AU2021106279A4 - Method for establishing hplc-elsd fingerprints of shenlingbaizhu pills and standard fingerprints thereof - Google Patents
Method for establishing hplc-elsd fingerprints of shenlingbaizhu pills and standard fingerprints thereof Download PDFInfo
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- 239000009765 shen ling bai zhu Substances 0.000 title claims abstract description 77
- 239000006187 pill Substances 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000000105 evaporative light scattering detection Methods 0.000 claims abstract description 40
- 239000012085 test solution Substances 0.000 claims abstract description 19
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 14
- 230000014759 maintenance of location Effects 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 9
- 229960000583 acetic acid Drugs 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 239000012362 glacial acetic acid Substances 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- RWYFURDDADFSHT-RBBHPAOJSA-N diane Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1.C1=C(Cl)C2=CC(=O)[C@@H]3CC3[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)C)[C@@]1(C)CC2 RWYFURDDADFSHT-RBBHPAOJSA-N 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 4
- 230000003247 decreasing effect Effects 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000002483 medication Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 15
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 description 7
- 229960004949 glycyrrhizic acid Drugs 0.000 description 7
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 description 7
- 235000019410 glycyrrhizin Nutrition 0.000 description 7
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 239000004378 Glycyrrhizin Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 229930182494 ginsenoside Natural products 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000132012 Atractylodes Species 0.000 description 3
- 241000202807 Glycyrrhiza Species 0.000 description 3
- 235000001453 Glycyrrhiza echinata Nutrition 0.000 description 3
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 description 3
- 235000017382 Glycyrrhiza lepidota Nutrition 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 229940010454 licorice Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 230000000087 stabilizing effect Effects 0.000 description 3
- 244000141331 Amomum villosum Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 244000131316 Panax pseudoginseng Species 0.000 description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000008434 ginseng Nutrition 0.000 description 2
- 229940089161 ginsenoside Drugs 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 2
- 244000077995 Coix lacryma jobi Species 0.000 description 1
- 240000004270 Colocasia esculenta var. antiquorum Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 235000014647 Lens culinaris subsp culinaris Nutrition 0.000 description 1
- 244000043158 Lens esculenta Species 0.000 description 1
- VTAJIXDZFCRWBR-UHFFFAOYSA-N Licoricesaponin B2 Natural products C1C(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2)C(O)=O)C)(C)CC2)(C)C2C(C)(C)CC1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O VTAJIXDZFCRWBR-UHFFFAOYSA-N 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 244000248825 Peltandra virginica Species 0.000 description 1
- 235000001188 Peltandra virginica Nutrition 0.000 description 1
- 235000006751 Platycodon Nutrition 0.000 description 1
- 244000274050 Platycodon grandiflorum Species 0.000 description 1
- 241001619461 Poria <basidiomycete fungus> Species 0.000 description 1
- 235000008599 Poria cocos Nutrition 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- PWAOOJDMFUQOKB-WCZZMFLVSA-N ginsenoside Re Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@](C)(CCC=C(C)C)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O PWAOOJDMFUQOKB-WCZZMFLVSA-N 0.000 description 1
- AOGZLQUEBLOQCI-UHFFFAOYSA-N ginsenoside-Re Natural products CC1OC(OCC2OC(OC3CC4(C)C(CC(O)C5C(CCC45C)C(C)(CCC=C(C)C)OC6OC(CO)C(O)C(O)C6O)C7(C)CCC(O)C(C)(C)C37)C(O)C(O)C2O)C(O)C(O)C1O AOGZLQUEBLOQCI-UHFFFAOYSA-N 0.000 description 1
- 239000001685 glycyrrhizic acid Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229930189914 platycodon Natural products 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
Classifications
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- 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
- G01N30/12—Preparation by evaporation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/53—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke
- G01N21/532—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke with measurement of scattering and transmission
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
OF THE DISCLOSURE
The present disclosure discloses a method for establishing an PLC-ELSD
fingerprint of Shenlingbaizhu pills. The method includes preparation of test solution,
determination of HPLC chromatographic conditions and preparation of HPLC-ELSD
standard fingerprints. The present disclosure also discloses an HPLC-ELSD fingerprint
of Shenlingbaizhu pills prepared by the method, the fingerprint has 24 common peaks,
and the relative retention times tR are respectively: 0.252, 0.690, 0.706, 0.947, 0.972,
1.000, 1.170, 1.189, 1.246, 1.386, 1.483, 1.531, 1.565, 1.592, 1.617, 1.652, 1.709, 1.800,
2.074, 2.171, 2.243, 2.485, 2.622. The quality detection method of the present
disclosure has simple operation, high stability, good reproducibility, and the obtained
fingerprint has many characteristic peaks, which can effectively characterize the quality
of Shenlingbaizhu pills. By comparing the common peaks of standard fingerprints, the
quality of Shenlingbaizhu pills can be accurately evaluated, which is beneficial to
comprehensively monitor the product quality and ensure the safety and effectiveness of
clinical medications.
ABSTRACT DRAWING
71
'is
FIG.1
1
Description
[01] The present disclosure relates to a method for establishing an HPLC-ELSD fingerprint of a compound Chinese medicine of Shenlingbaizhu pills and an HPLC-ELSD standard fingerprint thereof, belonging to the field of traditional Chinese medicine preparation analysis.
[02] Shenlingbaizhu pills are included in the first part of "Chinese Pharmacopoeia (2015 Edition)", which are prepared from 10 Chinese medicines including ginseng, tuckahoe, bran-fried atractylodes, yam, fried white lentils, lotus seeds, bran-fried coix seed, amomum villosum, platycodon, and licorice, and have the functions of invigorating the spleen and replenishing qi. They are used for fatigued body and lack of strength, lack of food and loose stools, and have a definite curative effect, which are included in the "National Essential Medicines List (2018 Edition)".
[03] The current quality control methods of Shenlingbaizhu pills mainly include identification and content determination. The identification uses microscopic identification of poria, bran-fried atractylodes, yam, and amomum villosum. Thin layer chromatography is used to identify ginseng and atractylodes. The content determination uses the high performance liquid chromatography to determine the content of glycyrrhizic acid in licorice. The determination of the composition is relatively simple, and its quality cannot be fully and systematically reflected.
[04] As a quality control technology, the fingerprint of traditional Chinese medicine can control the quality of medicines comprehensively, and has the characteristics of systemicity and integrity. At present, the quality of Shenlingbaizhu pills is controlled by fingerprints, and there are no patent publications and literature reports at home and abroad. There have been related reports on thefingerprints of Shenlingbaizhu san with different dosage forms of Shenlingbaizhu pills. Liu Caijun and others have established the HPLC characteristic fingerprints of Shenlingbaizhu powder. The test solution is extracted with 70 % methanol and then n-butanol, which requires troublesome processing steps, and can easily lead to the loss of some characteristic components. The established fingerprints have almost no chromatographic peaks after 30 minutes, and there are relatively few common peaks in the whole (Liu Caijun, Zhu Qin. Study on HPLC Characteristic Fingerprint of Shenlingbaizhu Powder and Simultaneous Determination of Its Five Indicative Components [J]. China Pharmaceutical Industry, 2018, 27(15): 12-16.). Li Jinglin et al. have established the fingerprint of Shenlingbaizhu powder based on the UPLC method. The fingerprints obtained are poorly separated, and the characteristic components of licorice at the selected detection wavelength could not be effectively detected (Li Jinglin, Wang Mei, Shi Yajun, etc. Study on fingerprint and multi-component quality of Shenlingbaizhu powder based on UPLC method [J]. Chinese Pharmacist, 2018, 22(2): 214-218.). Wang Xuemei et al.
have only determined the ginsenosides in Shenlingbaizhu powder based on HPLC fingerprint detection, and do not conduct a systematic study on the fingerprint of Shenlingbaizhu powder (Wang Xuemei, Li Yanjiao, Song Yanqing. Determination of Ginsenosides in Shenlingbaizhu Powder Based on HPLC fingerprint detection [J]. World Traditional Chinese Medicine, 2018, 13 (11): 2876-2883.).
[05] In view of the above shortcomings, it is necessary to establish a HPLC fingerprint measurement method for Shenlingbaizhu pills to comprehensively evaluate and control the quality of Shenlingbaizhu pills, thereby ensuring the stability of product quality and the effectiveness and safety of clinical medications.
[06] The purpose of the present disclosure is to address the deficiencies of the existing quality control methods for Shenlingbaizhu pills and to provide a method for establishing an HPLC-ELSD fingerprint of Shenlingbaizhu pills and the HPLC-ELSD standard fingerprint thereof. It is characterized by preparing Shenlingbaizhu pills into a test solution, and obtaining the HPLC-ELSD standard fingerprint of Shenlingbaizhu pills after HPLC separation and testing, so as to provide reliable basis for authenticity identification and internal quality of Shenlingbaizhu pills.
[07] The method for establishing the HPLC-ELSD fingerprint of Shenlingbaizhu pills of the present disclosure includes the following steps:
[08] 1) Preparation of a test solution: taking Shenlingbaizhu pills, placing in an Erlenmeyer flask with stoppers, adding a 90 % methanol solution thereto, soaking the pills overnight, ultrasonically treating the resulting mixture for 20 min-40 min, cooling naturally, filtering, and taking the filtrate to obtain the test solution;
[09] 2) Determination of HPLC chromatographic conditions: precisely drawing the test solution, separating and detecting by HPLC, using acetonitrile and a 0.1 %-0.3
% glacial acetic acid solution as the mobile phase, and using a gradient elution;
[10] 3) Preparation of fingerprint: according to the chromatographic conditions of step 2), analyzing the Shenlingbaizhu pills sample to obtain the HPLC chromatogram, and comparing the chromatogram to obtain the HPLC-ELSD standard fingerprint of Shenlingbaizhu pills consisting of the common characteristic peaks of the samples.
[11] Preferably, the test solution is prepared according to the following steps: accurately weighing 5 g of Shenlingbaizhu pills, placing in an Erlenmeyer flask with stoppers, accurately adding 20 ml of methanol with a volume concentration of 90 %, soaking the pills overnight, ultrasonically treating the resulting mixture for 30 min, cooling naturally, filtering, and taking the filtrate to obtain the test solution.
[12] The HPLC chromatographic conditions are as follows: using a Diane Acclaim Ci8 column, the particle size of the fillers of 5 m, a column length of 250 mm, a column inner diameter of 4.6 mm; using acetonitrile as a mobile phase A, using a 0.2 % glacial acetic acid solution as the mobile phase B, using a gradient elution: 0-55 min, increasing the volume percentage of mobile phase A from 5 % to 80 %, decreasing the volume percentage of mobile phase B from 95 % to 20 %; 55 min-60 min, increasing the volume percentage of mobile phase A from 80 % to 100 %, decreasing the volume percentage of mobile phase B from 20 % to 0 %; flow rate: 0.8 ml/min; column temperature: 30°C; using an evaporative light scattering detector: the temperature of a drift tube of 100°C, and gas flow rate of 3 L/min;
[13] According to the above chromatographic conditions, 11 batches of Shenlingbaizhu pills samples are analyzed, and the HPLC-ELSD chromatograms of 11 batches of samples are obtained. The chromatograms of 11 batches of samples are analyzed and compared to obtain the HPLC-ELSD standard fingerprint of Shenlingbaizhu pills consisting of the common characteristic peaks of the samples.
[14] The present disclosure also provides the HPLC-ELSD standard fingerprint of Shenlingbaizhu pills obtained by the above method. The specific steps are as follows: 11 batches of Shenlingbaizhu pills samples are prepared according to the above method into the test solution, separated and detected by HPLC, and analyzed using the software of "Similarity Evaluation System for TCM Chromatographic Fingerprints 2012 edition" recommended by the National Pharmacopoeia Commission to obtain the HPLC-ELSD standard fingerprint of Shenlingbaizhu pills.
[15] The HPLC-ELSD standard fingerprint of Shenlingbaizhu pills has 24 common peaks, and the relative retention times tR are respectively: 0.252, 0.690, 0.706, 0.947, 0.972, 1.000, 1.170, 1.189, 1.246, 1.386, 1.483, 1.531, 1.565, 1.592, 1.617, 1.652, 1.709, 1.800, 2.074, 2.171, 2.243, 2.485, 2.622.
[16] Compared with the prior art, the method for establishing the HPLC-ELSD fingerprint of Shenlingbaizhu pills provided by the present disclosure has high precision and good reproducibility. By comparing the presence or absence of common peaks in the obtained fingerprints, the quality of Shenlingbaizhu pills can be fully monitored, effectively ensuring the quality of the finished product. The fingerprint pays attention to the overall appearance characteristics, avoiding the one-sidedness of determining the quality of Shenlingbaizhu pills due to the determination of individual chemical components. In addition, most of the characteristic chromatographic peaks under the chromatographic conditions of the present disclosure have achieved good baseline separation, the method has good stability and many characteristic peaks, can comprehensively and accurately evaluate the quality of Shenlingbaizhu pills, and is suitable for authenticity identification and product quality control of Shenlingbaizhu pills.
[17] FIG. 1 is an HPLC-ELSD standard fingerprint of Shenlingbaizhu pills (1-24 are 24 common peaks);
[18] FIG. 2 is an overlay of HPLC-ELSD fingerprints of 11 batches of Shenlingbaizhu pills. DETAILED DESCRIPTION OF THE EMBODIMENTS
[19] The present disclosure will be further described below in conjunction with specific embodiments.
[20] Example 1 Establishment of HPLC-ELSD standard fingerprint of Shenlingbaizhu pills
[21] 1 Instruments and reagents
[22] 1.1 Instrument
[23] Agilent 1260 High Performance Liquid Chromatograph (USA): DAD detector, quaternary low pressure gradient pump, Agilent Open Lab chromatography workstation.
[24] 1.2 Reagents
[25] Shenlingbaizhu pills were provided by Shandong Kongshengtang Pharmaceutical Co., Ltd., as shown in Table 1. Acetonitrile was chromatographically pure, water was redistilled water, and the rest of the reagents were analytical pure.
[26] Table 1 Sample batch number of Shenlingbaizhu pills
[271 sample batch number sample batch number Sl 1702084 S7 1804001 S2 1704087 S8 1805080 S3 1707030 S9 1810046 S4 1808086 SlO 1811056 S5 1709086 Sll 1901111 S6 1710029
[28] 2 Methods and results
[29] 2.1 Chromatographic conditions: using a Diane Acclaim Cis column (4.6 x 250 mm, 5 m); using acetonitrile as a mobile phase A, a glacial acetic acid aqueous solution with a volume percentage of 0.2 % as a mobile phase B, and performing gradient elution as follows:
[30] time (min) mobile phase A (volume mobile phase B (volume percentage) percentage) 0-55 5%--80% 95%--20% 55-60 80%--100o% 20 %->+0%
[31] Flow rate of 0.8 ml/min; column temperature of 30°C; injection volume of 10 pl; evaporative light scattering detector: stabilizing the drift tube at 100°C, gas flow rate of 3 L/min.
[32] 2.2 Preparation of test solution: 5 g of Shenlingbaizhu pills were accurately weighed, placed in an Erlenmeyer flask with stoppers, 20 ml of 90 % methanol was added thereto, the pills were soaked overnight, the resulting mixture was ultrasonically treated for 30 min, cooled naturally, filtered, and the subsequent filtrate was taken to get the test solution.
[33] 2.3 Establishment offingerprints
[34] The HPLC-ELSD fingerprints of 11 batches of Shenlingbaizhu pills were determined, then analyzed and compared to obtain the HPLC-ELSD standard fingerprints of Shenlingbaizhu pills consisting of common characteristic peaks (as shown in FIG. 1, 2). Among them, taking Glycyrrhizin peak No. 6 as the reference peak, the relative retention time tR of the 24 common peaks in the standard fingerprint spectrum was calculated as: 0.252, 0.690, 0.706, 0.947, 0.972, 1.000, 1.170, 1.189, 1.246, 1.386, 1.483, 1.531, 1.565, 1.592, 1.617, 1.652, 1.709, 1.800, 2.074, 2.171, 2.243, 2.485, 2.622.
[35] Among them, peak No. 6 is glycyrrhizin, peak No. 7 is ginsenoside Rgi and ginsenoside Re, peak No. 11 is ginsenoside RbI, and peak No. 17 is glycyrrhizin.
[36] The HPLC-ELSD fingerprints of 11 batches of Shenlingbaizhu pills were imported into the software of " Similarity Evaluation System for TCM Chromatographic Fingerprints 2012 edition" recommended by the Pharmacopoeia Committee for analysis, and the chromatographic peaks were matched. Taking Glycyrrhizin peak No. 6 as the reference peak, 24 common peaks were identified as characteristic peaks constituting the fingerprint of Shenlingbaizhu pills. The relative retention times of the common peaks of the samples are shown in Table 2. The relative peak areas of the common peaks of the 11 batches of Shenlingbaizhu pills are shown in Table 3. The similarity calculation results of the 11 batches of Shenlingbaizhu pills and the standard fingerprint are: 0.992, 0.993, 0.992, 0.992, 0.997, 0.988, 0.999, 0.999, 0.992, 0.993, 0.987.
[37] Table 2 The relative retention time (tR) of the common peaks of 11 batches of Shenlingbaizhu pills
[381 No. Si S2 S3 S4 S5 S6 S7 S8 S9 S1O Sll 1 0.251 0.251 0.251 0.251 0.252 0.252 0.252 0.252 0.252 0.252 0.252 2 0.693 0.691 0.692 0.691 0.692 0.689 0.689 0.689 0.689 0.689 0.689 3 0.707 0.704 0.706 0.704 0.705 0.706 0.706 0.706 0.705 0.706 0.706 4 0.947 0.946 0.947 0.946 0.947 0.947 0.948 0.948 0.947 0.947 0.947 5 0.972 0.970 0.972 0.970 0.972 0.972 0.972 0.972 0.972 0.972 0.972 6 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 7 1.168 1.168 1.170 1.168 1.170 1.170 1.171 1.171 1.171 1.171 1.171 8 1.189 1.187 1.189 1.187 1.189 1.189 1.189 1.189 1.189 1.189 1.190 9 1.246 1.244 1.246 1.244 1.246 1.246 1.246 1.246 1.246 1.246 1.246 10 1.399 1.398 1.400 1.398 1.400 1.400 1.400 1.400 1.249 1.401 1.401 11 1.481 1.480 1.483 1.481 1.484 1.484 1.484 1.485 1.485 1.486 1.485 12 1.620 1.518 1.521 1.519 1.522 1.522 1.522 1.523 1.524 1.524 1.524 13 1.564 1.563 1.565 1.563 1.566 1.566 1.566 1.566 1.567 1.567 1.567 14 1.589 1.589 1.591 1.589 1.593 1.593 1.593 1.594 1.594 1.594 1.594 15 1.616 1.615 1.617 1.615 1.618 1.617 1.617 1.618 1.618 1.618 1.618 16 1.649 1.649 1.651 1.649 1.653 1.653 1.653 1.654 1.655 1.655 1.654 17 1.707 1.706 1.709 1.707 1.710 1.709 1.709 1.710 1.710 1.710 1.710 18 1.798 1.797 1.801 1.798 1.801 1.800 1.800 1.801 1.802 1.802 1.801 19 2.073 2.071 2.074 2.071 2.075 2.074 2.074 2.075 2.076 2.076 2.076 20 2.116 2.114 2.117 2.115 2.119 2.118 2.117 2.117 2.119 2.118 2.118 21 2.170 2.168 2.171 2.168 2.172 2.170 2.171 2.172 2.173 2.173 2.173 22 2.242 2.240 2.243 2.240 2.245 2.243 2.243 2.244 2.245 2.245 2.245 23 2.484 2.482 2.485 2.481 2.486 2.483 2.484 2.485 2.487 2.487 2.487 24 2.621 2.619 2.623 2.618 2.624 2.621 2.622 2.623 2.625 2.625 2.625
[39] Table 3 Relative peak area (S) of the common peaks of 11 batches of Shenlingbaizhu pills
[40]
No. Si S2 S3 S4 S5 S6 S7 S8 S9 S1O Sll 1 1.345 1.178 1.219 0.711 0.890 0.649 0.959 0.993 1.227 1.300 1.634 2 0.186 0.163 0.214 0.083 0.102 0.070 0.151 0.161 0.115 0.184 0.152 3 0.027 0.025 0.028 0.020 0.024 0.018 0.032 0.030 0.031 0.031 0.037 4 0.059 0.047 0.075 0.026 0.035 0.028 0.053 0.052 0.073 0.075 0.082 5 0.891 0.571 0.914 0.225 0.280 0.238 0.421 0.426 0.490 0.685 0.612 6 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 7 1.174 1.099 1.020 0.469 0.600 0.435 0.734 0.694 0.654 0.954 0.880 8 0.272 0.177 0.298 0.073 0.087 0.072 0.152 0.148 0.181 0.252 0.222 9 0.208 0.207 0.221 0.250 0.243 0.253 0.269 0.260 0.293 0.312 0.297 10 0.101 0.115 0.136 0.082 0.094 0.084 0.075 0.072 0.102 0.084 0.094 11 3.466 3.082 3.389 1.716 2.132 1.540 2.247 2.304 2.369 3.390 3.009 12 0.191 0.187 0.180 0.096 0.122 0.096 0..136 0.129 0.140 0.178 0.171 13 0.130 0.118 0.125 0.071 0.085 0.046 0.074 0.075 0.072 0.105 0.084 14 0.335 0.324 0.327 0.138 0.131 0.109 0.244 0.230 0.165 0.335 0.321 15 0.050 0.053 0.057 0.065 0.061 0.058 0.065 0.058 0.076 0.082 0.070 16 0.558 0.404 0.493 0.260 0.329 0.228 0.350 0.339 0.344 0.420 0.384 17 3.058 2.365 3.316 1.807 1.816 1.685 2.255 2.091 2.771 2.524 2.200 18 0.110 0.066 0.118 0.033 0.036 0.033 0.068 0.063 0.089 0.093 0.074 19 0.048 0.049 0.059 0.051 0.054 0.067 0.085 0.093 0.111 0.138 0.139 20 0.105 0.055 0.075 0.046 0.062 0.053 0.074 0.082 0.126 0.189 0.186 21 0.030 0.039 0.035 0.055 0.054 0.048 0.096 0.104 0.100 0.153 0.155 22 0.466 0.187 0.440 0.036 0.044 0.048 0.082 0.118 0.126 0.168 0.154 23 0.091 0.061 0.071 0.030 0.028 0.056 0.112 0.119 0.154 0.172 0.173 24 0.041 0.052 0.049 0.079 0.072 0.113 0.154 0.161 0.182 0.212 0.234
[41] 2.4 Methodological investigation
[42] 2.4.1 Precision test
[43] The sample with batch number 1901111 was taken to prepare the sample solution according to the method under item 2.2, and the sample was continuously injected for 6 times. With peak No. 6 as the reference peak, the RSD values of the relative retention time and relative peak area of common peaks 1 to 24 were calculated to be less than 3 %. Meanwhile, the similarity of each chromatograms fingerprint calculated by the similarity evaluation software is more than 0.99, indicating that the precision of the instrument is good.
[44] 2.4.2 Stability test
[45] The sample with batch number 1901111 was taken, the solution was prepared according to the method under item 2.2, and the samples were injected at 0, 2, 4, 6, 8, 24 h, respectively. With peak No. 6 as the reference peak, the RSD values of the relative retention time and relative peak area of common peaks 1 to 24 were calculated to be less than 3 %. Meanwhile, the similarity of each chromatograms fingerprint calculated by the similarity evaluation software is more than 0.99, indicating that the solution of the test sample is stable within 24 h.
[46] 2.4.3 Reproducibility test
[47] The sample with batch number 1901111 was taken and accurately weighed for 6 parts, respectively, the solution was prepared according to the method under item 2.2, and the samples were injected, respectively. With peak No. 6 as the reference peak, the RSD values of the relative retention time and relative peak area of common peaks 1 to 24 were calculated to be less than 3 %. Meanwhile, the similarity of each chromatograms fingerprint calculated by the similarity evaluation software is more than 0.99, indicating that the method had good reproducibility.
[48] Example 2 Establishment of HPLC-ELSD standard fingerprint of Shenlingbaizhu pills
[49] 1 Instruments and reagents
[50] 1.1 Instrument
[51] Agilent 1260 High Performance Liquid Chromatograph (USA): DAD detector, quaternary low pressure gradient pump, Agilent Open Lab chromatography workstation.
[52] 1.2 Reagents
[53] Shenlingbaizhu pills were provided by Shandong Kongshengtang Pharmaceutical Co., Ltd., as shown in Table 4. Acetonitrile was chromatographically pure, water was redistilled water, and the rest of the reagents were analytical pure.
[54] Table 4 Sample batch number of Shenlingbaizhu pills
[55] sample batch number sample batch number Sl 1702084 S7 1804001 S2 1704087 S8 1805080 S3 1707030 S9 1810046 S4 1808086 SlO 1811056 S5 1709086 Sll 1901111 S6 1710029
[56] 2 Methods and results
[57] 2.1 Chromatographic conditions: using a Diane Acclaim Cis column (4.6 x 250 mm, 5 m); using acetonitrile as a mobile phase A, a glacial acetic acid aqueous solution with a volume percentage of 0.1 % as a mobile phase B, and performing gradient elution as follows:
[58] time (min) mobile phase A (volume mobile phase B (volume percentage) percentage) 0-55 5%--80% 95%--20% 55-60 80%--100o% 20 %->+0%
[59] Flow rate of 0.8 ml/min; column temperature of 30°C; injection volume of 10 pl; evaporative light scattering detector: stabilizing the drift tube at 100°C, gas flow rate of 3 L/min.
[60] 2.2 Preparation of test solution: 5 g of Shenlingbaizhu pills were accurately weighed, placed in an Erlenmeyer flask with stoppers, 20 ml of 90 % methanol was added thereto, the pills were soaked overnight, the resulting system was ultrasonically treated for 20 min, cooled naturally, filtered, and the subsequent filtrate was taken to get the test solution.
[61] 2.3 Establishment offingerprints
[62] The HPLC-ELSD fingerprints of 11 batches of Shenlingbaizhu pills were determined, then analyzed and compared to obtain the HPLC-ELSD standard fingerprints of Shenlingbaizhu pills consisting of common characteristic peaks. Among them, taking Glycyrrhizin peak No. 6 as the reference peak, the relative retention timetR of the 24 common peaks in the standard fingerprint spectrum was calculated as: 0.251, 0.692, 0.705, 0.944, 0.973, 1.000, 1.172, 1.188, 1.245, 1.387, 1.484, 1.530, 1.564, 1.594, 1.616, 1.653, 1.711, 1.802, 2.076, 2.116, 2.172, 2.245, 2.483, 2.624.
[63] Example 3 Establishment of HPLC-ELSD standard fingerprint of Shenlingbaizhu pills
[64] 1 Instruments and reagents
[65] 1.1 Instrument
[66] Agilent 1260 High Performance Liquid Chromatograph (USA): DAD detector, quaternary low pressure gradient pump, Agilent Open Lab chromatography workstation.
[67] 1.2 Reagents
[68] Shenlingbaizhu pills were provided by Shandong Kongshengtang Pharmaceutical Co., Ltd., as shown in Table 5. Acetonitrile was chromatographically pure, water was redistilled water, and the rest of the reagents were analytical pure.
[69] Table 5 Sample batch number of Shenlingbaizhu pills
[701 sample batch number sample batch number Sl 1702084 S7 1804001 S2 1704087 S8 1805080 S3 1707030 S9 1810046 S4 1808086 SlO 1811056 S5 1709086 Sll 1901111 S6 1710029
[71] 2 Methods and results
[72] 2.1 Chromatographic conditions: using a Diane Acclaim Cis column (4.6 x 250 mm, 5 m); using acetonitrile as a mobile phase A, a glacial acetic acid aqueous solution with a volume percentage of 0.3 % as a mobile phase B, and performing gradient elution as follows:
[73] time (min) mobile phase A (volume mobile phase B (volume percentage) percentage) 0-55 5%--80% 95%--20% 55-60 80%--100o% 20 %->+0%
[74] Flow rate of 0.8 ml/min; column temperature of 30°C; injection volume of 10 pl; evaporative light scattering detector: stabilizing the drift tube at 100°C, gas flow rate of 3 L/min.
[75] 2.2 Preparation of test solution: 5 g of Shenlingbaizhu pills were accurately weighed, placed in an Erlenmeyer flask with stoppers, 20 ml of 90 % methanol was added thereto, the pills were soaked overnight, the resulting system was ultrasonically treated for 40 min, cooled naturally, filtered, and the subsequent filtrate was taken to get the test solution.
[76] 2.3 Establishment offingerprints
[77] The HPLC-ELSD fingerprints of 11 batches of Shenlingbaizhu pills were determined, then analyzed and compared to obtain the HPLC-ELSD standard fingerprints of Shenlingbaizhu pills consisting of common characteristic peaks. Among them, taking Glycyrrhizin peak No. 6 as the reference peak, the relative retention time t of the 24 common peaks in the standard fingerprint spectrum was calculated as: 0.253, 0.688, 0.705, 0.949, 0.974, 1.000, 1.170, 1.191, 1.245, 1.388, 1.482, 1.529, 1.566, 1.593, 1.618, 1.654, 1.710, 1.801, 2.073, 2.118, 2.170, 2.244, 2.484, 2.621.
[78] The above are only the preferred embodiments of the present disclosure, and the description is relatively specific and detailed, but it should not be understood as a limitation to the patent scope of the present disclosure. It should be pointed out that for those of ordinary skill in the art, without departing from the principle and concept of the present disclosure, several modifications and improvements can be made, and these should also fall within the protection scope of the present disclosure.
Claims (5)
1. A method for establishing an HPLC-ELSD fingerprint of Shenlingbaizhu pills, wherein comprising the following steps: 1) Preparation of a test solution: taking different batches of Shenlingbaizhu pills, adding a methanol solution, soaking the pills overnight, ultrasonically treating for 20 min-40 min, cooling naturally, filtering, and taking the filtrate to obtain the test solution; 2) Determination of HPLC chromatographic conditions: using a Diane Acclaim Ci8 column, using acetonitrile as a mobile phase A, a 0.1 %-0.3 % glacial acetic acid solution as the mobile phase B, using a gradient elution: 0-55 min, increasing the volume percentage of mobile phase A from 5 % to 80 %, decreasing the volume percentage of mobile phase B from 95 % to 20 %; 55 min-60 min, increasing the volume percentage of mobile phase A from 80 % to 100 %, decreasing the volume percentage of mobile phase B from 20 % to 0 %; a flow rate of 0.8 ml/min; the column temperature of 30°C; using an evaporative light scattering detector: the temperature of a drift tube of 100°C, and the gas flow rate of 3 L/min; 3) Preparation of fingerprint: according to the chromatographic conditions of step 2), analyzing and comparing the sample solution of Shenlingbaizhu pills to obtain the HPLC-ELSD standard fingerprint of Shenlingbaizhu pills consisting of the common characteristic peaks of the samples.
2. The method according to claim 1, wherein the test solution of the step 1) is prepared as follows: accurately weighing 5 g of Shenlingbaizhu pills, and accurately adding 20 mL of methanol with the volume percentage of 90 %, soaking the pills overnight, ultrasonically treating for 30 min, cooling naturally, filtering, and taking the filtrate to obtain the test solution;
3. The method for establishing an HPLC-ELSD fingerprint of Shenlingbaizhu pills according to claim 1, wherein the mobile phase B of the step 2) is a 0.2 % glacial acetic acid solution.
4. The method for establishing an HPLC-ELSD fingerprint of Shenlingbaizhu pills according to any one of claims 1-3, wherein the number of the samples is 11 batches; wherein the HPLC-ELSD fingerprint of Shenlingbaizhu pills is evaluated by using the software of "Similarity Evaluation System for TCM Chromatographic Fingerprints 2012 edition".
5. A standard fingerprint established by the method for establishing an HPLC-ELSD fingerprint of Shenlingbaizhu pills according to any one of claims 1-4, wherein the standard fingerprint has 24 common peaks, and the relative retention times tR are respectively: 0.252, 0.690, 0.706, 0.947, 0.972, 1.000, 1.170, 1.189, 1.246, 1.386, 1.483, 1.531, 1.565, 1.592, 1.617, 1.652, 1.709, 1.800, 2.074, 2.171, 2.243, 2.485, 2.622.
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