Method for constructing characteristic fingerprint of Xiaoxumingtang based on UPLC wavelength switching method and its application

Abstract

The present invention provides a method for constructing a characteristic fingerprint of Xiaoxumingtang based on UPLC wavelength switching method and its application, which relates to the technical field of quality control of traditional Chinese medicine. The fingerprint of the present invention is constructed based on the UPLC wavelength switching method. The detection wavelength selected in the fingerprint construction is: 0~(Rt 黄芩苷 ‑0.6)min detection wavelength 210nm; (Rt 黄芩苷 ‑0.6)~(Rt 黄芩苷 +0.6)min detection wavelength 360nm; (Rt 黄芩苷 The detection wavelength is 290 nm, and the detection time is 10 min. By selecting the above-mentioned UPLC detection wavelength and ultra-performance liquid chromatography conditions, the characteristic fingerprint of Xiaoxuming Decoction of the present invention can calibrate 22 common chromatographic peaks and identify 18 chromatographic peaks within 40 minutes. This enables a more comprehensive quality evaluation of Xiaoxuming Decoction in a short time, and can provide a scientific basis for the quality evaluation and control of Xiaoxuming Decoction.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine quality control, and in particular to a method for constructing a characteristic fingerprint of Xiaoxuming Decoction based on a UPLC wavelength switching method and an application thereof. Background Art

[0002] Xiaoxumingtang (Xiaopin Fang) was first described in Chen Yanzhi's "Xiaopin Fang" during the Jin and Southern and Northern Dynasties. It was used to treat stroke and became widely circulated after being included in "Qianjin Yaofang" (Prescriptions for Emergencies) and "Waitai Miyao" (Secret Prescriptions for the Secret of the Imperial Court). The recipe consists of one liang each of ephedra, stephania tetrandra, ginseng, scutellaria baicalensis, cinnamon heart, licorice, peony root, ligusticum chuanxiong, and apricot kernel, one aconite root, one and a half liang of saposhnikovia divaricata, and five liang of fresh ginger. It is primarily used to treat symptoms of stroke, including facial paralysis, muscle and vein spasms, hemiplegia, tongue stiffness, inability to speak, or a drowsy demeanor. Before the Tang and Song dynasties, physicians generally considered stroke to be caused by "internal deficiency and pathogenic invasion." This recipe, considered the premier remedy for wind-related diseases, boasted tonifying and dispelling pathogenic factors. It was widely used and was considered the "most essential of all decoctions," hence its widespread application. In recent years, the traditional Chinese medical community generally believes that blood stasis is the underlying cause of stroke. Xiaoxumingtang, with ephedra, cinnamon twig, and dried ginger as its main ingredients, warms and disperses cold-stagnated blood stasis in the meridians, dissolving blood stasis to treat stroke, rather than specifically targeting qi-tonifying or yang-warming properties. Modern understanding of the pathogenesis of stroke and the interpretation of Xiaoxuemiantang (Xiaoxuemiantang) has gained a unique perspective, providing a more comprehensive understanding. This has not only strengthened contemporary appreciation for this prescription but also significantly expanded and developed its original foundation. Modern clinical use focuses on stroke and its sequelae, cerebral infarction, facial neuritis, and Alzheimer's disease.

[0003] "Xiaoxuemiantang" is included in the "Catalogue of Ancient Classic Prescriptions (First Batch)". In recent years, in order to clarify the material basis of Xiaoxuemiantang in treating diseases and ensure the effectiveness and safety of clinical medication, many scholars have conducted research on the origin of the medicinal base of Xiaoxuemiantang, its harvesting, processing and preparation, as well as the pharmacology, pharmacodynamics and pharmacokinetics of its chemical composition and effective components, and continuously improved its quality standards, laying the foundation for the development of classic prescriptions. At present, research on Xiaoxuemiantang mainly focuses on pharmacology and clinical application. There is little research on the material basis, extraction process, multi-index component content determination and characteristic spectrum of its decoction, and it lacks systematicity. There is also little research on how to measure the consistency of the quality of large-scale production preparations and traditional decoctions.

[0004] A traditional Chinese medicine fingerprint is a chromatogram or spectrum that identifies the chemical characteristics of certain Chinese medicinal materials or preparations after appropriate processing and analytical methods. It is a comprehensive and quantifiable quality assessment method that effectively reflects the integrity and comprehensive effects of traditional Chinese medicine components. Its rapidity and accuracy make it widely used in traditional Chinese medicine analysis, identification, and quality control.

[0005] Xiaoxumingtang (Xiaoxumingtang) has a very complex composition. Current methods for fingerprinting Xiaoxumingtang using high-performance liquid chromatography (HPLC) and gas chromatography (GC) are time-consuming and produce a limited number of characteristic peaks, making it difficult to efficiently and comprehensively evaluate its quality. Therefore, developing a simple, highly accurate, and reproducible fingerprinting method that can capture as many of the ingredients as possible is crucial for quality control and evaluation.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The first purpose of the present invention is to provide a method for constructing a characteristic fingerprint of Xiaoxumiantang based on the UPLC (ultra-performance liquid chromatography) wavelength switching method. The characteristic fingerprint of Xiaoxumiantang constructed by this method can perform a more comprehensive quality evaluation of Xiaoxumiantang, and it takes a short time, which can improve the efficiency of quality detection. At the same time, the method is simple, accurate and reproducible.

[0008] The second object of the present invention is to provide an application of a method for constructing a characteristic fingerprint of Xiaoxumingtang based on the UPLC wavelength switching method.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0010] The present invention provides a method for constructing a characteristic fingerprint of Xiaoxumingtang based on a UPLC wavelength switching method, comprising the following steps:

[0011] Preparation of reference solution: dissolve baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance, and amygdalin reference substance in a solvent to obtain a mixed reference solution;

[0012] Preparation of test solution: Dissolve the Xiaoxumingtang reference sample in solvent to obtain the test solution;

[0013] Fingerprint construction: The test solution and the reference solution were measured by UPLC wavelength switching method to obtain the fingerprint.

[0014] The UPLC wavelength switching method uses timed wavelength switching technology to measure the absorbance values ​​of components at different wavelengths in different time periods. The detection wavelengths are:

[0015] 0~(Rt 黄芩苷 -0.6)min detection wavelength is 210nm;

[0016] (Rt 黄芩苷 -0.6)~(Rt 黄芩苷 +0.6)min detection wavelength is 360nm;

[0017] (Rt 黄芩苷+0.6)~40min detection wavelength is 290nm;

[0018] The Rt 黄芩苷 It is the time when the chromatogram reaches the baicalin peak.

[0019] Furthermore, the raw materials for preparing the Xiaoxuemiantang benchmark sample include: ephedra, stephania tetrandra, ginseng, scutellaria baicalensis, cinnamon bark, fried licorice, white peony root, chuanxiong, stewed bitter almonds, heishunpian, siler, and ginger.

[0020] Furthermore, the preparation method of the Xiaoxumingtang reference sample includes:

[0021] Ephedra, Stephania tetrandra, ginseng, scutellaria baicalensis, cinnamon bark, stir-fried licorice, white peony root, ligusticum chuanxiong, stewed bitter almond, heishunpian, saposhnikovia divaricata, and ginger were mixed, crushed, decocted, and a decoction was obtained, which was then freeze-dried to obtain a Xiaoxuemiantang benchmark sample.

[0022] Furthermore, the elution mode of the ultra-high performance liquid chromatography of the UPLC wavelength switching method is: acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and gradient elution;

[0023] The gradient elution method is:

[0024]

[0025] Furthermore, the chromatographic conditions of the ultra-high performance liquid chromatograph of the UPLC wavelength switching method include: the chromatographic column is filled with octadecylsilane bonded silica gel, the column temperature is 25-35° C.; the flow rate of the mobile phase is 0.3-0.5 ml / min;

[0026] Preferably, the chromatographic conditions of the ultra-high performance liquid chromatograph of the UPLC wavelength switching method include: the chromatographic column is filled with octadecylsilane bonded silica gel, the column temperature is 30° C.; the flow rate of the mobile phase is 0.5 ml / min;

[0027] Preferably, the chromatographic column is Agilent InfinityLab Poroshell 120EC-C18, with a column length of 150 mm, an inner diameter of 3.0 mm, and a particle size of 2.7 μm.

[0028] Furthermore, the preparation method of the reference solution includes:

[0029] Take appropriate amounts of baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance, and amygdalin reference substance, add them to 80% methanol to prepare solutions containing 0.3 mg, 20 μg, 20 μg, 0.1 mg, and 0.15 mg per ml respectively, as reference solution.

[0030] Furthermore, the preparation method of the test solution includes:

[0031] Take 0.5 g of Xiaoxumingtang reference sample, add it to 25 ml of 80% methanol, heat and reflux to extract for 30 minutes, filter, and take the filtrate to obtain the test solution.

[0032] Furthermore, the method for constructing the characteristic fingerprint of Xiaoxumingtang also includes:

[0033] The steps of taking at least 10 batches of Xiaoxuemiantang benchmark samples, preparing test sample solutions respectively, collecting fingerprints, importing them into the Chinese medicine chromatographic fingerprint similarity evaluation system of the National Pharmacopoeia Committee, and generating the characteristic reference fingerprint of Xiaoxuemiantang.

[0034] The present invention provides an application of the method for constructing a characteristic fingerprint of Xiaoxumiantang based on the UPLC wavelength switching method in the overall quality control of Xiaoxumiantang.

[0035] Furthermore, the application includes:

[0036] (A) providing a sample of Xiaoxumingtang to be tested, and obtaining a fingerprint of the Xiaoxumingtang sample according to the method for constructing a characteristic fingerprint of Xiaoxumingtang based on the UPLC wavelength switching method;

[0037] (B) Compare the obtained fingerprint of the Xiaoxumingtang sample with the characteristic fingerprint of the Xiaoxumingtang control. If the similarity is ≥0.9, the quality of the Xiaoxumingtang sample is qualified.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention provides a method for constructing a characteristic fingerprint of Xiaoxumingtang based on the UPLC wavelength switching method. In view of the fact that the chemical composition of Xiaoxumingtang is complex, the number of chromatographic peaks is large, and the ultraviolet absorption wavelengths of the chromatographic peaks vary greatly, the present invention has studied and found that the number of chromatographic peaks at the wavelength of 210nm is the largest, but the chromatographic peak signal of baicalin is strong, resulting in an uncoordinated ratio of the chromatographic peaks of the entire spectrum. The present application then uses a derivative channel to perform wavelength timing switching, that is, initially using 210nm to examine the chemical components in medicinal flavors such as ephedra and bitter almonds, and switching the wavelength to 360nm at the chromatographic peak of baicalin to reduce the signal intensity of baicalin, and then switching again. The wavelength was increased to 290 nm to better detect cinnamaldehyde from cinnamon bark, and the fingerprint construction method of the present application was able to calibrate 22 common chromatographic peaks within 40 min, and identify 18 chromatographic peaks, namely: ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, paeoniflorin, cimicifugoside, ferulic acid, liquiritin, 1,2,3,4,6-O-pentagalloylglucose, 5-O-methylvisaminol glycoside, fangchinoline, tetrandrine, baicalin, melaleucain A-7-O-β-D-glucuronide, cinnamaldehyde, baicalin, baicalein, wogonin and 6-gingerol.

[0040] Therefore, the characteristic fingerprint of Xiaoxuemiantang constructed in this application can conduct a more comprehensive quality evaluation of Xiaoxuemiantang in a short time, which can improve the efficiency of quality detection. At the same time, the method is simple, accurate and reproducible, which can provide a scientific basis for the quality evaluation and control of Xiaoxuemiantang.

[0041] The method for constructing the characteristic fingerprint of Xiaoxumingtang based on the UPLC wavelength switching method provided by the present invention can be widely used in the overall quality control process of Xiaoxumingtang. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 The characteristic fingerprint of the Xiaoxumingtang reference sample provided in Example 2 of the present invention;

[0044] Figure 2 The chromatogram of the reference substance provided in Example 3 of the present invention;

[0045] Figure 3Ultra-high performance liquid chromatography (UPLC) analysis of different extraction solvents provided in Example 3 of the present invention;

[0046] Figure 4 Ultra-high performance liquid chromatography (UPLC) analysis of different extraction methods provided in Example 3 of the present invention;

[0047] Figure 5 Ultra-high performance liquid chromatography (UPLC) analysis at different extraction times provided in Example 3 of the present invention;

[0048] Figure 6 Ultra-high performance liquid chromatography (UPLC) analysis of different solvent dosages provided in Example 3 of the present invention;

[0049] Figure 7 This is a full wavelength scanning spectrum of the Xiaoxumingtang reference sample prepared in Example 1 of the present invention;

[0050] Figure 8a The ultra-high performance liquid chromatography (UPLC) spectrum with a detection wavelength of 210 to 280 nm provided in Example 3 of the present invention;

[0051] Figure 8b The ultra-high performance liquid chromatography (UPLC) spectrum at a detection wavelength of 290 to 360 nm provided in Example 3 of the present invention;

[0052] Figure 9 The wavelength switching diagrams of different derivative channels provided in Example 3 of the present invention;

[0053] Figure 10 The investigation patterns of different chromatographic columns provided in Example 3 of the present invention;

[0054] Figure 11a The investigation spectrum of acetonitrile-water and acetonitrile-0.1 formic acid mobile phases provided in Example 3 of the present invention;

[0055] Figure 11b The investigation spectrum of acetonitrile-phosphoric acid mobile phase of different concentrations provided in Example 3 of the present invention;

[0056] Figure 12a The investigation spectrum for the column temperature of 25-30°C provided in Example 3 of the present invention;

[0057] Figure 12b The investigation spectrum for the column temperature of 31-35°C provided in Example 3 of the present invention;

[0058] Figure 13a The investigation graphs for flow rates of 0.45 ml / min and 0.48 ml / min provided in Example 3 of the present invention;

[0059] Figure 13bThe investigation graphs for flow rates of 0.50 ml / min and 0.51 ml / min provided in Example 3 of the present invention;

[0060] Figure 13c The investigation graphs for flow rates of 0.52 ml / min and 0.55 ml / min provided in Example 3 of the present invention;

[0061] Figure 14 The investigation graphs of different injection volumes provided in Example 3 of the present invention;

[0062] Figure 15 The blank mobile phase, blank solvent, and double retention time inspection spectrum provided in Example 3 of the present invention;

[0063] Figure 16 The original spectrum of the precision investigation provided in Example 4 of the present invention;

[0064] Figure 17 The spectrum after precision inspection matching provided in Example 4 of the present invention;

[0065] Figure 18 This is the original stability test spectrum provided in Example 4 of the present invention;

[0066] Figure 19 The stability test matched spectrum provided in Example 4 of the present invention;

[0067] Figure 20 The original spectrum of the repeatability study provided in Example 4 of the present invention;

[0068] Figure 21 The reproducibility test and matching pattern provided in Example 4 of the present invention;

[0069] Figure 22 The original spectrum of the intermediate precision inspection provided in Example 4 of the present invention;

[0070] Figure 23 This is the intermediate precision matching spectrum provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0071] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0072] According to one aspect of the present invention, a method for constructing a characteristic fingerprint of Xiaoxumingtang based on UPLC wavelength switching method comprises the following steps:

[0073] Preparation of reference solution: dissolve baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance, and amygdalin reference substance in a solvent to obtain a reference solution;

[0074] Preparation of test solution: Dissolve the Xiaoxumingtang reference sample in solvent to obtain the test solution;

[0075] Fingerprint construction: The test solution and the reference solution were measured by UPLC wavelength switching method to obtain the fingerprint.

[0076] The UPLC wavelength switching method uses timed wavelength switching technology to measure the absorbance values ​​of components at different wavelengths in different time periods. The detection wavelengths are:

[0077] 0~(Rt 黄芩苷 -0.6)min detection wavelength is 210nm;

[0078] (Rt 黄芩苷 -0.6)~(Rt 黄芩苷 +0.6)min detection wavelength is 360nm;

[0079] (Rt 黄芩苷 +0.6)~40min detection wavelength is 290nm;

[0080] The Rt 黄芩苷 It is the time when the chromatogram reaches the baicalin peak.

[0081] The present invention provides a method for constructing a characteristic fingerprint of Xiaoxumingtang based on the UPLC wavelength switching method. In view of the fact that Xiaoxumingtang contains multiple ingredients such as polysaccharides, organic acids, phenols, triterpenoid saponins, flavonoids, alkaloids, etc., its chemical composition is complex, the number of chromatographic peaks is large, and the ultraviolet absorption wavelengths of each chromatographic peak vary greatly. The present invention has found in the study of constructing the characteristic fingerprint of Xiaoxumingtang using the UPLC wavelength switching method that the number of chromatographic peaks at the wavelength of 210nm is the largest, but the baicalin chromatographic peak signal is strong, resulting in an uncoordinated ratio of the chromatographic peaks of the entire spectrum. The present application then uses a derivative channel for timed wavelength switching, that is, initially using 210nm to examine the chemical components in medicinal flavors such as ephedra and bitter almonds, and switching the wavelength to 360nm at the baicalin chromatographic peak to reduce the baicalin signal intensity, and then switching the wavelength to 290nm to better detect the baicalin signal. Cinnamaldehyde of cinnamon bark, and thus the fingerprint construction method of the present application can calibrate 22 common chromatographic peaks within 40 minutes, and identify 18 chromatographic peaks, namely: ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, paeoniflorin, cimicifugoside, ferulic acid, liquiritin, 1,2,3,4,6-O-pentagalloylglucose, 5-O-methylvisaminol glycoside, fangchinoline, tetrandrine, baicalin, melaleucain A-7-O-β-D-glucuronide, cinnamaldehyde, baicalin, baicalein, wogonin and 6-gingerol.

[0082] Therefore, the characteristic fingerprint of Xiaoxuemiantang constructed in this application can conduct a more comprehensive quality evaluation of Xiaoxuemiantang in a short time, which can improve the efficiency of quality detection. At the same time, the method is simple, accurate and reproducible, which can provide a scientific basis for the quality evaluation and control of Xiaoxuemiantang.

[0083] In a preferred embodiment of the present invention, the raw materials for preparing the Xiaoxu Ming Tang reference sample include: ephedra, stephania tetrandra, ginseng, scutellaria baicalensis, cinnamon bark, fried licorice, white peony root, chuanxiong, stewed bitter almonds, heishunpian, siler, and ginger.

[0084] As a preferred embodiment, the fried licorice and heishunpian used in the composition of the Xiaoxuemiantang benchmark sample of the present application are not only more consistent with the research records in ancient books than the existing Xiaoxuemiantang composition that uses licorice and white aconite as medicines, but the Xiaoxuemiantang benchmark sample also has a better chromatographic separation effect.

[0085] In the above preferred embodiment, the preparation method of the Xiaoxumingtang reference sample includes:

[0086] Ephedra, Stephania tetrandra, ginseng, scutellaria baicalensis, cinnamon bark, stir-fried licorice, white peony root, ligusticum chuanxiong, stewed bitter almond, heishunpian, saposhnikovia divaricata, and ginger were mixed, crushed, decocted, and a decoction was obtained, which was then freeze-dried to obtain a Xiaoxuemiantang benchmark sample.

[0087] Preferably, the preparation method of the Xiaoxumingtang reference sample comprises:

[0088] (1) Prepare the following raw materials for Xiaoxuemiantang: Ephedra 13.8g, Stephania tetrandra 13.8g, Ginseng 13.8g, Scutellaria baicalensis 13.8g, Cinnamon bark 13.8g, Fried Licorice root 13.8g, White Peony root 13.8g, Chuanxiong rhizome 13.8g, Boiled Bitter Apricot kernel 13.8g, Heishunpian 15g, Saposhnikovia divaricata 20.7g, Ginger 69g.

[0089] (2) The above medicinal materials were crushed into coarse particles of 5 to 8 mm. Ephedra sinica was taken and placed in a 5L casserole. 2400 ml of water was added and the mixture was soaked for 30 minutes. The mixture was heated and decocted using a fully automatic electric decoction pot heater. The mixture was boiled for three times and the foam was removed (decocted three times, each time for 15 minutes, and the foam was removed during the decoction). The remaining eleven herbs including Stephania tetrandra were added and the decoction was continued for 135 minutes (101 minutes on high heat and 34 minutes on low heat). The mixture was filtered through a 300-mesh filter cloth while hot to obtain 600 ml ± 50 ml of the standard decoction. The decoction was freeze-dried to obtain the Xiaoxuemiantang standard sample (powder) with a yield of 12% to 18%.

[0090] In a preferred embodiment of the present invention, the elution mode of the ultra-high performance liquid chromatography of the UPLC wavelength switching method is: acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and gradient elution is performed;

[0091] The gradient elution method is:

[0092]

[0093] In a preferred embodiment of the present invention, the chromatographic conditions of the ultra-high performance liquid chromatograph of the UPLC wavelength switching method include:

[0094] The chromatographic column was filled with octadecylsilane bonded silica gel, the column temperature was 25-35°C, and the flow rate of the mobile phase was 0.3-0.5 ml / min.

[0095] Preferably, the chromatographic column is Agilent InfinityLab Poroshell 120EC-C18, with a column length of 150 mm, an inner diameter of 3.0 mm, and a particle size of 2.7 μm.

[0096] In a preferred embodiment of the present invention, the method for preparing the reference solution comprises:

[0097] Take appropriate amounts of baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance, and amygdalin reference substance, add them to 80% methanol to prepare solutions containing 0.3 mg, 20 μg, 20 μg, 0.1 mg, and 0.15 mg per ml respectively, as reference solution.

[0098] In a preferred embodiment of the present invention, the method for preparing the test solution comprises:

[0099] Take 0.5 g of Xiaoxumingtang reference sample, add it to 25 ml of 80% methanol, heat and reflux to extract for 30 minutes, filter, and take the filtrate to obtain the test solution.

[0100] In a preferred embodiment of the present invention, the method for constructing the characteristic fingerprint of Xiaoxumingtang further comprises:

[0101] Take at least 10 batches of Xiaoxuemiantang benchmark samples, prepare test solutions respectively, collect fingerprints, and import them into the Chinese medicine chromatographic fingerprint similarity evaluation system of the National Pharmacopoeia Committee to generate the characteristic reference fingerprint of Xiaoxuemiantang.

[0102] According to one aspect of the present invention, the method for constructing the characteristic fingerprint of Xiaoxumiantang based on the UPLC wavelength switching method is applied in the overall quality control of Xiaoxumiantang.

[0103] The method for constructing the characteristic fingerprint of Xiaoxumingtang based on the UPLC wavelength switching method provided by the present invention can be widely used in the overall quality control process of Xiaoxumingtang.

[0104] In a preferred embodiment of the present invention, the application includes:

[0105] (A) providing a sample of Xiaoxumingtang to be tested, and obtaining a fingerprint of the Xiaoxumingtang sample according to the method for constructing a characteristic fingerprint of Xiaoxumingtang based on the UPLC wavelength switching method;

[0106] (B) Compare the obtained fingerprint of the Xiaoxumingtang sample with the characteristic fingerprint of the Xiaoxumingtang control. If the similarity is ≥0.9, the quality of the Xiaoxumingtang sample is qualified.

[0107] The technical solution of the present invention will be further described below with reference to embodiments.

[0108] The instruments and reagents used in the following examples are as follows:

[0109] Instrument: High performance liquid chromatography, Waters Acquity Arc HPLC TM , Waters AcquityHUPLCClass; CNC ultrasonic cleaner: KQ-500DE, power 500 W, frequency 40 kHz, Kunshan Ultrasonic Instrument Co., Ltd.; analytical balances: BT25S, BS2202S and BS124S, Sartorius, Germany.

[0110] Reagents: methanol, chromatographic grade, batch number 213476, Fisher reagent; acetonitrile, chromatographic grade, batch number 20101212G109, OCEANPAK; water, Millipore Mingche-D, prepared by ultrapure water integrated system; phosphoric acid, analytical grade, batch number C12815421, MACKLIN reagent; formic acid, analytical grade, batch number A2106152, Aladdin reagent.

[0111] Reference substances: ephedrine hydrochloride, batch number 171241-201809, purity 100%; pseudoephedrine hydrochloride, batch number 171237-201510, purity 99.8%; fangchinoline, batch number 110793-202108, purity 96.3%; tetrandrine, batch number 110711-201810, purity 99.6%; baicalin, batch number 110715-201720, purity 93. 5%; amygdalin, batch number 110820-202109, purity 93.1%; paeoniflorin, batch number 110736-202145, purity 94.6%; cimicifugoside, batch number 111522-201913, purity 94.6%; ferulic acid, batch number 110773-201915, purity 99.4%; cinnamaldehyde, batch number 110710-201720, purity 98.7%; 6-gingerol 111833-201806, purity 99.9%; liquiritin, batch number 111610-201908, purity 95.0%; 5-O-methylvisamidoside, batch number 111523-201811, purity 97.4%, all purchased from China Food and Drug Inspection Institute; baicalein, batch number MUST-16031618, purity 98.58%; wogonin, batch number MUST-16 062507, purity 99.86%; wogonin, MUST-16111611, purity 99.06%; melaleuca alternifolia A-7-O-β-D-glucuronide, batch number MUST-16090102, purity 98.00%; 1,2,3,4,6-O-pentagalloylglucose, batch number MUST-14020710, purity ≥98%, were purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0112] Reference sample for the study: freeze-dried powder of Xiaoxu Ming Tang prepared by China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd., batch number XXMT01.

[0113] Example 1 Preparation of Xiaoxumingtang Reference Sample

[0114] A method for preparing a Xiaoxumingtang reference sample comprises the following steps:

[0115] (1) Prepare the raw materials of Xiaoxumingtang according to the following quality:

[0116] Ephedra 13.8g, Stephania tetrandra 13.8g, Ginseng 13.8g, Scutellaria baicalensis 13.8g, Cinnamon bark 13.8g, Fried Licorice root 13.8g, White Peony root 13.8g, Chuanxiong 13.8g, Boiled Bitter Apricot kernel 13.8g, Heishun Pian 15g, Saposhnikovia divaricata 20.7g, Ginger 69g.

[0117] (2) The above medicinal materials were crushed into coarse particles of 5 to 8 mm. Ephedra sinica was taken and placed in a 5L casserole. 2400 ml of water was added and the mixture was soaked for 30 minutes. The mixture was heated and decocted using a fully automatic electric decoction pot heater. The mixture was boiled for three times and the foam was removed (decocted three times, each time for 15 minutes, and the foam was removed during the decoction). The remaining eleven herbs including Stephania tetrandra were added and the decoction was continued for 135 minutes (101 minutes on high heat and 34 minutes on low heat). The mixture was filtered through a 300-mesh filter cloth while hot to obtain 600 ml ± 50 ml of the standard decoction. The decoction was freeze-dried to obtain the Xiaoxuemiantang standard sample (powder) with a yield of 12% to 18%.

[0118] Example 2

[0119] A method for obtaining a characteristic fingerprint of Xiaoxumingtang, comprising the following steps:

[0120] 1. Preparation of reference solution:

[0121] Take appropriate amount of baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance, and amygdalin reference substance, accurately weigh them, and add 80% methanol to make solutions containing 0.3 mg, 20 μg, 20 μg, 0.1 mg, and 0.15 mg per 1 ml as reference solution.

[0122] 2. Preparation of test solution:

[0123] Take 0.5 g of the Xiaoxumingtang reference sample powder prepared in Example 1, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 80% methanol, weigh the weight, heat and reflux for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0124] 3. Fingerprint map construction:

[0125] Accurately pipette 2 μl of the reference solution and the test solution respectively, inject them into the ultra-high performance liquid chromatograph, and measure them using the UPLC wavelength switching method. Record the chromatogram for 40 minutes to obtain the fingerprint.

[0126] The chromatographic conditions are as follows:

[0127] Octadecylsilane bonded silica gel was used as the packing material (Agilent Infinity Lab Poroshell 120EC-C18, column length 150 mm, inner diameter 3.0 mm, particle size 2.7 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in Table 1; the flow rate was 0.5 ml / min; and the column temperature was 30°C.

[0128] The detection wavelength was PDA full wavelength scan (200-400 nm), and the derivatization channel was timed wavelength switching according to the gradient specified in Table 2. The theoretical plate number calculated based on the baicalin peak should be no less than 10,000.

[0129] Table 1 Gradient elution table:

[0130]

[0131] Table 2 Wavelength switching table:

[0132]

[0133]

[0134] Figure 1 This is the characteristic fingerprint of the Xiaoxumingtang benchmark sample detected in this example.

[0135] Figure 1 Among the 22 common peaks, peak 1: ephedrine hydrochloride, peak 2: pseudoephedrine hydrochloride, peak 4: amygdalin, peak 5: paeoniflorin, peak 6: cimicifugoside, peak 7: ferulic acid, peak 9: liquiritin, peak 11: 1,2,3,4,6-O-pentagalloylglucose, peak 12: 5-O-methylvisaminol glycoside, peak 13: fangchinoline, peak 14: tetrandrine, peak 15: baicalin, peak 17: melaleucain A-7-O-β-D-glucuronide, peak 18: cinnamaldehyde, peak 19: wogonin, peak 20: baicalein, peak 21: wogonin, peak 22: 6-gingerol.

[0136] Depend on Figure 1 It can be seen that the fingerprint of the test sample should present 22 chromatographic peaks, of which 5 peaks should correspond to the retention time of the corresponding reference peaks; there is a common peak match, and according to the similarity evaluation system of the chromatographic fingerprint of traditional Chinese medicine, the similarity between the fingerprint of the test sample and the fingerprint of the control shall not be less than 0.90.

[0137] Example 3 Test solution preparation and chromatographic conditions investigation

[0138] (1) Selection and investigation of reference objects:

[0139] According to the preparation method of the reference solution in Example 2, reference solutions of ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, paeoniflorin, cimicifugoside, 1,2,3,4,6-O-pentagalloylglucose, fangchinoline, tetrandrine, baicalin, wogonin, cinnamaldehyde, 6-gingerol, ferulic acid, 5-O-methylvisaminol glycoside, liquiritin, melaleucatin A-7-O-β-D-glucuronide, wogonin, and baicalein were prepared. Subsequently, the fingerprint detection method in Example 2 was used for detection, and the corresponding chromatogram is shown in FIG. Figure 2 .

[0140] Figure 2 The chromatogram is the reference substance detected in this example.

[0141] Figure 2 Among the 22 common peaks, peak 1: ephedrine hydrochloride, peak 2: pseudoephedrine hydrochloride, peak 4: amygdalin, peak 5: paeoniflorin, peak 6: cimicifugoside, peak 7: ferulic acid, peak 9: liquiritin, peak 11: 1,2,3,4,6-O-pentagalloylglucose, peak 12: 5-O-methylvisaminol glycoside, peak 13: fangchinoline, peak 14: tetrandrine, peak 15: baicalin, peak 17: melaleucain A-7-O-β-D-glucuronide, peak 18: cinnamaldehyde, peak 19: wogonin, peak 20: baicalein, peak 21: wogonin, peak 22: 6-gingerol.

[0142] Depend on Figure 2 It can be seen that Figure 2 The chromatographic peaks of amygdalin, paeoniflorin, fangchinoline, tetrandrine and baicalin are obvious, and the peak shapes and separation effects with adjacent chromatographic peaks are good. Therefore, amygdalin, paeoniflorin, fangchinoline, tetrandrine and baicalin were determined to be used as reference substances for the fingerprint of this product.

[0143] (2) Investigation of the preparation method of the test solution

[0144] (1) Investigation of extraction solvent:

[0145] It is planned to investigate different extraction solvents: water, 20% methanol, 50% methanol, 80% methanol, and methanol.

[0146] The test method is as follows: take 0.5g of the powder of the Xiaoxumingtang reference sample prepared in Example 1, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of the corresponding solvent, weigh it, ultrasonically treat it (power 500W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate. Each extraction solvent was tested in parallel according to the fingerprint spectrum construction method of Example 2. The results are shown in Figure 2. Figure 3 .

[0147] Figure 3 Ultra-high performance liquid chromatography (UPLC) analysis of different extraction solvents provided in this example.

[0148] Depend on Figure 3 It can be seen that samples extracted with water, 20% methanol and 50% methanol are difficult to filter; methanol has a weak extraction effect on highly polar components and the chromatographic peak shape is not good; 80% methanol has a large number of chromatographic peaks and is easy to filter, so the present invention uses 80% methanol as the extraction solvent.

[0149] (2) Investigation of extraction methods:

[0150] It is planned to investigate two extraction methods: ultrasound and reflux.

[0151] The test method is as follows: 0.5g of the powder of the Xiaoxumingtang reference sample prepared in Example 1 is taken, accurately weighed into four portions, and placed in stoppered conical flasks. 25ml of 80% methanol is accurately added and weighed. 1-2 portions are ultrasonically treated (power 500W, frequency 40kHz) for 30 minutes, and 3-4 portions are heated under reflux for 30 minutes. The mixture is taken out, cooled, and weighed again. The lost weight is supplemented with 80% methanol, shaken, filtered, and the filtrate is obtained. The results are shown in FIG. Figure 4 .

[0152] Figure 4 Ultra-high performance liquid chromatography (UPLC) analysis of different extraction methods provided in this example.

[0153] Depend on Figure 4 The results showed that the number and area of ​​chromatographic peaks obtained by the two extraction methods were similar. Considering that the experimental operation should be as simple as possible, the fingerprint and the determination of the content of Stephania tetrandra should share the same sample solution. Therefore, the reflux extraction method was selected as the extraction method for the fingerprint of this product.

[0154] (3) Extraction time investigation:

[0155] It is planned to investigate different extraction times of 15 minutes, 30 minutes, and 60 minutes.

[0156] The test method is as follows: 0.5g of the powdered Xiaoxumingtang standard sample prepared in Example 1 was accurately weighed and placed in a stoppered conical flask. 25ml of 80% methanol was accurately added and weighed. The sample was heated under reflux for the appropriate time. The sample was removed, cooled, and weighed again. The weight loss was supplemented with 80% methanol. The sample was shaken well, filtered, and the filtrate was obtained. Two replicates were prepared for each extraction time. The results are shown in Table 1. Figure 5 .

[0157] Figure 5 Ultra-high performance liquid chromatography (UPLC) analysis of the samples at different extraction times is provided in this example.

[0158] Depend on Figure 5The results showed that different extraction times had little effect on the number and area of ​​chromatographic peaks in the fingerprint. Therefore, a moderate extraction time of 30 minutes was selected.

[0159] (4) Investigation of solvent dosage:

[0160] It is planned to investigate different solvent dosages of 20ml, 25ml and 50ml.

[0161] The test method is as follows: 0.5g of the powder of the Xiaoxumingtang reference sample prepared in Example 1 is accurately weighed and placed in a stoppered conical flask. 20ml, 25ml, and 50ml of 80% methanol are accurately added, and the weight is weighed. The mixture is heated under reflux for 30 minutes, removed, cooled, and weighed again. The weight loss is supplemented with 80% methanol, shaken, filtered, and the filtrate is obtained. Each solvent is used in duplicate. The results are shown in FIG. Figure 6 .

[0162] Figure 6 Ultra-high performance liquid chromatography (UPLC) analysis of different solvent dosages provided in this example.

[0163] Depend on Figure 6 The results show that different solvent dosages have little effect on the number of chromatographic peaks in the fingerprint of this product. When the solvent dosage is 25 ml, the peak area is more appropriate. Therefore, the solvent dosage is selected as 25 ml.

[0164] Through the above investigation and research, the preparation method of the test solution for the fingerprint of the Xiaoxumingtang benchmark sample was finally determined as follows:

[0165] Take 0.5 g of the Xiaoxumingtang reference sample powder prepared in Example 1, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 80% methanol, weigh the weight, heat and reflux for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0166] (III) Chromatographic conditions and system suitability test

[0167] (1) Detection wavelength selection:

[0168] Figure 7 This is the full wavelength scanning spectrum of the Xiaoxumingtang reference sample prepared in Example 1.

[0169] Figure 8a The ultra-high performance liquid chromatography (UPLC) spectrum with a detection wavelength of 210 to 280 nm provided in Example 3 of the present invention;

[0170] Figure 8b This is an ultra-high performance liquid chromatography (UPLC) analysis spectrum at a detection wavelength of 290 to 360 nm provided in Example 3 of the present invention.

[0171] The diode array detector was used to detect the sample at full wavelength and collect the three-dimensional spectrum (see Figure 7 ), and simultaneously collect spectra at wavelengths of 210nm, 230nm, 254nm, 280nm, 290nm, 320nm, and 360nm (see Figure 8a-8b ).

[0172] The results showed that the chemical composition of this product was complex, with a large number of chromatographic peaks, and the ultraviolet absorption wavelengths of the chromatographic peaks varied greatly. The number of chromatographic peaks was the largest at a wavelength of 210 nm, but the chromatographic peak signal of baicalin was strong, resulting in an unbalanced ratio of chromatographic peaks in the entire spectrum. Therefore, the derivative channel method was used for timed wavelength switching, that is, 210 nm was initially used to examine the chemical components in medicinal flavors such as ephedra and bitter almonds. When it reached the chromatographic peak of baicalin, the wavelength was switched to 360 nm to reduce the signal intensity of baicalin, and then the wavelength was switched to 290 nm to better detect cinnamaldehyde from cinnamon.

[0173] Based on the above research, the fingerprint spectrum detection wavelength of this product selects PDA full wavelength scanning (200nm~400nm), and simultaneously collects 210nm, 290nm, and 360nm spectra. After the detection, the derivative channel is used for timed wavelength switching. The switching wavelength gradient is shown in Table 3:

[0174] Table 3:

[0175]

[0176] Note: The Rt baicalin is the time when the detection chromatogram reaches the baicalin peak.

[0177] The results after the derivative channel wavelength switching process are shown in Figure 9 .

[0178] Figure 9 This is a wavelength switching diagram of different derivative channels provided in this embodiment.

[0179] (2) Chromatographic column inspection:

[0180] Three different batches of Agilent InfinityLab Poroshell 120EC-C18 (3.0 mm × 150 mm, 2.7 μm) columns were tested. The results showed that all three columns provided good separation performance, demonstrating the stability and reproducibility of this column model across batches.

[0181] Figure 10 This is an investigation graph of different chromatographic columns provided in this example.

[0182] (3) Investigation of mobile phase:

[0183] Five mobile phases, including acetonitrile-0.05% phosphoric acid solution, acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.2% phosphoric acid solution, acetonitrile-water, and acetonitrile-0.1% formic acid solution, were investigated. The gradient elution table is shown in Table 4 (mobile phase A is acetonitrile, mobile phase B is water, 0.05% to 0.2% phosphoric acid solution, and 0.1% formic acid solution).

[0184] Table 4:

[0185]

[0186]

[0187] Figure 11a The investigation spectrum of acetonitrile-water and acetonitrile-0.1 formic acid mobile phases provided in Example 3 of the present invention;

[0188] Figure 11b The investigation spectrum of acetonitrile-phosphoric acid mobile phase of different concentrations provided in Example 3 of the present invention;

[0189] See the results Figure 11a-11b Among them, acetonitrile-0.1% phosphoric acid solution has a better separation effect on each chromatographic peak, so acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase.

[0190] (4) Investigation of column temperature:

[0191] Seven column temperatures of 25°C, 28°C, 29°C, 30°C, 31°C, 32°C, and 35°C were selected for investigation. The results are shown in the figure. Figure 12a-12b .

[0192] Figure 12a The investigation spectrum for the column temperature of 25-30°C provided in Example 3 of the present invention;

[0193] Figure 12b This is an investigation graph for a column temperature of 31-35°C provided in Example 3 of the present invention.

[0194] The results showed that the column temperature had a great influence on the separation effect of each chromatographic peak. When the temperature fluctuated between 29℃ and 30℃, the separation effect of each chromatographic peak was better. The column temperature of 30℃ was selected for subsequent methodological experimental studies.

[0195] (5) Investigation of flow rate:

[0196] Six flow rates of 0.45ml / min, 0.48ml / min, 0.50ml / min, 0.51ml / min, 0.52ml / min and 0.55ml / min were selected for investigation. The results are shown in Figures 13a to 13c .

[0197] Figure 13aThe investigation graphs for flow rates of 0.45 ml / min and 0.48 ml / min provided in Example 3 of the present invention;

[0198] Figure 13b The investigation graphs for flow rates of 0.50 ml / min and 0.51 ml / min provided in Example 3 of the present invention;

[0199] Figure 13c The investigation graphs for flow rates of 0.52 ml / min and 0.55 ml / min provided in Example 3 of the present invention;

[0200] The results showed that when the flow rate fluctuated in the range of 0.48ml / min to 0.50ml / min, the separation effect of each chromatographic peak was good, and the flow rate of 0.50ml / min was selected for subsequent methodological experimental research.

[0201] (6) Investigation of injection volume:

[0202] Four injection volumes of 1μl, 2μl, 3μl, and 4μl were selected for investigation. The results are shown in Figure 14 .

[0203] Figure 14 This is a graph showing the investigation of different injection volumes provided in this example.

[0204] The results showed that the separation effect of each chromatographic peak was good and the peak shape was good in 2-3 μl. The injection volume of 2 μl was selected for subsequent experimental studies.

[0205] (7) Specificity and integrity assessment:

[0206] The interference of blank mobile phase and blank solvent on the test solution was investigated, and the double retention time was investigated. The results are shown in Figure 15 .

[0207] Figure 15 The blank mobile phase, blank solvent, and double retention time inspection graph provided in this example

[0208] The results showed that the blank mobile phase and blank solvent had no interference on the fingerprint of the test solution, and the test sample spectrum at twice the retention time proved that the test sample had no other chromatographic peaks after 40 minutes.

[0209] Through the above investigations, the chromatographic conditions for the fingerprint of the Xiaoxumingtang benchmark sample were determined as follows: an Agilent InfinityLab Poroshell 120EC-C18 column (3.0 mm × 150 mm, 2.7 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; a flow rate of 0.5 ml / min; a column temperature of 30°C; a PDA full wavelength scan (200-400 nm) for detection, with timed wavelength switching of the derivatization channel according to the gradient specified in the table below. The number of theoretical plates, calculated based on the baicalin peak, should be no less than 10,000.

[0210] Table 5 Gradient elution table:

[0211]

[0212] Table 6 Wavelength switching table:

[0213]

[0214] Example 4 Methodological Investigation

[0215] (1) Precision test:

[0216] Take the freeze-dried powder of Xiaoxumingtang (batch number XXMT01) prepared by China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd. and prepare it according to the preparation method of the test solution in the main text. Inject the sample 6 times continuously and detect the fingerprint. The chromatogram is shown in Figures 16 and 17 ,The similarity was calculated using the fingerprint similarity evaluation software (2012 edition), and the results are shown in Table 7.

[0217] Note: The freeze-dried powder of Xiaoxumingtang with batch number XXMT01 is a reference sample for research, and its preparation method and raw material composition are the same as those in Example 1.

[0218] Figure 16 The original spectrum for precision investigation provided in this embodiment.

[0219] Figure 17 The precision test matched spectrum provided in this example (refer to the spectrum: S1) Time window: 0.10; method for generating the reference spectrum: average.

[0220] Table 7 Precision inspection results:

[0221]

[0222] The results showed that the similarity of each chromatogram reached above 0.90, which met the requirements of fingerprint spectrum and indicated that the instrument had good precision.

[0223] (2) Stability test

[0224] Take the freeze-dried powder of Xiaoxumingtang (batch number XXMT01) prepared by China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd. and prepare it according to the preparation method of the test solution in the text. The fingerprint spectrum was tested at 0, 2, 6, 12, 18, and 24 hours respectively. The results are shown in Figure 18 、 Figure 19 .

[0225] Figure 18 This is the original stability analysis graph provided in this example.

[0226] Figure 19 This is the stability test matched spectrum provided in this example. (Refer to the spectrum: S1) Time window: 0.10 Method for generating the control spectrum: average.

[0227] The similarity was calculated using the fingerprint similarity evaluation software (2012 edition), and the results are shown in Table 8.

[0228] Table 8 Stability investigation results:

[0229]

[0230] The results showed that the similarity of each chromatogram reached above 0.90, which met the requirements of the fingerprint, indicating that the test solution was stable for at least 24 hours.

[0231] (3) Repeatability test:

[0232] Take the freeze-dried powder of Xiaoxumingtang (batch number XXMT01) prepared by China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd., and prepare 6 test samples according to the preparation method of the test solution in the main text. The fingerprint spectrum is tested. The results are shown in Figures 20 and 21 .

[0233] Figure 20 The original atlas for the repeatability investigation provided in this embodiment.

[0234] Figure 21 The matched spectrum for the repeatability test provided in this example. (Refer to the spectrum: S1) Time window: 0.10 Method for generating the control spectrum: average.

[0235] The similarity was calculated using the fingerprint similarity evaluation software (2012 edition), and the results are shown in Table 9.

[0236] Table 9 Repeatability test results:

[0237]

[0238] The results showed that the similarity of each chromatogram reached above 0.90, which met the requirements of fingerprint, indicating that the method had good repeatability.

[0239] (4) Intermediate precision test:

[0240] Take this product (batch number XXMT01) and prepare 6 test solutions according to the test solution preparation method by different analysts on different dates. Use Waters Arc, Waters Class 1# and Waters Class 2# three analytical instruments to test. The results are shown in Figures 22 and 23 ,The similarity results of the three instruments are shown in Table 10.

[0241] Figure 22 The intermediate precision provided for this embodiment examines the original spectrum.

[0242] Figure 23 The intermediate precision test matched spectrum provided in this example. (Refer to the spectrum: S1) Time window: 0.10 Method for generating the reference spectrum: average.

[0243] Table 10 Intermediate precision test results:

[0244]

[0245] The results showed that the intermediate precision similarities of the three instruments were all greater than 0.90, indicating that the fingerprint method had good applicability.

[0246] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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 characteristic fingerprint of Xiaoxumingtang based on UPLC wavelength switching method, characterized in that: The following steps are involved: Preparation of reference solution: dissolve baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance, and amygdalin reference substance in a solvent to obtain a mixed reference solution; Preparation of test solution: Dissolve the Xiaoxumingtang reference sample in 80% methanol to obtain the test solution; Fingerprint construction: The test solution and the reference solution were measured by UPLC wavelength switching method to obtain the fingerprint. The UPLC wavelength switching method uses timed wavelength switching technology to measure the absorbance values ​​of components at different wavelengths in different time periods. The detection wavelengths are: 0~(Rt 黄芩苷 -0.6)min detection wavelength is 210nm; (Rt 黄芩苷 -0.6)~(Rt 黄芩苷 +0.6)min detection wavelength is 360nm; (Rt 黄芩苷 +0.6)~40min detection wavelength is 290nm; The Rt 黄芩苷 It is the time when the chromatogram reaches the baicalin peak; The UPLC wavelength switching method is carried out by using an ultra-high performance liquid chromatography (ULC) instrument with a gradient elution method using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The gradient elution method is: The chromatographic conditions of the ultra-high performance liquid chromatograph of the UPLC wavelength switching method include: The chromatographic column is filled with octadecylsilane bonded silica gel, the column temperature is 25-35° C., and the flow rate of the mobile phase is 0.3-0.5 ml / min.

2. The method according to claim 1, characterized in that The raw materials for preparing the Xiaoxuemiantang benchmark sample include: ephedra, stephania tetrandra, ginseng, scutellaria baicalensis, cinnamon bark, fried licorice, white peony root, chuanxiong, stewed bitter almonds, heishunpian, siler, and ginger.

3. The method according to claim 2, characterized in that The preparation method of the Xiaoxumingtang reference sample comprises: Ephedra, Stephania tetrandra, ginseng, scutellaria baicalensis, cinnamon bark, stir-fried licorice, white peony root, ligusticum chuanxiong, stewed bitter almond, heishunpian, saposhnikovia divaricata, and ginger were mixed, crushed, decocted, and a decoction was obtained, which was then freeze-dried to obtain a Xiaoxuemiantang benchmark sample.

4. The method according to claim 1, wherein The chromatographic column is Agilent Infinity Lab Poroshell 120EC-C18, with a column length of 150 mm, an inner diameter of 3.0 mm, and a particle size of 2.7 μm.

5. The method according to claim 1, wherein The preparation method of the reference substance solution comprises: Take appropriate amounts of baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance, and amygdalin reference substance, add them to 80% methanol to prepare solutions containing 0.3 mg, 20 μg, 20 μg, 0.1 mg, and 0.15 mg per ml respectively, as reference solution.

6. The method according to claim 1, characterized in that The preparation method of the test solution comprises: Take 0.5 g of Xiaoxumingtang reference sample, add it to 25 ml of 80% methanol, heat and reflux to extract for 30 minutes, filter, and take the filtrate to obtain the test solution.

7. The method according to claim 1, characterized in that The method for constructing the characteristic fingerprint of Xiaoxumingtang also includes: Take at least 10 batches of Xiaoxuemiantang benchmark samples, prepare test solutions respectively, collect fingerprints, and import them into the Chinese medicine chromatographic fingerprint similarity evaluation system of the National Pharmacopoeia Committee to generate the characteristic reference fingerprint of Xiaoxuemiantang.

8. Use of the method for constructing the characteristic fingerprint of Xiaoxumiantang based on the UPLC wavelength switching method according to any one of claims 1 to 7 in the overall quality control of Xiaoxumiantang.

9. The use according to claim 8, characterized in that The applications include: (A) providing a sample of Xiaoxumingtang to be tested, and obtaining a fingerprint of the Xiaoxumingtang sample according to the method for constructing a characteristic fingerprint of Xiaoxumingtang based on the UPLC wavelength switching method according to any one of claims 1 to 7; (B) Compare the obtained fingerprint of the Xiaoxumingtang sample with the characteristic fingerprint of the Xiaoxumingtang control. If the similarity is ≥0.9, the quality of the Xiaoxumingtang sample is qualified.

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