures 9a to 9d The detection wavelengths are 230nm, 274nm, 327nm and 345nm respectively.

[0075] Fig.10 The HPLC chromatogram (recorded for 120 minutes) of the fingerprint integrity inspection of Longqing Tablets (batch number 150120) in Effect Example 1;

[0076] Figures 11a to 11dThis is the HPLC chromatogram of the negative interference experiment of Longqing Tablets (batch number 150120) in Effect Example 2. Figures 11a to 11d The detection wavelengths are 230nm, 274nm, 327nm and 345nm respectively. DETAILED DESCRIPTION

[0077] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0078] The experimental materials used in the examples are as follows:

[0079] 1. Instrument

[0080] MS105 electronic balance, Mettler;

[0081] AL204IC electronic balance, Mettler;

[0082] KQ-400VDE dual-frequency CNC ultrasonic cleaning instrument, Kunshan Ultrasonic Instrument Company;

[0083] Waters e2695 / 2998 high performance liquid chromatograph, Waters Company, USA.

[0084] 2. Reagents and Drug Testing

[0085] 2.1 Test products

[0086] Longqing tablets (batch numbers 150120, 150186, 150058, FK15482, GD15554, GD15553, GA15514, 150093, GD15539, 150015, 150233, GA15522, 150024) are produced by Longshunrong Pharmaceutical Factory of Tianjin Sino-Singapore Pharmaceutical Group Co., Ltd.

[0087] Longqing Capsule A (batch numbers 20150704, 20190804) was purchased from Liangjianhao Pharmacy.

[0088] Longqing Capsule B (batch numbers 835036, 418024) was purchased from Liangjianhao Pharmacy.

[0089] 2.2 Medicinal Materials

[0090] Alisma orientalis (batch number Y08324), Plantago (batch number Y03098), Patrinia scabra (batch number Y08334), Lonicera japonica (batch number Y11375), Paeonia suffruticosa (batch number Y08333), Hedyotis diffusa (batch number Y09347), Paeonia rubra (batch number Y11390), Agrimonia echinense (batch number Y08327), Coptis chinensis (batch number Y10362), and Phellodendron chinense (batch number Y08323) were all provided by Longshunrong Pharmaceutical Factory of Tianjin Sino-Singapore Pharmaceutical Group Co., Ltd.

[0091] 2.3 Reference substances

[0092] Paeonol reference substance (batch number 110708-200506), the content is 99.0%. Bamatin hydrochloride reference substance (batch number 110732-201510), the content is 87.4%. Berberine hydrochloride reference substance (batch number 110713-201613), the content is 86.8%. Paeoniflorin reference substance (batch number 110736-201741), the content is 95.7%. Chlorogenic acid reference substance (batch number 110753-201716), the content is 99.3%. Jatrorrhizine hydrochloride reference substance (batch number 110733-201609), the content is 89.5%. Coptisine hydrochloride reference substance (batch number 112026-201802), the content is 94.0%. Phellodendron hydrochloride reference substance (batch number 111895-201805), the content is 94.9%. The above reference substances are all for content determination and are purchased from China Food and Drug Inspection Institute.

[0093] 2.4 Reagents and Drugs

[0094] Methanol and acetonitrile were of chromatographic grade, formic acid and ethanol were of analytical grade, and water was purified water.

[0095] Example 1

[0096] The prescription of Longqing Tablets is composed of Alisma orientalis, Plantago, Patrinia scabra, Honeysuckle, Paeonia suffruticosa, Hedyotis diffusa, Paeonia lactiflora, Agrimoniae scabra, Coptis chinensis, and Phellodendron amurense. The quality standards of medicinal materials and the content determination components and detection wavelengths specified therein are shown in Table 1.

[0097] Table 1 The standard collection of medicinal materials for Longqing Tablets and their index components and detection wavelengths

[0098] Medicinal Materials Standard collection Indicator ingredients Detection wavelength (nm) Alisma orientalis Chinese Pharmacopoeia 2015 Edition 23-Acetyl alismatol B 203 Plantago Chinese Pharmacopoeia 2015 Edition Geniposidic acid 254 Patrinia paniculata Sichuan Provincial Standards for Traditional Chinese Medicine - - honeysuckle Chinese Pharmacopoeia 2015 Edition Chlorogenic acid 327 Peony bark Chinese Pharmacopoeia 2015 Edition Paeonol 274 Hedyotis diffusa Shandong Province Traditional Chinese Medicine Standards - - Red Peony Root Chinese Pharmacopoeia 2015 Edition Paeoniflorin 230 Agrimony Chinese Pharmacopoeia 2015 Edition - - Coptis chinensis Chinese Pharmacopoeia 2015 Edition Berberine, etc. 345 Cork Chinese Pharmacopoeia 2015 Edition Berberine, phellodendronine 265、284

[0099] Note: “-” indicates that there is no content determination item in the quality standard.

[0100] It is obvious from the table that the detection wavelengths of the index components of various medicinal materials are quite different. It is easy to miss the information characteristics of the sample by using a single wavelength detection. Multi-wavelength detection is more effective in showing the subtle differences of the samples and can effectively solve this problem. The photodiode array detector is used to perform UV scanning on each chromatographic peak, and the 3D UV image of the HPLC is shown in Figure 1a The chromatograms of different detection wavelengths at 210nm-400nm were extracted at intervals of about 20nm for comparison. Figures 1b to 1k shown.

[0101] Combining the detection wavelength of the index components of the prescription drugs and the chromatogram information at different detection wavelengths, it can be found that 230nm, 274nm, 327nm, and 345nm are used as detection wavelengths. The information of the compounds on the chromatogram is relatively rich, the baseline is stable, and it has good representativeness within a certain wavelength range around it. At the same time, it has a corresponding relationship with the detection wavelengths of the index components of the main medicinal flavors of the prescription of Longqing Tablets, such as paeoniflorin (230nm), chlorogenic acid (327nm), paeonol (274nm), and berberine hydrochloride (345nm). Therefore, these four wavelengths were selected as the detection wavelengths for data statistics and similarity analysis.

[0102] Example 2-1

[0103] Take Longqing tablets, crush them, pass them through a No. 3 sieve, take out about 1.0 g, accurately weigh them, put them in a stoppered conical flask, accurately add 25 mL of 60% ethanol, weigh them, treat them with ultrasound for 30 minutes, take them out, let them cool to room temperature, make up for the weight loss with 60% ethanol, filter, and take the filtrate to obtain the product.

[0104] A water-polar solvent system was used for gradient elution. Considering that the sample contained a large amount of alkaloids, an appropriate amount of acid was added to the mobile phase to improve separation. A 0.1% formic acid solution-acetonitrile was used as the mobile phase system, and elution was performed on a chromatographic column (Aglient Extend C18 250mm×4.6mm, 5μm) filled with octadecylsilane bonded silica gel. The flow rate was 1.0mL / min, and the detection wavelengths were 230nm, 274nm, 327nm and 345nm, respectively. The elution conditions are shown in Table 2c. The chromatogram is shown in Figure 2a to Figure 2d As shown, Figure 2a to Figure 2d Each chromatogram in the figure is the elution conditions shown in Tables 2a, 2b and 2c from bottom to top, wherein 2c is the elution condition of this embodiment.

[0105] Table 2c

[0106] Time (min) Acetonitrile (%) 0.1% formic acid solution (%) 0 5 95 50 45 55 60 100 0

[0107] The method of this embodiment is used to detect Longqing Tablets (batch number 150120), and the chromatogram is as follows Figure 2e As shown, Figure 2e In the figure, (a) is detected at a wavelength of 345 nm, (b) is detected at a wavelength of 327 nm, (c) is detected at a wavelength of 274 nm, and (d) is detected at a wavelength of 230 nm.

[0108] Example 2-2

[0109] The difference between Example 2-2 and Example 2-1 is only the elution procedure, which is shown in Table 2a. Figure 2a to Figure 2d As shown, Figure 2a to Figure 2d The chromatogram in the middle shows the elution conditions shown in Tables 2a, 2b and 2c from bottom to top, wherein 2a is the elution condition of this embodiment.

[0110] Table 2a

[0111] Time (min) Acetonitrile (%) 0.1% formic acid solution (%) 0 5 95 50 40 60 60 100 0

[0112] Example 2-3

[0113] The difference between Example 2-3 and Example 2-1 is only the elution procedure. The elution procedure is shown in Table 2d.

[0114] Table 2d

[0115] Time (min) Acetonitrile (%) 0.1% formic acid solution (%) 0 3 97 50 50 50 60 100 0

[0116] Embodiment 2-4

[0117] The difference between Example 2-4 and Example 2-1 is only the elution procedure. The elution procedure is shown in Table 2e.

[0118] Table 2e

[0119] Time (min) Acetonitrile (%) 0.1% formic acid solution (%) 0 7 93 50 40 60 60 100 0

[0120] Embodiment 2-5

[0121] The difference between Example 2-5 and Example 2-1 is only the mobile phase. In this example, mobile phase A is acetonitrile and mobile phase B is 0.2% formic acid solution.

[0122] Embodiment 2-6

[0123] The difference between Example 2-6 and Example 2-1 is only the mobile phase. In this example, mobile phase A is acetonitrile and mobile phase B is 0.5% formic acid solution.

[0124] Comparative Example 1

[0125] The difference between Comparative Example 1 and Example 2-1 is only in the mobile phase and elution procedure. 0.1% glacial acetic acid solution-acetonitrile is used as the mobile phase system, and the elution procedure is shown in Table 2a. Figure 2a to Figure 2d As shown, Figure 2a to Figure 2dThe chromatograms in the middle are the elution conditions shown in Tables 2a, 2b and 2c from bottom to top, wherein 2b is the elution condition of this comparative example.

[0126] Table 2b

[0127] Time (min) Acetonitrile (%) 0.1% glacial acetic acid solution (%) 0 5 95 50 90 10 60 100 0

[0128] Comparison of the experimental results of Examples 2-1 to 2-6 and Comparative Example 1:

[0129] Example 2-1 and Example 2-2 are two chromatographic schemes using the same mobile phase and different elution procedures: The chromatograms of Example 2-1 and Example 2-2 are shown in FIG. Figure 2a to Figure 2d As shown, Figure 2a to Figure 2d The top chromatogram in each chromatogram is the chromatogram of Example 2-2 (elution procedure shown in Table 2a), and the bottom chromatogram is the chromatogram of Example 2-1 (elution procedure shown in Table 2c). Comparing the two chromatograms, it can be seen that the chromatogram obtained by the elution procedure of Example 2-1 (elution procedure shown in Table 2a) has fewer impurity peaks, the main chromatographic peaks have better symmetry and higher separation; most of the main chromatographic peaks have wider peak widths.

[0130] The acid added to the mobile phase B of Comparative Example 1 is glacial acetic acid, and the elution procedure is different from that of Examples 2-1 and 2-2. The chromatogram of Comparative Example 1 is shown in FIG. Figure 2a to Figure 2d As shown, Figure 2a to Figure 2d The chromatogram in the middle is the chromatogram of Comparative Example 1 (elution procedure shown in Table 2b). It can be seen from the figure that compared with Examples 2-1 and 2-2, the chromatographic conditions of Comparative Example 1 have a shorter retention time, poorer separation of the main peaks, and more impurity peaks. These characteristics indicate that the chromatographic conditions of Comparative Example 1 cannot meet the requirements of separating the main peaks and cannot construct a fingerprint spectrum.

[0131] Based on the experimental results of Examples 2-1 and 2-2 and Comparative Example 1, the inventors also tried the chromatographic conditions of Examples 2-3 to 2-6:

[0132] The only difference between Examples 2-3 and 2-4 and Example 2-1 is the different elution procedures. Compared with the elution procedure of Example 2-1, the chromatograms of Examples 2-3 (elution procedure shown in Table 2d) and 2-4 (elution procedure shown in Table 2e) are similar to the chromatogram obtained by the elution procedure of Example 2-2. There are more impurity peaks than the chromatogram of Example 2-1, but far less than the impurity peaks of Comparative Example 1. It is still possible to filter out interference while ensuring that sufficient compound information is obtained. Therefore, the elution conditions of Examples 2-3 and 2-4 can also realize the technical solution of the present invention.

[0133] Furthermore, the inventors also attempted to study the effect of formic acid content on the chromatogram by adjusting the formic acid content in mobile phase B. The inventors tried to adjust the formic acid concentration in mobile phase B to 0.2% v / v (Example 2-5) and 0.5% v / v (Example 2-6), and found that as the formic acid concentration increased, the number of impurity peaks could not be further reduced, and the interfering components could not be further eliminated. The chromatogram effect was similar to that of Example 2-2; therefore, the formic acid concentration in mobile phase B was better at 0.1% v / v.

[0134] Example 3

[0135] Take Longqing Tablets (batch number 150120), crush them, pass them through a No. 3 sieve, take out about 1.0 g, accurately weigh them, divide them into four parallel portions, place them in stoppered conical flasks, accurately add 25 mL each of pure water, 30% ethanol, 60% ethanol, and anhydrous ethanol, weigh them, heat and reflux for 30 minutes, take them out, let them cool to room temperature, make up for the weight loss with the extraction solvent, filter, and take the filtrate to obtain the product.

[0136] Accurately pipette 10 μL of each of the above test solutions and inject them into a high performance liquid chromatograph. Measure according to the chromatographic conditions of Example 1. The chromatogram is shown in FIG. Figure 3a~3d , Figure 3a~3d (a) is anhydrous ethanol, (b) is 60% ethanol, (c) is 30% ethanol, and (d) is water. The results show that 60% ethanol has the best extraction effect, can obtain more chromatographic peaks, and display more comprehensive information. Therefore, 60% ethanol is preferably used as the extraction solvent for Longqing Tablets.

[0137] Example 4

[0138] Take Longqing Tablets (batch number 150120), crush them, pass them through a No. 3 sieve, take out about 1.0 g, accurately weigh them, place them in two parallel portions, put them in a stoppered conical flask, accurately add 25 mL of 60% ethanol, weigh the weight, heat to reflux and ultrasonically treat for 30 minutes each, take them out, let them cool to room temperature, make up for the weight loss with 60% ethanol, filter, and take the filtrate to obtain the product.

[0139] Accurately pipette 10 μL of each test solution and inject it into the HPLC. Measure according to the proposed chromatographic conditions. The chromatogram is shown in Figures 4a to 4d , Figures 4a to 4d (a) is a heating reflux treatment, and (b) is an ultrasonic treatment. The results show that the chromatographic peaks obtained by heating reflux and ultrasonic treatment are basically the same, so the simpler ultrasonic treatment method is preferred as the extraction method.

[0140] The pretreatment method for preparing the Longqing tablets in the following Examples 5 to 15 is as follows: take the Longqing tablets, crush them, pass them through a No. 3 sieve, take out about 1.0 g, accurately weigh them, put them in a stoppered conical flask, accurately add 25 mL of 60% ethanol, weigh them, ultrasonically treat them for 30 minutes, take them out, let them cool to room temperature, make up the weight loss with 60% ethanol, filter them, and take the filtrate to obtain the product.

[0141] The chromatographic conditions were as follows: octadecylsilane bonded silica gel as filler (chromatographic column Aglient Extend C18 250 mm × 4.6 mm, 5 μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution as specified in Table 1c; detection wavelengths of 230 nm, 274 nm, 327 nm, and 345 nm; flow rate of 1.0 mL / min.

[0142] Example 5

[0143] Identification of chromatographic peaks: Chlorogenic acid (concentration 0.2016 mg / mL), paeoniflorin (concentration 0.5020 mg / mL), jatrorrhizine hydrochloride (concentration 0.2495 mg / mL), coptisine hydrochloride (concentration 0.2060 mg / mL), palmatine hydrochloride (concentration 0.3594 mg / mL), berberine hydrochloride (concentration 0.4400 mg / mL), paeonol (concentration 0.4672 mg / mL) reference solutions were prepared using methanol as solvent. The reference solutions and the test solution were subjected to high performance liquid chromatography analysis to confirm the main chromatographic peaks in the liquid fingerprint of the test sample.

[0144] Chromatograms of the test sample of Longqing Tablets and the reference sample of berberine hydrochloride are shown in Figure 5a and 5b , the full wavelength scan of the chromatographic peak is shown in Figure 5c and 5d As can be seen from the figure, the retention time of the chromatographic peak of the berberine hydrochloride reference substance (Rt=34.363) is basically consistent with the chromatographic peak (Rt=34.231) at the corresponding position in the chromatogram of the Longqing Tablets test substance, and is consistent with the overall trend and maximum absorption wavelength of the full wavelength scan of the chromatographic peak, which can confirm that the chromatographic peak is berberine hydrochloride. The confirmation method of other chromatographic peaks is the same. The HPLC chromatograms of other reference substances are as follows Figures 5e to 5k As shown, the full wavelength (210-400nm) scanning diagram of the chromatographic peaks of other reference substances and the corresponding positions in the chromatogram of the Longqing Tablet test substance is shown in Figure 5l~5w shown.

[0145] According to the above experimental results, the chromatographic peak was finally confirmed to be Figure 5xIn the figure, (a) is detected at a wavelength of 345 nm, (b) is detected at a wavelength of 327 nm, (c) is detected at a wavelength of 274 nm, and (d) is detected at a wavelength of 230 nm. In order to accurately describe the positions of the chromatographic peaks in Longqing Tablets, the berberine hydrochloride peak with a larger peak area and better separation was selected as the reference peak.

[0146] Example 6

[0147] Chromatographic peak medicinal material ownership:

[0148] 1. Preparation of Longqing Tablets test solution: Take Longqing Tablets, crush them, take about 1g, accurately weigh, and place them in 100mL conical flasks, accurately add 50mL of 60% ethanol, weigh the weight, ultrasonically treat for 30min, take out, let it cool to room temperature, weigh the weight again, make up the lost weight with 60% ethanol, filter, and obtain.

[0149] Preparation of medicinal material test solution: Weigh each medicinal ingredient in the prescription according to the prescribed amount, extract according to the extraction process specified in the quality standard of Longqing Tablets, concentrate and dry the extracts to obtain each medicinal material extract, accurately weigh, place in a 100mL conical flask, accurately add 50mL of 60% ethanol, weigh, ultrasonically treat for 30 minutes, take out, let it cool to room temperature, weigh again, make up the lost weight with 60% ethanol, filter, and obtain.

[0150] 2. Determination and results: Accurately pipette 10 μL of each of the above test solution, inject into the high performance liquid chromatograph, and determine according to the above chromatographic conditions. Compare the chromatograms of Longqing Tablets and medicinal materials, and combine the UV full wavelength scanning of each chromatographic peak in the chromatogram and the identification results of the chemical components of the chromatographic peaks in the fingerprint spectrum to attribute the medicinal material sources of each chromatographic peak in the chromatogram of Longqing Tablets. The results are shown in the table below and the chromatograms are shown in Figure 6a~6d As shown in the chromatogram, from bottom to top they are: Coptis chinensis, Phellodendron amurense, Paeonia suffruticosa, Paeonia lactiflora, Flos Lonicerae, Herba Patriniae, Herba Agrimoniae, Herba Hedyotis diffusae, Rhizoma Alismatis, Semen Plantago, and Longqing Tablets.

[0151] Table 3 The medicinal materials attribution of the chromatographic peaks in the fingerprint of Longqing Tablets

[0152]

[0153]

[0154] Note: “+” indicates that the chromatographic peak exists at this detection wavelength.

[0155] Example 7

[0156] Common peak calibration: 13 batches of Longqing tablets (batch numbers 150120, 150186, 150058, FK15482, GD15554, GD15553, GA15514, 150093, GD15539, 150015, 150233, GA15522, 150024) were prepared according to the determined preparation method of the test solution, and the sample was analyzed according to the above chromatographic conditions, and the chromatograms of 13 batches of samples at 230nm, 274nm, 327nm and 345nm were recorded for 60min. According to the information given by the 13 batches of liquid chromatograms of the test products, the number of common peaks was determined, and the proportion of the non-common peak area to the total peak area was calculated. Each common peak was numbered 1, 2, ..., N in sequence, and the berberine hydrochloride peak with good stability and high content was selected as the reference peak. The number of common peaks, peak area ratios and reference peak numbers at each detection wavelength are shown in Table 4, and the chromatograms are shown in Figures 7a to 7d , the batch numbers of the samples of Longqing Tablets in the figure are as follows: S1: 150120; S2: 150186; S3: 150058; S4: FK15482; S5: GD15554; S7: GD15553; S7: GA15514; S8: 150093; S9: GC15539; S10: 150015; S11: 150233; S12: GA15522; S13: 150024.

[0157] Table 4 Common peaks and reference peaks of Longqing Tablets at various detection wavelengths

[0158] Detection wavelength Number of common peaks Non-common peak area ratio (%) Reference peak number 230nm 19 14.5 16(S) 274nm 16 14.1 15(S) 327nm 18 5.6 17(S) 345nm 16 7.7 15(S)

[0159] Example 8

[0160] Relative retention time and relative ratio range of common peaks: Taking the berberine hydrochloride peak as the reference peak, the relative retention time ratio and relative ratio range of peak areas of 13 batches of Longqing Tablets were calculated at four different detection wavelengths of 230nm, 274nm, 327nm and 345nm. The results are shown in Tables 5 to 12. The results show that the relative retention time of each Longqing Tablet test sample has good reproducibility, and the RSD of the relative retention time of each common peak is less than 1.0%.

[0161] Table 5 Relative retention time of common peaks (230nm)

[0162]

[0163] Table 6 Relative ratio of common peak areas (230nm)

[0164]

[0165]

[0166] Table 7 Common peak relative retention time (274nm)

[0167]

[0168] Table 8 Relative ratio of total peak area (274nm)

[0169]

[0170] Table 9 Common peak relative retention time (327nm)

[0171]

[0172]

[0173] Table 10 Common peak area relative ratio (327nm)

[0174]

[0175] Table 11 Common peak relative retention time (345nm)

[0176]

[0177] Table 12 Common peak area relative ratio (345nm)

[0178]

[0179]

[0180] Example 9

[0181] Comparison of fingerprints and similarity calculations:

[0182] The chromatograms of 13 batches of Longqing Tablets were calculated and processed by the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.130723 Version” to obtain the HPLC reference fingerprint of Longqing Tablets. Figures 8a to 8d The similarity between the fingerprints of each batch of Longqing Tablets tested and the control was calculated to be ≥0.99. The similarity results are shown in Table 13.

[0183] Table 13 Similarity results of 13 batches of Longqing tablets and the control fingerprint

[0184]

[0185] The similarity calculation results showed that the similarities between the HPLC fingerprints of 13 batches of Longqing Tablets samples and the control fingerprints were all greater than 0.99, indicating that the fitted HPLC control fingerprint "R" could more comprehensively describe the information of the effective ingredients in Longqing Tablets, and the 13 batches of samples had little difference in chemical composition. However, it can be seen from the relative peak area calculation results that there were certain differences in the content of each component in the 13 batches of Longqing Tablets.

[0186] Example 10

[0187] Determination of Longqing Capsules: The established Longqing Tablets multi-wavelength reference fingerprint detection method was used to determine the commercially available Longqing Capsules samples and examine the similarity results. Take 4 batches of Longqing Capsules (batch numbers 20150704, 20190804, 835036, 418024), take out the contents, crush, pass through a No. 3 sieve, take about 1.0 g, accurately weigh, place in a stoppered conical flask, accurately add 25 mL of 60% ethanol, weigh the weight, ultrasonically treat for 30 minutes, take out, place at room temperature, make up for the weight loss with 60% ethanol, filter, take the filtrate, and determine according to the above chromatographic conditions. The fingerprint was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.130723 Version" and compared with the Longqing Tablets liquid chromatography reference fingerprint. The similarity between the test sample and the reference fingerprint of each batch of Longqing Capsules was calculated. The results are shown in Table 14 and the chromatograms are shown in Table 14. Figures 9a to 9d , the batch numbers of the Longqing Tablet samples in the figure are as follows: S2: 20190804; S3: 20150704; S4: 418024; S5: 835036; R is the reference fingerprint of Longqing Tablet.

[0188] Table 14 Similarity results between the Longqing Capsule spectrum and the control fingerprint spectrum

[0189]

[0190] The similarity calculation results showed that the similarity between the fingerprint of Longqing Capsules and the control fingerprint of Longqing Tablets was higher than 0.97 at each wavelength, and some samples were higher than 0.99 at a wavelength of 345nm, indicating that Longqing Capsules and Longqing Tablets had a high similarity, but lower than the similarity between batches of Longqing Tablets samples.

[0191] Effect Example 1

[0192] Fingerprint integrity: Take 10μl of the test solution of Longqing Tablets, inject it into the liquid chromatograph, detect it according to the proposed method, record the chromatogram for 2 hours, and show that there is no chromatographic peak after 60 minutes. The chromatogram is shown in Fig.10 In the figure, (a) is detected at a wavelength of 345 nm, (b) is detected at a wavelength of 327 nm, (c) is detected at a wavelength of 274 nm, and (d) is detected at a wavelength of 230 nm. Therefore, the chromatogram recording time is determined to be 60 minutes.

[0193] Effect Example 2

[0194] Fingerprint analysis method validation - negative interference test: Take 60% ethanol used in the preparation of the test sample, measure it according to the above chromatographic conditions, and compare it with the test sample solution. The chromatogram is shown in Figures 11a to 11d In the figure, (a) is the sample of Longqing Tablets, and (b) is the blank solvent. The results show that at four different detection wavelengths of 230nm, 274nm, 327nm and 345nm, the solvent blank has no interference with the sample detection.

[0195] Effect Example 3

[0196] Verification of fingerprint analysis method - precision: Take Longqing Tablets (batch number 150120), crush, pass through No. 3 sieve, take about 1.0g, accurately weigh, place in a stoppered conical flask, accurately add 25mL of 60% ethanol, weigh, ultrasonically treat for 30 minutes, take out, place at room temperature, make up the weight loss with 60% ethanol, filter, take the filtrate, and measure according to the proposed chromatographic conditions for 6 consecutive measurements. Record and analyze the relative retention time and relative peak area of ​​each common peak at four different detection wavelengths of 230nm, 274nm, 327nm and 345nm. Take the calculation results at the detection wavelength of 327nm as an example, see Table 15 and Table 16 for details.

[0197] Table 15 Precision test for verification of fingerprint analysis method of Longqing Tablets - relative retention time (327nm)

[0198]

[0199] Table 16 Precision test for verification of fingerprint analysis method of Longqing Tablets - relative ratio of peak area (327nm)

[0200]

[0201]

[0202] The results showed that the relative retention time of each chromatographic peak of the Longqing Tablets test solution at each detection wavelength was basically consistent (RSD≤5%), the relative peak area of ​​each peak was basically consistent (RSD≤5%), and the precision test met the requirements.

[0203] Effect Example 4

[0204] Verification of fingerprint analysis method - repeatability: Take Longqing Tablets (batch number 150120), crush, pass through No. 3 sieve, take about 1.0g, accurately weigh, parallel 6 portions, place in a stoppered conical flask, accurately add 25mL of 60% ethanol, weigh the weight, ultrasonically treat for 30 minutes, take out, place at room temperature, make up the weight loss with 60% ethanol, filter, take the filtrate, and measure according to the proposed chromatographic conditions. Record and analyze the relative retention time and relative peak area of ​​each common peak at four different detection wavelengths of 230nm, 274nm, 327nm and 345nm. Take the calculation results at the detection wavelength of 327nm as an example, see Table 17 and Table 18.

[0205] Table 17 Repeatability test for verification of the fingerprint analysis method of Longqing Tablets - relative retention time (327nm)

[0206]

[0207] Table 18 Repeatability test for verification of the fingerprint analysis method of Longqing Tablets - relative ratio of peak area (327nm)

[0208]

[0209] The results showed that the relative retention time of each chromatographic peak of the Longqing Tablets test sample at each detection wavelength was basically consistent (RSD≤5%), the relative peak area of ​​each peak was basically consistent (RSD≤5%), and the repeatability test met the requirements.

[0210] Effect Example 5

[0211] Verification of fingerprint analysis method - stability: Take the test solution of this product (batch number 120150) and store it at room temperature. Measure it at 0, 4, 8, 12, 16, 20 and 24 hours respectively, and record and analyze the relative retention time and relative peak area of ​​each common peak at four different detection wavelengths of 230nm, 274nm, 327nm and 345nm. Take the calculation results at the detection wavelength of 327nm as an example, see Table 19 and Table 20.

[0212] Table 19: Stability test for verification of fingerprint analysis method of Longqing Tablets - relative retention time (327nm)

[0213]

[0214] Table 20 Verification of stability test of fingerprint analysis method of Longqing tablets - relative ratio of peak area (327nm)

[0215]

[0216] The results showed that the relative retention time of each chromatographic peak of the Longqing Tablets test solution at each detection wavelength was basically consistent (RSD≤5%), and the relative peak area of ​​each peak was basically consistent (RSD≤5%), indicating that the test solution was stable within 24 hours.

[0217] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a fingerprint of a Longqing preparation, characterized in that: The method comprises measuring a test solution and a reference solution by high performance liquid chromatography to obtain a fingerprint spectrum, confirming the main chromatographic peaks in the high performance liquid chromatography spectrum of the test solution and determining the reference peaks according to the chromatographic peaks in the high performance liquid chromatography spectrum of the reference solution, and obtaining the relative retention time of the main chromatographic peaks relative to the reference peaks; and providing fingerprints of different batches of Longqing preparations, calibrating common peaks and synthesizing reference fingerprints; The reference substance solution includes solutions containing the following ingredients respectively: chlorogenic acid, paeoniflorin, jatrorrhizine hydrochloride, coptisine hydrochloride, palmatine hydrochloride, berberine hydrochloride and paeonol; The test solution is prepared as follows: the Longqing preparation is made into powder, extracted with ethanol and water solution, the weight loss is supplemented with the extraction solvent, and then the filtrate is taken as the test solution; The fingerprint spectrum of the Longqing preparation includes a fingerprint spectrum under the condition of a detection wavelength of 230nm, a fingerprint spectrum under the condition of a detection wavelength of 274nm, a fingerprint spectrum under the condition of a detection wavelength of 327nm, and a fingerprint spectrum under the condition of a detection wavelength of 345nm; The control fingerprint spectrum with a detection wavelength of 230 nm contains 19 common peaks, the control fingerprint spectrum with a detection wavelength of 274 nm contains 16 common peaks, the control fingerprint spectrum with a detection wavelength of 327 nm contains 18 common peaks, and the control fingerprint spectrum with a detection wavelength of 345 nm contains 16 common peaks; in the control fingerprint spectrum, the berberine hydrochloride peak is used as the reference peak; The analysis conditions of HPLC were: Chromatographic column: The chromatographic column uses octadecylsilane bonded silica gel as filler; Mobile phase: Mobile phase A is acetonitrile, mobile phase B is formic acid aqueous solution; The gradient elution procedure is as follows, based on volume percentage: 0 min, mobile phase A 5 ± 2%, mobile phase B 95 ± 2%; 50 min, mobile phase A 45 ± 5%, mobile phase B 55 ± 5%; 60 min, mobile phase A 100%, mobile phase B 0%; The Longqing preparation is Longqing tablets or Longqing capsules.

2. The construction method according to claim 1, characterized in that: The fingerprint spectrum of the Longqing preparation consists of a fingerprint spectrum with a detection wavelength of 230nm, a fingerprint spectrum with a detection wavelength of 274nm, a fingerprint spectrum with a detection wavelength of 327nm and a fingerprint spectrum with a detection wavelength of 345nm.

3. The construction method according to claim 1, characterized in that: The gradient elution procedure is as follows, based on volume percentage: 0 min, mobile phase A 5 ± 1%, mobile phase B 95 ± 1%; 50 min, mobile phase A 45 ± 1%, mobile phase B 55 ± 1%; 60 min, mobile phase A 100%, mobile phase B 0%.

4. The construction method according to claim 3, characterized in that: The gradient elution procedure is as follows, based on volume percentage: 0 min, mobile phase A 5%, mobile phase B 95%; 50 min, mobile phase A 45%, mobile phase B 55%; 60 min, mobile phase A 100%, mobile phase B 0%.

5. The construction method according to claim 1, characterized in that: In terms of volume percentage, mobile phase B is 0.1%~0.2% formic acid solution.

6. The construction method according to claim 5, characterized in that: In terms of volume percentage, mobile phase B was 0.1% formic acid solution.

7. The construction method according to claim 1, characterized in that: The chromatographic column is Aglient Extend C18 250 mm × 4.6 mm, 5 μm.

8. The construction method according to claim 1, characterized in that: In volume percentage, the ethanol aqueous solution is 30% to 60% ethanol aqueous solution.

9. The construction method according to claim 8, characterized in that: In terms of volume percentage, the ethanol aqueous solution is 60% ethanol aqueous solution.

10. The construction method according to claim 1, characterized in that: The extraction is carried out by ultrasonic treatment or heating reflux.

11. The construction method according to claim 1, characterized in that: The concentration of the chlorogenic acid reference solution is 0.1800~0.2100 mg / mL.

12. The construction method according to claim 11, characterized in that: The concentration of the chlorogenic acid control solution was 0.2016 mg / mL.

13. The construction method according to claim 1, characterized in that: The concentration of the paeoniflorin reference solution was 0.4000~0.5050 mg / mL.

14. The construction method according to claim 13, characterized in that: The concentration of the paeoniflorin control solution was 0.5020 mg / mL.

15. The construction method according to claim 1, characterized in that: The concentration of the jatrorrhizine hydrochloride reference solution is 0.2200~0.2500 mg / mL.

16. The construction method according to claim 15, characterized in that: The concentration of the jatrorrhizine hydrochloride control solution was 0.2495 mg / mL.

17. The construction method according to claim 1, characterized in that: The concentration of the coptisine hydrochloride reference solution is 0.1900~0.2100 mg / mL.

18. The construction method according to claim 17, characterized in that: The concentration of the coptisine hydrochloride reference solution was 0.2060 mg / mL.

19. The construction method according to claim 1, characterized in that: The concentration of the bamipine hydrochloride reference solution is 0.3000~0.3600 mg / mL.

20. The construction method according to claim 19, characterized in that: The concentration of the palmatine hydrochloride control solution was 0.3594 mg / mL.

21. The construction method according to claim 1, characterized in that: The concentration of the berberine hydrochloride reference solution is 0.3700~0.4500 mg / mL.

22. The construction method according to claim 21, characterized in that: The concentration of the berberine hydrochloride control solution was 0.4400 mg / mL.

23. The construction method according to claim 1, characterized in that: The concentration of the paeonol reference solution is 0.4500~0.4700 mg / mL.

24. The construction method according to claim 23, characterized in that: The concentration of the paeonol control solution was 0.4672 mg / mL.

25. The construction method according to claim 1, characterized in that: The samples to be tested are analyzed by high performance liquid chromatography, and the samples to be tested include a test solution and a reference solution; The test solution is prepared as follows: the Longqing preparation is made into powder, a 60% ethanol aqueous solution is added, ultrasonic extraction is performed, a 60% ethanol aqueous solution is used to make up for the weight loss, and then the filtrate is taken as the test solution; The reference solution includes: a chlorogenic acid reference solution with a concentration of 0.2016 mg / mL, a paeoniflorin reference solution with a concentration of 0.5020 mg / mL, a jatrorrhizine hydrochloride reference solution with a concentration of 0.2495 mg / mL, a coptisine hydrochloride reference solution with a concentration of 0.2060 mg / mL, a palmatine hydrochloride reference solution with a concentration of 0.3594 mg / mL, a berberine hydrochloride reference solution with a concentration of 0.4400 mg / mL, and a paeonol reference solution with a concentration of 0.4672 mg / mL; the reference solution uses methanol as a solvent; the analysis conditions of the high performance liquid chromatography are: Mobile phase A was acetonitrile, and mobile phase B was 0.1% formic acid solution; The gradient elution procedure is as follows, based on volume percentage: 0 min, mobile phase A 5%, mobile phase B 95%; 50 min, mobile phase A 45%, mobile phase B 55%; 60 min, mobile phase A 100%, mobile phase B 0%; The chromatographic column was Aglient Extend C18 250 mm × 4.6 mm, 5 μm; The detection wavelengths are 230nm, 274nm, 327nm and 345nm respectively.

26. The construction method according to claim 1, characterized in that: The relative retention time RSD of the common peak is no more than 1%; The control fingerprint with a detection wavelength of 230 nm contains 19 common peaks, peak 16 is the reference peak, and the average relative retention times of the common peaks are as follows: peak 1: 0.345; peak 2: 0.353; peak 3: 0.370; peak 4: 0.499; peak 5: 0.511; peak 6: 0.559; peak 7: 0.644; peak 8: 0.655; peak 9: 0.720; peak 10: 0.734; peak 11: 0.855; peak 12: 0.864; peak 13: 0.873; peak 14: 0.885; peak 15: 0.979; peak 16: 1.000; peak 17: 1.060; peak 18: 1.234; peak 19: 1.513; The control fingerprint with a detection wavelength of 274 nm contains 16 common peaks, peak 15 is the reference peak, and the average relative retention times of the common peaks are as follows: peak 1: 0.124; peak 2: 0.251; peak 3: 0.345; peak 4: 0.371; peak 5: 0.499; peak 6: 0.553; peak 7: 0.560; peak 8: 0.644; peak 9: 0.655; peak 10: 0.855; peak 11: 0.864; peak 12: 0.873; peak 13: 0.885; peak 14: 0.979; peak 15: 1.000; peak 16: 1.234; The control fingerprint with a detection wavelength of 327 nm contains 18 common peaks, peak 17 is the reference peak, and the average relative retention times of the common peaks are as follows: peak 1: 0.251; peak 2: 0.323; peak 3: 0.345; peak 4: 0.371; peak 5: 0.405; peak 6: 0.499; peak 7: 0.644; peak 8: 0.683; peak 9: 0.711; peak 10: 0.720; peak 11: 0.757; peak 12: 0.855; peak 13: 0.864; peak 14: 0.873; peak 15: 0.885; peak 16: 0.979; peak 17: 1.000; peak 18: 1.234; The control fingerprint spectrum with a detection wavelength of 345 nm contains 16 common peaks, peak 15 is the reference peak, and the average relative retention times of the common peaks are as follows: peak 1: 0.251; peak 2: 0.323; peak 3: 0.345; peak 4: 0.371; peak 5: 0.499; peak 6: 0.644; peak 7: 0.683; peak 8: 0.710; peak 9: 0.720; peak 10: 0.855; peak 11: 0.864; peak 12: 0.873; peak 13: 0.885; peak 14: 0.979; peak 15: 1.000; peak 16: 1.

234.

27. Use of the method for constructing the fingerprint of the Longqing preparation according to any one of claims 1 to 26 in evaluating the Longqing preparation, wherein the Longqing preparation is Longqing tablets or Longqing capsules.

28. The use according to claim 27, characterized in that The fingerprint of the Longqing preparation to be evaluated is compared with the reference fingerprint, and the similarity between the fingerprint of the Longqing preparation to be evaluated and the reference fingerprint is calculated.

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