CN117849254A - Method for evaluating quality of medicinal garlic by utilizing ultra-high performance liquid chromatography fingerprint - Google Patents
Method for evaluating quality of medicinal garlic by utilizing ultra-high performance liquid chromatography fingerprint Download PDFInfo
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to the field of medicines, and particularly discloses a method for evaluating the quality of medicinal garlic by utilizing an ultra-high performance liquid chromatography fingerprint. The construction method of the medicinal garlic fingerprint comprises the following steps: a step of preparing a sample solution and a reference solution by using ultrapure water and an ultra-high performance liquid chromatography step. The method is simple, chromatographic analysis conditions are easy to realize, the precision is high, the stability and reproducibility of analysis results are good, and compared with other test analysis methods in the prior art, the method shortens the online analysis time and saves the material cost and the time cost; meanwhile, the detection method of the invention uses alliin as a reference peak, determines and identifies 18 common peaks, has rich and comprehensive fingerprint composition, and is a reliable, simple and convenient medicinal garlic quality evaluation method.
Description
Technical Field
The invention relates to the field of medicines, in particular to a method for evaluating the quality of medicinal garlic by utilizing an ultra-high performance liquid chromatography fingerprint.
Background
Garlic (Allium sativum l.), also known as garlic, cucurbit, thyme, etc., is an underground bulb part of garlic of the family liliaceae, is pungent in taste, warm in nature, enters spleen, stomach and lung meridians, is native to the northeast and middle asia of the iran, is used to make various dishes worldwide, and is one of the earliest-documented edible and medicinal crops in our country. Because the bactericidal and bacteriostatic effects are the strongest of the plant medicinal materials, the plant medicinal materials are known as penicillin growing in the ground and natural broad-spectrum antibacterial drugs.
Garlic meeting pharmacopoeia standards is called medicinal garlic. Due to the difference of the growth regions of garlic, garlic used as a traditional Chinese medicinal material has the problems of unstable quality control, inaccuracy, difficulty in qualitative and quantitative analysis and the like. Therefore, in order to improve the medicinal quality, optimization of the quality control method of the medicinal material is urgently needed.
Therefore, the invention provides a new technical method for quality control and evaluation of medicinal garlic by researching the fingerprint of medicinal garlic by utilizing UPLC technology for the first time.
Disclosure of Invention
The invention provides a method for evaluating the quality of medicinal garlic by utilizing an ultra-high performance liquid chromatography fingerprint spectrum, a medicinal garlic medicinal material standard fingerprint spectrum obtained by the method and application of the medicinal garlic medicinal material standard fingerprint spectrum in optimized screening, quality identification and control of medicinal garlic medicinal materials. The construction method solves the problem that the integral quality of the medicinal garlic can not be effectively reflected and controlled in the component analysis of medicinal garlic medicinal materials, and makes up the defects of the existing quality control technology.
According to one aspect of the invention, a construction method of an ultra-high performance liquid chromatography fingerprint of a medicinal garlic medicinal material is provided, comprising the following steps:
step one: preparation of test solution:
taking about 10.0g of peeled fresh garlic, precisely weighing, adding 30mL of ultrapure water, carrying out medium-high fire microwave alliinase killing for 1-10 min,and (5) cooling. Homogenizing for 5min, pouring the slurry into a 50mL centrifuge tube, flushing a homogenizer with 10mL ultrapure water, mixing the washing liquid into the 50mL centrifuge tube, ultrasonically extracting for 5-15 min, and centrifuging (4000-7000 r.min) -1 ) Transferring the supernatant into a 100mL volumetric flask for 5-15 min, adding 20mL of water into the precipitate, shaking up fully, centrifuging by ultrasound again (0-2 times), merging the supernatant into the 100mL volumetric flask, adding ultrapure water to fix the volume to a scale, shaking up, taking 1.2mL of the solution into a 10mL volumetric flask, and using the ultrapure water to fix the volume to serve as a sample solution;
step two: preparation of a mixed control solution:
an appropriate amount of amino acid reference substances were precisely weighed, placed in the same 25mL volumetric flask, dissolved in water and scaled to obtain reference substance solutions (Table 1).
TABLE 1 concentration of amino acids in the Mixed label
Step three: derivatization of sample solutions:
preparing a derivatizing reagent: triethylamine acetonitrile solution (1 mol.L) -1 ): weighing 13.5mL of triethylamine, placing in a 100mL volumetric flask, adding acetonitrile for dilution, fixing the volume scale, and shaking uniformly to obtain the product. Phenyl isothiocyanate acetonitrile solution (0.1 mol.L) -1 ): weighing 1.2mL of phenyl isothiocyanate, placing in a 100mL volumetric flask, adding acetonitrile for dilution, fixing the volume scale, and shaking uniformly to obtain the product.
Derivatization reaction: accurately transferring 2mL of reference substance solution (or sample solution or ultrapure water solution), and adding 0.1 mol.L of reference substance solution respectively -1 Phenyl isothiocyanate acetonitrile solution 1mL,1 mol.L -1 1mL of triethylamine acetonitrile solution, shaking and mixing uniformly, standing at room temperature for 30min, adding 4mL of n-hexane, shaking and mixing uniformly fully, and centrifuging (6000 r.min) -1 Removing the upper layer solution, extracting with n-hexane 4mL again, centrifuging (6000 r.min) for 5min at 25deg.C -1 Absorbing the lower solution at 25 ℃ for 5min, and filtering with a 0.45 mu m filter membrane to obtain a reference substance derivatization solution (or a sample derivatization solution or a negative control derivatization solution).
Step four: determining chromatographic conditions of the UPLC:
ACE Excel 1.7SuperC18 column (100 mm. Times.2.1 mm,1.7 μm); column temperature: 25-40 ℃; flow rate: 0.1 to 0.6 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Measurement wavelength: 210-310 nm; sample injection volume: 0.5-2 mu L; mobile phase: mobile phase a: weighing 8.2g of sodium acetate trihydrate, placing in a 1000mL beaker, adding 600mL of ultrapure water, stirring for dissolution, adjusting the pH value to 6.5 with glacial acetic acid, adding 45mL of acetonitrile, mixing uniformly, and filtering with a 0.45 μm filter membrane; mobile phase B is methanol: acetonitrile: water = 20:60:20 (V/V/V); elution by gradient elution mode, gradient elution procedure: 0min 2min 13.6min 15.2min 16.8min 18min 20min mobile phase B (methanol: acetonitrile: water=20:60:20 (V/V)) 10 vol% →12 vol% →65 vol% →100 vol% →0 vol%.
Step five: ultra-high performance liquid chromatography assay:
precisely sucking the sample, the reference substance and the negative control derivatization solution in the third step, injecting the sample, the reference substance and the negative control derivatization solution into an ultra-high performance liquid chromatograph for measurement, and recording a chromatogram;
step six: establishing a fingerprint spectrum:
introducing the reference substance solution chromatogram and the sample solution chromatogram into fingerprint software, processing the chromatogram with the fingerprint software to obtain the fingerprint of medicinal Bulbus Allii, and comparing the chromatograms of the sample to be tested with standard fingerprint similarity.
Compared with the prior art, the method for evaluating the quality of the medicinal garlic by utilizing the ultra-high performance liquid chromatography fingerprint or the obtained standard fingerprint has the following remarkable advantages:
(1) The invention firstly utilizes UPLC technology to conduct fingerprint research on medicinal garlic, and provides a new analysis method for quality control of medicinal garlic.
(2) The medicinal garlic medicinal materials are treated as a whole, and the subtle differences among different medicinal materials can be found out by comparing the common peaks of the medicinal garlic medicinal materials, so that the method is suitable for identifying and controlling the authenticity, the production place and the quality of the medicinal garlic medicinal materials.
(3) The chromatographic condition of the invention is easy to realize, the precision is high, the stability and the repeatability are better, the detection wavelength adopted by the invention has more peaks, and the reflected information is complete.
(4) Meanwhile, the detection method of the invention uses alliin as a reference peak, determines and identifies 18 common peaks, and has rich and comprehensive fingerprint spectrum composition.
(5) Compared with the traditional thin-layer chromatography fingerprint spectrum and HPLC method, the method has the advantages of greatly shortening the on-line analysis time and saving the material cost and the time cost on the premise of not affecting the whole effect.
The traditional Chinese medicine fingerprint refers to the characteristic spectrum of each component group of the traditional Chinese medicine obtained by modern analysis techniques such as spectrum or chromatograph of a traditional Chinese medicine sample, and the characteristic spectrum has become one of the most effective means for identifying traditional Chinese medicine varieties and evaluating the quality of the traditional Chinese medicine accepted at home and abroad at present. One of the latest fingerprint spectrum testing methods at present is ultra-high performance liquid chromatography (Ultra Performance Liquid Chromatography, UPLC), which is a novel liquid chromatography technology in recent years, and adopts a small-particle-size chromatographic column filler of 1.7 mu m, so that the column efficiency is improved, and the column efficiency is kept constant in a wider flow rate range, thereby being beneficial to shortening the analysis time and improving the analysis flux by improving the flow rate of a mobile phase. UPLC has better separation efficiency, peak capacity and sensitivity than conventional high performance liquid chromatography (High Performance Liquid Chromatography, HPLC). However, in the technical field of analysis of medicinal garlic medicinal materials, quality control analysis and fingerprint spectrum reporting of medicinal garlic medicinal materials by UPLC are not yet available.
Drawings
FIG. 1 is a graph showing a 27 batch shared peak fingerprint of medicinal garlic UPLC; in the figure, A is a chromatogram of a mixed reference substance; b is a reference fingerprint spectrum R; c is blank reagent chromatogram (1 is aspartic acid, 2 is glutamic acid, 3 is serine, 4 is glycine, 5 is histidine, 6 is arginine, 7 is threonine, 8 is alanine, 9 is proline, 10 is alliin, 11 is tyrosine, 12 is valine, 13 is methionine, 14 is cystine, 15 is leucine, 16 is phenylalanine, 17 is tryptophan, and 18 is lysine.
Fig. 2 is a standard UPLC fingerprint of a medicinal garlic material of the present invention.
FIG. 3 is a superimposed chromatogram of UPLC fingerprints (S1-S27) and standard UPLC fingerprints (R) of 27 batches of medicinal garlic samples of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be further described with reference to examples to more clearly describe the advantages and features of the present invention. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions in form and details may be made therein without departing from the spirit and scope of the invention but are intended to be within the scope of the invention.
Referring to fig. 1 to 3, the application provides a method for constructing a fingerprint of a medicinal garlic material, which comprises the following steps:
(1) Preparation of test solution: taking about 10.0g of peeled fresh garlic, precisely weighing, adding 30mL of ultrapure water, carrying out medium-high fire microwave alliinase killing for 1-10 min, and cooling. Homogenizing for 5min, pouring the slurry into a 50mL centrifuge tube, flushing a homogenizer with 10mL ultrapure water, mixing the washing liquid into the 50mL centrifuge tube, ultrasonically extracting for 5-15 min, and centrifuging (4000-7000 r.min) -1 ) Transferring the supernatant into a 100mL volumetric flask for 5-15 min, adding 20mL of water into the precipitate, shaking up fully, centrifuging by ultrasound again (0-2 times), merging the supernatant into the 100mL volumetric flask, adding ultrapure water to fix the volume to a scale, shaking up, taking 1.2mL of the solution into a 10mL volumetric flask, and using the ultrapure water to fix the volume to serve as a sample solution;
(2) Preparation of a mixed control solution: an appropriate amount of amino acid reference substances were precisely weighed, placed in the same 25mL volumetric flask, dissolved in water and scaled to obtain reference substance solutions (Table 1).
TABLE 1 concentration of amino acids in the Mixed label
(3) Derivatization of sample solutions: preparing a derivatizing reagent:triethylamine acetonitrile solution (1 mol.L) -1 ): weighing 13.5mL of triethylamine, placing in a 100mL volumetric flask, adding acetonitrile for dilution, fixing the volume scale, and shaking uniformly to obtain the product. Phenyl isothiocyanate acetonitrile solution (0.1 mol.L) -1 ): weighing 1.2mL of phenyl isothiocyanate, placing in a 100mL volumetric flask, adding acetonitrile for dilution, fixing the volume scale, and shaking uniformly to obtain the product; derivatization reaction: accurately transferring 2mL of reference substance solution (or sample solution or ultrapure water solution), and adding 0.1 mol.L of reference substance solution respectively -1 Phenyl isothiocyanate acetonitrile solution 1mL,1 mol.L -1 1mL of triethylamine acetonitrile solution, shaking and mixing uniformly, standing at room temperature for 30min, adding 4mL of n-hexane, shaking and mixing uniformly fully, and centrifuging (6000 r.min) -1 Removing the upper layer solution, extracting with n-hexane 4mL again, centrifuging (6000 r.min) for 5min at 25deg.C -1 Absorbing the lower solution at 25 ℃ for 5min, and filtering with a 0.45 mu m filter membrane to obtain a reference substance derivatization solution (or a sample derivatization solution or a negative control derivatization solution).
(4) Determination of UPLC chromatographic conditions:
chromatographic column: ACE Excel 1.7SuperC18 (100 mm. Times.2.1 mm,1.7 μm);
measurement wavelength: 210-310 nm;
sample injection volume: 0.5-2 mu L;
column temperature: 25-40 ℃;
mobile phase: mobile phase a: weighing 8.2g of sodium acetate trihydrate, placing in a 1000mL beaker, adding 600mL of ultrapure water, stirring for dissolution, adjusting the pH value to 6.5 with glacial acetic acid, adding 45mL of acetonitrile, mixing uniformly, and filtering with a 0.45 μm filter membrane; mobile phase B is methanol: acetonitrile: water = 20:60:20 (V/V/V);
flow rate: 0.1 to 0.6 mL/min -1 ；
Elution by gradient elution mode, gradient elution procedure: 0min 2min 13.6min 15.2min 16.8min 18min 20min mobile phase B (methanol: acetonitrile: water=20:60:20 (V/V)) 10 vol% →12 vol% →65 vol% →100 vol% →0 vol%.
(5) Establishment of standard fingerprint
Preparing a plurality of batches of medicinal garlic medicines into test solution according to the step (3), simultaneously respectively injecting the mixed reference solution into an ultra-high performance liquid chromatograph, detecting according to the liquid chromatography condition of the step (4), respectively recording the patterns and data of the mixed reference solution and the test solution, respectively introducing the obtained patterns and data into software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012A edition) for analysis, setting one of the chromatograms of the test solution as a reference pattern, setting a standard pattern generation method as a median method, and simultaneously adopting a multi-point correction method to establish a standard fingerprint of the medicinal garlic medicines.
Preferably, the sample solution in step (1) is prepared as follows: taking about 10.0g of peeled fresh garlic, precisely weighing, adding 30mL of ultrapure water, carrying out medium-high fire microwave alliinase killing for 1.5min, and cooling. Homogenizing for 5min, pouring the slurry into a 50mL centrifuge tube, washing the homogenizer with 10mL ultrapure water, mixing the washing solution with the 50mL centrifuge tube, ultrasonically extracting for 10min, and centrifuging (6000 r.min) -1 ) Transferring the supernatant into a 100mL volumetric flask for 10min, adding 20mL of water into the precipitate, shaking uniformly, performing ultrasonic centrifugation again, combining the supernatant into the 100mL volumetric flask, adding ultrapure water to a scale, shaking uniformly, taking 1.2mL of the solution into the 10mL volumetric flask, and using the ultrapure water to a constant volume to serve as a sample solution.
Preferably, step (4) determination of UPLC chromatographic conditions: the chromatographic conditions were ACE Excel 1.7SuperC18 (100 mm. Times.2.1 mm,1.7 μm) column. Column temperature 30 ℃ and flow rate 0.2 mL-min -1 The method comprises the steps of carrying out a first treatment on the surface of the The measurement wavelength was 254nm; sample injection volume: 0.8. Mu.L; mobile phase a: weighing 8.2g of sodium acetate trihydrate, placing in a 1000mL beaker, adding 600mL of ultrapure water, stirring for dissolution, adjusting the pH value to 6.5 with glacial acetic acid, adding 45mL of acetonitrile, mixing uniformly, and filtering with a 0.45 μm filter membrane; mobile phase B is methanol: acetonitrile: water = 20:60:20 (V/V/V); the gradient elution procedure was: 0min 2min 2.4min 5.6min 7.2min 12.8min 13.6min 15.2min 16.8min 18min 20min mobile phase B (methanol: acetonitrile: water=20:60:20 (V/V)) 10 vol% →12 vol% →15 vol% →19 vol% →31 vol% →55 vol% →65 vol% →100 vol% →0 vol%.
The standard fingerprint consists of 18 common peaks, and the peak 10 is taken as a reference peak, and the relative retention time and the relative standard deviation RSD of the 18 common peaks are shown in the following table 2:
table 218 relative retention times of common peaks and RSD
By comparison, of the 18 common characteristic peaks, peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is glycine, peak 5 is histidine, peak 6 is arginine, peak 7 is threonine, peak 8 is alanine, peak 9 is proline, peak 10 is alliin, peak 11 is tyrosine, peak 12 is valine, peak 13 is methionine, peak 14 is cystine, peak 15 is leucine, peak 16 is phenylalanine, peak 17 is tryptophan, and peak 18 is lysine.
(6) Quality detection of medicinal garlic medicinal material to be detected
Preparing a to-be-detected test solution from the medicinal garlic to be detected according to the steps (1) and (3), preparing a mixed reference substance solution from 18 amino acids according to the steps (2) and (3), respectively injecting the prepared to-be-detected test solution and the mixed reference substance solution into an ultra-high performance liquid chromatograph, detecting according to the ultra-high performance liquid chromatograph conditions of the step (4), respectively recording the patterns and data of the test solution and the mixed reference substance solution, comparing the obtained patterns and data with the standard fingerprint patterns established in the step (5), calculating the similarity, identifying the number of common absorption peaks, and determining the similarity.
In the specific implementation process, the following environments are adopted:
1. instrument and materials
1.1 instruments
H-class ultra-high performance liquid chromatograph (Waters, USA); WP-UPS-40 laboratory ultra pure water machine (Sichuan Wo Teer Water treatment equipments Co., ltd.); ES225SM-DR model one ten thousandth balance; AR124CN model ten-thousandth balance; FW-100 type high-speed universal pulverizer (Yongguangming medical instruments works in Beijing); DHG-9145A type electrothermal blowing drying oven (Shanghai-Heng science instruments Co., ltd.); SHB-III type circulating water multipurpose vacuum pump (zheng great wall department industry and trade limited); SX-5-12-T type box-type resistance furnace; DZKW-0-2 electric heating constant temperature water bath (Yongguangming medical instruments Co., ltd., beijing); KDM type temperature-regulating electrothermal jacket (Yongxing instrumentation factory in the city of eastern mountain); DLSB-5/20 low temperature coolant circulation pump (Zhengzhou great wall Co., ltd.); g-type silica gel plate (Qingdao ocean chemical Co., ltd.); WK001 type microwave oven (nanotechnology corporation); FSH-2A adjustable high speed refiner (Jiangsu gold instrument technologies limited); KQ-500DE ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); centrifuge (Germany Ai Bende); XH-C vortex mixer (medical instruments, kingtan).
1.2 reagents
The 27 batches (numbered G1-G27) of medicinal garlic were collected in 27 different provinces and cities in China, the corresponding numbers and production places of the medicinal materials are shown in Table 3, and the information of the reference substances is shown in Table 4.
TABLE 3 medicinal garlic 27 lot source information
TABLE 4 control information
2. Method and results
2.1 preparation of control solution
An appropriate amount of amino acid reference substances were precisely weighed, placed in the same 25mL volumetric flask, dissolved in water and scaled to obtain reference substance solutions (Table 1).
TABLE 1 concentration of amino acids in the Mixed label
2.2 preparation of sample solution
Taking about 10.0g of peeled fresh garlic, precisely weighing,30mL of ultrapure water is added, and the alliinase is killed by medium-high fire microwaves for 1.5min and cooled. Homogenizing for 5min, pouring the slurry into a 50mL centrifuge tube, washing the homogenizer with 10mL ultrapure water, mixing the washing solution with the 50mL centrifuge tube, ultrasonically extracting for 10min, and centrifuging (6000 r.min) -1 ) Transferring the supernatant into a 100mL volumetric flask for 10min, adding 20mL of water into the precipitate, shaking uniformly, performing ultrasonic centrifugation again, combining the supernatant into the 100mL volumetric flask, adding ultrapure water to a scale, shaking uniformly, taking 1.2mL of the solution into the 10mL volumetric flask, and using the ultrapure water to a constant volume to serve as a sample solution.
2.3 derivatization reactions
Preparing a derivatizing reagent: triethylamine acetonitrile solution (1 mol.L) -1 ): weighing 13.5mL of triethylamine, placing in a 100mL volumetric flask, adding acetonitrile for dilution, fixing the volume scale, and shaking uniformly to obtain the product. Phenyl isothiocyanate acetonitrile solution (0.1 mol.L) -1 ): weighing 1.2mL of phenyl isothiocyanate, placing in a 100mL volumetric flask, adding acetonitrile for dilution, fixing the volume scale, and shaking uniformly to obtain the product.
Derivatization reaction: accurately transferring 2mL of reference substance solution (or sample solution or ultrapure water solution), and adding 0.1 mol.L of reference substance solution respectively -1 Phenyl isothiocyanate acetonitrile solution 1mL,1 mol.L -1 1mL of triethylamine acetonitrile solution, shaking and mixing uniformly, standing at room temperature for 30min, adding 4mL of n-hexane, shaking and mixing uniformly fully, and centrifuging (6000 r.min) -1 Removing the upper layer solution, extracting with n-hexane 4mL again, centrifuging (6000 r.min) for 5min at 25deg.C -1 Absorbing the lower solution at 25 ℃ for 5min, and filtering with a 0.45 mu m filter membrane to obtain a reference substance derivatization solution (or a sample derivatization solution or a negative control derivatization solution).
2.4 chromatographic conditions
The column was an ACE Excel 1.7SuperC18 column (100 mm. Times.2.1 mm,1.7 μm), mobile phase A: weighing 8.2g of sodium acetate trihydrate, placing in a 1000mL beaker, adding 600mL of ultrapure water, stirring for dissolution, adjusting the pH value to 6.5 with glacial acetic acid, adding 45mL of acetonitrile, mixing uniformly, and filtering with a 0.45 μm filter membrane; mobile phase B is methanol: acetonitrile: water = 20:60:20 (V/V/V); the detection wavelength is 254nm; the flow rate is 0.2 mL-min -1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature is 35 ℃; sample injection amount is 0.8 mu L; the gradient elution procedure was: 0 min-2 min-2.4 min-5.6 min-7.2 min-12 min8min, 13.6min, 15.2min, 16.8min, 18min, 20min, mobile phase B (methanol: acetonitrile: water=20:60:20 (V/V)) 10 vol% →12 vol% →15 vol% →19 vol% →31 vol% →55 vol% →65 vol% →100 vol% →0 vol%.
2.5 precision investigation 1 part of medicinal garlic (G1) is taken, precisely weighed, a sample solution is prepared according to the method under the item "2.1.2", the sample solution is derivatized according to the method under the item "2.1.3", the continuous measurement is carried out for 6 times according to the chromatographic condition under the item "2.1.4", 10 peak alliin is taken as a reference peak (S), and the relative retention time RSD of each common peak is calculated to be less than 0.30%, and the relative peak area RSD is less than 1.41%, thus indicating that the instrument precision is good.
2.6 repeatability investigation A test solution is prepared by taking 6 parts of medicinal garlic (G1), precisely weighing, preparing the test solution in parallel according to the method under the item "2.1.2", derivatizing the test solution under the item "2.1.3", respectively sampling and measuring according to the chromatographic condition under the item "2.1.4", taking 10-peak alliin as a reference peak (S), and calculating the relative retention time RSD of each common peak to be less than 0.07%, and the relative peak area RSD to be less than 1.44%, thus showing that the method has good repeatability.
2.7 stability investigation 1 part of medicinal garlic (G1) is taken, precisely weighed, a sample solution is prepared according to a method under the item "2.1.2", the sample solution is derivatized according to the item "2.1.3", sample injection measurement is carried out according to chromatographic conditions under the item "2.1.4" at 0, 2, 4, 8, 12 and 24 hours respectively, 10-peak alliin is taken as a reference peak (S), and the relative retention time RSD of each common peak is calculated to be less than 0.97%, the relative peak area RSD is less than 1.44%, so that the sample derivative solution has good stability in 24 hours.
2.8 establishment of UPLC fingerprint and identification of common peaks
Taking 27 batches of medicinal garlic medicines, preparing a test solution according to the method under the item "2.1.2", derivatizing the test solution under the item "2.1.3", carrying out sample injection measurement according to the chromatographic condition under the item "2.1.4", and recording a chromatogram. Processing 27 batches of sample chromatograms by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012A edition), taking a G1 sample chromatogram as a reference chromatogram, and generating a control fingerprint R by using an average method, wherein the time window width is 0.1. The 18 common peaks are marked by comparison of the reference substances, and can be respectively identified as aspartic acid (peak 1), glutamic acid (peak 2), serine (peak 3), glycine (peak 4), histidine (peak 5), arginine (peak 6), threonine (peak 7), alanine (peak 8), proline (peak 9), alliin (peak 10), tyrosine (peak 11), valine (peak 12), methionine (peak 13), cystine (peak 14), leucine (peak 15), phenylalanine (peak 16), tryptophan (peak 17) and lysine (peak 18). The UPLC fingerprint superposition diagram and the reference fingerprint R of 27 batches of medicinal garlic medicines are shown in figure 3, and the chromatogram of the mixed reference substance, the chromatogram of the blank reagent and the common peak assignment are shown in figure 1.
2.9 similarity evaluation (similarity assessment, SA)
The similarity evaluation of 27 batches of medicinal garlic medicines is carried out by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012A edition), and the similarity of 27 batches of medicinal garlic medicines is 0.892-0.995 by taking a reference fingerprint R as a reference, wherein the similarity evaluation result is shown in Table 6. The alliin peak of No. 10 is taken as a reference peak (S), and the relative retention time RSD of the common peaks of 27 batches of medicinal garlic medicinal material samples is less than 1.46 percent and the relative peak area RSD is 54.90-138.01 percent. The chemical compositions of the medicinal garlic medicines in different producing areas are similar, but the mass fractions of the common chemical components among the samples are different to a certain extent.
TABLE 2 relative retention time and RSD of 18 common peaks in fingerprint of medicinal garlic
TABLE 5 relative peak areas and RSD of 18 common peaks in fingerprint of medicinal Bulbus Allii
TABLE 6 pharmaceutical Garlic fingerprint 27 Lot similarity
3. Conclusion(s)
According to the similarity evaluation result, the chemical component types of the medicinal garlic medicaments in different batches are basically consistent, but the contents are obviously different. In conclusion, the fingerprint spectrum method and the application of the medicinal garlic medicinal material established by the invention carry out quality analysis on medicinal garlic medicinal materials with different batches of different enterprises on the whole, and provide a scientific evaluation method and a scientific monitoring means for quality control of the preparation.
The foregoing examples illustrate embodiments of the invention and are described in more detail, but are not to be construed as limiting the scope of the invention. It should be noted that variations and modifications can be made by those skilled in the art without departing from the spirit of the invention, which falls within the scope of the invention.
Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A method for evaluating the quality of medicinal garlic by utilizing ultra-high performance liquid chromatography fingerprint is characterized by comprising the following steps:
step one: preparation of test solution: taking about 10.0g of peeled fresh garlic, precisely weighing, adding 30mL of ultrapure water, carrying out medium-high fire microwave alliinase killing for 1-10 min, and cooling. Homogenizing for 5min, pouring the slurry into a 50mL centrifuge tube, flushing a homogenizer with 10mL ultrapure water, mixing the washing liquid into the 50mL centrifuge tube, ultrasonically extracting for 5-15 min, and centrifuging (4000-7000 r.min) -1 ) Transferring the supernatant into a 100mL volumetric flask for 5-15 min, adding 20mL of water into the precipitate, shaking up fully, centrifuging by ultrasound again (0-2 times), merging the supernatant into the 100mL volumetric flask, adding ultrapure water to fix the volume to a scale, shaking up, taking 1.2mL of the solution into a 10mL volumetric flask, and using the ultrapure water to fix the volume to serve as a sample solution;
step two: preparation of a mixed control solution: weighing an amino acid reference substance, placing the amino acid reference substance into the same 25mL volumetric flask, adding water for dissolution, and fixing the volume to a scale, wherein the amino acid reference substance is used as a reference substance solution, and the amino acid is as follows:
step three: derivatization of sample solutions:
preparing a derivatizing reagent: triethylamine acetonitrile solution (1 mol.L) -1 ): weighing 13.5mL of triethylamine, placing in a 100mL volumetric flask, adding acetonitrile for dilution, fixing volume scales, and shaking uniformly to obtain the product; phenyl isothiocyanate acetonitrile solution (0.1 mol.L) -1 ): weighing 1.2mL of phenyl isothiocyanate, placing in a 100mL volumetric flask, adding acetonitrile for dilution, fixing the volume scale, and shaking uniformly to obtain the product;
derivatization reaction: accurately transferring 2mL of reference substance solution (or sample solution or ultrapure water solution), and adding 0.1 mol.L of reference substance solution respectively -1 Phenyl isothiocyanate acetonitrile solution 1mL,1 mol.L -1 1mL of triethylamine acetonitrile solution, shaking and mixing uniformly, standing at room temperature for 30min, adding 4mL of n-hexane, shaking and mixing uniformly fully, and centrifuging (6000 r.min) -1 Removing the upper layer solution, extracting with n-hexane 4mL again, centrifuging (6000 r.min) for 5min at 25deg.C -1 Absorbing the lower layer solution at 25 ℃ for 5min, and filtering with a 0.45 mu m filter membrane to obtain a reference substance derivatization solution (or a sample derivatization solution or a negative control derivatization solution);
step four: determining chromatographic conditions of the UPLC:
ACE Excel 1.7SuperC18 column (100 mm. Times.2.1 mm,1.7 μm); column temperature: 25-40 ℃; flow rate: 0.1 to 0.6 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Measurement wavelength: 210-310 nm; sample injection volume: 0.5-2 mu L; mobile phase: mobile phase a: weighing 8.2g of sodium acetate trihydrate, placing in a 1000mL beaker, adding 600mL of ultrapure water, stirring for dissolution, adjusting the pH value to 6.5 with glacial acetic acid, adding 45mL of acetonitrile, mixing uniformly, and filtering with a 0.45 μm filter membrane; mobile phase B is methanol: acetonitrile: water = 20:60:20 (V/V/V); elution by gradient elution mode, gradient elution procedure: 0 min- > 2 min-13.6 min- > 15.2 min- > 16.8 min- > 18 min- > 20min, mobile phase B (methanol: acetonitrile: water=20:60:20 (V/V)) 10% by volume- > 12% by volume-65 massVolume% → 100% by volume% → 0% by volume "→ 0% by volume;
step five: ultra-high performance liquid chromatography assay:
precisely sucking the sample, the reference substance and the negative control derivatization solution in the third step, injecting the sample, the reference substance and the negative control derivatization solution into an ultra-high performance liquid chromatograph for measurement, and recording a chromatogram;
step six: establishing a fingerprint spectrum:
introducing the reference substance solution chromatogram and the sample solution chromatogram into fingerprint software, processing the chromatogram with the fingerprint software to obtain the fingerprint of medicinal Bulbus Allii, and comparing the chromatograms of the sample to be tested with standard fingerprint similarity.
2. The method for evaluating the quality of medicinal garlic by utilizing the ultra-high performance liquid chromatography fingerprint spectrum according to claim 1, wherein the preparation of the test solution is carried out according to the following method:
taking about 10.0g of peeled fresh garlic, precisely weighing, adding 30mL of ultrapure water, carrying out medium-high fire microwave alliinase killing for 1.5min, and cooling. Homogenizing for 5min, pouring the slurry into a 50mL centrifuge tube, washing the homogenizer with 10mL ultrapure water, mixing the washing solution with the 50mL centrifuge tube, ultrasonically extracting for 10min, and centrifuging (6000 r.min) -1 ) Transferring the supernatant into a 100mL volumetric flask for 10min, adding 20mL of water into the precipitate, shaking uniformly, performing ultrasonic centrifugation again, combining the supernatant into the 100mL volumetric flask, adding ultrapure water to a scale, shaking uniformly, taking 1.2mL of the solution into the 10mL volumetric flask, and using the ultrapure water to a constant volume to serve as a sample solution. Accurately transferring 2mL of the sample solution, and adding 0.1 mol.L respectively -1 Phenyl isothiocyanate acetonitrile solution 1mL,1 mol.L -1 1mL of triethylamine acetonitrile solution, shaking and mixing uniformly, standing at room temperature for 30min, adding 4mL of n-hexane, shaking and mixing uniformly fully, and centrifuging (6000 r.min) -1 Removing the upper layer solution, extracting with n-hexane 4mL again, centrifuging (6000 r.min) for 5min at 25deg.C -1 And sucking the lower layer solution at 25 ℃ for 5min, and filtering with a 0.45 mu m filter membrane to obtain the sample derivatization solution.
3. The advantage according to claim 1The method for evaluating the quality of the medicinal garlic by using the ultra-high performance liquid chromatography fingerprint is characterized in that the chromatographic condition in the fourth step is an ACE Excel SuperC18 (100 mm multiplied by 2.1mm,1.7 mu m) chromatographic column; column temperature 30 ℃ and flow rate 0.2 mL-min -1 The method comprises the steps of carrying out a first treatment on the surface of the The measurement wavelength was 254nm; sample injection volume: 0.8. Mu.L; mobile phase a: weighing 8.2g of sodium acetate trihydrate, placing in a 1000mL beaker, adding 600mL of ultrapure water, stirring for dissolution, adjusting the pH value to 6.5 with glacial acetic acid, adding 45mL of acetonitrile, mixing uniformly, and filtering with a 0.45 μm filter membrane; mobile phase B is methanol: acetonitrile: water = 20:60:20 (V/V/V) gradient elution was used.
4. The method for evaluating the quality of medicinal garlic by utilizing the ultra-high performance liquid chromatography fingerprint according to claim 1, wherein the fingerprint software in the step six is a "traditional Chinese medicine chromatography fingerprint similarity evaluation System".
5. The method for evaluating the quality of medicinal garlic by utilizing the ultra-high performance liquid chromatography fingerprint spectrum according to claim 1 to 4, wherein the method comprises 18 common peaks, which are respectively identified as follows: peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is glycine, peak 5 is histidine, peak 6 is arginine, peak 7 is threonine, peak 8 is alanine, peak 9 is proline, peak 10 is alliin, peak 11 is tyrosine, peak 12 is valine, peak 13 is methionine, peak 14 is cystine, peak 15 is leucine, peak 16 is phenylalanine, peak 17 is tryptophan, and peak 18 is lysine.
6. The fingerprint of medicinal garlic herb according to claim 5, comprising 18 common peaks, wherein 10-peak alliin is used as reference peak S, and the relative retention time of each common peak is as follows:
wherein each common peak has a relative retention time RSD value of not more than 1.5%.
7. A standard fingerprint of a medicinal garlic material obtained by the method for evaluating the quality of a medicinal garlic using an ultra-high performance liquid chromatography fingerprint according to any one of claims 1 to 6.
8. A standard fingerprint of medicinal garlic medicine is characterized in that: the fingerprint spectrum of the ultra-high performance liquid chromatography UPLC comprises 18 common characteristic peaks, and alliin 10 is taken as a reference peak, and the relative retention time is respectively as follows: 0.19 (±5%), 0.47 (+ -5%), 0.50 (+ -5%), 0.59 (+ -5%), 0.68 (+ -5%), 0.73 (+ -5%), 0.76 (+ -5%), 0.79 (+ -5%), 0.94 (+ -5%), 1.00 (+ -5%), 1.10 (+ -5%), 1.17 (+ -5%), 1.20 (+ -5%), 1.31 (+ -5%), 1.66 (+ -5%), 1.68 (+ -5%), 1.72 (+ -5%), and 1.76 (+ -5%).
9. Use of a standard fingerprint of a medicinal garlic material according to claim 7 or 8 in the detection of a medicinal garlic material and a preparation containing a medicinal garlic material.
10. A method for evaluating the quality of medicinal garlic, which is characterized in that the method comprises comparing the fingerprint of the medicinal garlic with the standard fingerprint of the medicinal garlic according to claim 7 or 8, wherein the similarity is more than 0.85, and the fingerprint of the medicinal garlic is obtained by using UPLC.
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