CN115308352B - Quality control method of herba Aristolochiae Mollissimae sample - Google Patents
Quality control method of herba Aristolochiae Mollissimae sample Download PDFInfo
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- CN115308352B CN115308352B CN202210997228.7A CN202210997228A CN115308352B CN 115308352 B CN115308352 B CN 115308352B CN 202210997228 A CN202210997228 A CN 202210997228A CN 115308352 B CN115308352 B CN 115308352B
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- 238000003908 quality control method Methods 0.000 title claims abstract description 27
- BBFQZRXNYIEMAW-UHFFFAOYSA-N aristolochic acid I Chemical compound C1=C([N+]([O-])=O)C2=C(C(O)=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-N 0.000 claims abstract description 113
- 239000000523 sample Substances 0.000 claims abstract description 112
- 239000000243 solution Substances 0.000 claims abstract description 79
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 claims abstract description 73
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims abstract description 73
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 claims abstract description 73
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 claims abstract description 73
- 235000005493 rutin Nutrition 0.000 claims abstract description 73
- 229960004555 rutoside Drugs 0.000 claims abstract description 73
- 239000013558 reference substance Substances 0.000 claims abstract description 70
- BBFQZRXNYIEMAW-UHFFFAOYSA-M Aristolochate I Natural products C1=C([N+]([O-])=O)C2=C(C([O-])=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-M 0.000 claims abstract description 67
- IKGXIBQEEMLURG-NVPNHPEKSA-N rutin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-NVPNHPEKSA-N 0.000 claims abstract description 67
- 239000012488 sample solution Substances 0.000 claims abstract description 62
- 238000001228 spectrum Methods 0.000 claims abstract description 45
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- 238000004445 quantitative analysis Methods 0.000 claims abstract description 7
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 claims abstract description 6
- 239000000463 material Substances 0.000 claims description 91
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 45
- 230000014759 maintenance of location Effects 0.000 claims description 45
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 238000010828 elution Methods 0.000 claims description 18
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- 238000004809 thin layer chromatography Methods 0.000 claims description 14
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 13
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000012467 final product Substances 0.000 claims description 2
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- 238000000034 method Methods 0.000 abstract description 72
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- 238000011084 recovery Methods 0.000 abstract description 9
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 249
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 34
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 9
- 239000000377 silicon dioxide Substances 0.000 description 9
- 244000185386 Thladiantha grosvenorii Species 0.000 description 8
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- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 5
- 241000041628 Aristolochia mollissima Species 0.000 description 5
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- 238000010438 heat treatment Methods 0.000 description 5
- 238000010992 reflux Methods 0.000 description 5
- 239000003643 water by type Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 239000012925 reference material Substances 0.000 description 4
- 238000013112 stability test Methods 0.000 description 4
- 102100028292 Aladin Human genes 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
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- -1 aristolochic acid terpene ester Chemical class 0.000 description 3
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- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
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- FCSKOFQQCWLGMV-UHFFFAOYSA-N 5-{5-[2-chloro-4-(4,5-dihydro-1,3-oxazol-2-yl)phenoxy]pentyl}-3-methylisoxazole Chemical compound O1N=C(C)C=C1CCCCCOC1=CC=C(C=2OCCN=2)C=C1Cl FCSKOFQQCWLGMV-UHFFFAOYSA-N 0.000 description 1
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- 229940076810 beta sitosterol Drugs 0.000 description 1
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
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- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/95—Detectors specially adapted therefor; Signal analysis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a quality control method of a sought bone wind sample, which comprises the following steps: (1) preparing a reference solution and a test solution; respectively preparing reference substance solutions of reference substances by taking rutin and aristolochic acid I as reference substances of reference substances; (2) Respectively sucking the reference substance solution and the sample solution for ultra-high performance liquid chromatography analysis to construct a characteristic spectrum of the sample to be detected, wherein the characteristic spectrum comprises at least 9 characteristic peaks, one characteristic peak exists in the characteristic peaks corresponding to a rutin reference substance peak, and one characteristic peak exists corresponding to an aristolochic acid I reference substance peak; (3) Carrying out qualitative analysis on the sample to be detected through the characteristic spectrum obtained in the step (2); and (3) taking rutin as a content measurement index, taking aristolochic acid I as a measurement index, and carrying out quantitative analysis on a sample to be detected. The method is simple to operate, good in repeatability and stability, reliable in recovery rate, low in detection cost and high in detection efficiency through methodological verification.
Description
Technical Field
The invention relates to a medicine quality control method, in particular to a quality control method for a comprehensively controlled sought-bone wind sample.
Background
The Aristolochia Mollissimae is dried whole herb of Aristolochia Mollissimae Aristolochia mollissima Hance (pharmacopoeia Committee of the health department of the people' S republic of China. Chinese pharmacopoeia [ S ]. People health Press 1977:251). Aristolochia contains amino acids, aristololactone, aristolochic acid I, aristolochic acid terpene ester I, beta-sitosterol and silver-bag lactone B, and in addition, contains aristolochic acid sesquiterpene ester and 39 known compounds (Peng Guoping, lifeichang, wang Ying, etc. the ester compound VII of sesquiterpene of aristolochic acid in aristolochia mollissima [ J ]. Pharmaceutical report, 1996,31 (6): 446-450). It is pungent and bitter in flavor and neutral in nature. Has effects of dispelling pathogenic wind, dredging collaterals, and relieving pain, and can be used for treating rheumatalgia, joint pain, etc. (Wang Xiulan, hong Zongchao, sun Wei, etc.. Momordica Charantia quality standard research [ J ]. Hubei journal of Chinese medicine, 2018,40 (05): 54-58).
The research of modern pharmacological actions and clinical application shows that the Momordica grosvenori has better curative effects on anti-inflammatory and analgesic effects (Chen Duobao, xu Bing, li Bing, etc. Momordica grosvenori has anti-inflammatory and analgesic effects (J. Journal of basic Chinese medicine, 2001 (01): 9-10), antirheumatic effects (Wang Xue, liu Weizhong, shen Yunxiu, etc. Momordica grosvenori has the research of the chemical composition of Momordica grosvenori [ J ]. Shandong agricultural science, 2010 (04): 34-36), the treatment of rheumatoid arthritis (Guo Chunhui, wang Dongqiao, yang Zhen. Momordica grosvenori has the external application of powder for treating osteoarthritis (131 cases J ]. J. Journal of Chinese medicine, 2001 (01): 16-17), sciatica (Zhang Shiyou. Momordica grosvenori has the treatment of sciatica [ J ]. Henan barker medical wine, 1976 (01): 48), malaria, traumatic hemorrhage, acute carbuncle and various suppurative infections (Xia Houguo, li Xiaohua, fan Xiaona. Determination of trace elements in Momordica grosvenori has better curative effects on medicines (J. National medicine, 2010,21 (03): 9-750).
The traditional Chinese medicine formula granule is prepared by extracting single traditional Chinese medicine decoction pieces with water, concentrating, drying and granulating, is a new form of decoction piece innovation, has the characteristics of convenient carrying and simple use compared with the traditional decoction piece decoction, and can meet the requirements of modern fast-paced work and life. In recent years, the traditional Chinese medicine formula particles are rapidly developed, and the national standard of the formula particles of a plurality of traditional Chinese medicine varieties is continuously discharged, so that great contribution is made to standardizing the production of the traditional Chinese medicine formula particles and promoting the modernization of traditional Chinese medicines. The herba Aristolochiae Mollissimae formula granule is prepared by extracting herba Aristolochiae Mollissimae medicinal materials by modern process, concentrating, drying, and granulating, retains clinical effects of herba Aristolochiae Mollissimae, and simultaneously has portability.
The quality control means of the medicinal materials of the Aristolochia Mollissimae are partially reported at present. In 1996, the Chinese medicinal materials standard of Henan province and Henan local Chinese medicinal materials standard are adopted as the standard for controlling the quality of the medicinal materials of the Aristolochia Mollissimae by adopting the characteristics, stem cross section and powder microscopic identification and physical and chemical identification (Henan province Chinese medicinal materials standard of Henan province [ S ]. Zhengzhou: china farmer publishing society, 1992:39-40). Liu Chaodeng A RP-HPLC method is established to quantitatively study aristolochic acid A in herba Aristolochiae Mollissimae (Liu Chao, chen Yun, zhao Xiaolei, etc. A RP-HPLC method is used to measure aristolochic acid A content [ J ]. Chinese herbal medicine, 2003 (12): 87-89). Wang Xiulan et al establish quality standards for the medicinal materials of Aristolochia, and studied the properties, identification (microscopic and lamellar), various examinations (moisture, total ash, acid insoluble ash, extract), content determination, etc. (Wang Xiulan, hong Zongchao, sun Wei, etc.. Aristolochia medicinal materials quality standards research [ J ]. Hubei J.Chinese medicine, 2018,40 (05): 54-58). The quality control of the medicinal materials of the Aristolochia Mollissimae is not partially researched in the Chinese pharmacopoeia of the 2020 edition at present, but a comprehensive, effective and systematic quality evaluation method and standard are not established, for example, the characteristic spectrum of the medicinal materials of the Aristolochia Mollissimae is not analyzed and researched temporarily, the characteristic spectrum of the medicinal materials of the Aristolochia Mollissimae is concentrated in the aspect of content determination, the effective components in the Aristolochia Mollissimae are not quantitatively analyzed and researched, and the establishment of a validity evaluation means and a control method of the overall quality of the Aristolochia Mollissimae have a plurality of defects. In addition, the existing literature data show that the quality control research of the herba Aristolochiae Mollissimae is mainly focused on medicinal materials, and the research on the quality control of the herba Aristolochiae Mollissimae formula particles is not searched. Therefore, the invention comprehensively controls the overall profile, the effective components and the toxic components of the herba Aristolochiae Mollissimae formula particle by combining various analysis means, and ensures the effectiveness and the safety of the product quality.
Disclosure of Invention
The invention aims to: the quality control method for the sought-bone wind sample is good in reproducibility and stability, reliable in recovery rate, low in detection cost and high in detection efficiency.
The technical scheme is as follows: the invention discloses a quality control method of a sought bone wind sample, which comprises the following steps:
(1) Selecting a sought bone wind sample, and preparing a sample solution; preparing a reference substance solution of a reference medicinal material by taking the herba Aristolochiae Mollissimae as the reference medicinal material; respectively preparing reference substance solutions of reference substances by taking rutin and aristolochic acid I as reference substances of reference substances;
preferably, 0.2 g-1.0 g of the herba Aristolochiae Mollissimae formula particles are ground, water or 10% -70% methanol is added for extraction, and the subsequent filtrate is taken to obtain a sample solution;
preferably, 0.5g to 2g of the reference medicinal material of the herba Aristolochiae Mollissimae is taken, 30 to 70 percent of methanol is added for extraction, and the subsequent filtrate is taken as the reference solution of the reference medicinal material; adding appropriate amount of rutin control and aristolochic acid I control into methanol water solution for dissolving, and taking as reference solution;
(2) Respectively sucking the reference substance solution and the sample solution for ultra-high performance liquid chromatography analysis to construct a characteristic spectrum of the sample to be detected, wherein the characteristic spectrum comprises at least 9 characteristic peaks, one characteristic peak exists in the characteristic peaks corresponding to a rutin reference substance peak, and one characteristic peak exists corresponding to an aristolochic acid I reference substance peak;
Preferably, the solution of the reference substance and the solution of the sample are sucked into the sample and measured in an amount of 0.5. Mu.l to 2. Mu.l;
(3) Carrying out qualitative analysis on the sample to be detected through the characteristic spectrum obtained in the step (2); and (3) taking rutin as a content measurement index, taking aristolochic acid I as a measurement index, and carrying out quantitative analysis on a sample to be detected.
Further, the method further comprises the step of carrying out qualitative analysis on the sample to be detected through a thin layer chromatography, and judging whether spots of the sample to be detected and the reference medicinal material on the thin layer plate correspond to each other or not by using the reference medicinal material of the herba Aristolochiae Mollissimae as a reference; if yes, carrying out the steps (1) - (3), otherwise, determining that the sample to be tested is a disqualified sample.
Wherein, the preparation method comprises the steps of adding an organic solvent into 0.2 g-1.0 g of the herba Aristolochiae Mollissimae formula particles for extraction, and obtaining a subsequent filtrate to obtain a sample solution; extracting the reference medicinal material of the herba Aristolochiae Mollissimae with an organic solvent in an amount of 0.5-2 g, and collecting the filtrate to obtain a reference solution of the reference medicinal material.
Further, the developing agent includes toluene, acetone, and formic acid. Preferably, the thin layer plate is silica gel G, and the developing agent is toluene-acetone-formic acid (9:1:0.5).
In the step (1), the extractant for preparing the reference substance solution and/or the sample solution is water or methanol water solution with the volume concentration of 10-70%.
Furthermore, the reference substance solution is prepared, the usage amount of the corresponding extractant per gram of the herba Aristolochiae Mollissimae is 5 ml-50 ml, and the extraction time is 15-60 minutes.
Further, in the step (1), the concentration of the rutin reference substance solution is 20-80 mug/ml; the concentration of the reference substance solution of the aristolochic acid I reference substance is 3-15 mug/ml.
Further, the conditions of the ultra performance liquid chromatography are as follows: the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler; acetonitrile is taken as a mobile phase A, 0.05 to 0.2 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is adopted: 0-3 min,5% A, 3-20 min, 5-25% A, 20-25 min, 25-43% A, 25-30 min, 43-60% A.
Further, the detection wavelength is 220-320 nm; the flow rate is 0.25 ml-0.35 ml per minute; the column temperature is 30-40 ℃; an ultraviolet detector is used.
Further, the ultra-high performance liquid chromatography characteristic spectrum of the herba Aristolochiae Mollissimae sample has 9 characteristic peaks; and corresponds to 9 characteristic peak retention times in the chromatogram of the reference substance of the control medicinal material; calculating the relative retention time of peaks 4, 6, 7, 8 and S peak by taking the corresponding peak of the rutin reference object as the S peak, wherein the relative retention time is within +/-10% of a specified value, and the specified value is: 0.63 (Peak 4), 1.14 (Peak 6), 1.55 (Peak 7), 1.58 (Peak 8).
Further, the herba Aristolochiae Mollissimae sample is herba Aristolochiae Mollissimae medicinal material, herba Aristolochiae Mollissimae decoction pieces, herba Aristolochiae Mollissimae preparation intermediate or herba Aristolochiae Mollissimae preparation finished product.
The invention establishes a group of rapid, comprehensive and specific quality detection methods for the Aristolochia prescription granule, which comprises a set of thin layer identification method and a set of liquid phase method for simultaneously carrying out the characteristic spectrum, the rutin content measurement and the aristolochic acid I limit measurement. Compared with Wang Xiulan et al (Wang Xiulan, hong Zongchao, sun Wei, etc.. The quality standard research of the medicinal materials of the Aristolochia Mollissimae [ J ]. J.J.Hubei. J.Chinese medicine, 2018,40 (05): 54-58), the quality standard research of the medicinal materials of the Aristolochia Mollissimae has the advantages that:
(1) The quality standard of the Humata prescription granule is thoroughly studied for the first time.
(2) The thin layer identification method uses reference medicinal materials, and through optimizing developing agent, the number of chromatographic spots is more, and the optimized developing agent toluene-acetone-formic acid (9:1:0.5) can analyze different polarity components, so that the quality information is more comprehensive, and the method accords with the overall quality control characteristic of the traditional Chinese medicine.
(3) The invention adopts a set of chromatographic methods to realize qualitative and quantitative control of the sought bone wind. The method is more beneficial to the overall quality control of the herba Aristolochiae Mollissimae formula particles, and simultaneously takes into account the safety, the effectiveness and the overall quality evaluation of the traditional Chinese medicines.
(4) The characteristic spectrum meeting the overall characteristic requirement of the sought bone wind is established for the first time. A characteristic map containing 9 characteristic peaks is established, 2 peaks are identified, and meanwhile, a reference medicinal material is used as a reference substance, so that the consistency of the formula particles and the medicinal materials can be reflected.
(5) The selection of the effectiveness and safety indexes is more targeted. Rutin is used as a content index component, has the effects of resisting inflammation, easing pain and the like, and can fully characterize the effects of resisting inflammation and relieving pain of the herba Aristolochiae Mollissimae. The rutin content is higher, the components are more stable, and the method has better guiding significance for process control and product quality stability of the herba Aristolochiae Mollissimae formula particles; because aristolochic acid I has renal toxicity, the invention also conducts limited study on aristolochic acid I. Provides a technical control means for the quality of the raw materials, intermediates and formula particles of the herba Aristolochiae Mollissimae, and provides a powerful guarantee for the industrialization and quality control of the herba Aristolochiae Mollissimae.
(6) Compared with the content control in the prior art (HPLC method), the invention adopts the ultra-high liquid chromatography, is more rapid and effective, and is more beneficial to the industrialized application of the Aristolochia Mollissimae formula particles.
(7) The invention adopts ultra-high liquid chromatography and thin layer chromatography, reasonably optimizes chromatographic conditions, establishes an organic combination of characteristic spectrum, content measurement, limit measurement and thin layer identification methods of the sought-after wind formula particles, wherein the characteristic spectrum and the thin layer identification belong to qualitative methods, the content measurement and the limit measurement belong to quantitative methods, main effective components can be quantitatively analyzed, the thin layer identification operation is convenient, equipment is simple, the cost is lower, different polar components can be analyzed by optimizing a developing agent, the characteristic spectrum method depends on liquid phase equipment, the artificial interference is less, the detection sensitivity is higher, the method is mainly suitable for analyzing medium polar components and low polar components, and the characteristic spectrum and the content measurement are mutually complemented.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
(1) The method is a more comprehensive, simple, quick and effective quality control method of the sought-bone wind formula particles, performs quality control from thin layer identification, characteristic spectrum, content measurement and limited measurement at a plurality of angles, takes safety and effectiveness into consideration, and provides more powerful guarantee for industrialization of the sought-bone wind formula particles;
(2) The method is simple to operate, has good repeatability and stability, reliable recovery rate, low detection cost and high detection efficiency through methodological verification, and provides an important guarantee for the production of the standard Aristolochia-Mollissimae formula particles.
Drawings
Fig. 1 is a characteristic spectrum of the sought bone wind formula particle, peak 5 (S): rutin; peak 9: aristolochic acid I;
fig. 2 is a characteristic spectrum of a midbody of the sought bone wind, peak 5 (S): rutin; peak 9: aristolochic acid I;
fig. 3 is a characteristic spectrum of the medicinal material of the herba Aristolochiae Mollissimae/decoction piece, peak 5 (S): rutin; peak 9: aristolochic acid I;
FIG. 4 is a chromatogram of a control, a control drug, and a herba Aristolochiae Mollissimae formulation;
FIG. 5 UPLC chromatograms of the sought bone wind formulation particles at different detection wavelengths;
FIG. 6 rutin full wavelength scan;
FIG. 7 shows a full wavelength scan of aristolochic acid I;
FIG. 8 UPLC diagrams of different extraction solvent surveys;
FIG. 9 is a UPLC diagram of different extraction modes;
FIG. 10 UPLC diagrams examined at different extraction times;
FIG. 11 UPLC plots of different extraction volume surveys;
FIG. 12 is a view of a characteristic spectrum chromatographic column of the Aristolochia Mollissimae formula particle;
FIG. 13 is a graph showing the characteristic spectrum column temperature investigation of the sought bone wind formula particle;
FIG. 14 is a graph of the characteristic spectrum flow velocity investigation of the sought bone wind formula particle;
FIG. 15 is a linear relationship diagram of rutin control;
FIG. 16 shows a linear relationship of aristolochic acid I control;
FIG. 17 TLC patterns of different spotting amounts of the sought-bone wind formula particles, wherein 1: 1 μl of test sample; 2: 2 μl of test sample; 3: 5 μl of test sample; 4: 10 μl of test sample; s1: 1 μl of control medicinal material; s2: 2 μl of control medicinal material; s3: 5 μl of control medicinal material; s4: control drug 10 μl;
fig. 18 TLC profile of a specific experiment of the sought bone wind formulation particles, wherein 1-3: a herba Aristolochiae Mollissimae formula granule; 4: a negative control; s: herba Aristolochiae Mollissimae is used as reference material;
FIG. 19 shows TLC patterns of the Aristolochia Mollissimae granule under different temperature conditions, wherein 1-3: a herba Aristolochiae Mollissimae formula granule; s: herba Aristolochiae Mollissimae is used as reference material;
FIG. 20 TLC patterns of the Aristolochia Mollissimae granule formulations under different humidity conditions, wherein 1-3: a herba Aristolochiae Mollissimae formula granule; s: herba Aristolochiae Mollissimae is used as reference material;
FIG. 21 shows TLC patterns of thin-layer plate sought-bone wind formula particles of different manufacturers, wherein 1-3: a herba Aristolochiae Mollissimae formula granule; s: the herba Aristolochiae Mollissimae control medicinal material (Qingdao marine chemical Co., ltd. (left), qingdao Kang Yexin pharmaceutical silica gel desiccant Co., ltd. (right), shanghai A Ding Shenghua technology Co.);
fig. 22 thin layer identification chromatograms of different batches of the sought-bone wind formula particle samples, 1-3: a herba Aristolochiae Mollissimae formula granule; s: herba Aristolochiae Mollissimae is used as reference material;
FIG. 23 separation effect of different gradients;
FIG. 24 is a graph comparing extraction efficiencies of different extraction solvents;
FIG. 25 is a graph comparing extraction efficiencies of different extraction modes;
FIG. 26 is a graph comparing extraction efficiency at different extraction times;
FIG. 27 is a graph comparing extraction efficiency for different extraction volumes;
FIG. 28 Mount Speare wind formula particle specificity test;
FIG. 29 is a view of a sought after wind formula particle integrity test;
FIG. 30 Mount Speare wind formula particle specificity test;
fig. 31 TLC profile of the sought-after wind formula particles (different test preparation methods), wherein 1: a herba Aristolochiae Mollissimae granule (method one); 2: a herba Aristolochiae Mollissimae granule (method II); 3: a herba Aristolochiae Mollissimae formula granule (method III); s1: the herba Aristolochiae Mollissimae is used as reference medicine (method one); s2: herba Aristolochiae Mollissimae is used as reference (method II).
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
The embodiment provides a quality control method of a herba Aristolochiae Mollissimae formula sample, wherein the herba Aristolochiae sample can be herba Aristolochiae medicinal materials, herba Aristolochiae Mollissimae decoction pieces, herba Aristolochiae Mollissimae preparation intermediates or herba Aristolochiae Mollissimae preparation finished products, and the quality control method comprises the following steps of:
1. thin layer authentication:
performing qualitative analysis on the sample to be detected by using a thin-layer chromatography, and judging whether spots of the sample to be detected and the reference medicinal material on the thin-layer plate correspond to each other or not by using the reference medicinal material as a reference; if so, carrying out characteristic spectrum, content measurement and limit measurement, otherwise, determining that the sample to be measured is an unqualified sample.
Specifically, 0.2 g-1.0 g of the herba Aristolochiae Mollissimae particles are ground, 50ml of methanol is added, ultrasonic treatment is carried out for 30 minutes, cooling is carried out, filtration is carried out, filtrate is evaporated to dryness, and 1ml of methanol is added into residues to be dissolved to be used as a test solution. And adding 30ml of methanol into 0.5-2 g of the herba Aristolochiae Mollissimae reference medicinal material to prepare a reference medicinal material solution. Sucking 5 μl of each of the above two solutions, respectively spotting on the same silica gel G thin layer plate, spreading with toluene-acetone-formic acid (9:1:0.5) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).
2. Feature profile, content determination and limit determination:
(1) Selecting a sought bone wind sample, and preparing a sample solution; preparing a reference substance solution of a reference medicinal material by taking the herba Aristolochiae Mollissimae as the reference medicinal material; respectively preparing reference substance solutions of reference substances by taking rutin and aristolochic acid I as reference substances of reference substances;
preparation of reference solution: taking 0.5 g-2 g g g of the herba Aristolochiae Mollissimae reference medicine, placing the herba Aristolochiae Mollissimae reference medicine into a conical flask with a plug, adding 10 ml-25 ml of 30% -70% methanol, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 15-60 minutes, cooling, filtering, and taking the subsequent filtrate as reference medicine solution. Reference substance preparation: taking appropriate amounts of rutin reference substance and aristolochic acid I reference substance, precisely weighing, adding methanol to prepare solution containing rutin 20-80 μg and aristolochic acid I3-15 μg per 1ml, and taking as reference substance solution.
Preparation of test solution: about 0.2-1.0 g of the Aristolochia Mollissimae formula particles are taken, precisely weighed, placed in a conical bottle with a plug, precisely added with 30% -70% of methanol 10-25 ml, sealed, weighed, subjected to ultrasonic treatment (power is 250W and frequency is 40 kHz) for 15-60 minutes, cooled, weighed again, and subjected to shaking and filtering to obtain a subsequent filtrate, wherein the subsequent filtrate is obtained after 30% -70% of methanol is used for supplementing the lost weight.
Alternatively, in the step 1), 30% -70% of the methanol as the extractant may be replaced by the same amount of water.
(2) Respectively absorbing the reference substance solution and the sample solution for ultra-high performance liquid chromatography analysis to construct a characteristic spectrum of a sample to be detected, wherein the characteristic spectrum of the Aristolochia prescription granule comprises at least 9 characteristic peaks corresponding to the retention time of the 9 characteristic peaks in the reference substance chromatogram of the reference medicinal material, one characteristic peak corresponding to the rutin reference substance peak exists in the characteristic peaks, and one characteristic peak corresponding to the aristolochic acid I reference substance peak exists;
respectively carrying out ultra-high performance liquid chromatography on the sample solution and the reference solution prepared in the step (1) to obtain a corresponding ultra-high performance liquid chromatogram; precisely sucking 0.5-2 μl of each of the reference solution and the sample solution, and measuring with a liquid chromatograph.
Wherein, chromatographic condition parameters: the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler; acetonitrile is taken as a mobile phase A, 0.05 to 0.2 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is adopted; the flow rate is 0.25 ml-0.35 ml per minute; the column temperature is 30-40 ℃; an ultraviolet detector is adopted; the detection wavelength is 220-320 nm.
Preferably, it is: octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the flow rate is 0.3ml per minute; the column temperature is 35 ℃; the detection wavelength was 320nm.
Optionally, the herba Aristolochiae Mollissimae granule can be replaced by other herba Aristolochiae Mollissimae sample, such as herba Aristolochiae Mollissimae medicinal material, herba Aristolochiae Mollissimae decoction pieces, herba Aristolochiae Mollissimae preparation intermediate or herba Aristolochiae Mollissimae preparation final product.
And (3) selecting the midbody of the gristle wind as a gristle wind sample, and repeating the step (1), wherein the characteristic spectrum of the constructed midbody of the gristle wind is shown in figure 2.
Selecting the herba Aristolochiae Mollissimae medicinal material/decoction pieces as herba Aristolochiae Mollissimae sample, and repeating step (1), wherein the characteristic map of the constructed herba Aristolochiae Mollissimae medicinal material/decoction pieces is shown in figure 3.
As shown in fig. 4, the characteristic spectrum of the particles of the formulation of the aristolochia mollissima is shown in 9 characteristic peaks in the chromatogram of the solution of the test sample and corresponds to 9 characteristic peak retention times in the chromatogram of the reference substance of the reference medicinal material, wherein peak 5 and peak 9 are consistent with the peak retention time of the reference substance of the rutin reference substance and the peak corresponding to the rutin reference substance is an S peak, and the relative retention time of peak 4, 6, 7, 8 and the S peak is calculated, the relative retention time is within +/-10% of the specified value, and the specified value is: 0.63 (Peak 4), 1.14 (Peak 6), 1.55 (Peak 7), 1.58 (Peak 8).
(3) Carrying out qualitative analysis on the sample to be detected through the characteristic spectrum obtained in the step (2); and (3) taking rutin as a content measurement index, taking aristolochic acid I as a measurement index, and carrying out quantitative analysis on a sample to be detected. Full wavelength scans of rutin and aristolochic acid I are shown in FIGS. 6 and 7, respectively.
The quality control method provided by the embodiment comprises a set of thin layer identification method and a set of liquid phase method for simultaneously carrying out limit measurement, characteristic spectrum and rutin content measurement on aristolochic acid I. A group of rapid, comprehensive and specific quality detection methods for the Hurricane prescription granule are established. Quality control is performed from a plurality of angles of thin layer identification, limit measurement, characteristic spectrum and content measurement, and a more powerful guarantee is provided for industrialization of the Hurricane Aristolochia prescription granule.
Example 1
Taking 3 batches of the herba Aristolochiae Mollissimae formula particles and 15 batches of herba Aristolochiae Mollissimae medicinal materials as herba Aristolochiae Mollissimae formula samples, and respectively performing quality control measurement according to the following steps:
1. thin layer authentication:
3, carrying out thin-layer identification on the group of the herba Aristolochiae Mollissimae formula particles, and judging whether to enter the step 2 according to the result; and (3) directly carrying out characteristic spectrum, content measurement and limit measurement on 15 batches of the medicinal materials of the herba Aristolochiae Mollissimae without carrying out thin-layer identification. Performing qualitative analysis on the sample to be detected by using a thin-layer chromatography, and judging whether spots of the sample to be detected and the reference medicinal material on the thin-layer plate correspond to each other or not by using the reference medicinal material as a reference; if so, carrying out characteristic spectrum, content measurement and limit measurement, otherwise, determining that the sample to be measured is an unqualified sample.
3 thin layer identification processes of the group of the Aristolochia Mollissimae formula particles: grinding herba Aristolochiae Mollissimae granule 0.5g, adding methanol 50ml, ultrasonic treating for 30 min, cooling, filtering, evaporating filtrate to dryness, and dissolving residue with methanol 1ml to obtain test solution. And adding 30ml of methanol into 1g of the herba Aristolochiae Mollissimae reference medicine to prepare a reference medicine solution. Sucking 5 μl of each of the above two solutions, respectively spotting on the same silica gel G thin layer plate, spreading with toluene-acetone-formic acid (9:1:0.5) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).
The results are shown in FIG. 22. As can be seen from the figure, the chromatogram of the herba Aristolochiae Mollissimae formula granule and the chromatogram of the control medicinal material show spots of the same color at the corresponding positions. And 3 batches of the herba Aristolochiae Mollissimae formula particles enter the step 2 for continuous measurement.
2. Feature profile, content determination and limit determination:
(1) Selecting a sought bone wind sample, and preparing a sample solution; preparing a reference substance solution of a reference medicinal material by taking the herba Aristolochiae Mollissimae as the reference medicinal material; respectively preparing reference substance solutions of reference substances by taking rutin and aristolochic acid I as reference substances of reference substances;
preparation of reference solution: taking 1g of the herba Aristolochiae Mollissimae reference medicine, placing into a conical flask with a plug, adding 25ml of 70% methanol, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, filtering, and taking the subsequent filtrate as reference medicine solution.
Reference substance preparation: taking appropriate amounts of rutin control and aristolochic acid I control, precisely weighing, and adding methanol to obtain solution containing rutin 40 μg and aristolochic acid I6 μg per 1ml, as reference solution.
Preparation of test solution of the Aristolochia Mollissimae prescription granule: about 0.3g of the Aristolochia Mollissimae formula granule is taken, precisely weighed, placed in a conical flask with a plug, precisely added with 15ml of 70% methanol, sealed, weighed, subjected to ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooled, weighed again, complemented with 70% methanol to obtain the loss weight, shaken uniformly, filtered, and the subsequent filtrate is taken to obtain the Aristolochia Mollimae powder.
Preparing a test solution of the medicinal materials of the herba Aristolochiae Mollissimae: about 1g of the powder of the medicinal material of the herba Aristolochiae Mollissimae is precisely weighed, placed in a conical bottle with a plug, precisely added with 25ml of 70% methanol, sealed, weighed, subjected to ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooled, weighed again, complemented with 70% methanol to reduce the weight, shaken uniformly, filtered, and the subsequent filtrate is taken to obtain the product.
(2) Respectively carrying out ultra-high performance liquid chromatography on the sample solution and the reference solution prepared in the step 1) to obtain a corresponding ultra-high performance liquid chromatogram; precisely sucking 1 μl of each of the reference solution and the sample solution, and measuring with a liquid chromatograph.
Wherein, chromatographic condition parameters:
octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the flow rate is 0.3ml per minute; the column temperature is 35 ℃; the detection wavelength was 320nm.
As shown in fig. 4, the characteristic spectrum of the granules of the formulation of the aristolochia mollissima comprises 9 characteristic peaks in the chromatogram of the solution of the test sample and corresponds to 9 characteristic peak retention times in the chromatogram of the reference substance of the reference medicinal material, wherein peak 5 and peak 9 are consistent with the peak retention times of the reference substances of the rutin reference substance and the aristolochic acid I reference substance, the peak corresponding to the rutin reference substance is an S peak, the relative retention times of peak 4, 6, 7, 8 and the S peak are calculated, the relative retention times are within +/-10% of the specified value, and the specified value is: 0.63 (Peak 4), 1.14 (Peak 6), 1.55 (Peak 7), 1.58 (Peak 8).
The test solutions were assayed as follows.
TABLE 1 results of determination of Mount Howling granule samples (relative retention time)
TABLE 2 determination of 15 samples of Aristolochia Mollissimae (relative retention time)
(3) Comparing the ultra-high performance liquid chromatogram obtained in the step (2) with the characteristic chromatogram obtained in the step (1), and carrying out qualitative analysis on the sample;
And (3) taking rutin as a content measurement index, taking aristolochic acid I as a measurement index, and carrying out quantitative analysis on the sample.
The rutin content in the samples was measured, and the experimental results are shown in Table 3 and Table 4.
TABLE 3 determination of rutin content in Mount Hospital formula particles
TABLE 4 determination of rutin content in Aristolochia Mollissimae medicinal materials
The content of aristolochic acid I in the sample was measured, and the experimental results are shown in Table 5 and Table 6.
TABLE 5 determination of aristolochic acid I content in Moistolochia-containing formulation particles
Table 6 determination of content of aristolochic acid I in the medicinal materials of the Aristolochia prescription
From the above test results, from the qualitative analysis, the characteristic patterns of 3 batches of the herba Aristolochiae Mollissimae formula particles and 15 batches of herba Aristolochiae Mollissimae medicinal materials all meet the requirements of the characteristic patterns in the step (2). From the quantitative analysis, the rutin content of 3 batches of the herba Aristolochiae Mollissimae formula particles and 15 batches of herba Aristolochiae medicinal materials is stable, and the aristolochic acid I content is 0.05%, so that the effectiveness and the safety of the herba Aristolochiae Mollissimae formula particles and the medicinal materials are ensured.
Example 2 establishment of characteristic Spectrum method of Mount Hospita formula particle and methodological study
The embodiment is used for confirming an optimal scheme established by a characteristic spectrum in a quality control method of a sought-bone wind sample, and specifically comprises comparison of schemes of chromatographic conditions, wavelength selection and preparation of test sample solutions, and selection of the optimal chromatographic conditions and the test sample preparation schemes; and the optimal scheme is respectively inspected for specificity, integrity, precision, intermediate precision, stability, repeatability and durability.
1. Instrument and reagent
Agilent-1290 ultra-high performance liquid chromatograph, waters-H-class ultra-high performance liquid chromatograph; KQ-250B ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); electronic analytical balance (Metrehler-Tolyduo instruments (Shanghai), temperature-controlled water bath (Nantong Huatai laboratory instruments Co., ltd.), HY-4 oscillator (Kingtan Kexing instruments Co., ltd.), pure water system (Millipore Co.), TGL-16C centrifuge (Shanghai Anting scientific instruments Co., ltd.), acetonitrile (chromatographic purity, thermo Fisher Co.), phosphoric acid (chromatographic purity, aladin Co.), methanol (chromatographic purity, thermo Fisher Co., ltd.), water as ultrapure water, and other reagents as analytical purity.
The Mount Hospital control was purchased from Shanghai HongYongsheng biotechnology Co., ltd., number 410016-202004.
Rutin (100080-202012), aristolochic acid I (110746-201611) reference substances are purchased from Chinese food and drug verification institute.
The Mount Hospita formula granule is provided by Jiang Yintian river pharmaceutical Co.
2. Determination of detection wavelength
Taking a proper amount of the product, grinding, taking about 0.3g, placing into a conical flask with a plug, precisely adding 15ml of 70% methanol, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate. Chromatograms of the samples at 220nm,254nm and 320nm were collected as shown in fig. 5.
The result shows that the number of peaks is more under the wavelength of 320nm, the response is larger, and the base line is also smoother, so that 320nm is selected as the detection wavelength.
3. Optimization of chromatographic conditions
Method 1: octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the following table; the flow rate is 0.3ml per minute; the column temperature is 35 ℃; the detection wavelength was 320nm.
Method 2: octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the following table; the flow rate is 0.3ml per minute; the column temperature is 35 ℃; the detection wavelength was 320nm.
Method 3: octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the following table; the flow rate is 0.3ml per minute; the column temperature is 35 ℃; the detection wavelength was 320nm.
The results show (Table above, FIG. 23) that method 3 has the best separation effect, symmetrical peak shape and moderate retention time, and therefore, this method selects method 3 for subsequent study.
4. Preparation of test solutions
4.1 investigation of different extraction solvents
Grinding the materials, taking about 0.3g, precisely weighing in parallel 6 groups, placing into a conical flask with a plug, precisely adding 15ml of water, 10% of methanol, 30% of methanol, 50% of methanol, 70% of methanol and 15ml of methanol respectively, sealing, weighing, respectively performing ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with corresponding solvent, shaking uniformly, filtering, and collecting the subsequent filtrate. 1 μl of each sample solution was precisely aspirated and injected into a liquid chromatograph, and the ratio of the characteristic peak area to the sample amount was calculated by measuring under the above chromatographic conditions, and the results are shown in Table 7, FIG. 8 and FIG. 24.
TABLE 7 comparison of extraction efficiency for different extraction solvents (peak area/sample size)
As can be seen from the table above: besides lower extraction efficiency of methanol, the extraction results of other methanol with different concentrations as an extraction solvent are closer, and the extraction efficiency of 70% methanol on the content index rutin is considered to be higher, so that the extraction solvent is consistent with the content measurement extraction solvent, and the extraction solvent is determined to be 70% methanol through comprehensive consideration.
4.2 investigation of different extraction methods
Grinding the materials, taking about 0.3g, precisely weighing in parallel 3 groups, placing into a conical flask with a plug, precisely adding 15ml of 70% methanol, sealing, weighing, respectively performing ultrasonic treatment (power 250W, frequency 40 kHz), shaking extraction, heating and refluxing for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and collecting the subsequent filtrate. 1. Mu.l of each sample solution was precisely aspirated and injected into a liquid chromatograph, and the characteristic peak area/sample amount was calculated by measuring under the above-mentioned chromatographic conditions, and the results are shown in Table 8, FIG. 9 and FIG. 25.
TABLE 8 comparison of extraction efficiency for different extraction modes (Peak area/sample size)
As can be seen from the table above: the extraction efficiency of the different extraction modes is not greatly different, and the extraction method is determined to be ultrasonic treatment in consideration of the simplicity of operation.
4.3 investigation of different extraction times
Taking proper amount of the product, grinding, taking about 0.3g, precisely weighing in 4 groups, placing into a conical flask with a plug, precisely adding 15ml of 70% methanol, sealing, weighing, respectively performing ultrasonic treatment (power 250W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes and 60 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product. 1. Mu.l of each sample was precisely aspirated and injected into a liquid chromatograph, and the characteristic peak area/sample amount was calculated by measuring under the above-mentioned chromatographic conditions, and the results are shown in Table 9, FIG. 10 and FIG. 26.
TABLE 9 comparison of extraction efficiency at different extraction times (peak area/sample size)
As can be seen from the table above: the characteristic peak-to-peak area/sample size difference in the sought-bone wind formula particles is small in different times of ultrasonic treatment, the ultrasonic treatment is sufficient in 15 minutes of extraction, and the extraction time is determined to be 30 minutes in order to ensure the extraction efficiency.
4.4 investigation of different extraction volumes
Taking proper amount of the product, grinding, taking about 0.3g, precisely weighing in parallel 3 groups, placing into a conical flask with a plug, precisely adding 10ml, 15ml, 20ml and 25ml of 70% methanol, sealing, weighing, respectively performing ultrasonic treatment (power is 250W and frequency is 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate. 1 μl of each sample solution was precisely sucked up and injected into a liquid chromatograph, and the characteristic peak area/sample weight was calculated by measuring the sample solution under the above chromatographic conditions, and the extraction volume/15 was calculated, and the results are shown in table 10, fig. 11 and fig. 27.
Table 10 comparison of extraction efficiency for different extraction volumes (peak area/sample size extraction volume/15)
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As seen from the table above: the extraction efficiency of the different extraction volumes is not greatly different, and the extraction volume is determined to be 15ml from the standpoint of saving solvent and sufficient extraction.
4.5 determination of the method for preparing the sample solution
According to the research result, the preparation method of the sample solution for determining the characteristic spectrum of the Aristolochia Mollissimae prescription granule comprises the following steps: grinding herba Aristolochiae Mollissimae formula granule, precisely weighing about 0.3g, placing into conical flask with plug, precisely adding 70% methanol 15ml, sealing, weighing, ultrasonic treating (power 250W, frequency 40 kHz) for 30 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the filtrate.
5. Feature atlas methodology study
5.1 specificity investigation
The result of liquid chromatograph is shown in figure 28 below with the sample solution of the herba Aristolochiae Mollissimae prescription granule and the negative solution of herba Aristolochiae Mollissimae.
The experimental results show that: the negative solution has no chromatographic peak at the retention time of each characteristic peak in the chromatogram of the sample solution, which indicates that the solvent has no interference to the determination of the formulation particles of the herba Aristolochiae Mollissimae, and the determination of the formulation particles of the herba Aristolochiae Mollissimae by the method has specificity.
5.2 integrity inspection
The elution time was extended under the specific chromatographic conditions, and whether or not the residual impurity peak affected the subsequent sample under the specific chromatographic conditions was examined, and the results are shown in fig. 29.
The experimental result shows that the elution time is prolonged, no impurity peak exists, and the chromatographic condition basically meets the principle of maximum information quantity and has no influence on the analysis of subsequent samples.
5.3 precision investigation
The same batch of samples are taken, the sample solution is prepared into a test solution according to the preparation method of the test solution, the sample is continuously injected for 6 times, 1 mu l of sample solution is injected each time, the retention time of characteristic peaks is recorded, the relative retention time is calculated according to the text requirement, and the result is shown in Table 11.
TABLE 11 precision experimental results (relative retention time)
The results show that the relative retention time of each characteristic peak has RSD less than 1% and the precision is good.
5.4 intermediate precision investigation
The same lot of samples were taken, a test solution was prepared according to the test solution preparation method, 3 parts were processed by two laboratory workers A, B respectively according to the requirements under the text items, 1 μl was sampled on Agilent, waters instruments respectively, the characteristic peak retention time was recorded, and the relative retention time was calculated according to the text requirements, and the results were shown in Table 12.
Table 12 intermediate precision investigation (relative retention time)
The results show that: the sample has characteristic peaks except 1-4, the relative retention time RSD is less than 2%, and the intermediate precision test is good.
5.5 stability investigation
Taking the same batch of samples, preparing test solution according to the preparation method of the test solution, injecting 1 μl of the test solution at 0, 2, 4, 8, 12 and 24 hours respectively, recording the retention time of the characteristic peaks, and calculating the relative retention time according to the text requirement, wherein the result is shown in Table 13.
TABLE 13 stability investigation (relative retention time)
The results show that the relative retention time of each characteristic peak is less than 1% and the stability of the test solution is good within 24 hours.
5.6 repeatability investigation
The same batch of samples are taken, 6 groups are paralleled, test solutions are prepared according to the preparation method of the test solution, 1 μl of each sample is injected, the retention time of the characteristic peak is recorded, the relative retention time is calculated according to the text requirement, and the result is shown in Table 14.
TABLE 14 repeatability test (relative retention time)
The results show that the reproducibility of the method is good.
5.7 durability inspection
(1) Chromatographic column inspection
Taking the same batch of samples, preparing test sample solutions according to the preparation method of the text test sample solutions, and respectively adopting AcclaimTM RSLC 120C18 (Thermo, 2.1mm multiplied by 100mm,2.2 μm); porosill 120EC-C18 (Agilent, 2.1 mm. Times.100 mm,1.9 μm); ACQUITY UPLC BEH C18 (Waters, 2.1X100 mm,1.7 μm) three columns were each injected with 1. Mu.l, and the corresponding chromatograms were recorded, and the results are shown in the figure.
The results show that: the relative retention times of peaks 1 to 3 are greatly affected by the different columns, so the characteristic spectrum detection of the particles of the sought-after wind formula recommends using a column ACQUITY UPLC BEH C (Waters, 2.1×100mm,1.7 μm).
(2) Column temperature investigation
Taking the same batch of samples, preparing a test sample solution according to the preparation method of the text test sample solution, examining three temperatures of 30 ℃, 35 ℃ and 40 ℃, respectively injecting 1 μl, recording the retention time of characteristic peaks, and calculating the relative retention time according to the text requirements, wherein the results are shown in fig. 13 and table 15.
Table 15 column temperature investigation (relative retention time)
The results show that the column temperature is in the range of 30-40 ℃ and the measurement results of the samples are relatively stable and all meet the retention time requirements specified by the method, and the durability is good.
(3) Flow rate investigation
Taking the same batch of samples, preparing a test sample solution according to the preparation method of the text test sample solution, examining three flow rates of 0.25ml, 0.30ml and 0.35ml per minute, respectively injecting 1 μl, recording the retention time of characteristic peaks, and calculating the relative retention time according to the text requirements, wherein the results are shown in fig. 14 and table 16.
TABLE 16 flow rate investigation (relative retention time)
The results show that the flow rate is in the interval of 0.25 ml/min-0.35 ml/min, the measurement results of the samples are relatively stable, and all the flow rate meets the retention time requirements specified by the method, so that the durability is good.
Example 3 establishment of method for determining the content of particles in a Aristolochia Mollissimae formulation and methodological study
1 instrument and reagents
Waters-H-class ultra-high performance liquid chromatograph; an Agilent-1290 ultra-high performance liquid chromatograph; KQ-250B ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); electronic analytical balances (mertrer-tolido instruments (Shanghai); temperature-controlled water bath (Nantong Huatai laboratory instruments Co., ltd.); HY-4 oscillator (Jintan Kexing Instrument Co.); pure water systems (Millipore corporation); AS165W type centrifuge (Sunswang (Shanghai) commercial Co., ltd.); acetonitrile (chromatographic purity, thermo Fisher); methanol (chromatographic purity, thermo Fisher); phosphoric acid (chromatographic purity, aladin company); the water is ultrapure water; the other reagents were all analytically pure.
Rutin (100080-202012) reference was purchased from chinese food and drug assay institute;
the Mount Hospita formula granule is provided by Jiang Yintian river pharmaceutical Co.
2 inspection of reference source and purity
Rutin is purchased from Chinese food and drug inspection institute, with number 100080-202012, and is used for content measurement, the content is 91.6%, and no treatment is required before use.
3 determination of detection wavelength
The rutin control solution was scanned at full wavelength in the experiment, and the ultraviolet absorption diagram was recorded, see FIG. 6.
As a result, rutin has a large absorption at a wavelength of 256nm or 354nm, and the detection wavelength of 320nm is selected as the detection wavelength for rutin content measurement by comprehensively considering the combination of the characteristic spectrum detection wavelength.
Determination of 4 chromatographic conditions
By referring to the characteristic spectrum measuring method of the herba Aristolochiae Mollissimae, the content measuring method of rutin in the herba Aristolochiae Mollissimae formula granule is established, and the chromatographic conditions are as follows:
octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the flow rate is 0.3ml per minute; the column temperature is 35 ℃; the detection wavelength was 320nm.
5 preparation of sample solution
5.1 investigation of different extraction solvents
Grinding proper amount of herba Aristolochiae Mollissimae formula granule, taking about 0.3g, 6 groups in parallel, 2 parts of each group in parallel, precisely weighing, placing into a conical flask with a plug, precisely adding 15ml of water, 10% of methanol, 30% of methanol, 50% of methanol, 70% of methanol and 15ml of methanol respectively, sealing, weighing, respectively performing ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the reduced weight with corresponding solvent, shaking, filtering, and collecting subsequent filtrate. 1 μl of each sample solution was precisely aspirated, and the mixture was injected into a liquid chromatograph to calculate the rutin content, and the results are shown in FIG. 8 and Table 17.
TABLE 17 comparison of different extraction solvents
The results showed that: the 70% methanol extraction efficiency was high, so the extraction solvent was determined to be 70% methanol.
5.2 investigation of different extraction methods
Grinding proper amount of herba Aristolochiae Mollissimae formula granule, taking about 0.3g, 3 groups in parallel, 2 parts in each group in parallel, precisely weighing, placing into a conical flask with a plug, precisely adding 15ml of 70% methanol, sealing, weighing, respectively performing ultrasonic treatment (power 250W, frequency 40 kHz), shaking extraction, heating and refluxing for 30 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the subsequent filtrate. 1 μl of each sample solution was precisely aspirated, and the mixture was injected into a liquid chromatograph to calculate the rutin content, and the results are shown in FIG. 9 and Table 18.
Table 18 comparison of different extraction methods
The results showed that: the extraction efficiency of ultrasonic, shaking and heating reflux is equivalent, and the extraction method is determined to be ultrasonic treatment in consideration of the convenience of operation.
5.3 investigation of different extraction times
Grinding proper amount of herba Aristolochiae Mollissimae formula granule, taking about 0.3g, 4 groups in parallel, 2 parts in each group in parallel, precisely weighing, placing into a conical flask with a plug, precisely adding 15ml of 70% methanol, sealing, weighing, respectively performing ultrasonic treatment (power 250W, frequency 40 kHz) for 15 min, 30 min, 45 min and 60 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the subsequent filtrate. 1 μl of each sample solution was precisely aspirated, and the mixture was injected into a liquid chromatograph to calculate the rutin content, and the results are shown in FIG. 10 and Table 19.
TABLE 19 comparison of different extraction times
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The results showed that: the different extraction times have no obvious effect on the rutin content, and the extraction time is determined to be 30 minutes from the standpoint of complete extraction and time saving.
5.4 investigation of different extraction volumes
Grinding proper amount of herba Aristolochiae Mollissimae formula granule, precisely weighing about 0.3g, 4 groups in parallel, 2 parts in each group, placing into conical flask with plug, precisely adding 70% methanol 10ml, 15ml, 20ml and 25ml, sealing, weighing, respectively performing ultrasonic treatment (power 250W and frequency 40 kHz) for 30 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the filtrate. 1 μl of each sample solution was precisely aspirated, and the mixture was injected into a liquid chromatograph to calculate the rutin content, and the results are shown in FIG. 11 and Table 20.
Table 20 comparison of different extraction volumes
The results showed that: the extraction efficiency of different extraction volumes is equivalent, and the extraction volume is determined to be 15ml from the comprehensive consideration of full extraction and solvent saving.
The preparation method of the test sample for determining the content of the sought-bone wind particles finally comprises the following steps of:
grinding proper amount of herba Aristolochiae Mollissimae formula granule, precisely weighing about 0.3g, placing into conical flask with plug, precisely adding 70% methanol 15ml, sealing, weighing, ultrasonic treating (power 250W, frequency 40 kHz) for 30 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the filtrate.
6 methodological verification
6.1 linearity
And respectively precisely sucking 0.1 mu l, 0.2 mu l, 0.5 mu l, 1.0 mu l, 1.5 mu l and 2.0 mu l of rutin reference substance mixed solution (concentration 73.24 mu g/mL), injecting into a liquid chromatograph, measuring according to the chromatographic conditions, taking a peak area integral value as an ordinate and a sample injection quantity (mu g) as an abscissa, drawing a standard curve, and obtaining a rutin regression equation, wherein the results are shown in tables 21 and 22 and figure 15.
Table 21 regression equation
Table 22 relation between sample injection amount and peak area of rutin control
The results show that: the rutin sample injection amount is within the range of 0.007324-0.146480 mug, and the sample injection amount and the peak area value have good linear relation.
6.2 precision test
6.2.1 Instrument precision test
Precisely sucking the sample solution, injecting into a liquid chromatograph, measuring under the chromatographic conditions, continuously injecting sample for 6 times, recording peak area, and calculating relative standard deviation, wherein the result is shown in the table below.
Meter 23 Instrument precision test
The results show that: the precision test of the instrument is good (RSD is less than or equal to 2.0%).
6.2.2 repeatability test
The preparation method of the test solution comprises the steps of taking proper amount of the herba Aristolochiae Mollissimae prescription granule, grinding, taking about 0.3g, precisely weighing, and preparing 6 parts in parallel, preparing the test solution according to the preparation method of the test solution, respectively injecting 1 μl, measuring peak area of rutin, calculating content and RSD, and the result is shown in Table 24.
Table 24 results of repeatability test of samples
The results show that: the repeatability test is good (RSD%. Ltoreq.2.0%).
6.2.3 intermediate precision test
The amount of the powder of the formulation of the herba Aristolochiae Mollissimae was appropriately measured, and the powder was ground to about 0.3g, and 3 parts of the powder were processed by two laboratory workers A, B according to the above-mentioned requirements, and 1. Mu.l of the powder was sampled on Waters-UPLC and Agilent-UPLC, and the peak area value of rutin was measured and the content and RSD thereof were calculated, and the results are shown in Table 25.
Intermediate precision test of Table 25
The results show that: the intermediate precision is good.
6.3 accuracy test
Taking three groups of samples with known content (rutin content 1.94 mg/g) with proper amount, grinding, taking about 0.15g, and accurately weighing in 3 parts, and respectively adding reference substances of 50%, 100% and 150% of each component contained in 0.15g sample. Sample solutions were prepared and recovered by the above-mentioned sample solution preparation method, 1. Mu.l each was sampled, and the recovery, average recovery, RSD were calculated by the following formula, and the results are shown in Table 26.
Table 26 rutin accuracy results
The results show that: the recovery rate of rutin is between 99.98 percent and 106.15 percent, and the accuracy test result is good.
6.4 specificity test
The auxiliary materials added in the herba Aristolochiae Mollissimae formula granule are maltodextrin, silicon dioxide and magnesium stearate. The experiment examines the influence of a negative sample of the lack of the herba Aristolochiae Mollissimae on the determination of the content of the herba Aristolochiae Mollissimae formula particles. Taking a negative sample of the herba Aristolochiae Mollissimae, and preparing a negative sample solution according to a sample preparation method.
And (3) taking the test solution, the negative control solution and the rutin control solution of the Aristolochia Mollissimae prescription granule, and injecting the test solution, the negative control solution and the rutin control solution into a liquid chromatograph.
The experimental results showed (fig. 28): the negative chromatogram has no chromatographic peak at the retention time corresponding to the reference substance, which indicates that the auxiliary material and the solvent have no interference to the determination of rutin, and the determination of the rutin content in the herba Aristolochiae Mollissimae formula granule by the method has specificity.
6.5 integrity test
On the determined chromatographic conditions, the elution gradient when the acetonitrile proportion is highest is maintained, the elution time is doubled, and whether residual impurity peaks affect the subsequent samples or not under the determined chromatographic conditions is examined, and the result is shown in fig. 29.
The experimental result shows that the chromatographic condition basically meets the principle of maximum information amount and has no influence on the analysis of the subsequent samples by prolonging the elution time by one time and having no impurity peak.
6.6 durability test
6.6.1 stability test
Preparing test solution according to the preparation method of the test solution, sampling 1 μl of the test solution at 0, 2, 4, 8, 12 and 24 hours respectively, measuring peak area value, and calculating RSD.
Table 27 stability test results
The results show that: the sample test solution has good stability within 24 hours.
6.6.2 different flow Rate investigation
The test solution is prepared by the preparation method of the test solution, and the rutin content at the flow rates of 0.25ml/min, 0.30ml/min and 0.35ml/min is examined, and the results are shown in FIG. 14 and Table 28.
Table 28 investigation of different flow rates
The results show that: the rutin chromatographic peaks have better separation degree, similar content and better durability at the three flow rates.
Investigation of 6.6.3 different column temperatures
The test solution is prepared by the preparation method of the test solution, and the rutin content at three temperatures of 30 ℃, 35 ℃ and 40 ℃ is examined, and the results are shown in figure 13 and table 29.
Investigation of Table 29 different column temperatures
The results show that: the separation degree of rutin chromatographic peaks is good at the three column temperatures, the content is similar, and the durability is good.
6.6.4 chromatographic column investigation
Preparing test solution by the preparation method of the test solution, and respectively examining AcclaimTM RSLC 120C18 (Thermo, 2.1mm×100mm,2.2 μm); porosill 120EC-C18 (Agilent, 2.1 mm. Times.100 mm,1.9 μm); ACQUITY UPLC BEH C18 (Waters, 2.1X100 mm,1.7 μm) the rutin content of the Mount Hospita formula particles under 3 different chromatographic column conditions, the results are shown in FIG. 12, table 30.
Table 30 comparison of different chromatographic columns
The results show that: the chromatographic columns of three different types have good rutin separation effect, and have small influence on the content results, and the method has universal adaptability.
Example 4 establishment of method for determining limit of particles in Aristolochia Mollissimae formulation and methodological study
1 instrument and reagents
Waters-H-class ultra-high performance liquid chromatograph; an Agilent-1290 ultra-high performance liquid chromatograph; KQ-250B ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); electronic analytical balances (mertrer-tolido instruments (Shanghai); temperature-controlled water bath (Nantong Huatai laboratory instruments Co., ltd.); HY-4 oscillator (Jintan Kexing Instrument Co.); pure water systems (Millipore corporation); AS165W type centrifuge (Sunswang (Shanghai) commercial Co., ltd.); acetonitrile (chromatographic purity, thermo Fisher); methanol (chromatographic purity, thermo Fisher); phosphoric acid (chromatographic purity, aladin company); the water is ultrapure water; the other reagents were all analytically pure.
Aristolochic acid I (110746-201611) control was purchased from China food and drug testing institute;
the Mount Hospita formula granule is provided by Jiang Yintian river pharmaceutical Co.
2 inspection of reference source and purity
Aristolochic acid I is purchased from Chinese food and drug inspection institute and is numbered 110746-201611 for content measurement, the content is 98.9%, and the aristolochic acid I is not required to be treated before use.
3 determination of detection wavelength
Experiments the aristolochic acid I control solution was scanned throughout the wavelength and its uv absorbance profile was recorded, see figure 7.
As a result, it was found that aristolochic acid I was absorbed more strongly at 250nm and 322nm, and that 320nm was selected as the detection wavelength for the content measurement of aristolochic acid I, considering the combination of the characteristic spectrum detection wavelength of 320nm.
Determination of 4 chromatographic conditions
The method for measuring the content of aristolochic acid I in the aristolochic acid formula particles is established by referring to the characteristic spectrum measuring method of the aristolochic acid, and chromatographic conditions are as follows:
octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the flow rate is 0.3ml per minute; the column temperature is 35 ℃; the detection wavelength was 320nm.
5 preparation of sample solution
5.1 investigation of different extraction solvents
Grinding proper amount of herba Aristolochiae Mollissimae formula granule, taking about 0.3g, 6 groups in parallel, 2 parts of each group in parallel, precisely weighing, placing into a conical flask with a plug, precisely adding 15ml of water, 10% of methanol, 30% of methanol, 50% of methanol, 70% of methanol and 15ml of methanol respectively, sealing, weighing, respectively performing ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the reduced weight with corresponding solvent, shaking, filtering, and collecting subsequent filtrate. 1 μl of each test solution was precisely aspirated, and the solution was injected into a liquid chromatograph to calculate the content of aristolochic acid I, and the results are shown in FIG. 8 and Table 31.
TABLE 31 comparison of different extraction solvents
The results showed that: the extraction efficiency of other methanol with different concentrations is close to that of water and 10% methanol, and the peak symmetry factor and the theoretical plate number are combined, so that the extraction solvent is determined to be 70% methanol.
5.2 investigation of different extraction methods
Grinding proper amount of herba Aristolochiae Mollissimae formula granule, taking about 0.3g, 3 groups in parallel, 2 parts in each group in parallel, precisely weighing, placing into a conical flask with a plug, precisely adding 15ml of 70% methanol, sealing, weighing, respectively performing ultrasonic treatment (power 250W, frequency 40 kHz), shaking extraction, heating and refluxing for 30 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the subsequent filtrate. 1 μl of each test solution was precisely aspirated, and the solution was injected into a liquid chromatograph to calculate the aristolochic acid I content, and the results are shown in FIG. 9 and Table 32.
Table 32 comparison of different extraction methods
The results showed that: the extraction efficiency of ultrasonic, shaking and heating reflux is equivalent, and the extraction method is determined to be ultrasonic treatment in consideration of the convenience of operation.
5.3 investigation of different extraction times
Grinding proper amount of herba Aristolochiae Mollissimae formula granule, taking about 0.3g, 4 groups in parallel, 2 parts in each group in parallel, precisely weighing, placing into a conical flask with a plug, precisely adding 15ml of 70% methanol, sealing, weighing, respectively performing ultrasonic treatment (power 250W, frequency 40 kHz) for 15 min, 30 min, 45 min and 60 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the subsequent filtrate. 1 μl of each test solution was precisely aspirated, and the solution was injected into a liquid chromatograph to calculate the aristolochic acid I content, and the results are shown in FIG. 10 and Table 33.
TABLE 33 comparison of different extraction times
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The results showed that: the different extraction times have no significant effect on the content of aristolochic acid I, and the extraction time is thus determined to be 30 minutes from the viewpoint of complete extraction and time saving.
5.4 investigation of different extraction volumes
Grinding proper amount of herba Aristolochiae Mollissimae formula granule, precisely weighing about 0.3g, 4 groups in parallel, 2 parts in each group, placing into conical flask with plug, precisely adding 70% methanol 10ml, 15ml, 20ml and 25ml, sealing, weighing, respectively performing ultrasonic treatment (power 250W and frequency 40 kHz) for 30 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the filtrate. 1 μl of each test solution was precisely aspirated, and the solution was injected into a liquid chromatograph to calculate the aristolochic acid I content, and the results are shown in FIG. 11 and Table 34.
Table 34 comparison of different extraction volumes
The results showed that: the extraction efficiency of different extraction volumes is equivalent, and the extraction volume is determined to be 15ml from the comprehensive consideration of full extraction and solvent saving.
The preparation method of the test sample for determining the content of the sought-bone wind particles finally comprises the following steps of:
grinding proper amount of herba Aristolochiae Mollissimae formula granule, precisely weighing about 0.3g, placing into conical flask with plug, precisely adding 70% methanol 15ml, sealing, weighing, ultrasonic treating (power 250W, frequency 40 kHz) for 30 min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the filtrate.
6 methodological verification
6.1 linearity
And respectively precisely sucking 0.1 mu l, 0.2 mu l, 0.5 mu l, 1.0 mu l, 1.5 mu l and 2.0 mu l of aristolochic acid I reference substance mixed solution (with the concentration of 12.26 mu g/mL), injecting into a liquid chromatograph, measuring according to the chromatographic conditions, taking a peak area integral value as an ordinate and a sample injection amount (mu g) as an abscissa, drawing a standard curve, and obtaining aristolochic acid I regression equations, wherein the results are shown in tables 35-36 and FIG. 16.
Table 35 regression equation
Table 36 sample injection amount and peak area relation of aristolochic acid I control
The results show that: the sample injection amount of aristolochic acid I is within the range of 0.001226-0.024520 mug, and the sample injection amount and the peak area value have good linear relation.
6.2 precision test
6.2.1 Instrument precision test
Precisely sucking the sample solution, injecting into a liquid chromatograph, measuring under the chromatographic conditions, continuously injecting sample for 6 times, recording peak area, and calculating relative standard deviation, wherein the result is shown in the table below.
Precision test of meter 37 instrument
The results show that: the precision test of the instrument is good (RSD is less than or equal to 2.0%).
6.2.2 repeatability test
The preparation method of the test solution comprises the steps of taking a proper amount of the aristolochic acid prescription granule, grinding, taking about 0.3g, precisely weighing, and preparing 6 parts in parallel, preparing the test solution according to the preparation method of the test solution, respectively, injecting 1 μl of the test solution, measuring the peak area of aristolochic acid I, and calculating the content and RSD (reactive power distribution), wherein the result is shown in Table 38.
Results of the repeatability test of the sample of Table 38
The results show that: the repeatability test is good (RSD%. Ltoreq.2.0%).
6.2.3 intermediate precision test
The amount of the Aristolochia-free formulation particles was appropriately determined, the particles were ground, about 0.3g was obtained, 3 parts were treated by two laboratory workers A, B according to the above requirements, 1. Mu.l of the mixture was sampled on Waters-UPLC and Agilent-UPLC, and the peak area value of aristolochic acid I was measured and the content and RSD were calculated, and the results are shown in Table 39.
Intermediate precision test of Table 39
The results show that: the intermediate precision is good.
6.3 accuracy test
Taking three groups of samples with known content (aristolochic acid I content 0.029%) and grinding, taking about 0.15g, parallel 3 parts, precisely weighing, and adding reference substances of 50%, 100% and 150% of each component contained in 0.15g sample respectively. Sample solutions were prepared and recovered by the above-mentioned sample solution preparation method, 1. Mu.l each was sampled, and the recovery rate, average recovery rate, RSD, was calculated by the following formula, and the results are shown in Table 40.
TABLE 40 aristolochic acid I accuracy results
The results show that: the recovery rate of aristolochic acid I is between 96.79% and 103.66%, and the accuracy test result is good.
6.4 specificity test
The auxiliary materials added in the herba Aristolochiae Mollissimae formula granule are maltodextrin, silicon dioxide and magnesium stearate. The experiment examines the influence of a negative sample of the lack of the herba Aristolochiae Mollissimae on the determination of the content of the herba Aristolochiae Mollissimae formula particles. Taking a negative sample of the herba Aristolochiae Mollissimae, and preparing a negative sample solution according to a sample preparation method.
And (3) taking the test solution of the Aristolochia prescription granule, the negative control solution and the Aristolochia acid I control solution, and injecting the solution into a liquid chromatograph.
The experimental results showed (fig. 28): the negative chromatogram has no chromatographic peak at the retention time corresponding to the reference substance, which indicates that the auxiliary materials and the solvent have no interference to the determination of the aristolochic acid I, and the determination of the content of the aristolochic acid I in the aristolochic acid prescription granule by the method has specificity.
6.5 integrity test
On the determined chromatographic conditions, the elution gradient when the acetonitrile proportion is highest is maintained, the elution time is doubled, and whether residual impurity peaks affect the subsequent samples or not under the determined chromatographic conditions is examined, and the result is shown in fig. 29.
The experimental result shows that the chromatographic condition basically meets the principle of maximum information amount and has no influence on the analysis of the subsequent samples by prolonging the elution time by one time and having no impurity peak.
6.6 durability test
6.6.1 stability test
Preparing test solution according to the preparation method of the test solution, sampling 1 μl of the test solution at 0, 2, 4, 8, 12 and 24 hours respectively, measuring peak area value, and calculating RSD.
Table 41 stability test measurement results
The results show that: the sample test solution has good stability within 24 hours.
6.6.2 different flow Rate investigation
The aristolochia mollissima prescription granule is taken, a test solution is prepared according to the preparation method of the test solution, and the aristolochic acid I content at the flow rates of 0.25ml/min, 0.30ml/min and 0.35ml/min is examined, and the results are shown in figure 14 and table 42.
Table 42 investigation of different flow rates
The results show that: the separation degree of the aristolochic acid I chromatographic peak is good, the content is similar, and the durability is good under the three flow rates.
Investigation of 6.6.3 different column temperatures
The Aristolochia-containing granule is prepared by preparing the above test solution, and examining the aristolochic acid I content at 30deg.C, 35deg.C and 40deg.C, and the results are shown in figure 13 and Table 43.
Examination of Table 43 different column temperatures
The results show that: the separation degree of the aristolochic acid I chromatographic peak is good, the content is similar and the durability is good under the three column temperatures.
6.6.4 chromatographic column investigation
Preparing test solution by the preparation method of the test solution, and respectively examining AcclaimTM RSLC 120C18 (Thermo, 2.1mm×100mm,2.2 μm); porosill 120EC-C18 (Agilent, 2.1 mm. Times.100 mm,1.9 μm); ACQUITY UPLC BEH C18 (Waters, 2.1X100 mm,1.7 μm) the content of aristolochic acid I in the granules of the Momordica grosvenori formulation under 3 different chromatographic column conditions, the results are shown in FIG. 12, table 44.
Table 44 comparison of different chromatographic columns
The results show that: the chromatographic columns of three different types have good separation effect on aristolochic acid I, and have small influence on content results, and the method has universal adaptability.
Example 5 establishment of a method for identifying a thin layer of Mount Hospita formulation particle and methodological study
1 instrument and reagent
Instrument: a thin-layer automated imager (CAMAG TLC VISUALIZER), an ME204E ten-thousandth balance (Metrele-Toril), a KQ-250E ultrasonic cleaner (Kunshan ultrasonic electronics Co., ltd.).
Reagent: methanol (national medicine group chemical reagent limited), ethanol (national medicine group chemical reagent limited), toluene (national medicine group chemical reagent limited), acetone (national medicine group chemical reagent limited), formic acid (national medicine group chemical reagent limited) are all analytically pure; silica gel G thin layer plate (Qingdao ocean chemical Co., ltd., qingdao Kang Yexin medical silica gel desiccant Co., shanghai A Ding Shenghua technology Co., ltd.).
The herba Aristolochiae Mollissimae reference medicine (lot number: 410016-202004) is purchased from Shanghai Hongshai Yongsheng biotechnology Co.
The Mount Hospita formula granule is provided by Jiang Yintian river pharmaceutical Co.
2 thin layer authentication conditions
Adopting silica gel G plate as thin layer plate, using toluene-acetone-formic acid (9:1:0.5) as developing agent, developing, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).
3 preparation of solution
3.1 preparation of sample solutions
The method comprises the following steps: taking 0.5g of the product, grinding, adding 50ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a sample solution.
The second method is as follows: taking 0.5g of the product, grinding, adding 50ml of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a sample solution.
And a third method: taking 0.5g of the product, grinding, adding 50ml of ethyl acetate, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1ml of methanol into the residue to dissolve the residue to obtain a sample solution.
3.2 preparation of control drug solution
The method comprises the following steps: taking 1g of herba Aristolochiae Mollissimae reference medicine (batch number: 410016-202004), adding 100ml of water, boiling for 30 min, cooling, filtering, evaporating filtrate to dryness, adding 50ml of methanol into residue, performing ultrasonic treatment for 30 min, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residue to dissolve to obtain reference medicine solution.
The second method is as follows: 1g of a herba Aristolochiae Mollissimae reference medicinal material (batch No. 410016-202004) is added with 50ml of methanol, ultrasonic treatment is carried out for 30 minutes, filtering is carried out, filtrate is evaporated to dryness, and 1ml of methanol is added into residues to dissolve the residues to obtain a reference medicinal material solution.
3.3 preparation of negative control solution
Taking 1g of negative sample of the sought after bone deficiency, and preparing a negative control solution by the same method I in the preparation method of the test sample solution.
Determination of 4 thin layer authentication methods
4.1 investigation of the preparation methods of the sample solution and the control solution
Preparing herba Aristolochiae Mollissimae formula granule into test solution according to the above three test solution preparation methods, preparing corresponding reference medicinal solution from herba Aristolochiae reference medicinal materials according to the above two reference medicinal solution preparation methods, respectively spotting on silica gel G thin layer plate, developing according to the above thin layer chromatography developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The results are shown in FIG. 31 below.
The graph shows that the sample solution prepared by the first method and the second method and the reference medicinal material solution prepared by the second method have rich spots and can be in one-to-one correspondence. In consideration of unification of the preparation methods of the sample solution and the control medicinal material solution, the first method for selecting the sample is used as the sample treatment method for the thin layer identification, and the second method for controlling the medicinal material is used as the control medicinal material treatment method for the thin layer identification.
4.2 investigation of different spotting amounts
Preparing test solution and control medicinal solution from herba Aristolochiae Mollissimae formula granule and herba Aristolochiae Mollissimae control medicinal material respectively according to certain preparation method, respectively spotting on the same silica gel G thin layer plate according to text thin layer chromatography condition with different spotting amounts, spreading with toluene-acetone-formic acid (9:1:0.5) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The results are shown in FIG. 17.
As can be seen from the graph, when the sample application amount of the sample solution and the control medicinal material solution is 5 mu l, the fluorescent spots in the corresponding positions of the sample chromatogram and the control medicinal material chromatogram are relatively clear and correspond to each other, so that the sample application amount of the sample solution and the control medicinal material solution is 5 mu l.
Determination of a 5-thin layer authentication method
According to the research results, the thin layer identification method for determining the Aristolochia Mollissimae prescription granule comprises the following steps:
taking 0.5g of the product, grinding, adding 50ml of methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a sample solution. And adding 30ml of methanol into 1g of the herba Aristolochiae Mollissimae reference medicine to prepare a reference medicine solution. Sucking 5 μl of each of the above two solutions, respectively spotting on the same silica gel G thin layer plate, spreading with toluene-acetone-formic acid (9:1:0.5) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test sample, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the control medicinal material.
6 methodological verification
6.1 sample specificity experiments
Sample solution, control medicinal material solution and negative control solution of herba Aristolochiae Mollissimae granule are respectively spotted on the same silica gel G thin layer plate, and are spread with toluene-acetone-formic acid (9:1:0.5) as developing agent, taken out, air dried, and detected under ultraviolet lamp (365 nm). The results are shown in FIG. 18.
The figure shows that the chromatogram of the test sample of the herba Aristolochiae Mollissimae prescription granule and the chromatogram of the control medicinal material show fluorescent spots with the same color at the corresponding positions, and the negative control has no interference, which indicates that the thin layer identification method has good specificity.
6.2 durability test
6.2.1 investigation of different temperatures
Taking sample solution and control medicinal material solution, respectively at normal temperature and low temperature, and high temperature, spreading with toluene-acetone-formic acid (9:1:0.5) as developing agent on the same silica gel G thin layer plate, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The results are shown in FIG. 19.
The graph shows that the test sample chromatogram and the control medicinal material chromatogram of the herba Aristolochiae Mollissimae formula granule show spots with the same color at the corresponding positions under different temperature conditions, and the separation effect is good. Experimental results show that the temperature has no influence on the thin-layer identification of the Aristolochia Mollissimae prescription granule, and the thin-layer identification method has good durability on different temperatures.
6.2.2 investigation of different humidity
Taking sample solution and control medicinal material solution, respectively under different humidity conditions, spreading with toluene-acetone-formic acid (9:1:0.5) as developing agent on the same silica gel G thin layer plate, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The results are shown in FIG. 20.
The figure shows that the chromatogram of the test sample and the chromatogram of the reference medicinal material of the test sample of the herba Aristolochiae Mollissimae prescription granule show spots with the same color at the corresponding positions under different humidity conditions, and the spots are basically separated. Experimental results show that the humidity has no influence on the thin-layer identification of the Aristolochia Mollissimae formula particles, and the thin-layer identification method has good durability on different humidity.
Investigation of 6.2.3 different brands of laminates
Sample solution and control medicinal material solution are taken, respectively spotted on silica gel G thin layer plates of different brands, and are spread with toluene-acetone-formic acid (9:1:0.5) as developing agent, taken out, air-dried, and inspected under ultraviolet lamp (365 nm). The results are shown in FIG. 21.
As can be seen, in the different brands of silica gel G plates, spots with the same color appear on the corresponding positions of the chromatogram of the test sample and the chromatogram of the reference medicinal material in the herba Aristolochiae Mollissimae formula granule, and the spot separation degree is good. Experimental results show that the silica gel G plates of different brands have no influence on the thin layer identification result of the sought-bone wind formula particles, and the thin layer identification method has good durability on different thin layer plates.
Claims (5)
1. A quality control method of a sought-after wind sample, comprising the steps of:
(1) Selecting a sought bone wind sample, and preparing a sample solution; preparing a reference substance solution of a reference medicinal material by taking the herba Aristolochiae Mollissimae as the reference medicinal material; respectively preparing reference substance solutions of reference substances by taking rutin and aristolochic acid I as reference substances of reference substances; wherein, the extractant for preparing the reference substance solution and the sample solution is water or methanol water solution with the volume concentration of 10-70 percent;
(2) Respectively sucking the reference substance solution and the sample solution for ultra-high performance liquid chromatography analysis to construct a characteristic spectrum of the sample to be detected, wherein the characteristic spectrum comprises at least 9 characteristic peaks, one characteristic peak exists in the characteristic peaks corresponding to a rutin reference substance peak, and one characteristic peak exists corresponding to an aristolochic acid I reference substance peak; the conditions for the ultra performance liquid chromatography are as follows: the column was ACQUITY UPLC BEH C, 2.1X100 mm,1.7 μm; acetonitrile is used as a mobile phase A, 0.05% -0.2% phosphoric acid solution is used as a mobile phase B, and gradient elution is adopted: 0-3 min,5% of A, 3-20 min, 5-25% of A, 20-25 min, 25-43% of A, 25-30 min, 43-60% of A; the detection wavelength is 220-320 nm; the flow rate is 0.25 ml-0.35 ml per minute; the column temperature is 30-40 ℃; an ultraviolet detector is adopted;
wherein, the characteristic spectrum of the ultra-high performance liquid chromatography of the herba Aristolochiae Mollissimae sample has 9 characteristic peaks and corresponds to the retention time of 9 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; calculating the relative retention time of the peaks 4, 6, 7, 8 and the S peak by taking the peak corresponding to the rutin reference object as the S peak, wherein the relative retention time is within +/-10% of a specified value, the specified value of the peak 4 is 0.63, the specified value of the peak 6 is 1.14, the specified value of the peak 7 is 1.55 and the specified value of the peak 8 is 1.58;
(3) Carrying out qualitative analysis on the sample to be detected through the characteristic spectrum obtained in the step (2); and (3) taking rutin as a content measurement index, taking aristolochic acid I as a measurement index, and carrying out quantitative analysis on a sample to be detected.
2. The quality control method of a sample of the herba Aristolochiae Mollissimae according to claim 1, further comprising performing qualitative analysis on the sample to be tested by thin-layer chromatography, and judging whether spots of the sample to be tested and the reference medicinal material on the thin-layer plate correspond one by using the herba Aristolochiae Mollissimae reference medicinal material as a reference; if yes, carrying out the steps (1) - (3), otherwise, judging that the sample to be tested is an unqualified sample.
3. The quality control method of a sample of the collarbone wind according to claim 1, wherein the amount of the extractant used per gram of the collarbone wind is 5ml to 50ml, and the extraction time is 15 minutes to 60 minutes.
4. The quality control method of a herba Aristolochiae Mollissimae sample according to claim 1, wherein in the step (1), the concentration of the rutin reference substance solution is 20-80 μg/ml; the concentration of the reference solution of the aristolochic acid I reference substance is 3-15 mug/ml.
5. The quality control method of a herba Aristolochiae Mollissimae sample according to claim 1, wherein the herba Aristolochiae Mollissimae sample is herba Aristolochiae Mollissimae medicinal material, herba Aristolochiae Mollissimae decoction pieces, herba Aristolochiae Mollissimae preparation intermediate or herba Aristolochiae Mollissimae preparation final product.
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| 不同产地马兜铃蜜炙前后HPLC指纹图谱分析;章莹 等;中国药学杂志;第52卷(第16期);1397-1402 * |
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