Method for detecting chemical components in Xiaozhi decoction reference sample

Abstract

The invention relates to the technical field of detection methods, and provides a method for detecting chemical components in a Xiaozhi decoction reference sample, which adopts high performance liquid chromatography to detect the components in the Xiaozhi decoction reference sample. Chromatographic conditions during high performance liquid chromatography detection are as follows: a mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.1-0.3%; gradient elution is adopted during elution. According to the technical scheme, the problems of insufficient repeatability and stability during detection of the components in the Xiaozhi decoction in related technologies are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of detection methods, and specifically, to a method for detecting chemical components in a reference sample of Xiao Xuming Decoction. Background Art

[0002] Xiao Xuming Decoction was first recorded in "Xiaopin Prescription" by Chen Yanzhi in the Eastern Jin Dynasty. Before the Tang and Song dynasties, it was regarded as "the most important among all decoctions" because of its remarkable effect in treating stroke. From the "Key Information Table of Ancient Classical Famous Prescriptions", it can be known that the prescription of Xiao Xuming Decoction comes from "Emergency Prescriptions Worth a Thousand Gold" by Sun Simiao, and it is mainly composed of 12 herbs including Ephedra, Stephania Tetrandra Root, Ginseng, Scutellaria Baicalensis, Cinnamon Twig, Licorice Root, Paeony Root, Chuanxiong Rhizome, Apricot Kernel, Aconite Root, Ledebouriella Root, and Fresh Ginger. It has the functions of expelling wind and dispelling cold, supplementing qi and warming yang; it is mainly used for treating yang deficiency, sudden onset of stroke, muscle spasms, and hemiplegia. Xiao Xuming Decoction has remarkable curative effects in treating stroke and its sequelae, Alzheimer's disease, rheumatoid arthritis and other diseases.

[0003] Classical famous prescriptions refer to prescriptions recorded in ancient Chinese medical classics with definite curative effects, still widely used today, and having obvious characteristics and advantages. These prescriptions have been used until now, and their clinical application value has been widely respected and affirmed by Chinese medical scholars of all dynasties. However, most famous prescriptions are decoctions, which cannot achieve large-scale modern industrial production. The pharmacology and toxicology of modern traditional Chinese medicines are relatively complex, and there are many ways to investigate the extraction process. As recorded in "Zhang Yuan, Li Ningxin. Suggestions for the Research and Development of the Protection of Chinese Herbal Medicine Classical Famous Prescriptions [N]. People's Political Consultative Conference News, 2021: 006", it is recommended to refer to the concept of standard decoction proposed for oral preparations by Japanese Kampo medicines, and establish the material benchmark and biological benchmark of the standard decoction to ensure the safety, effectiveness and stability of clinical use. "Requirements for Declaration Materials of Traditional Chinese Medicine Compound Preparations of Ancient Classical Famous Prescriptions and Their Reference Samples (Draft for Comment)" points out that to achieve the quality control of compound preparations, quality research should be carried out on medicinal materials, cut pieces, standard decoctions and reference samples to determine the key quality attributes.

[0004] Therefore, it is necessary to establish a method for determining the characteristic fingerprint and the content of index components of Xiao Xuming Decoction with excellent repeatability and stability, analyze the transfer law of the amount values of index components between cut pieces and reference samples, formulate the quality standard of the reference sample of Xiao Xuming Decoction, achieve the quality control of the reference sample of Xiao Xuming Decoction, and provide a reference for the research and production of Xiao Xuming Decoction and its related preparations. Summary of the Invention

[0005] The present invention provides a method for detecting chemical components in a reference sample of Xiao Xuming Decoction, which solves the problem of insufficient repeatability and stability in detecting the components in Xiao Xuming Decoction in related technologies.

[0006] The technical solution of the present invention is as follows: The present invention provides a method for detecting chemical components in the reference sample of Xiao-Xu-Ming decoction, and high performance liquid chromatography is used to detect the components in the reference sample of Xiao-Xu-Ming decoction; When the high performance liquid chromatography is used for detection, the chromatographic conditions include: The mobile phase includes mobile phase A and mobile phase B; mobile phase A is acetonitrile, and mobile phase B is an aqueous phosphoric acid solution with a volume fraction of 0.1% - 0.3%; Gradient elution is adopted during elution, and the elution program of the gradient elution is: 0 - 5 min, the volume fraction of mobile phase A is 4%; 5 - 12 min, the volume fraction of mobile phase A is 4% → 8%; 12 - 17 min, the volume fraction of mobile phase A is 8% → 9%; 17 - 18 min, the volume fraction of mobile phase A is 9% → 11%; 18 - 52 min, the volume fraction of mobile phase A is 11% → 15%; 52 - 55 min, the volume fraction of mobile phase A is 15% → 20%; 55 - 90 min, the volume fraction of mobile phase A is 20% → 27%; 90 - 105 min, the volume fraction of mobile phase A is 27% → 50%; 105 - 110 min, the volume fraction of mobile phase A is 50% → 75%; 110 - 120 min, the volume fraction of mobile phase A is 75%; 120 - 121 min, the volume fraction of mobile phase A is 75% → 4%.

[0007] As a further technical solution, the chromatographic conditions further include: the detection wavelengths are 210 nm and 245 nm; the column temperature is 29 - 31 °C; the flow rate is 0.8 - 1.2 mL·min -1 ; the injection volume is 5 - 15 μL.

[0008] As a further technical solution, the column temperature is 30 °C, the flow rate is 1 mL·min -1 , and the injection volume is 10 μL.

[0009] As a further technical solution, the chromatographic conditions further include: the chromatographic column is a Waters X Bridge C18 chromatographic column.

[0010] As a further technical solution, the method for detecting chemical components in the reference sample of Xiao-Xu-Ming decoction includes the following steps: Preparation of the test solution: Mix the powder of the reference sample of Xiao-Xu-Ming decoction and a methanol solution, filter, and take the filtrate to obtain the test solution; Preparation of the mixed reference solution: Mix the mixed reference substances and methanol to obtain the mixed reference solution; Perform high performance liquid chromatography detection on the test solution and the mixed reference solution respectively to obtain the characteristic chromatogram of the reference sample of Xiao-Xu-Ming decoction.

[0011] As a further technical solution, the mixed reference substance includes paeoniflorin, prim-O-glucosylcimifugin, ferulic acid, 5-O-methylvisammioside, wogonoside, baicalin, baicalein, ammonium glycyrrhizinate, tetrandrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, fangchinoline, and 6-gingerol.

[0012] As a further technical solution, the characteristic chromatogram of the Xiaoxuming Decoction reference sample has a total of 18 characteristic peaks, and 13 characteristic peaks are identified; among them, peak 1 is ephedrine hydrochloride, peak 2 is pseudoephedrine hydrochloride, peak 3 is paeoniflorin, peak 4 is prim-O-glucosylcimifugin, peak 5 is ferulic acid, peak 9 is 5-O-methylvisammioside, peak 10 is fangchinoline, peak 11 is tetrandrine, peak 12 is baicalin, peak 14 is wogonoside, peak 15 is baicalein, peak 16 is ammonium glycyrrhizinate, and peak 18 is 6-gingerol.

[0013] As a further technical solution, the volume fraction of the methanol solution is 25% - 100%. Preferably, the volume fraction of the methanol solution is 75%.

[0014] As a further technical solution, the mass-volume ratio of the powder of the Xiaoxuming Decoction reference sample to the methanol solution is 0.1 - 0.5 g:20 mL.

[0015] As a further technical solution, the mass-volume ratio of the powder of the Xiaoxuming Decoction reference sample to the methanol solution is 0.3 g:20 mL.

[0016] As a further technical solution, the mass-volume ratio of the mixed reference substance to methanol is 0.5 - 0.6 mg / mL.

[0017] As a further technical solution, when preparing the test solution, the mixing includes one of ultrasonic mixing and reflux mixing.

[0018] As a further technical solution, when preparing the test solution, when the mixing is ultrasonic mixing, the ultrasonic frequency is 30 - 50 kHz, the ultrasonic power is 150 - 250 W, and the ultrasonic time is 20 - 50 min.

[0019] As a further technical solution, when the mixing is ultrasonic mixing, the ultrasonic frequency is 40 kHz, the ultrasonic power is 200 W, and the ultrasonic time is 30 min.

[0020] As a further technical solution, the preparation method of the reference sample powder of Xiao Xuming Decoction includes the following steps: separately crush Ephedrae Herba, Stephaniae Tetrandrae Radix, Ginseng Radix, Scutellariae Radix, Cinnamomi Cortex, Paeoniae Radix Alba, Glycyrrhizae Radix et Rhizoma, Chuanxiong Rhizoma, Armeniacae Semen Amarum, Saposhnikoviae Radix, Aconiti Lateralis Radix Praeparata, and Zingiberis Rhizoma Recens; mix Ephedrae Herba and water, boil, cool, add the remaining medicinal materials, boil, decoct, filter, cool, and dry to obtain the reference sample powder of Xiao Xuming Decoction.

[0021] The working principle and beneficial effects of the present invention are as follows: In the present invention, the characteristic chromatogram and the determination method for the contents of multiple index components of the reference sample of the classical famous prescription Xiao Xuming Decoction are established. The effective components of 8 medicinal materials, namely Ephedrae Herba, Paeoniae Radix Alba, Saposhnikoviae Radix, Chuanxiong Rhizoma, Stephaniae Tetrandrae Radix, Scutellariae Radix, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens, are characterized in the characteristic chromatogram. There are 18 characteristic peaks in the characteristic chromatograms of 15 batches of reference samples. 13 characteristic peaks, such as ephedrine hydrochloride and pseudoephedrine hydrochloride, are identified, and the similarity is between 0.998 and 0.999.

[0022] The detection method established by the present invention detects that the content of ephedrine hydrochloride in the reference sample of Xiao Xuming Decoction is 1.29 - 2.02 mg·g -1 and the content of pseudoephedrine hydrochloride is 0.50 - 1.82 mg·g -1 the content of paeoniflorin is 7.34 - 10.93 mg·g -1 the content of baicalin is 19.33 - 26.81 mg·g -1 the content of wogonoside is 3.76 - 6.00 mg·g -1 the content of ammonium glycyrrhizinate is 1.19 - 3.27 mg·g -1 the content of prim-O-glucosylcimifugin is 1.38 - 2.12 mg·g -1 the content of tetrandrine is 0.17 - 0.48 mg·g -1 the content of baicalein is 0.35 - 0.64 mg·g -1 .

[0023] The gradient elution program during the detection of the present invention has the advantages of good separation effect for the effective components in the test solution and no impurity peaks in the chromatogram of the test solution.

[0024] The characteristic chromatogram and the multi-component content determination method established by the present invention are simple and feasible, with good repeatability and stability, laying a foundation for the quality evaluation of the reference sample and preparation of the classical famous prescription Xiao Xuming Decoction. Description of the Drawings

[0025] The present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0026] Figure 1 It is the high performance liquid chromatography characteristic chromatogram of Xiao Xuming Decoction in Example 1 of the present invention; Figure 2 It is the superimposed chromatogram of the characteristic chromatograms of 15 batches of Xiao Xuming Decoction reference samples at a detection wavelength of 210 nm in Example 1 of the present invention; Figure 3 It is the superimposed chromatogram of the characteristic chromatograms of 15 batches of Xiao Xuming Decoction reference samples at a detection wavelength of 245 nm in Example 1 of the present invention; Figure 4 It is the characteristic chromatogram of the reference solution and the test solution of Xiao Xuming Decoction at a detection wavelength of 210 nm in Example 1 of the present invention, In the figure, A is the reference solution and B is the test solution; Figure 5 It is the characteristic chromatogram of the reference solution and the test solution of Xiao Xuming Decoction at a detection wavelength of 245 nm in Example 1 of the present invention, In the figure, A is the reference solution and B is the test solution; Figure 6 It is the HPLC chromatogram for the specificity investigation of Xiao Xuming Decoction at a detection wavelength of 245 nm in Example 1 of the present invention, In the figure, A is the reference solution, B is the test solution of Xiao Xuming Decoction, C is the negative sample of Paeonia lactiflora, D is the negative sample of Scutellaria baicalensis, E is the negative sample of Saposhnikovia divaricata, and F is the negative sample of Glycyrrhiza uralensis; Figure 7 It is the HPLC chromatogram for the specificity investigation of Xiao Xuming Decoction at a detection wavelength of 210 nm in Example 1 of the present invention, In the figure, A is the reference solution, B is the test solution of Xiao Xuming Decoction, C is the negative sample of Stephania tetrandra, and D is the negative sample of Ephedra sinica; Figure 8 It is the chromatogram of the test solution of Xiao Xuming Decoction extracted with a 25% methanol aqueous solution by volume in Example 1 of the present invention; Figure 9 It is the chromatogram of the test solution of Xiao Xuming Decoction extracted with a 50% methanol aqueous solution by volume in Example 1 of the present invention; Figure 10 It is the chromatogram of the test solution of Xiao Xuming Decoction extracted with a 75% methanol aqueous solution by volume in Example 1 of the present invention; Figure 11 It is the chromatogram of the test solution of Xiao Xuming Decoction extracted with 100% methanol by volume in Example 1 of the present invention; Figure 12 It is the chromatogram of the test solution of Xiao Xuming Decoction extracted by different extraction methods in Example 1 of the present invention, In the figure, A is ultrasonic extraction and B is reflux extraction; Figure 13 It is the chromatogram of the test solution detected at different detection wavelengths in Example 1 of the present invention, In the figure, A is the detection wavelength of 210 nm, B is the detection wavelength of 240 nm, and C is the detection wavelength of 245 nm; Figure 14 This is the chromatogram of the test solution of Comparative Example 1 of the present invention; Figure 15 This is the chromatogram of the test solution of Comparative Example 2 of the present invention; Figure 16 This is the chromatogram of the test solution of Comparative Example 3 of the present invention. Detailed implementation mode

[0027] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0028] The instruments and reagents used in the following embodiments: The high-performance liquid chromatograph is Waters e 2695 (Waters Corporation, USA); Reference substances: paeoniflorin (batch number 110736 - 202145, mass fraction 100%), prim-O-glucosylcimifugin (batch number 111522 - 201913, mass fraction 94.6%), ammonium glycyrrhizinate (batch number 110731 - 201619, mass fraction 93.0%), tetrandrine (batch number 110711 - 201810, mass fraction 99.6%), baicalein (batch number 111595 - 201607, mass fraction 98.5%), baicalin (batch number 110715 - 202122, mass fraction 94.2%), wogonoside (batch number 112002 - 201702, mass fraction 98.5%), ephedrine hydrochloride (batch number 171241 - 201809, mass fraction 100%), pseudoephedrine hydrochloride (batch number 171237 - 201510, mass fraction 99.8%), ferulic acid (batch number 110773 - 201915, mass fraction 99.4%), fangchinoline (batch number 110793 - 202108, mass fraction 100%), 5-O-methylvisammioside (batch number 111523 - 201811, mass fraction 97.4%), 6-gingerol (batch number 111833 - 202007, mass fraction 99.3%); Methanol, acetonitrile (chromatographic grade, Merck); The water is self-made ultrapure water in the laboratory; other reagents are all of analytical grade; The medicinal materials used in Xiao Xuming Decoction were purchased from Anguo Xinhua Chinese Medicinal Materials Co., Ltd. and identified by the Hebei Institute for Drug and Medical Device Control. Scutellaria baicalensis is the dried root of the Labiatae plant Scutellaria baicalensis Georgi Scutellaria aicalensis Georgi ; Cinnamon is the dried bark of the Lauraceae plant Cinnamomum cassia Presl Cinnamomum cassia Presl ; Licorice is the dried root and rhizome of the Leguminosae plant Glycyrrhiza uralensis Fisch Glycyrrhiza uralensis Fisch. ; White peony root is the dried root of the Ranunculaceae plant Paeonia lactiflora Pall Paeonia lactiflora Pall. ; Chuanxiong rhizome is the dried rhizome of the Umbelliferae plant Ligusticum chuanxiong Hort Ligusticum chuanxiong Hort ; Bitter apricot seed is the dried ripe seed of the Rosaceae plant Armeniaca sibirica (L.) Lam Prunus armeniaca L.var.ansu Maxim ; Saposhnikovia root is the dried root of the Umbelliferae plant Saposhnikovia divaricata (Turcz.) Schischk Saposhnikovia divaricate (Turcz.) Schischk. ; Ginger is the fresh rhizome of the Zingiberaceae plant Zingiber officinale Rosc Zingiber officinale Rosc ; Ginseng is the dried root and rhizome of the Araliaceae plant Panax ginseng C. A. Mey Panax ginseng C. A. Mey. ; Ephedra is the dried herbaceous stem of the Ephedraceae plant Ephedra sinica Stapf Ephedra sinica Stapf ; Stephania root is the dried root of the Menispermaceae plant Stephania tetrandra S. Moore Stephania tetrandra S.Moore ; Prepared aconite root is the processed product of the daughter root of the Ranunculaceae plant Aconitum carmichaelii Debx Aconitum carmichaelii Debx. . All of them comply with the provisions of the 2020 edition of the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the "Chinese Pharmacopoeia"). According to the requirements of the Key Information Table of Ancient Classic Famous Prescriptions, the prepared aconite root is black processed aconite. Beijing Chunfeng Pharmaceutical Co., Ltd. processed each batch of the prepared aconite root into decoction pieces according to the processing methods of each medicinal material determined in the previous research, and detected them according to the requirements of the 2020 edition of the Chinese Pharmacopoeia. All of them comply with the regulations According to the random number table method, the 12 kinds of decoction pieces in Xiao Xuming Decoction were randomly combined and sorted to form 15 batches of reference samples of the formula of Xiao Xuming Decoction. The batch numbers of the corresponding decoction pieces for the 15 batches of formulas are shown in Tables 1-2

[0029] Table 1 Batch numbers of the medicinal material decoction pieces of 15 batches of reference samples of Xiao Xuming Decoction

[0030] Table 2 Batch numbers of the medicinal material decoction pieces of 15 batches of reference samples of Xiao Xuming Decoction

[0031] Example 1 1. The 15 batches of reference samples of Xiao Xuming Decoction were prepared according to the following method Weigh 13.8 g of Ephedrae Herba, 13.8 g of Stephaniae Tetrandrae Radix, 13.8 g of Ginseng Radix, 13.8 g of Scutellariae Radix, 13.8 g of Cinnamomi Cortex, 13.8 g of Paeoniae Radix Alba, 13.8 g of Glycyrrhizae Radix Preparata, 13.8 g of Chuanxiong Rhizoma, 13.8 g of Armeniacae Semen Amarum (processed), 20.7 g of Saposhnikoviae Radix, 15 g of Aconiti Lateralis Praeparata, and 69 g of fresh Ginger Rhizome. Crush them separately into coarse powder particles that can pass through a 4-mesh sieve for standby. Then, place Ephedrae Herba in a casserole, add 2400 mL of water, soak for 30 min, and boil over high heat (1200 W). Remove the foam 3 times using the "remove fire and control temperature" method. Then turn off the heat, cool the medicinal liquid to room temperature, add the remaining medicinal materials, and soak for 30 min. After soaking, bring to a boil over high heat and then adjust to low heat (600 W) and decoct for 90 min. Filter while it is hot through a 200-mesh gauze, cool to room temperature, and make up the volume to 600 mL. Mix well and then freeze-dry to obtain the reference sample powder of Xiao Xuming Decoction.

[0032] 2. Chromatographic conditions Chromatographic column: Waters X Bridge C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase: acetonitrile (A) - 0.2% phosphoric acid aqueous solution (B); The elution program for gradient elution is as follows: 0 - 5 min, the volume fraction of mobile phase A is 4%; 5 - 12 min, the volume fraction of mobile phase A is 4% → 8%; 12 - 17 min, the volume fraction of mobile phase A is 8% → 9%; 17 - 18 min, the volume fraction of mobile phase A is 9% → 11%; 18 - 52 min, the volume fraction of mobile phase A is 11% → 15%; 52 - 55 min, the volume fraction of mobile phase A is 15% → 20%; 55 - 90 min, the volume fraction of mobile phase A is 20% → 27%; 90 - 105 min, the volume fraction of mobile phase A is 27% → 50%; 105 - 110 min, the volume fraction of mobile phase A is 50% → 75%; 110 - 120 min, the volume fraction of mobile phase A is 75%; 120 - 121 min, the volume fraction of mobile phase A is 75% → 4%; Detection wavelength is 210 nm or 245 nm; Column temperature is 30 °C; Flow rate is 1.0 mL·min -1 ; Injection volume is 10 μL.

[0033] 3. Preparation of test solution Precisely weigh 0.3 g of the freeze-dried powder of the reference sample of Xiao Xuming Decoction, precisely add 20 mL of 75% methanol solution, weigh, tightly stopper, ultrasonically treat for 30 min, and make up for the weight loss. Filter through a 0.45 μm microporous filter membrane, and take the subsequent filtrate to obtain the test solution.

[0034] 4. Preparation of Reference Substance Solution Accurately weigh ephedrine hydrochloride, pseudoephedrine hydrochloride, paeoniflorin, prim-O-glucosylcimifugin, wogonoside, baicalin, baicalein, ammonium glycyrrhizinate, tetrandrine, add methanol to prepare a mixed reference substance solution containing 0.05, 0.034, 0.18, 0.038, 0.101, 0.482, 0.015, 0.047, 0.015 mg per 1 mL respectively, as the reference substance reference solution.

[0035] 5. Content Determination and Value Transfer Analysis of 15 Batches of Reference Samples of Xiao Xuming Decoction Take 15 batches of reference samples of Xiao Xuming Decoction, and prepare 15 batches of test solution of Xiao Xuming Decoction respectively according to the above-mentioned preparation method of test solution, with 2 parallel portions for each batch; carry out HPLC analysis on the test solution successively according to the above-mentioned chromatographic conditions, calculate the contents of each index component in 15 batches of reference samples of Xiao Xuming Decoction, as shown in Table 3, and calculate the value transfer from the cut crude drugs to the reference samples, and the results are shown in Table 4.

[0036] Table 3 Contents of Index Components in 15 Batches of Reference Samples of Xiao Xuming Decoction

[0037] Table 4 Transfer Rates from Cut Crude Drugs to Reference Samples of 15 Batches of Xiao Xuming Decoction

[0038] According to the above content determination results, it can be known that the content of ephedrine hydrochloride in 15 batches of reference samples of Xiao Xuming Decoction is 1.29 - 2.02 mg·g -1 , the content of pseudoephedrine hydrochloride is 0.50 - 1.82 mg·g -1 , the content of paeoniflorin is 7.34 - 10.93 mg·g - 1 1. The content of baicalin is 19.33 - 26.81 mg·g -1 , the content of wogonoside is 3.76 - 6.00 mg·g -1 , the content of ammonium glycyrrhizinate is 1.19 - 3.27 mg·g -1 , the content of prim-O-glucosylcimifugin is 1.38 - 2.12 mg·g -1 , the content of tetrandrine is 0.17 - 0.48 mg·g -1 , the content of baicalein is 0.35 - 0.64 mg·g -1 .

[0039] The content fluctuation range of the required indicator components generally does not exceed 70% - 130% of the mean. Therefore, according to Table 3, the content of each indicator component in the 15 batches of Xiao Xuming Decoction reference samples, except for pseudoephedrine hydrochloride, tetrandrine, and ammonium glycyrrhizinate, is stable within the qualified range. The reason for the content of pseudoephedrine hydrochloride, tetrandrine, and ammonium glycyrrhizinate not being within the range is that different sources of Chinese medicinal materials will affect their quality.

[0040] 6. Similarity evaluation of characteristic fingerprints of 15 batches of Xiao Xuming Decoction reference samples Take 15 batches of Xiao Xuming Decoction reference samples, prepare 15 batches of test solution of Xiao Xuming Decoction according to the above preparation method of test solution, and perform HPLC detection according to the above chromatographic conditions to obtain the high - performance liquid chromatography characteristic fingerprints of 15 batches of Xiao Xuming Decoction. Import the characteristic fingerprints of 15 batches of Xiao Xuming Decoction reference samples into the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2008 version) in sequence. Using the chromatogram of the test solution of Xiao Xuming Decoction numbered S1 as the reference chromatogram, adopt the median method, with a time window width of 0.1, perform multi - point calibration and chromatographic peak matching, generate a reference chromatogram and calculate the similarity. The results are shown in Table 5, and the high - performance liquid chromatography characteristic fingerprints of Xiao Xuming Decoction are shown in Figure 1 , and 18 characteristic peaks are determined; the superimposed map of the characteristic fingerprints of 15 batches of Xiao Xuming Decoction reference samples is shown in Figures 2 - 3 .

[0041] Table 5 Similarity of characteristic fingerprints of 15 batches of Xiao Xuming Decoction reference samples

[0042] As can be seen from Table 5, the similarity of 15 batches of Xiao Xuming Decoction reference samples is greater than 0.998.

[0043] 7. Identification and attribution of common peaks in the characteristic fingerprints of Xiao Xuming Decoction reference samples Compare the test solution with the mixed reference solution. The characteristic fingerprints of the reference solution and the test solution of Xiao Xuming Decoction at a detection wavelength of 210 nm are shown in Figure 4 , and the characteristic fingerprints of the reference solution and the test solution of Xiao Xuming Decoction at a detection wavelength of 245 nm are shown in Figure 5 . Figures 4 - 5There are a total of 18 characteristic peaks, and 13 chromatographic peaks are identified: peaks 1 - 2 come from Ephedrae Herba; peak 3 comes from Paeoniae Radix Alba; peaks 4 and 9 come from Saposhnikoviae Radix; peak 5 comes from Chuanxiong Rhizoma; peaks 10 - 11 come from Stephaniae Tetrandrae Radix; peaks 12 - 15 come from Scutellariae Radix; peak 16 comes from Glycyrrhizae Radix; peak 18 comes from Zingiberis Rhizoma Recens. Peak 1 is ephedrine hydrochloride, peak 2 is pseudoephedrine hydrochloride, peak 3 is paeoniflorin, peak 4 is prim-O-glucosylcimifugin, peak 5 is ferulic acid, peak 9 is 5-O-methylvisammioside, peak 10 is fangchinoline, peak 11 is tetrandrine, peak 12 is baicalin, peak 14 is wogonoside, peak 15 is baicalein, peak 16 is ammonium glycyrrhizinate, and peak 18 is 6-gingerol.

[0044] Based on the relative retention time results of 15 batches of reference samples of Xiao Xuming Decoction, the identification criteria for the characteristic chromatogram of the reference samples of Xiao Xuming Decoction were determined. There should be 18 characteristic peaks in the test sample's characteristic chromatogram. Using peak 12 as the reference peak (S peak), the relative retention times of each characteristic peak and the S peak were calculated as shown in Tables 6 - 7, and the deviation of their relative retention times should be within ±10% of the specified values. The specified values are 0.220 (peak 1), 0.228 (peak 2), 0.457 (peak 3), 0.509 (peak 4), 0.522 (peak 5), 0.591 (peak 6), 0.612 (peak 7), 0.648 (peak 8), 0.852 (peak 9), 0.869 (peak 10), 0.930 (peak 11), 1.147 (peak 13), 1.219 (peak 14), 1.366 (peak 15), 1.491 (peak 16), 1.506 (peak 17), 1.530 (peak 18).

[0045] Table 6 Relative Retention Times of Characteristic Peaks in 15 Batches of Reference Samples of Xiao Xuming Decoction

[0046] Table 7 Relative Retention Times of Characteristic Peaks in 15 Batches of Reference Samples of Xiao Xuming Decoction

[0047] 8. Methodological Investigation 8.1 Methodological Investigation of the Characteristic Chromatogram of Xiao Xuming Decoction 8.1.1 Precision Take the same test sample solution and perform HPLC analysis according to the chromatographic conditions in "2. Chromatographic Conditions". Inject the sample continuously 6 times and record the chromatogram. Using peak 12 (baicalin) as the reference peak at 245 nm and 210 nm, calculate the relative retention time of each peak and calculate RSD , and the results are shown in Table 8.

[0048] 8.1.2 Stability Take the same test sample solution and perform HPLC analysis according to the chromatographic conditions in item "2. Chromatographic Conditions". Inject samples at 0, 2, 4, 8, 12, and 24 h respectively, and record the chromatograms. Using peak 12 (baicalin) as the reference peak at 245 nm and 210 nm, calculate the relative retention time of each peak and calculate RSD , and the results are shown in Table 8.

[0049] 8.1.3 Repeatability Precisely weigh 0.15 g, 0.3 g, and 0.45 g of the reference sample powder of the same batch, three portions each. Prepare the test sample solution according to the method in item "3. Preparation of Test Sample Solution", and perform HPLC analysis successively according to the chromatographic conditions in item "2. Chromatographic Conditions", and record the chromatograms. Using peak 12 (baicalin) as the reference peak at 245 nm and 210 nm, calculate the relative retention time of each peak and calculate RSD , and the results are shown in Table 8.

[0050] Table 8 Methodology Investigation of the Characteristic Chromatogram of Xiao Xuming Decoction

[0051] 8.2 Methodology Investigation of the Content Determination of the Reference Sample of Xiao Xuming Decoction 8.2.1 Specificity Prepare a negative reference sample without ephedra, stephania root, scutellaria root, licorice root, white peony root, and ledebouriella root according to the reference sample preparation method determined in item "1. Preparation of 15 Batches of Reference Samples of Xiao Xuming Decoction as Follows". Take the mixed reference solution, test sample solution, and negative solution, and perform HPLC analysis according to the chromatographic conditions determined in item "2. Chromatographic Conditions", and record the chromatograms, as shown in Figures 6 - 7 .

[0052] 8.2.2 Linear Relationship Precisely measure the mixed stock solution of the reference substance, and dilute it to obtain reference solutions with a series of concentrations. Perform HPLC analysis according to the chromatographic conditions in item "2. Chromatographic Conditions" to investigate the linear relationship between the peak area and the concentration, as shown in Table 9.

[0053] 8.2.3 Precision Take the same test sample solution and perform HPLC analysis according to item "2. Chromatographic Conditions". Inject samples continuously for 6 times and record the chromatograms. Measure the peak areas of each target component respectively and calculate RSD , and the results are shown in Table 9.

[0054] 8.2.4 Stability Take the same test sample solution and perform HPLC analysis according to the chromatographic conditions in item "2. Chromatographic Conditions". Inject samples at 0, 2, 4, 8, 12, and 24 h respectively, measure the peak areas of each target component, and calculate RSD , and the results are shown in Table 9.

[0055] 8.2.5, Stability Precisely weigh 0.15 g, 0.3 g, and 0.45 g of the reference sample powder of the same batch, three portions each. Prepare the test solution according to the method of "3. Preparation of the Test Solution", and perform HPLC analysis successively according to the chromatographic conditions of "2. Chromatographic Conditions" to measure the peak areas of each index component and calculate RSD , and the results are shown in Table 9.

[0056] 8.2.6, Sample Recovery Rate Precisely weigh 9 portions of 0.15 g of the reference sample powder of the same batch as the repeatability test. Precisely measure appropriate amounts of the control mixture stock solution according to the ratios of 1:0.5, 1:1, and 1:1.5 of the known contents of each index component, and dilute to an appropriate concentration to prepare the control mixture solution according to the method of "4. Preparation of the Control Solution"; use the control mixture solution as the extraction solvent, prepare the test solution according to the method of "3. Preparation of the Test Solution", with 3 parallel portions for each concentration; and perform HPLC analysis successively according to the chromatographic conditions of "2. Chromatographic Conditions" to measure the contents of each index component and calculate their sample recovery rates, as shown in Table 9.

[0057] Table 9 Methodological Investigation on the Content Determination of the Reference Sample of Xiao Xuming Decoction

[0058] 9. Investigation on the Decoction Process of the Reference Sample of Xiao Xuming Decoction The preparation method of this formula recorded in "Qianjin Yaofang" is as follows: "Ephedra, Stephania Tetrandra, Ginseng, Scutellaria Baicalensis, Cinnamon Twig, Licorice Root, White Peony Root, Chuanxiong Rhizome, Apricot Kernel, each 1 liang, Aconite Root 1 piece, Saposhnikovia Root 1.5 liang, Fresh Ginger 5 liang. The above twelve flavors are cut into pieces, decocted with 12 liters of water. First, boil Ephedra for three boils, remove the foam, add the other herbs, and decoct to obtain 3 liters. Take in three doses, very effective." Combining with the content recorded in the article "Textual Research on the Key Information of the Prescription of the Ancient Classic Famous Prescription Xiao Xuming Decoction [J]. Pharmacology and Clinics of Chinese Materia Medica, 2022, 38(4): 179.", the decoction method of Xiao Xuming Decoction is determined as follows: "Add 2400 mL of water to 12 flavors of herbs, first boil Ephedra for 'three boils' and remove the foam, then add the remaining herbs, decoct to obtain 600 mL, and take warm in 3 times." Soak and decoct the herbs in the early stage of the experiment. Investigate the decoction time, and decoct the soaked herbs for 75, 90, and 105 minutes respectively. Take the contents of each component as the main basis to investigate the decoction time.

[0059] The results show that the contents of each index component in the reference sample after decocting for 90 minutes are the highest.

[0060] 10. Investigation on the Preparation Method of the Test Solution The preparation process of the test solution includes: Precisely weigh the freeze-dried powder of the reference sample of Xiaoxuming Decoction, precisely add methanol solution, weigh, stopper tightly, mix, and make up the weight. Filter through a 0.45 μm microporous membrane, and take the subsequent filtrate to obtain the solution.

[0061] 10.1 Extraction solvent In terms of volume fraction, four extraction solvents, namely 25% methanol aqueous solution, 50% methanol aqueous solution, 75% methanol aqueous solution, and 100% methanol, were investigated in this experiment; the results are as Figures 8 - 11 shown.

[0062] The results showed that the separation effect and content of the characteristic peaks of each active ingredient in the test solution of Xiaoxuming Decoction extracted with 75% methanol aqueous solution were the best. Therefore, 75% methanol was selected as the extraction solvent.

[0063] 10.2 Sampling amount Three sampling amounts of freeze-dried powder, 0.2 g, 0.3 g, and 0.4 g, were investigated; The results showed that within the range of 0.2 - 0.4 g, the content of the target components was proportional to the sampling amount.

[0064] 10.3 Extraction method According to the determined chromatographic conditions, two extraction methods, ultrasonic extraction and reflux extraction, were investigated, and the results are as Figure 12 shown.

[0065] For reflux extraction, reflux extraction was carried out at 70 °C for 30 min; For ultrasonic extraction, ultrasonic extraction was carried out at a power of 200 W and a frequency of 40 kHz for 30 min; The results showed that ultrasonic extraction was more complete than reflux extraction, and the repeatability of the samples by ultrasonic extraction was better.

[0066] 10.4 Extraction time Four extraction times, 20 min, 30 min, 40 min, and 50 min, were investigated in the experiment; The results showed that the content of each target component in the test solution of Xiaoxuming Decoction reached saturation after extraction for 30 min; Therefore, a sampling amount of 0.3 g was selected, 75% methanol was used as the extraction solvent, and ultrasonic extraction was carried out for 30 min to prepare the test solution.

[0067] 11 Wavelength selection The test solution was detected according to the determined method and full-wavelength scanning was carried out. The scanning range was 190 - 400 nm, and the results are as Figure 13 shown; The results showed that ephedrine hydrochloride and pseudoephedrine hydrochloride could be detected at 210 nm. The number of chromatographic peaks at 240 nm and 245 nm was relatively large and similar, but the peak of paeoniflorin had serious tailing at 240 nm. Therefore, the detection wavelengths were selected as 210 nm and 245 nm.

[0068] Comparative Example 1 The difference between this comparative example and Example 1 was only that the gradient elution program was as shown in Table 10 below; the chromatogram of the test solution was as Figure 14 shown.

[0069] Table 10 Gradient Elution Program

[0070] Comparative Example 2 The difference between this comparative example and Example 1 was only that the gradient elution program was as shown in Table 11 below; the chromatogram of the test solution was as Figure 15 shown.

[0071] Table 11 Gradient Elution Program

[0072] Comparative Example 3 The difference between this comparative example and Example 1 was only that the gradient elution program was as shown in Table 12 below; the chromatogram of the test solution was as Figure 16 shown.

[0073] Table 12 Gradient Elution Program

[0074] In Comparative Examples 1 to 3, by changing the gradient elution program, the effective components such as 5-O-methylvisamminol, fangchinoline, and tetrandrine in the test sample failed to achieve an ideal separation effect, and there were many miscellaneous peaks in the chromatogram. Therefore, the gradient elution programs of Comparative Examples 1 to 3 were abandoned, and the gradient elution program in Example 1 was selected.

[0075] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting chemical components in a reference sample of Xiao Xuming Decoction, characterized in that, The components in the reference sample of Xiao Xuming Decoction are detected by high performance liquid chromatography; When detected by the high performance liquid chromatography, the chromatographic conditions include: The mobile phase includes mobile phase A and mobile phase B; mobile phase A is acetonitrile, and mobile phase B is an aqueous phosphoric acid solution with a volume fraction of 0.1% - 0.3%; Gradient elution is adopted during elution, and the elution program of the gradient elution is: 0 - 5 min, the volume fraction of mobile phase A is 4%; 5 - 12 min, the volume fraction of mobile phase A is 4% → 8%; 12 - 17 min, the volume fraction of mobile phase A is 8% → 9%; 17 - 18 min, the volume fraction of mobile phase A is 9% → 11%; 18 - 52 min, the volume fraction of mobile phase A is 11% → 15%; 52 - 55 min, the volume fraction of mobile phase A is 15% → 20%; 55 - 90 min, the volume fraction of mobile phase A is 20% → 27%; 90 - 105 min, the volume fraction of mobile phase A is 27% → 50%; 105 - 110 min, the volume fraction of mobile phase A is 50% → 75%; 110 - 120 min, the volume fraction of mobile phase A is 75%; 120 - 121 min, the volume fraction of mobile phase A is 75% → 4%.

2. The detection method of chemical components in the reference sample of Xiao Xuming Decoction according to claim 1, characterized in that, The chromatographic conditions further include: the detection wavelength is 210 nm or 245 nm; the column temperature is 29~31 °C; the flow rate is 0.8~1.2 mL·min -1 ; the injection volume is 5~15 μL.

3. The detection method of chemical components in the reference sample of Xiao Xuming Decoction according to claim 1, characterized in that, It includes the following steps: Preparation of test solution: The powder of the reference sample of Xiao Xuming Decoction and a methanol solution are mixed and filtered, and the filtrate is taken to obtain the test solution; Preparation of mixed reference solution: The mixed reference substances and methanol are mixed to obtain the mixed reference solution; The test solution and the mixed reference solution are respectively subjected to high performance liquid chromatography detection to obtain the characteristic chromatogram of the reference sample of Xiao Xuming Decoction.

4. The detection method of chemical components in the reference sample of Xiao Xuming Decoction according to claim 3, characterized in that, The mixed reference substances include paeoniflorin, prim-O-glucosylcimifugin, ferulic acid, 5-O-methylvisammioside, wogonoside, baicalin, baicalein, ammonium glycyrrhizinate, tetrandrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, fangchinoline and 6-gingerol.

5. The detection method of chemical components in the reference sample of Xiao Xuming Decoction according to claim 3, characterized in that, The characteristic chromatogram of the reference sample of Xiao Xuming Decoction has 18 characteristic peaks, and 13 characteristic peaks are identified; among them, peak 1 is ephedrine hydrochloride, peak 2 is pseudoephedrine hydrochloride, peak 3 is paeoniflorin, peak 4 is prim-O-glucosylcimifugin, peak 5 is ferulic acid, peak 9 is 5-O-methylvisammioside, peak 10 is fangchinoline, peak 11 is tetrandrine, peak 12 is baicalin, peak 14 is wogonoside, peak 15 is baicalein, peak 16 is ammonium glycyrrhizinate, and peak 18 is 6-gingerol.

6. The detection method of chemical components in the reference sample of Xiao Xuming Decoction according to claim 3, characterized in that, The volume fraction of the methanol solution is 25% - 100%, Preferably, the volume fraction of the methanol solution is 75%.

7. The detection method of chemical components in the reference sample of Xiao Xuming Decoction according to claim 3, characterized in that, The mass-to-volume ratio of the powder of the reference sample of Xiao Xuming Decoction and the methanol solution is 0.1 - 0.5 g:20 mL; The mass-to-volume ratio of the mixed reference substances and methanol is 0.5 - 0.6 mg / mL.

8. The detection method of chemical components in the reference sample of Xiao Xuming Decoction according to claim 3, characterized in that During the preparation of the test solution, the mixing includes one of ultrasonic mixing and reflux mixing.

9. The detection method of chemical components in the reference sample of Xiao Xuming Decoction according to claim 8, characterized in that, During the preparation of the test solution, when the mixing is ultrasonic mixing, the ultrasonic frequency is 30 - 50 kHz, the ultrasonic power is 150 - 250 W, and the ultrasonic time is 20 - 50 min; Preferably, when the mixing is ultrasonic mixing, the ultrasonic frequency is 40 kHz, the ultrasonic power is 200 W, and the ultrasonic time is 30 min.

10. The detection method of chemical components in a reference sample of Xiao Xuming Decoction according to claim 3, characterized in that, The preparation method of the Xiaoxuming Decoction reference sample powder comprises the following steps: taking Ephedrae Herba, Stephaniae Tetrandrae Radix, Ginseng Radix, Scutellariae Radix, Cinnamomi Cortex, Paeoniae Radix Alba, Glycyrrhizae Radix, Chuanxiong Rhizoma, Armeniacae Semen Amarum, Saposhnikoviae Radix, Aconiti Lateralis Praeparata Radix, and Zingiberis Rhizoma Recens, respectively crushing them, then mixing Ephedrae Herba with water, boiling, cooling, adding the remaining medicinal materials and boiling, decocting, filtering, cooling, and drying to obtain the Xiaoxuming Decoction reference sample powder.

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