Construction method of Houpu Mahuang decoction fingerprint and its standard fingerprint and application

Abstract

The present invention provides a method for constructing a fingerprint of Houpu Mahuang Tang and its standard fingerprint and application, belonging to the technical field of drug detection, the construction method is to prepare a test solution from Houpu Mahuang Tang, and the obtained test solution is detected by HPLC-Q-TOF-MS method to obtain the fingerprint of Houpu Mahuang Tang; the mobile phase A used in the high performance liquid chromatography in the HPLC-Q-TOF-MS is acetonitrile, and the mobile phase B is a formic acid aqueous solution with a concentration of 0.1-0.3wt%; and the standard fingerprint of Houpu Mahuang Tang is obtained, which is used for quality evaluation or control in the whole process of research / development / production / clinical application of Houpu Mahuang Tang. The construction method of the present invention can reflect the main chemical components in Houpu Mahuang Tang in the characteristic spectrum, and identify 18 chemical components, and the established standard fingerprint can more comprehensively reflect the quality information of Houpu Mahuang Tang.

Description

Technical Field

[0001] The invention relates to drug detection technology, and in particular to a method for constructing a fingerprint of Houpo Mahuang decoction, a standard fingerprint thereof and application thereof. Background Art

[0002] Houpo Mahuang Decoction comes from the Synopsis of the Golden Chamber written by Zhang Zhongjing: 5 liang of Houpo, 4 liang of Mahuang, gypsum as big as an egg, half a liter of almond, half a liter of Pinellia, 2 liang of dried ginger, 2 liang of Asarum, 1 liter of wheat, and half a liter of Schisandra. The above nine ingredients are boiled with 1 dou 2 liters of water. First, boil the wheat until it is cooked, remove the dregs, add the other herbs, and boil until 3 liters are left. As a prescription included in the Catalog of Ancient Classic Prescriptions (First Batch), Houpo Mahuang Decoction has a definite effect. Many clinical studies have confirmed that this prescription can effectively relieve the clinical symptoms of patients with chronic bronchitis, chronic obstructive pulmonary disease and emphysema, and reduce the level of serum inflammatory factors. At present, there are no relevant research reports on the quality control of Houpo Mahuang Decoction, and the quality attributes are unclear, which limits its further preparation development.

[0003] Characteristic spectrum is a chromatogram or spectrum for analyzing a certain type or several types of components in the substance being tested. At present, characteristic spectrum has become an internationally recognized effective ingredient for controlling the quality of traditional Chinese medicine or natural medicine, and is a hot issue in the current traditional Chinese medicine industry. In recent years, people have gradually realized that the quality control method formed only for a certain chemical component can no longer properly reflect the intrinsic quality of the product. The characteristic spectrum of traditional Chinese medicine, as a multi-index quality control model, can better control the intrinsic quality of the product. Therefore, by constructing the fingerprint spectrum of Houpu Mahuang Decoction, the quality of Houpu Mahuang Decoction can be effectively controlled. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for constructing a fingerprint of Houpu Mahuang Decoction and its standard fingerprint and application.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A method for constructing a fingerprint of Houpu Mahuang Decoction, the method comprising preparing a test solution from Houpu Mahuang Decoction, and detecting the obtained test solution by HPLC-Q-TOF-MS to obtain the fingerprint of Houpu Mahuang Decoction;

[0007] The mobile phase A used in the high performance liquid chromatography in the HPLC-Q-TOF-MS is acetonitrile, and the mobile phase B is a formic acid aqueous solution with a concentration of 0.1 to 0.3 wt%.

[0008] Furthermore, the elution mode of the high performance liquid chromatography is gradient elution;

[0009] The elution conditions of the gradient elution are:

[0010] 0-16 min, 5% mobile phase A, 95% mobile phase B;

[0011] 16-18 min, 5% → 9% mobile phase A, 95% → 91% mobile phase B;

[0012] 18-28 min, 9% → 10% mobile phase A, 91% → 90% mobile phase B;

[0013] 28-32 min, 10% mobile phase A, 90% mobile phase B;

[0014] 32-40 min, 10% → 14% mobile phase A, 90% → 86% mobile phase B;

[0015] 40-52 min, 14% → 16% mobile phase A, 86% → 84% mobile phase B;

[0016] 52-63 min, 16% → 20% mobile phase A, 84% → 80% mobile phase B;

[0017] 63-73 min, 20% → 33% mobile phase A, 80% → 67% mobile phase B;

[0018] 73-80 min, 33% → 70% mobile phase A, 67% → 30% mobile phase B;

[0019] 80-90 min, 70% mobile phase A, 30% mobile phase B.

[0020] Furthermore, the detection wavelength of the HPLC is 280 nm.

[0021] Furthermore, the column temperature of the HPLC is 34-36° C. and the flow rate is 0.8-1.2 mL / min.

[0022] Furthermore, the mass spectrometry conditions of the HPLC-Q-TOF-MS are:

[0023] The dryer (N2) flow rate was 10.00 L / min; the temperature was 320°C; the nebulizer pressure was 45 psig; the capillary voltage was 4000 V; the fragmentor was 170 V, the skimmer was 65 V, the OCT 1RF Vpp, 750 V; the acquisition frequency was 3 spectra / s; and the ion mode was positive ion mode.

[0024] Further, the test solution is prepared by dissolving 100 parts by weight of Houpo Mahuang Decoction in water and diluting the volume to 10 parts by volume;

[0025] The ratio of parts by weight to parts by volume is mg:mL.

[0026] Furthermore, a total of 18 characteristic peaks were obtained and identified in the fingerprint of Houpu Mahuang Decoction.

[0027] Furthermore, the relative retention times and corresponding chemical compositions of the 18 characteristic peaks are:

[0028] The chemical component corresponding to peak 1 is phenylalanine, with a retention time of 9.180;

[0029] The chemical component corresponding to peak 2 is norephedrine, with a retention time of 10.170;

[0030] The chemical component corresponding to peak 3 is demethylpseudoephedrine, with a retention time of 11.431;

[0031] The chemical component corresponding to peak 4 is ephedrine, with a retention time of 14.296;

[0032] The chemical component corresponding to peak 5 is pseudoephedrine, with a retention time of 15.661;

[0033] The chemical component corresponding to peak 6 is methylephedrine, with a retention time of 17.093;

[0034] The chemical component corresponding to peak 7 is methyl pseudoephedrine, with a retention time of 17.503;

[0035] The chemical component corresponding to peak 8 is magnocurine, with a retention time of 27.598;

[0036] The chemical component corresponding to peak 9 is amygdalin, with a retention time of 33.020;

[0037] The chemical component corresponding to peak 10 is magnolamine, with a retention time of 38.272;

[0038] The chemical component corresponding to peak 11 is rekulin, with a retention time of 42.092;

[0039] The chemical component corresponding to peak 12 is Oblongine, with a retention time of 43.286;

[0040] The chemical component corresponding to peak 13 is pavone, with a retention time of 48.913;

[0041] The chemical component corresponding to peak 14 is 6-shogaol, with a retention time of 82.204;

[0042] The chemical component corresponding to peak 15 is Schisandra chinensis lignans L, with a retention time of 82.613;

[0043] The chemical component corresponding to peak 16 is schisandrin A, with a retention time of 82.647;

[0044] The chemical component corresponding to peak 17 is honokiol, with a retention time of 84.523;

[0045] The chemical component corresponding to peak 18 is magnolol, and the retention time is 86.604.

[0046] A standard fingerprint of Houpo Mahuang Decoction obtained by the above construction method.

[0047] An application of the above standard fingerprint spectrum in the quality evaluation or control of the whole process of research / development / production / clinical application of Houpu Mahuang Decoction.

[0048] The method for constructing the fingerprint of Houpu Mahuang Decoction of the present invention and its standard fingerprint and application have the following beneficial effects:

[0049] The construction method of the present invention can reflect the main chemical components in Houpu Mahuang Decoction in the characteristic spectrum, and identify 18 chemical components. The established standard fingerprint spectrum can more comprehensively reflect the quality information of Houpu Mahuang Decoction.

[0050] The present invention changes the elution system, utilizes the different physical and chemical properties of different effective components in Houpu Mahuang Decoction, and changes the detection wavelength, thereby achieving the purpose of separating and simultaneously detecting different effective components in Houpu Mahuang Decoction.

[0051] The present invention obtains a standard fingerprint spectrum with good separation, relatively stable baseline, more peaks and better ability to reflect the quality of Houpu Mahuang Decoction by adjusting the mobile phase and selecting a suitable elution gradient.

[0052] The detection wavelength of the present invention is significantly better than other detection wavelengths, and the standard fingerprint of Houpu Mahuang Decoction with better peaks can be obtained by using the detection wavelength of the present invention.

[0053] The present invention effectively optimizes the separation conditions by selecting appropriate process conditions such as chromatographic column, column temperature, flow rate, etc., thereby improving the peak stability of the standard fingerprint of Houpu Mahuang Decoction;

[0054] The present invention can identify 18 chemical components at one time by selecting appropriate mass spectrometry conditions, thus reducing the difficulty of chemical component identification;

[0055] The construction method of the present invention also has good feasibility, stability and reproducibility;

[0056] The standard fingerprint spectrum of Houpo Mahuang Decoction obtained by the present invention has 18 characteristic peaks, which can provide effective guarantee for the quality monitoring of Houpo Mahuang Decoction in the whole process of research, development, production and clinical application.

[0057] By using the presence or absence and characteristics of characteristic peaks in the standard fingerprint spectrum of Houpu Mahuang Decoction obtained by the present invention, the product quality of Houpu Mahuang Decoction can be fully monitored. By comparing the similarity of chromatographic fingerprint characteristics, the quality of Houpu Mahuang Decoction can be evaluated, and the stability and consistency can be examined, which makes up for the shortcomings of the current quality control method. At the same time, the stability of the production process of Houpu Mahuang Decoction can also be monitored to ensure that its quality is stable, uniform and controllable.

[0058] The standard fingerprint of Houpo Mahuang Decoction obtained by the present invention improves the quality monitoring standards of the finished and semi-finished products of Houpo Mahuang Decoction, effectively prevents the occurrence of product counterfeiting incidents, and ensures the normal production and circulation order of Houpo Mahuang Decoction; on the basis of the present invention, the correlation study between the fingerprint information and the pharmacodynamic activity information can also be carried out, so as to deeply clarify the correlation between the intrinsic chemical components of Houpo Mahuang Decoction and the efficacy of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is the fingerprint of Houpo Mahuang Decoction in Example 1 of the present invention;

[0060] Figure 2 are the primary and secondary mass spectra of ephedrine in Example 1 of the present invention;

[0061] Figure 3 are the primary and secondary mass spectra of magnocurine in Example 1 of the present invention;

[0062] Figure 4 are the primary and secondary mass spectra of amygdalin in Example 1 of the present invention;

[0063] Figure 5 are the primary and secondary mass spectra of Schisandrin A in Example 1 of the present invention;

[0064] Figure 6 are the primary and secondary mass spectra of magnolol in Example 1 of the present invention;

[0065] Figure 7 It is the standard fingerprint of Houpo Mahuang Decoction in Example 7 of the present invention;

[0066] Figure 8 It is the fingerprint of Houpo Mahuang Decoction in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0067] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0068] Example 1 A method for constructing a fingerprint of Houpu Mahuang Decoction

[0069] This example uses Houpo Mahuang Tang reference material to construct the fingerprint of Houpo Mahuang Tang. The specific preparation method of Houpo Mahuang Tang reference material is as follows:

[0070] Houpo Mahuang Decoction was prepared according to the method recorded in the Catalogue of Ancient Classic Prescriptions (First Batch), and the Houpo Mahuang Decoction reference material was obtained by concentration and freeze-drying.

[0071] This embodiment is a method for constructing a fingerprint of Hou Pu Ma Huang Tang, comprising the following specific steps:

[0072] 1) Preparation of test solution

[0073] Accurately weigh 100 mg of Houpo Mahuang Tang reference substance, place it in a 10 mL volumetric flask, add 8 mL of pure water, ultrasonically treat for 30 min, cool, make up to volume with pure water, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution.

[0074] 2) Fingerprint construction

[0075] The test solution was tested by HPLC-Q-TOF-MS to obtain the total ion current chromatogram of Houpu Mahuang Decoction, which is the fingerprint of Houpu Mahuang Decoction. Figure 1 ;

[0076] The HPLC conditions are as follows:

[0077] Chromatographic column: Shimadzu Shim-pack VP-ODS (5 μm, 4.6 × 250 mm, PN 228-34937-92);

[0078] The detection wavelength is 280nm;

[0079] The column temperature was 35°C;

[0080] The flow rate was 1.0 mL / min;

[0081] The injection volume was 20 μL;

[0082] Mobile phase A was acetonitrile, and mobile phase B was a 0.2 wt% formic acid aqueous solution;

[0083] The elution method is gradient elution, and the specific elution procedure is:

[0084] 0-16 min, 5% mobile phase A, 95% mobile phase B;

[0085] 16-18 min, 5% → 9% mobile phase A, 95% → 91% mobile phase B;

[0086] 18-28 min, 9% → 10% mobile phase A, 91% → 90% mobile phase B;

[0087] 28-32 min, 10% mobile phase A, 90% mobile phase B;

[0088] 32-40 min, 10% → 14% mobile phase A, 90% → 86% mobile phase B;

[0089] 40-52 min, 14% → 16% mobile phase A, 86% → 84% mobile phase B;

[0090] 52-63 min, 16% → 20% mobile phase A, 84% → 80% mobile phase B;

[0091] 63-73 min, 20% → 33% mobile phase A, 80% → 67% mobile phase B;

[0092] 73-80 min, 33% → 70% mobile phase A, 67% → 30% mobile phase B;

[0093] 80-90 min, 70% mobile phase A, 30% mobile phase B.

[0094] The mass spectrometry conditions were:

[0095] The dryer (N2) flow rate was 10.00 L / min; the temperature was 320°C; the nebulizer pressure was 45 psig; the capillary voltage was 4000 V; the fragmentor was 170 V, the skimmer was 65 V, the OCT 1RF Vpp, 750 V; the acquisition frequency was 3 spectra / s; and the ion mode was positive ion mode.

[0096] Based on the structural information of the compounds provided by mass spectrometry fragments, and with the assistance of reference substances and UV spectra, a total of 18 chemical components were identified. The total ion current chromatogram and the primary and secondary mass spectra of some representative compounds and the structural analysis process are as follows:

[0097] Combination Figure 1 and Figure 2 It can be seen that in the primary mass spectrum, the quasi-molecular ion peak is m / z166.1227[M+H] + , the quasi-molecular ion peak in the secondary mass spectrum loses a molecule of water, and obtains m / z 148.1118 [M + H-H2O] + On this basis, the methyl group is removed to form a fragment m / z 133.0881 [M + H-H2O-CH3] +, and the fragment ion m / z 117.0689 [M+H-H2O-NHCH3]+ after removing one molecule of water and then removing one molecule of aminomethyl. The ion peak signals in the above mass spectrum are consistent with the ephedrine reported in "Identification of Chemical Components of Mahuang Decoction Based on GC-MS and UPLC-Q-TOF-MS" (Li Rui et al., Chinese Journal of Traditional Chinese Medicine, 2014, 39(4): 704-709). Therefore, the chemical component corresponding to characteristic peak 4 is determined to be ephedrine.

[0098] Combination Figure 1 and Figure 3 It can be seen that the primary mass spectrum shows that its quasi-molecular ion peak is m / z314.1747[M+H] + The molecular ion peak in the secondary mass spectrum lost the N,N-dimethyl fragment, and the fragment ion m / z 269.1174 [M-C2H7N] was obtained. + At the same time, the molecular ion peak loses a phenol group to form a fragment m / z 175.0751 [M-C6H6O] + , and the molecular ion peak loses a C 12 H 17 O2 fragment, forming 4-benzylphenol fragment m / z 107.0484 [MC 12 H 17 O2] + The ion peak signals in the above mass spectrum are consistent with the magnocurarine reported in "Analysis of Chemical Components of Magnolia Bark Based on UFLC-Triple TOF-MS / MS Technology" (Zhao Hui et al., Chinese Journal of Pharmaceutical Sciences, 2017, 52(20): 1824-1831). Therefore, it is determined that the chemical component corresponding to characteristic peak 8 is magnocurarine.

[0099] Combination Figure 1 and Figure 4 It can be seen that the primary mass spectrum shows that its quasi-molecular ion peak is m / z475.1920 [M+NH4] + The secondary mass spectrum shows that after the quasi-molecular ion peak loses a molecule of water, it removes a molecule of benzaldehyde structure to obtain a fragment ion m / z 374.0992 [M+H-H2O-C6H5CHO] + At the same time, the secondary mass spectrum shows that after the quasi-molecular ion peak loses a molecule of water, it removes a molecule of almond nitrile to form a fragment m / z 347.0958 [M+H-H2O-C8H7NO] +The fragment ion peak signal in the above mass spectrum is consistent with the characteristic ion peak signal of amygdalin reported in "Study on Simultaneous Determination of Amygdalin and Paeoniflorin in Human Urine and Their Urinary Excretion by LC-MS / MS" (Li Xiaobing et al., Acta Pharmaceutica Sinica, 2015, 50(10):1330-1335). Therefore, it is determined that the chemical component corresponding to characteristic peak 9 is amygdalin.

[0100] Combination Figure 1 and Figure 5 It can be seen that the primary mass spectrum shows that its quasi-molecular ion peak is m / z433.2216[M+H] + In the secondary mass spectrum, the quasi-molecular ion peak removes one molecule of water and then one molecule of methoxy to obtain the fragment ion m / z 384.1912 [M+H-H2O-OCH3] + On this basis, a methyl group is removed to form a fragment m / z 369.1668 [M+H-H2O-OCH3-CH3] + On this basis, it continues to cleave and removes another methoxyl group to form a fragment m / z 338.1500 [M+H-H2O-(OCH3)2-CH3] + The ion peak signals in the above mass spectrum are consistent with the schisandrin A reported in "Analysis of Lignans in Schisandrae Chinensis by LC-MS / MS" (Tian Hongzhe et al., Natural Product Research and Development, 2012, 24(S1):32-35,40). Therefore, it is determined that the chemical component corresponding to characteristic peak 16 is schisandrin A.

[0101] Combination Figure 1 and Figure 6 It can be seen that the primary mass spectrum shows that its quasi-molecular ion peak is m / z267.1384[M+H] + The secondary mass spectrum shows that its quasi-molecular ion peak loses a C2H4 fragment, resulting in a fragment ion m / z 239.2257 [M+H-C2H4] + At the same time, its quasi-molecular ion peak loses a C3H5 fragment, resulting in a fragment ion m / z 226.0695 [M+H-C3H5] + , and the quasi-molecular ion peak loses a C4H6O fragment to form a fragment m / z 197.0578 [M+H-C4H6O] + The ion peak signals in the above mass spectrum are consistent with the characteristic signals of magnolia officinalis reported in "Analysis of Chemical Components of Magnolia Officinalis by UPLC / Q-TOF-MS / MS" (Han Liang et al., Chinese Traditional Patent Medicine, 2013, 35(4): 766-769). Therefore, it is determined that the chemical component corresponding to characteristic peak 18 is magnolia officinalis.

[0102] The structural identification results of the chemical components corresponding to the 18 characteristic peaks are shown in the table below.

[0103] Table 1 HPLC-Q-TOF-MS / MS structural identification of Houpu Mahuang Decoction

[0104]

[0105]

[0106]

[0107] Embodiment 2-3 Construction method of fingerprint of Houpu Mahuang Decoction

[0108] Examples 2 and 3 are respectively methods for constructing a fingerprint of Houpu Mahuang Decoction, and their steps are basically the same as those of Example 1, except that:

[0109] In Example 2, 0.1 wt % formic acid aqueous solution was used as mobile phase B, the column temperature was 36° C., and the flow rate was 0.8 mL / min.

[0110] In Example 3, 0.3 wt % formic acid aqueous solution was used as mobile phase B, the column temperature was 34° C., and the flow rate was 1.2 mL / min.

[0111] The contents of other parts of Examples 2 to 3 are the same as those of Example 1, and the results are also basically the same as those of Example 1.

[0112] Example 4 Standard fingerprint of Houpo Mahuang Decoction

[0113] The fingerprint of 10 batches of Houpo Mahuang Decoction reference materials were determined by the method for constructing the fingerprint of Houpo Mahuang Decoction in Example 1, and the fingerprints of the 10 batches of Houpo Mahuang Decoction were synthesized by using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" of the National Pharmacopoeia Committee to generate a standard fingerprint of Houpo Mahuang Decoction consisting of 18 characteristic peaks. The specific characteristic peaks are the same as those in Example 1, see Figure 7 .

[0114] The standard fingerprint of Houpu Mahuang Decoction obtained in this example can be used for quality evaluation or control in the whole process of research / development / production / clinical application of Houpu Mahuang Decoction.

[0115] Experimental Example 1: Investigation of Fingerprint Methodology

[0116] This experimental example investigates the construction method of the fingerprint of Houpo Mahuang Decoction in Example 1. The specific investigation method refers to the "Technical Guide for the Study of Fingerprints of Traditional Chinese Medicine Injections". The obtained chromatogram is converted into AIA format and then input into the similarity evaluation software. The methodological investigation is carried out by comparing the similarity of the obtained spectra, mainly investigating the precision, reproducibility and stability of the construction method.

[0117] 1. Precision

[0118] According to the construction method in Example 1, Houpo Mahuang Decoction was used to prepare a precision test solution, and the same precision test solution was continuously injected 6 times for fingerprint detection. The RSD of the relative retention time of each characteristic peak was calculated with chromatographic peak 1 as a reference. The specific results are shown in Table 3:

[0119] Table 3 Relative retention time results (precision)

[0120] Injection times 1 2 3 4 5 6 RSD t1 / t1 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ t2 / t1 1.108 1.109 1.108 1.108 1.109 1.107 0.07％ t3 / t1 1.245 1.246 1.246 1.244 1.243 1.247 0.12％ t4 / t1 1.557 1.559 1.558 1.555 1.556 1.554 0.12％ t5 / t1 1.707 1.705 1.704 1.706 1.709 1.706 0.10％ t6 / t1 1.862 1.864 1.860 1.861 1.863 1.859 0.10％ t7 / t1 1.909 1.907 1.904 1.910 1.902 1.908 0.16％ t8 / t1 3.006 3.009 3.011 3.001 2.998 3.008 0.17％ t9 / t1 3.596 3.599 3.594 3.597 3.596 3.595 0.05％ t10 / t1 4.170 4.165 4.166 4.163 4.168 4.167 0.06％ t11 / t1 4.586 4.584 4.585 4.582 4.586 4.587 0.04％ t12 / t1 4.714 4.712 4.717 4.715 4.713 4.716 0.04％ t13 / t1 5.331 5.326 5.329 5.327 5.325 5.329 0.04％ t14 / t1 8.957 8.951 8.952 8.961 8.950 8.949 0.05％ t15 / t1 9.004 8.996 8.993 8.996 8.992 8.995 0.05％ t16 / t1 9.010 9.003 8.996 8.999 9.009 8.999 0.06％ t17 / t1 9.215 9.206 9.198 9.211 9.207 9.203 0.06％ t18 / t1 9.436 9.429 9.431 9.438 9.430 9.428 0.04％

[0121] It can be seen from Tables 3 and 4 that the RSD of the relative retention time of the characteristic peaks in the chromatogram obtained by continuous injection of 6 times using the construction method of Example 1 is between 0.00% and 0.17%, indicating that the construction method of the present invention has good precision and meets the requirements of the fingerprint spectrum.

[0122] 2. Reproducibility

[0123] According to the construction method in Example 1, 6 reproducible test solutions were prepared in parallel from the same batch of Hou Pu Ma Huang Tang, and fingerprint detection was performed on the 6 reproducible test solutions respectively. The relative retention time and relative peak area RSD of each characteristic peak were calculated with chromatographic peak 1 as a reference. The specific results are shown in Table 4:

[0124] Table 4 Relative retention time results (reproducibility)

[0125] Injection times 1 2 3 4 5 6 RSD t1 / t1 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ t2 / t1 1.107 1.106 1.11 1.108 1.109 1.108 0.13％ t3 / t1 1.246 1.244 1.245 1.247 1.246 1.244 0.10％ t4 / t1 1.559 1.557 1.556 1.552 1.553 1.558 0.18％ t5 / t1 1.704 1.709 1.706 1.708 1.711 1.702 0.19％ t6 / t1 1.863 1.866 1.858 1.862 1.864 1.861 0.15％ t7 / t1 1.907 1.906 1.905 1.909 1.903 1.906 0.10％ t8 / t1 2.998 3.004 3.011 3.001 3.007 3.002 0.15％ t9 / t1 3.591 3.607 3.587 3.592 3.603 3.601 0.22％ t10 / t1 4.162 4.172 4.164 4.169 4.161 4.168 0.10％ t11 / t1 4.589 4.582 4.586 4.580 4.581 4.588 0.08％ t12 / t1 4.721 4.708 4.715 4.717 4.712 4.719 0.10％ t13 / t1 5.330 5.321 5.327 5.324 5.328 5.329 0.06％ t14 / t1 8.952 8.954 8.963 8.949 8.944 8.958 0.07％ t15 / t1 9.001 8.992 8.995 8.997 8.999 8.996 0.03％ t16 / t1 9.011 8.999 9.001 9.003 9.002 9.004 0.05％ t17 / t1 9.210 9.201 9.196 9.209 9.211 9.207 0.06％ t18 / t1 9.439 9.427 9.436 9.432 9.435 9.431 0.04％

[0126] It can be seen from Table 4 that the RSD of the relative retention time of the characteristic peaks in the chromatograms obtained by detecting 6 test solutions using the construction method in Example 1 is between 0.00% and 0.22%, indicating that the construction method of the present invention has good reproducibility and meets the requirements of the fingerprint spectrum.

[0127] 3. Stability

[0128] According to the test solution preparation method in Example 1, Houpo Mahuang Decoction was used to prepare a stability test solution. After 0, 6, 15, 18, and 24 hours of storage, the solution was analyzed according to the chromatographic conditions in Example 1. The specific results are shown in Table 5:

[0129] Table 5 Relative retention time results (stability)

[0130]

[0131]

[0132] It can be seen from Table 5 that the RSD of the relative retention time of the characteristic peaks in the chromatogram obtained by testing the test solution placed for different times using the chromatographic conditions of Example 1 is between 0.00% and 0.24%, indicating that the test solution prepared by the present invention can remain stable within 24 hours and meets the requirements of sample detection.

[0133] Experimental Example 2 Investigation of various process parameters in the construction method

[0134] Comparative Example 1 is a comparative test of the construction method in Example 1, the only difference being that:

[0135] In Comparative Example 1, the mobile phase A used was acetonitrile, and the mobile phase B was a 0.2 wt % phosphoric acid aqueous solution. The obtained spectrum is shown in Figure 8 It can be seen that peaks 2 and 3 cannot be separated, peaks 5 to 7 cannot be separated, and the peaks are relatively dense, making it difficult to perform mass spectrometry analysis.

[0136] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint of Houpu Mahuang Decoction, characterized in that: The construction method is to prepare a test solution from Houpo Mahuang Decoction, and the obtained test solution is detected by HPLC-Q-TOF-MS method to obtain the fingerprint of Houpo Mahuang Decoction; The mobile phase A used in the HPLC-Q-TOF-MS is acetonitrile, and the mobile phase B is a formic acid aqueous solution with a concentration of 0.1-0.3 wt%; The elution mode of the high performance liquid chromatography is gradient elution; The elution conditions of the gradient elution are: 0-16 min, 5% mobile phase A, 95% mobile phase B; 16-18 min, 5%→9% mobile phase A, 95%→91% mobile phase B; 18-28 min, 9%→10% mobile phase A, 91%→90% mobile phase B; 28-32 min, 10% mobile phase A, 90% mobile phase B; 32-40 min, 10%→14% mobile phase A, 90%→86% mobile phase B; 40-52 min, 14% → 16% mobile phase A, 86% → 84% mobile phase B; 52-63 min, 16%→20% mobile phase A, 84%→80% mobile phase B; 63-73 min, 20%→33% mobile phase A, 80%→67% mobile phase B; 73-80 min, 33%→70% mobile phase A, 67%→30% mobile phase B; 80-90 min, 70% mobile phase A, 30% mobile phase B.

2. The method for constructing the fingerprint of Houpu Mahuang Decoction according to claim 1, characterized in that: The detection wavelength of the high performance liquid chromatography is 280 nm.

3. The method for constructing the fingerprint of Houpu Mahuang Decoction according to claim 1, characterized in that: The column temperature of the HPLC is 34-36° C. and the flow rate is 0.8-1.2 mL / min.

4. The method for constructing the fingerprint of Houpu Mahuang Decoction according to claim 1, characterized in that: The mass spectrometry conditions of the HPLC-Q-TOF-MS are: The dryer was N2 with a flow rate of 10.00 L / min; the temperature was 320°C; the nebulizer pressure was 45 psig; the capillary voltage was 4000 V; the fragmentor was 170 V, the skimmer was 65 V, and the OCT 1 RF Vpp, 750 V; the acquisition frequency was 3 spectra / s; and the ion mode was positive ion mode.

5. The method for constructing the fingerprint of Hou Pu Ma Huang Tang according to claim 1, characterized in that: The test solution is prepared by dissolving 100 parts by weight of Houpo Mahuang Decoction in water and then adjusting the volume to 10 parts by volume; The ratio of parts by weight to parts by volume is mg:mL.

6. The method for constructing the fingerprint of Houpu Mahuang Decoction according to claim 1, characterized in that: A total of 18 characteristic peaks were obtained and identified in the fingerprint of Houpu Mahuang Decoction.

7. The method for constructing the fingerprint of Hou Pu Ma Huang Tang according to claim 6, characterized in that: The relative retention times and corresponding chemical compositions of the 18 characteristic peaks are: The chemical component corresponding to peak 1 is phenylalanine, with a retention time of 9.180 min; The chemical component corresponding to peak 2 is norephedrine, with a retention time of 10.170 min; The chemical component corresponding to peak 3 is demethyl pseudoephedrine, with a retention time of 11.431 min; The chemical component corresponding to peak 4 is ephedrine, with a retention time of 14.296 min; The chemical component corresponding to peak 5 is pseudoephedrine, with a retention time of 15.661 min; The chemical component corresponding to peak 6 is methylephedrine, with a retention time of 17.093 min; The chemical component corresponding to peak 7 is methyl pseudoephedrine, with a retention time of 17.503 min; The chemical component corresponding to peak 8 is magnocurarine, with a retention time of 27.598 min; The chemical component corresponding to peak 9 is amygdalin, with a retention time of 33.020 min; The chemical component corresponding to peak 10 is magnolamine, with a retention time of 38.272 min; The chemical component corresponding to peak 11 is rekulin, with a retention time of 42.092 min; The chemical component corresponding to peak 12 is Oblongine, with a retention time of 43.286 min; The chemical component corresponding to peak 13 is pavone, with a retention time of 48.913 min; The chemical component corresponding to peak 14 is 6-shogaol, with a retention time of 82.204 min; The chemical component corresponding to peak 15 is Schisandra chinensis lignans L, with a retention time of 82.613 min; The chemical component corresponding to peak 16 is schisandrin A, with a retention time of 82.647 min; The chemical component corresponding to peak 17 is honokiol, with a retention time of 84.523 min; The chemical component corresponding to peak No. 18 is magnolol, and the retention time is 86.604 min.

8. The standard fingerprint of Hou Pu Ma Huang Tang obtained by the construction method described in any one of claims 1 to 7.

9. Application of the standard fingerprint spectrum described in claim 8 in the quality evaluation or control of the whole process of research / development / production / clinical application of Houpu Mahuang Decoction.

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