Method for constructing Xiaosheng characteristic fingerprint spectrum based on UPLC wavelength switching method and application of Xiaosheng characteristic fingerprint spectrum

Abstract

The invention provides a method for constructing a Xiaozhi decoction characteristic fingerprint spectrum based on a UPLC wavelength switching method and application thereof, and relates to the technical field of traditional Chinese medicine quality control. The fingerprint spectrum is constructed based on a UPLC (Ultra Performance Liquid Chromatography) wavelength switching method; in the construction of the fingerprint spectrum, the detection wavelength is selected as follows: the detection wavelength of 0-(Rt baicale-0. 6) min is 210 nm; the detection wavelength of (Rt baicale-0. 6)-(Rt baicalin + 0. 6) min is 360 nm; and the detection wavelength of the (Rt baicalin + 0.6)-40 minutes is 290 nm. By selecting the UPLC detection wavelength and the ultra-high performance liquid chromatography conditions, 22 common chromatographic peaks can be calibrated and 18 chromatographic peaks can be identified in 40 minutes in the characteristic fingerprint of the Xiaoshenfen decoction, more comprehensive quality evaluation can be performed on the Xiaoshenfen decoction, the time is short, and a scientific basis can be provided for quality evaluation and control of the Xiaoshenfen decoction.

Description

Technical Field

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

[0002] Xiaoxumingtang was first mentioned in Xiaopinfang written by Chen Yanzhi during the Jin Dynasty and the Southern and Northern Dynasties. It was used to treat stroke. After being included in the Prescriptions for Emergencies and Prescriptions for Thousand Gold Pieces and Waitai Miyao, it became widely circulated. The recipe includes one liang each of ephedra, fangji, ginseng, scutellaria, cinnamon heart, licorice, peony, chuanxiong, and almond, one aconite, one and a half liang of saposhnikovia, and five liang of ginger. It is mainly used to treat symptoms such as crooked mouth and eyes, tight tendons and veins, hemiplegia, tongue stiffness and inability to speak, or depressed expression. Before the Tang and Song Dynasties, doctors believed that the cause of stroke was "internal deficiency and evil spirits". This recipe was the first recipe for treating wind, which had the function of nourishing and removing evil spirits. It was widely used and was called "the most important of all soups" at that time, so it was widely used. In recent years, the Chinese medicine community generally believes that blood stasis is the material basis of stroke. Xiaoxumingtang uses ephedra, cinnamon twig, and dried ginger as the main ingredients to warm and disperse the blood stasis of cold in the meridians and remove blood stasis to treat stroke, and it is not specifically for invigorating qi and warming yang. Modern people have a more comprehensive understanding of the pathogenesis of stroke and the explanation of Xiaoxumingtang from a unique perspective. This not only strengthens the contemporary emphasis on this prescription, but also allows Xiaoxumingtang to be greatly extended and developed on the original basis. Modern clinical practice mainly uses it for stroke and its sequelae, cerebral infarction, facial neuritis, Alzheimer's disease, etc.

[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 studied the origin of the Chinese medicinal base of Xiaoxuemiantang, the harvesting, processing and preparation, as well as the pharmacology, pharmacodynamics and pharmacokinetics of the chemical composition and effective components, and continuously improved its quality standards, laying the foundation for the development of classic prescriptions. At present, the research on Xiaoxuemiantang is mainly focused on the research of pharmacology and clinical application. There are few studies on the material basis, extraction process, multi-index component content determination and characteristic spectrum of its decoction, which 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] Traditional Chinese medicine fingerprint refers to a chromatogram or spectrum that can indicate the chemical characteristics of certain Chinese medicinal materials or Chinese medicinal preparations after proper treatment and using certain analytical methods. It is a comprehensive and quantifiable quality evaluation method that can effectively reflect the integrity and comprehensive effects of Chinese medicine components. It is widely used in traditional Chinese medicine analysis, identification and quality control due to its rapid and accurate characteristics.

[0005] The ingredients of Xiaoxumingtang are very complex. The fingerprint of Xiaoxumingtang established by high performance liquid chromatography and gas chromatography takes a long time and has a small number of characteristic peaks, making it difficult to evaluate the quality of Xiaoxumingtang efficiently and comprehensively. Therefore, it is very necessary to develop a simple, accurate, and repeatable characteristic spectrum detection method that can reflect as many ingredients as possible in Xiaoxumingtang for its 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 conduct a more comprehensive quality evaluation of Xiaoxumiantang in a short time, and 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 a UPLC wavelength switching method.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly 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 substance solution: dissolving baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance and amygdalin reference substance in a solvent to obtain a mixed reference substance solution;

[0012] Preparation of test solution: dissolve the Xiaoxumingtang reference sample in a solvent to obtain a 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 a timed wavelength switching technique to measure the absorbance values ​​at different wavelengths for components in different time periods. The detection wavelength is:

[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 chromatographic peak of baicalin.

[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, cinnamon, fried licorice, white peony root, ligusticum chuanxiong, stewed bitter almond, heishunpian, saposhnikovia divaricata and ginger were mixed, crushed and decocted to obtain a decoction, 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: using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B for 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 uses octadecylsilane bonded silica gel as a filler, the column temperature is 25 to 35° C.; the flow rate of the mobile phase is 0.3 to 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 uses octadecylsilane bonded silica gel as a filler, 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 InfinityLabPoroshell 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 comprises:

[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 make solutions containing 0.3 mg, 20 μg, 20 μg, 0.1 mg, and 0.15 mg per 1 ml, respectively, as reference substance solutions.

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

[0031] Take 0.5 g of Xiaoxumingtang standard 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 are as follows: taking at least 10 batches of Xiaoxumiantang benchmark samples, preparing test sample solutions respectively, collecting fingerprints, and importing them into the Chinese medicine chromatographic fingerprint similarity evaluation system of the State Pharmacopoeia Committee to generate the characteristic reference fingerprint of Xiaoxumiantang.

[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 Xiaoxumingtang sample fingerprint with the Xiaoxumingtang characteristic reference fingerprint. 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 complex chemical composition of Xiaoxumingtang, the large number of chromatographic peaks, and the large difference in the ultraviolet absorption wavelengths of the chromatographic peaks, 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 relatively 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 chromatographic peak of baicalin to reduce the baicalin signal intensity, and then switching again. The wavelength is increased to 290 nm so as to better detect cinnamaldehyde from cinnamon. Then, 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, cimicifuga glycoside, ferulic acid, glycyrrhizin, 1,2,3,4,6-O-pentagalloyl glucose, 5-O-methylvisaminol glycoside, fangchinoline, tetrandrine, baicalin, melaleucatin A-7-O-β-D-glucuronide, cinnamaldehyde, baicalin, baicalein, wogonin and 6-gingerol.

[0040] Therefore, the characteristic fingerprint of Xiaoxumiantang constructed in the present application can conduct a more comprehensive quality evaluation of Xiaoxumiantang 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 Xiaoxumiantang.

[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 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.

[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 inspection 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 inspection spectrum with a detection wavelength of 290 to 360 nm provided in Example 3 of the present invention;

[0052] Fig. 9 The wavelength switching spectrum of different derivative channels provided in Example 3 of the present invention;

[0053] Fig.10 The investigation spectrum of different chromatographic columns provided in Example 3 of the present invention;

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

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

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

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

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

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

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

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

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

[0063] Fig.16 The original spectrum of precision investigation provided by Example 4 of the present invention;

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

[0065] Fig.18 The original stability test spectrum provided in Example 4 of the present invention;

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

[0067] Fig. 20 The original spectrum of the repeatability investigation provided in Example 4 of the present invention;

[0068] Fig.21 The reproducibility test matched spectrum provided in Example 4 of the present invention;

[0069] Fig. 22 The original spectrum of the intermediate precision investigation provided in Example 4 of the present invention;

[0070] Fig.23 The intermediate precision inspection matched spectrum provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0071] 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.

[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 substance solution: dissolving baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance and amygdalin reference substance in a solvent to obtain a reference substance solution;

[0074] Preparation of test solution: dissolve the Xiaoxumingtang reference sample in a solvent to obtain a 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 a timed wavelength switching technique to measure the absorbance values ​​at different wavelengths for components in different time periods. The detection wavelength is:

[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 chromatographic peak of baicalin.

[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 are greatly different. The present invention studied and found in the construction of 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 to perform 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 signal from Cinnamaldehyde of cinnamon bark, and then 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, cimicifuga glycoside, ferulic acid, liquiritigenin, 1,2,3,4,6-O-pentagalloyl glucose, 5-O-methylvisaminol glycoside, fangchinoline, tetrandrine, baicalin, melaleucatin A-7-O-β-D-glucuronide, cinnamaldehyde, baicalin, baicalin, wogonin, and 6-gingerol.

[0082] Therefore, the characteristic fingerprint of Xiaoxumiantang constructed in the present application can conduct a more comprehensive quality evaluation of Xiaoxumiantang 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 Xiaoxumiantang.

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

[0084] As a preferred embodiment, the fried licorice and heishunpian used as medicines in the composition raw materials of the Xiaoxuemiantang benchmark sample of the present application are not only more in line 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 comprises:

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

[0087] Preferably, the method for preparing the Xiaoxumingtang reference sample comprises:

[0088] (1) Prepare the raw materials of Xiaoxuemiantang according to the following mass: ephedra 13.8 g, stephania tetrandra 13.8 g, ginseng 13.8 g, scutellaria baicalensis 13.8 g, cinnamon bark 13.8 g, stir-fried licorice root 13.8 g, white peony root 13.8 g, chuanxiong rhizome 13.8 g, stewed bitter almond 13.8 g, heishunpian 15 g, siler 20.7 g, ginger 69 g.

[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 foam was removed after three boils (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: using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B for gradient elution;

[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 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;

[0095] Preferably, the chromatographic column is Agilent InfinityLabPoroshell 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 make solutions containing 0.3 mg, 20 μg, 20 μg, 0.1 mg, and 0.15 mg per 1 ml, respectively, as reference substance solutions.

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

[0099] Take 0.5 g of Xiaoxumingtang standard 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 Xiaoxumiantang benchmark samples, prepare test solutions respectively, collect fingerprints, and import them into the Chinese medicine chromatographic fingerprint similarity evaluation system of the State Pharmacopoeia Committee to generate the characteristic reference fingerprint of Xiaoxumiantang.

[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 comprises:

[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 Xiaoxumingtang sample fingerprint with the Xiaoxumingtang characteristic reference fingerprint. 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 in conjunction with 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 500W, frequency 40kHz, Kunshan Ultrasonic Instrument Co., Ltd.; Analytical balance: 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%; cimicifuga glycosides, 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-methylvisaminol glycoside, 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, 98.00%; 1,2,3,4,6-O-pentagalloylglucose, batch number MUST-14020710, purity ≥98%, were all purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0112] Reference sample for research: Xiaoxuemiantang freeze-dried powder prepared by China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd., batch number XXMT01.

[0113] Example 1 Preparation of Xiaoxumingtang Standard Sample

[0114] A method for preparing a reference sample of Xiaoxumingtang 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, Ligusticum chuanxiong 13.8g, Boiled bitter almond 13.8g, Heishun tablets 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 foam was removed after three boils (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 comprises 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, weigh accurately, 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 substance solutions.

[0122] 2. Preparation of test solution:

[0123] Take 0.5 g of the Xiaoxumingtang reference sample powder prepared in Example 1, accurately weigh it, put 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, weigh it again, make up the lost weight with 80% methanol, shake well, filter, 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 spectrum.

[0126] The chromatographic conditions are as follows:

[0127] Octadecylsilane bonded silica gel was used as filler (Agilent InfinityLabPoroshell 120EC-C18, column length 150 mm, inner diameter 3.0 mm, particle size 2.7 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 0.5 ml per minute; the column temperature was 30°C;

[0128] The detection wavelength is PDA full wavelength scan (200-400 nm), and the derivative channel is timed wavelength switching according to the specified gradient in Table 2. The theoretical plate number calculated based on the baicalin peak should not be 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: cimicifuga glycoside, peak 7: ferulic acid, peak 9: liquiritigenin, peak 11: 1,2,3,4,6-O-pentagalloyl glucose, 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: baicalin, peak 21: wogonin, peak 22: 6-gingerol.

[0136] Depend on Figure 1 It can be seen that there should be 22 chromatographic peaks in the fingerprint of the test sample, of which 5 peaks should correspond to the retention times of the corresponding reference peaks; there is common peak matching, and according to the similarity evaluation system of Chinese medicine chromatographic fingerprint, 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] (I) Selection and investigation of reference objects:

[0139] According to the preparation method of the reference solution of Example 2, prepare reference solutions of ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, paeoniflorin, cimicifuga glycosides, 1,2,3,4,6-O-pentagalloyl glucose, fangchinoline, tetrandrine, baicalin, wogonin, cinnamaldehyde, 6-gingerol, ferulic acid, 5-O-methylvisaminol glycoside, licorice glycoside, melaleucatin A-7-O-β-D-glucuronide, wogonin, and baicalin. Then detect according to the fingerprint detection method of Example 2, 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: cimicifuga glycoside, peak 7: ferulic acid, peak 9: liquiritigenin, peak 11: 1,2,3,4,6-O-pentagalloyl glucose, 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: baicalin, 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] (II) 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: take 0.5g of the Xiaoxumingtang reference sample powder prepared in Example 1, accurately weigh it, put 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 map construction method of Example 2. The results are shown in 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 has a poor chromatographic peak shape; 80% methanol has a large number of chromatographic peaks and is easy to filter, so the present invention selects 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: take 0.5g of the powder of the Xiaoxumingtang reference sample prepared in Example 1, accurately weigh four portions, place them in stoppered conical bottles, accurately add 25ml of 80% methanol, weigh the weight, ultrasonically treat 1-2 portions (power 500W, frequency 40kHz) for 30 minutes, heat reflux extraction for 30 minutes for 3-4 portions, take them out, cool them, weigh them again, make up the lost weight with 80% methanol, shake them well, filter them, and take the filtrate to obtain the result. 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 show that the number and area of ​​chromatographic peaks obtained by the two extraction methods are not much different. Considering that the experimental operation should be as simple as possible, the fingerprint spectrum and the determination of the content of Stephania tetrandra should share the same test solution. Therefore, the reflux extraction method was selected as the extraction method for the fingerprint spectrum 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: take 0.5g of the powder of the Xiaoxumingtang standard sample prepared in Example 1, weigh it accurately, put it in a stoppered conical flask, add 25ml of 80% methanol accurately, weigh it, heat and reflux to extract for a corresponding time, 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. Two parallels are made for each extraction time. The results are shown in Figure 5 .

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

[0158] Depend on Figure 5It shows that different extraction times have little effect on the number and area of ​​chromatographic peaks in the fingerprint. Therefore, a relatively 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: take 0.5g of the powder of the Xiaoxumingtang standard sample prepared in Example 1, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml, 25ml, and 50ml of 80% methanol respectively, weigh it, heat and reflux to extract 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. Each amount of solvent is used in parallel in duplicate. The results are shown in 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 It shows 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 suitable. 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, put it in a stoppered conical flask, accurately add 25 ml of 80% methanol, weigh it, 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 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 inspection 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 survey spectrum with 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 three-dimensional spectra (see Figure 7 ), and simultaneously collect spectra at wavelengths of 210nm, 230nm, 254nm, 280nm, 290nm, 320nm, and 360nm (see Figure 8a to Figure 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 210nm, but the signal of the chromatographic peak of baicalin was strong, resulting in an uncoordinated ratio of chromatographic peaks in the entire spectrum. Therefore, the derivative channel method was used for timed wavelength switching, that is, 210nm was initially used to examine the chemical components in medicinal flavors such as ephedra and bitter almonds. At the chromatographic peak of baicalin, the wavelength was switched to 360nm to reduce the signal intensity of baicalin, and then the wavelength was switched to 290nm 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 chromatographic peak.

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

[0178] Fig. 9 The wavelength switching spectra of different derivative channels provided in this embodiment.

[0179] (2) Chromatographic column inspection:

[0180] Three different batches of Agilent InfinityLabPoroshell 120EC-C18 (3.0mm×150mm, 2.7μm) chromatographic columns were investigated. The results showed that the separation effects of the three columns were good, indicating that the chromatographic columns of this model are stable and reproducible between different batches.

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

[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 respectively. The gradient elution table is shown in Table 4 (mobile phase A is acetonitrile, mobile phase B is water, 0.05%~0.2% phosphoric acid solution, and 0.1% formic acid solution).

[0184] Table 4:

[0185]

[0186]

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

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

[0189] Results Figure 11a to Figure 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 is 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 Figure 12a to Figure 12b .

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

[0193] Figure 12b This is an investigation spectrum 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 it fluctuated in the range of 29℃~30℃, the separation effect of each chromatographic peak was better. The column temperature of 30℃ was selected for subsequent methodological experimental research.

[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 Figure 13a to Figure 13c .

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

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

[0199] Fig.13c The investigation graphs of 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 Fig.14 .

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

[0204] The results showed that 2-3 μl had good separation effect on each chromatographic peak and better peak shape. 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 Fig.15 .

[0207] Fig.15 The blank mobile phase, blank solvent, and double retention time inspection spectrum 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 sample spectrum with twice the retention time proved that the test sample had no other chromatographic peaks after 40 minutes.

[0209] Through the above investigation and research, the chromatographic conditions of the fingerprint of the Xiaoxumingtang benchmark sample were finally determined as follows: Agilent InfinityLabPoroshell 120EC-C18 (3.0mm×150mm, 2.7μm) as the chromatographic column; acetonitrile as the mobile phase A, 0.1% phosphoric acid solution as the mobile phase B, gradient elution as specified in the table below; flow rate of 0.5ml per minute; column temperature of 30℃; detection wavelength of PDA full wavelength scan (200-400nm), and timed wavelength switching of the derivatization channel as specified in the table below. The theoretical plate number 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 Xiaoxumingtang freeze-dried powder (batch number XXMT01) prepared by China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd., prepare it according to the preparation method of the test solution in the text, inject it 6 times continuously, and detect the fingerprint spectrum. The chromatogram is shown in Figure 16-17 , the similarity was calculated using the fingerprint similarity evaluation software (2012 edition), and the results are shown in Table 7.

[0217] Note: The Xiaoxumingtang freeze-dried powder of 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] Fig.16 The original spectrum for precision investigation provided for this embodiment.

[0219] Fig.17 The precision inspection matched spectrum provided in this embodiment. (Refer to the spectrum: S1) Time window: 0.10; Method for generating the reference spectrum: average.

[0220] Table 7 Precision investigation results:

[0221]

[0222] The results showed that the similarity of each chromatogram was 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 Xiaoxumingtang freeze-dried powder (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 Fig.18 , Fig.19 .

[0225] Fig.18 This is the original stability study graph provided in this example.

[0226] Fig.19 The matched spectrum for stability inspection provided in this embodiment. (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 similarities of the chromatograms were all above 0.90, meeting the requirements of the fingerprint, indicating that the test solution was stable for at least 24 hours.

[0231] (3) Repeatability test:

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

[0233] Fig. 20 The original spectrum for the repeatability investigation provided in this embodiment.

[0234] Fig.21 The matched spectrum after repeatability inspection provided in this embodiment. (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 was 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 for testing. The results are shown in Figure 22-23 ,The similarity results of the three instruments are shown in Table 10.

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

[0242] Fig.23 The intermediate precision inspection matched spectrum provided in this embodiment. (Refer to the spectrum: S1) Time window: 0.10 Method for generating the control 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 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 characteristic fingerprint of Xiaoxumingtang based on UPLC wavelength switching method, characterized in that: The following steps are involved: Preparation of reference substance solution: dissolving baicalin reference substance, fangchinoline reference substance, tetrandrine reference substance, paeoniflorin reference substance and amygdalin reference substance in a solvent to obtain a mixed reference substance solution; Preparation of test solution: dissolve the Xiaoxumingtang reference sample in a solvent to obtain a 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 a timed wavelength switching technique to measure the absorbance values ​​at different wavelengths for components in different time periods. The detection wavelength is: 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 chromatographic peak of baicalin.

2. The method according to claim 1, characterized in that The raw materials for preparing the Xiaoxuemiantang benchmark sample include: ephedra, tetrandra, ginseng, scutellaria, cinnamon, 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, cinnamon, fried licorice, white peony root, ligusticum chuanxiong, stewed bitter almond, heishunpian, saposhnikovia divaricata and ginger were mixed, crushed and decocted to obtain a decoction, which was then freeze-dried to obtain a Xiaoxuemiantang benchmark sample.

4. The method according to claim 1, characterized in that: The elution mode of the ultra-high performance liquid chromatography of the UPLC wavelength switching method is: using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B for gradient elution; The gradient elution method is:

5. The method according to claim 1, characterized in that: 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; Preferably, the chromatographic column is Agilent InfinityLabPoroshell 120EC-C18, with a column length of 150 mm, an inner diameter of 3.0 mm, and a particle size of 2.7 μm.

6. The method according to claim 1, characterized in that 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 make solutions containing 0.3 mg, 20 μg, 20 μg, 0.1 mg, and 0.15 mg per 1 ml, respectively, as reference substance solutions.

7. The method according to claim 1, characterized in that The preparation method of the test solution comprises: Take 0.5 g of Xiaoxumingtang standard 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.

8. 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 Xiaoxumiantang benchmark samples, prepare test solutions respectively, collect fingerprints, and import them into the Chinese medicine chromatographic fingerprint similarity evaluation system of the State Pharmacopoeia Committee to generate the characteristic reference fingerprint of Xiaoxumiantang.

9. Application of the method for constructing the characteristic fingerprint of Xiaoxumiantang based on UPLC wavelength switching method as described in any one of claims 1 to 8 in the overall quality control of Xiaoxumiantang.

10. The use according to claim 9, 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 as described in any one of claims 1 to 8; (B) Compare the obtained Xiaoxumingtang sample fingerprint with the Xiaoxumingtang characteristic reference fingerprint. If the similarity is ≥ 0.9, the quality of the Xiaoxumingtang sample is qualified.

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