CN111413423A - Construction method of cortex lycii UP L C characteristic map and detection method of cortex lycii - Google Patents
Construction method of cortex lycii UP L C characteristic map and detection method of cortex lycii Download PDFInfo
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Abstract
The invention discloses a method for constructing a cortex lycii UP L C characteristic spectrum, which comprises the steps of (1) respectively taking a cortex lycii contrast medicinal material, a cortex lycii ethyl contrast product and a cortex lycii methyl ethyl acetate contrast product, adding a solvent for extraction or dissolution to prepare a reference substance solution, (2) taking cortex lycii powder, extracting by using a methanol-acetic acid mixed solution to prepare a sample solution, (3) taking a preset amount of the reference substance solution and the sample solution, injecting into a liquid chromatograph, and carrying out gradient elution by using octadecylsilane bonded silica gel as a filling agent, methanol as a mobile phase A and trifluoroacetic acid aqueous solution as a mobile phase B on the liquid chromatograph to establish a cortex lycii UP L C characteristic spectrum.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine quality analysis and detection, in particular to a construction method of a cortex lycii UP L C characteristic map and a detection method for identifying different primordia and fake cortex periplocae of a cortex lycii medicinal material.
Background
Cortex lycii radicis is dry root bark of lycium barbarum L fungus mill or lycium barbarum L fungus bararum L of solanaceae, the root is dug in the early spring or late autumn, cleaned, stripped, dried, sweet in taste, cold, enters lung, liver and kidney channels, has the effects of cooling blood, removing dampness, clearing lung-heat and reducing internal heat, and is used for treating yin deficiency and tidal fever, bone steaming and night sweat, lung heat cough, hemoptysis, epistaxis and internal heat thirst, and is accepted by Shen nong Ben Cao Jing, Ming Yi Bie Lu and Ben Cao Jing, and the like, and is one of the traditional Chinese medicines commonly used clinically.
The Chinese pharmacopoeia stipulates that the basic source of the cortex lycii radicis comprises the medlar and Ningxia medlar. In the process of operating and using the traditional Chinese medicine, the cortex periplocae and the cortex lycii radicis are similar in appearance and are not easy to distinguish, and the cortex periplocae and the cortex lycii radicis are easy to be confused when in use. The two herbs are different in source, component, efficacy and other aspects. The cortex lycii radicis is derived from the medlar or Ningxia medlar, has similar components, mainly contains cinnamic acid, a large amount of phenolic substances, betaine, lycine A, lycine B, linoleic acid, linolenic acid and the like, and has the functions of sterilizing, disinfecting, regulating immunity, reducing blood pressure, blood sugar and heat. The cortex Periplocae Radicis mainly contains more than 10 kinds of cardiac glycosides, periplocin, and cortex Periplocae Radicis A, B, C, D, E, F, G, K, and also contains compounds such as methoxysalicylaldehyde and resinol. Periplocin is a type A cardiac glycoside, has quick response, short action time and difficult accumulation, but has narrow safety range, and is easy to cause toxic reaction when being used excessively, and the toxic mechanism is that periplocin is used excessively to strengthen and weaken the cardiac muscle, so that arrhythmia and cardiac fibrillation are caused to die. Xiangjiapi is toxic, mainly due to periplocin. If cortex periplocae is mistakenly used as cortex lycii radicis, serious consequences can be caused, and potential quality hazards exist.
The cortex lycii radicis medicinal material of the version of 2015, which is in Chinese pharmacopoeia, has microscopic and thin-layer identification items, does not have content measurement and characteristic spectrum items, cannot comprehensively evaluate the components of the cortex lycii radicis, and does not have a method for identifying different primitive cortex lycii radicis (medlar, Ningxia medlar) and counterfeit cortex periplocae. Domestic and foreign references have few studies on the quality of the above aspects. At present, no literature reports on the technical research of fingerprint/characteristic spectrum for identifying different primordia and counterfeit cortex of cortex lycii radicis medicinal materials, and the authenticity of the cortex lycii radicis medicinal materials cannot be effectively identified.
Disclosure of Invention
The invention aims to solve the technical problem of providing a construction method of a UP L C characteristic map of cortex lycii radicis, which has good reproducibility, accuracy and reliability, and can realize the identification of different primordia and counterfeit cortex of cortex lycii radicis through the established UP L C characteristic map.
The invention aims to solve the technical problem of providing a detection method for identifying different primordia and pseudo-product cortex periplocae of a cortex lycii radicis medicinal material, the detection method is simple, the different primordia and pseudo-product cortex periplocae of the cortex lycii radicis medicinal material can be quickly and conveniently identified, and the result is accurate.
In order to achieve the technical effects, the invention provides a method for constructing a characteristic map of cortex lycii radicis UP L C, which comprises the following steps:
(1) respectively extracting cortex Lycii reference material, cortex Lycii B reference substance, and kukoamine A reference substance with solvent to obtain reference substance solution;
(2) extracting cortex Lycii powder with methanol-acetic acid mixed solution to obtain sample solution;
(3) injecting preset amounts of reference substance solution and test solution into a liquid chromatograph, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, methanol as a mobile phase A and trifluoroacetic acid aqueous solution as a mobile phase B for gradient elution to establish a cortex lycii radicis UP L C characteristic map.
Specifically, the step (1) comprises the following steps:
taking cortex lycii radicis as a reference medicinal material, precisely weighing, placing into a volumetric flask with a plug, adding a mixed solution of 50% methanol and 0.5% acetic acid, carrying out ultrasonic treatment, cooling, adding a mixed solution of 50% methanol and 0.5% acetic acid, centrifuging, filtering, and taking a subsequent filtrate as a reference medicinal material solution;
accurately weighing cortex Lycii B reference substance and kukoamine reference substance, dissolving in methanol, and making into mixed solution containing 80-100 μ g of kukoamine per 1ml as reference substance solution.
Preferably, the sampling amount of the cortex lycii radicis control medicinal material is 0.5-1g, the capacity of the mixed solution of 50% methanol and 0.5% acetic acid is 20-30ml, and the capacity of the mixed solution of 50% methanol and 0.5% acetic acid is 5-10ml after ultrasonic treatment; in the mixed solution of 50% methanol and 0.5% acetic acid, the dosage ratio of 50% methanol to 0.5% acetic acid is (1-2) to (1-2).
The method has the advantages that the influence difference of extraction of different polar solvents on the number and the area of extraction peaks is large, and the peak extraction efficiency is comprehensively considered, so that the cortex lycii radicis medicinal material is extracted by adopting a mixed solution of 50% methanol and 0.5% acetic acid, a good characteristic map of the number and the area of the extraction peaks can be obtained, and the peak extraction efficiency can be ensured. More preferably, the dosage ratio of 50% methanol to 0.5% acetic acid in the mixed solution of 50% methanol and 0.5% acetic acid is 1: 1.
The ultrasonic treatment time is 20-40 minutes, the power is 400-600W, and the frequency is 30-50 KHz. The ultrasonic extraction under the conditions has small influence on the number of the extracted peaks and the total peak area/sample weighing amount of the characteristic map of the cortex lycii radicis, and is convenient to operate and high in efficiency.
The step (2) comprises the following steps:
taking cortex lycii radicis powder, sieving, precisely weighing, placing into a centrifuge tube, adding a mixed solution of 50% methanol and 0.5% acetic acid, carrying out ultrasonic treatment and centrifugation, taking supernatant, transferring into a measuring flask, repeatedly extracting for 2-3 times, washing residues with a proper amount of mixed solution of 50% methanol and 0.5% acetic acid, combining extracting solutions, adding a mixed solution of 50% methanol and 0.5% acetic acid, filtering, and taking a subsequent filtrate to obtain a sample solution.
Preferably, the sampling amount of the cortex lycii radicis powder is 0.1-0.3g, and the capacity of the mixed solution of 50% methanol and 0.5% acetic acid added for the first time is 6-10 ml;
in the mixed solution of 50% methanol and 0.5% acetic acid, the dosage ratio of 50% methanol to 0.5% acetic acid is (0.5-1.5): 1;
the method has the advantages that the influence difference of extraction of different polar solvents on the number and the area of extraction peaks is large, and the peak extraction efficiency is comprehensively considered, so that the cortex lycii radicis powder is extracted by adopting a mixed solution of 50% methanol and 0.5% acetic acid, a good characteristic map of the number and the area of the extraction peaks can be obtained, and the peak extraction efficiency can be ensured. Preferably, in the mixed solution of 50% methanol and 0.5% acetic acid, the dosage ratio of 50% methanol to 0.5% acetic acid is 1: 1.
the ultrasonic treatment time is 20-40 minutes, the power is 400-600W, and the frequency is 30-50 KHz. The ultrasonic extraction under the conditions has small influence on the number of the extracted peaks and the total peak area/sample weighing amount of the characteristic map of the cortex lycii radicis, and is convenient to operate and high in efficiency.
In the step (3), octadecylsilane chemically bonded silica is used as a filler, the column length is 100mm, the inner diameter is 2.1mm, the particle size is 1.7 mu m, the column temperature is 40-42 ℃, the flow rate is 0.25ml per minute, and the detection wavelength is 278 ion-285 nm.
Preferably, the detection wavelength is 280nm, and when 280nm is selected as the detection wavelength, the peak response and the information content are richer, the interference is smaller, and the separation degree is better.
The different column temperatures have great influence on the separation degree of each peak of the cortex lycii radicis characteristic map, and preferably, the column temperature is 40 ℃, and at the moment, the separation degree of each peak of the cortex lycii radicis characteristic map is better.
The mobile phase A is methanol, and the mobile phase B is trifluoroacetic acid aqueous solution with the concentration of 0.05-0.3%. Preferably, the mobile phase B is trifluoroacetic acid aqueous solution with the concentration of 0.10-0.15%. More preferably, the mobile phase B is trifluoroacetic acid aqueous solution with the concentration of 0.15%.
The gradient elution was performed according to the following procedure:
0-5 min, wherein the mobile phase A is 8% → 14%, and the mobile phase B is 92% → 86%;
5-14 min, wherein the mobile phase A is from 14% → 25%, and the mobile phase B is from 86% → 75%;
14-23 min, wherein the content of mobile phase A is 25% → 50%, and the content of mobile phase B is 75% → 50%;
23-26 min, wherein the mobile phase A is 50% → 90%, and the mobile phase B is 50% → 10%;
26-26.1 min, wherein the mobile phase A is 90% → 8%, and the mobile phase B is 10% → 92%;
26.1-30 min, 8% of mobile phase A and 92% of mobile phase B.
The separation degree and the peak shape of each peak of the characteristic spectrum of the cortex lycii radicis are greatly influenced by different acid flows, the mobile phase A is methanol, the mobile phase B is trifluoroacetic acid aqueous solution with the concentration of 0.15%, the peak shape and the separation degree of each peak are good, and the number of the peaks is more.
The cortex lycii UP L C characteristic map constructed by the invention comprises 7 characteristic peaks, wherein the peak corresponding to a kukoamine reference substance is an S peak, the relative retention time of each characteristic peak and the S peak is within +/-10% of a specified value, the specified values comprise 0.473 of peak 1, 0.833 of peak 2, 1.067 of peak 4, 1.508 of peak 5, 1.781 of peak 6 and 1.996 of peak 7.
It should be noted that the relative retention time of each characteristic peak and the S peak should be within ± 10% of the specified value, which includes: peak 1 was 0.473, peak 2 was 0.833, peak 4 was 1.067, peak 5 was 1.508, peak 6 was 1.781, peak 7 was 1.996, with the following meanings:
the relative retention time of characteristic peak 1 to the S peak should be within ± 10% of 0.473;
the relative retention time of characteristic peak 2 to the S peak should be within ± 10% of 0.833;
the relative retention time of characteristic peak 4 to the S peak should be within ± 10% of 1.067;
the relative retention time of characteristic peak 5 to the S peak should be within ± 10% of 1.508;
the relative retention time of the characteristic peak 6 and the S peak should be within + -10% of 1.781;
the relative retention time of characteristic peak 7 to the S peak should be within ± 10% of 1.996.
Correspondingly, the invention also provides a detection method for identifying different primordia and fake cortex periplocae of the cortex lycii radicis medicinal material, which comprises the following steps:
(1) respectively extracting cortex Lycii reference material, cortex Lycii B reference substance, and kukoamine A reference substance with solvent to obtain reference substance solution;
(2) extracting cortex Lycii powder with methanol-acetic acid mixed solution to obtain sample solution;
(3) injecting a preset amount of reference substance solution into a liquid chromatograph, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, methanol as a mobile phase A and trifluoroacetic acid aqueous solution as a mobile phase B for gradient elution to establish a cortex lycii radicis UP L C characteristic map;
(4) injecting a preset amount of a test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filler, methanol as a mobile phase A and a trifluoroacetic acid aqueous solution as a mobile phase B by the liquid chromatograph to obtain a test chromatogram;
(5) comparing the chromatogram of the test sample with the characteristic spectrum of the UP L C of the cortex lycii radicis, and judging different primordia and counterfeit products of the cortex lycii radicis medicinal material according to the relative retention time of each characteristic peak of the chromatogram of the test sample and/or the relative peak area of each characteristic peak.
The retention time RSD of characteristic peaks of different original or cortex lycii radicis medicinal materials mixed with counterfeit products is small, but the relative peak area difference of the characteristic peaks is large, and the RSD is larger than 20%. Therefore, the method can effectively identify different primordia of the cortex lycii radicis medicinal material and the fake product cortex periplocae of the cortex lycii radicis medicinal material through the relative peak area of each characteristic peak of the chromatogram of the test sample or the relative retention time and the relative peak area of each characteristic peak. The peak area of each characteristic peak reflects the mass conditions of different samples to a certain extent.
Preferably, the test sample chromatogram shows 7 characteristic peaks, and the 7 characteristic peaks correspond to 7 characteristic peaks in a cortex lycii UP L C characteristic map, wherein the peak corresponding to a kukoamine reference substance is an S peak, the relative retention time of each characteristic peak and the S peak of the test sample chromatogram is calculated, and the relative retention time is within +/-10% of a specified value, wherein the specified values comprise that the peak 1 is 0.473, the peak 2 is 0.833, the peak 4 is 1.067, the peak 5 is 1.508, the peak 6 is 1.781 and the peak 7 is 1.996;
calculating relative peak areas of the peak 2, the peak 5 and the S peak, and judging according to the relative peak areas and a specified range, wherein the specified range is as follows: peak 2 should not exceed 0.050 and peak 5 should not exceed 0.297;
if the peak 2 and the peak 5 are in the specified range, judging the Chinese wolfberry root-bark, wherein the Chinese wolfberry root-bark belongs to the original Chinese wolfberry root-bark;
if the peak 2 is not in the specified range and the peak 5 is in the specified range, the Chinese wolfberry is judged as the cortex lycii radicis, and the original root of the Chinese wolfberry belongs to Ningxia Chinese wolfberry;
and if the peak 5 is more than 0.800, judging the fake product of the cortex periplocae.
In the prior art, the identification and distinction of cortex lycii radicis and cortex periplocae mainly adopt the aspects of characters, microscopic identification and the like, but the characteristics of the aspects are not strong, and no quantitative index exists, however, the invention establishes the UP L C characteristic map of the cortex lycii radicis medicinal material, and has the following beneficial effects:
(1) the method utilizes a methanol-acetic acid mixed solution to extract the cortex lycii radicis, and injects the cortex lycii radicis into a liquid chromatograph, the liquid chromatograph takes octadecylsilane chemically bonded silica as a filler, takes methanol as a mobile phase A, takes trifluoroacetic acid aqueous solution as a mobile phase B to carry out gradient elution, establishes a UP L C characteristic map of the cortex lycii radicis medicinal material, simultaneously adopts UP L C-MS-MS to carry out confirmation of relevant material basic research, and calibrates 2 characteristic peaks, the UP L C characteristic map of the cortex lycii radicis medicinal material established by the method can fully display the characteristics of chemical components of the cortex lycii radicis medicinal material, has rich information quantity of the characteristic peaks, has good reproducibility, accuracy and reliability and good stability, can realize effective identification of the cortex lycii radicis medicinal material through the established UP L C characteristic map, can distinguish the cortex lycii radicis medicinal material with different origins and fake products thereof, and effectively control the quality of the cortex lycii radicis medicinal material.
(2) The method utilizes the UP L C characteristic spectrum of the cortex lycii radicis medicinal material, and can effectively identify different primordia and the pseudo-product cortex periplocae of the cortex lycii radicis medicinal material by calculating the relative retention time and the relative peak area of each characteristic peak of the chromatogram of the test sample.
Drawings
Fig. 1 is a characteristic map of the study of the extraction solvent of cortex lycii radicis medicinal material in example 1 of the present invention.
Fig. 2 is a characteristic map of the cortex lycii radicis medicinal material extraction mode in example 1 of the present invention.
Fig. 3 is a feature map of the study of the extraction time of cortex lycii radicis medicinal material in example 1 of the present invention.
FIG. 4 is an absorption spectrum of cortex Lycii with a wavelength of 200-400 nm detected in example 1 of the present invention.
Fig. 5 is a characteristic diagram of different detection wavelengths of cortex lycii radicis medicinal material in example 1 of the present invention.
Fig. 6 is a characteristic map of different mobile phases of cortex lycii radicis medicinal material in example 1 of the present invention.
Fig. 7 is a characteristic map of different column temperature studies of cortex lycii radicis medicinal materials in example 1 of the present invention.
Fig. 8 is a characteristic map of 18 batches of cortex lycii radicis medicinal material in example 1 of the present invention.
Fig. 9 is a cortex lycii radicis medicinal material comparison feature map established in example 1 of the present invention.
FIG. 10 chromatogram of cortex Lycii medicinal material and kukoamine A standard UP L C.
FIG. 11 chromatogram of cortex Lycii medicinal material and kukoamine B standard UP L C.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Example 1 construction method for identifying characteristic map of Chinese wolfberry root-bark medicinal material UP L C
1. Instrument and reagent
The apparatus includes Waters H-class ultra high performance liquid chromatograph, Waters PDA detector, Waters TUV detector, Empower workstation, Waters Acquity UP L C BEH C18(100 × 2.1.1 mm, 1.7 μm) chromatographic column, ten thousandth analytical balance (A L104, Mettler-Torlo Co., Ltd.), one hundred thousandth analytical balance (MS105DU, Mettler-Torlo Co., Ltd.), ultrasonic cleaning machine (KQ-500DE Kunshan ultrasonic Instrument Co., Ltd.), and ultra pure water system (Micky GmbH, MI LL IPORE Synergy UV).
Experimental reagent: acetonitrile (BCR corporation, usa, chromatographically pure); methanol (BCR corporation, usa, chromatographically pure); formic acid (alatin, analytically pure); phosphoric acid (alatin, analytical grade); trifluoroacetic acid (alatin, chromatographically pure); the water is ultrapure water.
Reagent testing: kukoamine control (P18D8F50977, shanghai-sourced leaf biology ltd); guardian B control (DST170724-105, Doudster Biotechnology Co., Ltd.). Cortex lycii radicis reference drug (121087 and 201306, China institute for testing food and drug); cortex lycii radicis medicinal material (batch number: DGP 05).
2. Chromatographic conditions and preparation of test solutions
2.1 chromatographic conditions
Chromatographic conditions and system applicability test: using octadecylsilane chemically bonded silica as filler, methanol as mobile phase A, and 0.15% trifluoroacetic acid water solution as mobile phase B, and performing gradient elution according to the specification in the following table 1; the column temperature was 40 ℃; flow rate 0.25ml per minute; the detection wavelength was 280 nm. The number of theoretical plates is not less than 10000 calculated according to the lycium bark ethyl peak;
TABLE 1 gradient elution Table
2.2 preparation of reference solutions
Preparation of reference solutions: taking about 0.5g of cortex lycii radicis as a reference medicinal material, precisely weighing, placing in a 25ml volumetric flask with a plug, adding about 20ml of a mixed solution of 50% methanol-0.5% acetic acid (1: 1), carrying out ultrasonic treatment (power 500W, frequency 40KHz) for 30 minutes, cooling, adding a mixed solution of 50% methanol-0.5% acetic acid (1: 1) to a scale, centrifuging, filtering, and taking a subsequent filtrate as a reference medicinal material solution; taking appropriate amount of cortex Lycii B reference substance and kukoamine reference substance, precisely weighing, adding methanol to dissolve, and making into mixed solution containing 100 μ g of kukoamine per 1ml as reference substance solution;
2.3 preparation of test solutions
Preparing a test solution: taking about 0.2g of sample powder, sieving the sample powder by a third sieve, precisely weighing, placing the sample powder into a 50ml centrifuge tube, adding about 7ml of a mixed solution of 50% methanol and 0.5% acetic acid (1: 1), carrying out ultrasonic treatment (power 500W and frequency 40KHz) for 30 minutes, centrifuging, taking supernatant, transferring the supernatant into a 25ml measuring flask, repeatedly extracting for 2 times, washing residues by using a mixed solution of a proper amount of 50% methanol and 0.5% acetic acid (1: 1), combining extracting solutions, adding a mixed solution of 50% methanol and 0.5% acetic acid (1: 1) to scale, filtering, and taking a subsequent filtrate to obtain the product.
2.4 assay
Precisely absorbing 1 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and establishing cortex Lycii reference characteristic chromatogram by using Chinese medicinal chromatogram fingerprint similarity evaluation system.
3. Examination of preparation method of test solution
3.1 examination of extraction solvent
Taking about 0.2g of the same cortex lycii radicis medicinal material powder (batch number: DGP05, sieving with a third sieve), precisely weighing, placing into a conical flask with a plug, and precisely adding different solvents: water, 50% methanol-0.5% acetic acid (1: 1), methanol, and 70% ethanol each 7ml, ultrasonic treating (power 500W, frequency 40KHz) for 30min, centrifuging, collecting supernatant, transferring into 25ml measuring flask, repeatedly extracting for 2 times, washing residue with appropriate amount of corresponding solvent, mixing extractive solutions, and adding corresponding solvent to scale. And (5) injecting sample according to specified chromatographic conditions for determination.
The results show that by comparing the characteristic maps of 4 kinds of extraction solvents, the difference of the influence of extraction of different polarity solvents on the number and area of extraction peaks is large, the peak extraction efficiency is comprehensively considered, and a mixed solution of 50% methanol and 0.5% acetic acid (1: 1) is selected as the extraction solvent, as shown in figure 1.
3.2 examination of extraction methods
The method selects two extraction modes of ultrasonic extraction and water bath reflux to compare.
Ultrasonic extraction: taking about 0.2g of sample powder, sieving the sample powder by a third sieve, precisely weighing, placing the sample powder into a 50ml centrifuge tube, adding about 7ml of a mixed solution of 50% methanol and 0.5% acetic acid (1: 1), carrying out ultrasonic treatment (power 500W and frequency 40KHz) for 30 minutes, centrifuging, taking supernatant, transferring the supernatant into a 25ml measuring flask, repeatedly extracting for 2 times, washing residues by using a mixed solution of a proper amount of 50% methanol and 0.5% acetic acid (1: 1), combining extracting solutions, adding a mixed solution of 50% methanol and 0.5% acetic acid (1: 1) to scale, filtering, and taking a subsequent filtrate to obtain the product.
Reflux extraction: taking about 0.2g of sample powder, sieving the sample powder by a third sieve, precisely weighing, placing the sample powder into a 50ml centrifuge tube, adding about 7ml of a mixed solution of 50% methanol and 0.5% acetic acid (1: 1), heating and refluxing for 30 minutes, centrifuging, taking supernatant, transferring the supernatant into a 25ml measuring flask, repeatedly extracting for 2 times, washing residues by using a mixed solution of a proper amount of 50% methanol and 0.5% acetic acid (1: 1), combining extracting solutions, adding a mixed solution of 50% methanol and 0.5% acetic acid (1: 1) to a scale, filtering, and taking a subsequent filtrate to obtain the product.
And respectively carrying out sample injection analysis according to the chromatographic conditions, and calculating the total characteristic peak area/sample weighing.
The result shows that the extraction mode has less influence on the number of the extracted peaks and the total peak area/sample weighing amount of the characteristic spectrum of the cortex lycii radicis by comparing the ultrasound with the reflux, and the ultrasound is selected as the extraction mode in consideration of convenient operation, as shown in figure 2.
3.3 extraction time study
Taking 0.2g of cortex lycii radicis medicinal material powder (batch number: DGP05, sieving with a third sieve), paralleling three parts, precisely weighing, placing in a 25ml conical flask with a plug, precisely adding 25ml of a mixed solution of 50% methanol and 0.5% acetic acid (1: 1), weighing, respectively performing ultrasonic treatment (power 500W, frequency 40kHz) for 20 minutes, 30 minutes and 40 minutes, cooling, complementing lost weight, shaking uniformly, centrifuging and filtering to obtain the cortex lycii radicis medicinal material powder. And (4) carrying out sample injection analysis according to the chromatographic conditions, and calculating the specific total characteristic peak area/sample weighing.
The result shows that the influence of different extraction times on the cortex lycii characteristic spectrum is compared, the difference between the number of peaks and the total peak area/sample weighing amount of the cortex lycii characteristic spectrum in 20-40 minutes by ultrasonic treatment is small, and 30 minutes by ultrasonic treatment is selected as the extraction time in consideration of time saving, and the figure is 3.
4. Chromatographic condition optimization
4.1 determination of the detection wavelength
Preparation of a test solution: taking about 0.2g of cortex lycii radicis medicinal material powder (batch number: DGP05, sieving by a third sieve), precisely weighing, placing in a 50ml centrifuge tube, adding about 7ml of mixed solution of 50% methanol-0.5% acetic acid (1: 1), carrying out ultrasonic treatment (power 500W, frequency 40KHz) for 30 minutes, centrifuging, taking supernatant, transferring to a 25ml measuring flask, repeatedly extracting for 2 times, washing residues with a proper amount of mixed solution of 50% methanol-0.5% acetic acid (1: 1), combining extracting solutions, adding a mixed solution of 50% methanol-0.5% acetic acid (1: 1) to a scale, filtering, taking a subsequent filtrate as a test solution, carrying out sample injection analysis according to the chromatographic conditions, and recording an absorption spectrum within the range of 200-400 nm.
The experimental results are as follows: by comparing chromatograms of different detection wavelengths, it is found that when 280nm is selected as the detection wavelength, peak response and information content are richer, interference is smaller, and separation degree is better, so that the 280nm is selected as the detection wavelength in comprehensive consideration, as shown in fig. 4 and 5.
4.2 optimization of the Mobile phase
Taking the same sample (batch number: DGP05) solution, selecting four mobile phases of methanol-0.15% trifluoroacetic acid aqueous solution, acetonitrile-0.15% trifluoroacetic acid aqueous solution, methanol-0.1% formic acid aqueous solution and methanol-0.1% phosphoric acid aqueous solution respectively, and comparing the separation effects of different mobile phases according to the chromatographic conditions except the proportion of the mobile phases by sample injection determination.
The experimental result shows that the separation degree and peak shape of each peak of the cortex lycii characteristic map are greatly influenced by different acid water solution flows compared with the chromatograms of 4 different mobile phases, the peak shape and separation effect of the 0.15% trifluoroacetic acid aqueous solution are better, the number of the peaks is more, and the methanol-0.15% trifluoroacetic acid aqueous solution is selected as the cortex lycii characteristic map mobile phase in comprehensive consideration, as shown in fig. 6.
4.3 investigation of different column temperatures
Sampling the same sample (lot number: DGP05) solution with the same chromatographic column and liquid chromatograph at different column temperatures (30 deg.C, 35 deg.C, 40 deg.C) according to the above chromatographic conditions, and recording chromatogram.
The result shows that the different column temperatures have great influence on the separation degree of each peak of the cortex lycii characteristic spectrum by comparing 3 chromatograms with different column temperatures, wherein the separation effect is better at the column temperature of 40 ℃, and the column temperature of the cortex lycii characteristic spectrum is selected to be 40 ℃ in comprehensive consideration, which is shown in figure 7.
5. Stability survey
Taking the same batch of samples, preparing sample solutions according to a preparation method of the test solution, respectively inserting needles for 0h, 2h, 4h, 8h, 12h, 16h and 24h, determining for 24 hours, respectively injecting 1 mu L sample, and inspecting the consistency of the relative retention time of the characteristic peaks, wherein the results are shown in Table 2.
TABLE 2 stability test results (relative Retention time)
TABLE 3 stability test results (relative peak area)
6. Determination of cortex lycii radicis medicinal material characteristic map
6.1 taking 18 batches of cortex lycii radicis (medlar) medicinal material samples, wherein the information of each batch of 0.2g of the medlar medicinal material samples in 18 batches is shown in the following table 4, measuring a sample characteristic spectrum according to the preparation method and chromatographic conditions, and the measuring results are shown in tables 5 and 6.
TABLE 418 batch cortex Lycii sample information
TABLE 518 batch cortex Lycii medicinal material characteristic map (relative retention time)
TABLE 618 GUADOUQI medicinal material characteristic map (relative peak area)
As a result: the retention time RSD of the characteristic peaks in the 18 samples was less than 3%. The relative peak areas of the characteristic peaks are greatly different, and the RSD value is between 20.02% and 82.51%, which shows that the relative peak area ratios of the components contained in different samples are greatly different, and the peak areas of the characteristic peaks reflect the quality conditions of the different samples to a certain extent.
6.2 construction of cortex Lycii reference characteristic map
The 18 batches of cortex lycii radicis medicinal material feature spectra adopt a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system, and through matching, 7 common peaks exist, and the spectra are shown in a figure 8. Establishing cortex lycii radicis medicinal material comparison characteristic map as shown in figure 9.
Determining that 7 characteristic peaks should be presented in the characteristic spectrum of the cortex lycii radicis medicinal material, and corresponding to 7 characteristic peaks in the chromatogram of the reference material of the reference medicament; calculating the relative retention time of each characteristic peak and the S peak, wherein the relative retention time is within +/-10% of a specified value, and the specified value comprises: peak 1 was 0.473, Peak 2 was 0.833, Peak 4 was 1.067, Peak 5 was 1.508, Peak 6 was 1.781, Peak 7 was 1.996.
It should be noted that the relative retention time of each characteristic peak and the S peak should be within ± 10% of the specified value, which includes: peak 1 was 0.473, peak 2 was 0.833, peak 4 was 1.067, peak 5 was 1.508, peak 6 was 1.781, peak 7 was 1.996, with the following meanings:
the relative retention time of characteristic peak 1 to the S peak should be within ± 10% of 0.473;
the relative retention time of characteristic peak 2 to the S peak should be within ± 10% of 0.833;
the relative retention time of characteristic peak 4 to the S peak should be within ± 10% of 1.067;
the relative retention time of characteristic peak 5 to the S peak should be within ± 10% of 1.508;
the relative retention time of the characteristic peak 6 and the S peak should be within + -10% of 1.781;
the relative retention time of characteristic peak 7 to the S peak should be within ± 10% of 1.996.
6.3 identification of different primordial substances and its counterfeit cortex Periplocae Radicis
6.3.1 comparison of characteristic spectra of different primordial (fructus Lycii, Ningxia fructus Lycii) cortex Lycii
The method comprises the steps of taking a cortex lycii radicis medicinal material DGP21, identifying the cortex lycii radicis medicinal material DGP21 as Ningxia wolfberry, preparing a sample solution according to a sample solution preparation method, injecting 1 mu L to obtain a characteristic map of the cortex lycii radicis (Ningxia wolfberry), comparing the characteristic map of the cortex lycii radicis (Ningxia wolfberry) with a cortex lycii radicis medicinal material comparison characteristic map generated by 18 batches of the cortex lycii radicis medicinal material, and displaying 7 characteristic peaks corresponding to chromatographic peaks of the cortex lycii radicis medicinal material comparison characteristic map, wherein peaks 3 and 4 are consistent with reference substance peak retention time of a reference substance, the relative peak areas of the characteristic peaks are as follows, wherein peak 1 is 0.103, peak 2 is 0.104, peak 3 is 1.000, peak 4 is 0.105, peak 5 is 0.047, peak 6 is 0.013, peak 7 is 0.019.18, the relative range of the relative peak area of the cortex lycii radicis 0.008-0.050, and the relative peak area of the relative peak of the cortex lycii radicis not different from the relative peak area of the initial cortex lycii radicis determined.
6.3.2 comparison of characteristic spectra of cortex Lycii and cortex Periplocae Radicis
The method comprises the steps of preparing a test solution according to a preparation method of the test solution by taking a cortex periplocae medicinal material XJP01 and cortex lycii radicis medicinal materials DGP14 and DGP15 doped with cortex periplocae, and injecting 1 mu L to obtain a characteristic map of the cortex periplocae and the cortex lycii radicis doped with the cortex periplocae and comparing the characteristic map with a cortex lycii radicis medicinal material comparison characteristic map generated by 18 batches of cortex lycii radicis medicinal materials.
6.4 identification of characteristic peaks of characteristic spectrum of cortex Lycii
6.4.1 instruments and reagents
UP L C-MS-MS high resolution mass spectrometer SYNAPT G2 HDMS ultra-high performance liquid phase time of flight high resolution mass spectrometry combined system (Waters Corporation, Milford, MA, USA), data processing system is Marker L ynx 4.1.1 workstation (Waters, Manchester, U.K.), AB135-S electronic analytical balance (Mettler-Torpedo), KQ-118B ultrasonic oscillator (Kunshan ultrasonic instruments, Inc.);
reagent testing: chromatographic acetonitrile and methanol were purchased from j.t.baker (phillips burg, NJ, USA), chromatographic grade formic acid, phosphoric acid, leucine enkephalin from Sigma Aldrich (MO, USA), and experimental ultrapure water (18.2M Ω) was prepared using a Milli-Q water purification system (Millipore, France).
6.4.2 chromatographic and Mass Spectrometry conditions
Chromatographic conditions are as follows: "chromatographic conditions" as in the examples.
Mass spectrum conditions: quadrupole series time-of-flight mass spectrometry; the mass spectrum parameters are as follows: ESI mode: positive and negative ion scanning, mass number mass-to-charge ratio range: 50-1200 m/z; flow rate N of drying gas2600L/h (positive ion mode)/600L/h (negative ion mode), a scanning time of 0.5s, a scanning time interval of 0.02s, a drying gas temperature of 400 ℃ (positive ion mode)/350 ℃ (negative ion mode), a capillary voltage of 3.0kV (positive ion mode)/2.5 kV (negative ion mode), a cone hole voltage of 40V, a data acquisition form of continuum, a sensitivity of normal, and a dynamic range of extended.
6.4.3 preparation of reference solutions: precisely weighing appropriate amount of cortex Lycii Frutus extract A and cortex Lycii Frutus extract B, respectively, dissolving in methanol to obtain control stock solutions with mass concentration of 1mg/ml, and filtering with 0.22 μm microporous membrane.
6.4.4 preparation of test solutions: taking about 20mg of sample, grinding into fine powder, placing into a 5ml centrifuge tube, adding 2ml of 50% methanol and 0.5% acetic acid water solution, performing ultrasonic treatment for 20min, centrifuging, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain test solution with concentration of about 10 mg/ml.
6.4.5 assay
Precisely sucking the reference solution and the sample solution respectively at a ratio of 1 μ L/2 μ L (positive/negative ion mode), injecting into a UP L C-MS-MS high-resolution mass spectrometer, and measuring.
6.4.6 analysis of results: according to the primary and secondary mass spectra and the analysis of related databases and the results of literature search, and comparison with a standard product, determining that the peak 3 is Kukoamine B and the peak 4 is Kukoamine A. The results are shown in FIGS. 10-11, Table 7.
TABLE 7 Peak 3-4 materials and structures
Example 2: detection method for identifying different primordia and pseudo-cortex periplocae of cortex lycii radicis medicinal material
The method comprises the following steps:
(1) preparation of test solution
Taking a cortex lycii radicis medicinal material sample 1 to be detected (batch number: DGP01), a sample 2 to be detected (batch number: DGP15) and a sample 3 to be detected (batch number: DGP21) powder (sieved by a three-number sieve) to be precisely weighed, putting the powder into a 50ml centrifuge tube, adding about 7ml of a mixed solution of 50% methanol-0.5% acetic acid (1: 1), carrying out ultrasonic treatment (power 500W and frequency 40KHz) for 30 minutes, centrifuging, taking supernatant, transferring the supernatant into a 25ml measuring flask, repeatedly extracting for 2 times, washing residues with a proper amount of mixed solution of 50% methanol-0.5% acetic acid (1: 1), combining extracting solutions, adding a mixed solution of 50% methanol-0.5% acetic acid (1: 1) to scales, filtering, and taking a subsequent filtrate to obtain the cortex lycii radicis medicinal material.
(2) And (3) measuring, namely precisely absorbing the test solution with 1 mu L each, injecting the test solution into a liquid chromatograph, and measuring to obtain the characteristic map of the cortex lycii radicis medicinal material to be measured, wherein the chromatographic conditions are the same as the item of the 2.1 chromatographic conditions in the example 1.
(3) And a result judgment method comprises the following steps: calculating relative peak areas of the peak 2, the peak 5 and the S peak, and judging the relative peak areas and a specified range; the specified ranges are: peak 2 should not exceed 0.050, Peak 5 should not exceed 0.297.
If the peak 2 and the peak 5 are in the specified range, judging the Chinese wolfberry root-bark, wherein the Chinese wolfberry root-bark belongs to the original Chinese wolfberry root-bark;
if the peak 2 is not in the specified range and the peak 5 is in the specified range, the Chinese wolfberry is judged as the cortex lycii radicis, and the original root of the Chinese wolfberry belongs to Ningxia Chinese wolfberry;
and if the peak 5 is more than 0.800, judging the fake product of the cortex periplocae.
(4) Comparing the relative peak area peaks 2 and 5 of the characteristic spectrum of the cortex lycii radicis medicinal material to be detected with the standard specification:
relative peak area of sample 1 to be tested (lot number: DGP 01): peak 2 was 0.022, and peak 5 was 0.110. Wherein peak 2 is less than 0.050, peak 5 is less than 0.297, and peak 2 and peak 5 are in the range meeting the regulation, so that the Chinese wolfberry is judged as the Chinese wolfberry and belongs to the original Chinese wolfberry.
Relative peak area of sample 2 to be tested (lot number: DGP 15): peak 5 is 23.777. Wherein peak 5 is greater than 0.800, and the product is judged to be counterfeit Chinese silkvine root bark.
Relative peak area of sample 3 to be tested (lot number: DGP 21): peak 1 was 0.103, peak 2 was 0.104, peak 3 was 1.000, peak 4 was 0.105, peak 5 was 0.047, peak 6 was 0.013, and peak 7 was 0.019. Wherein the peak 2 is more than 0.050, and the peak 5 is less than 0.297, and the Chinese wolfberry is judged as the lycium bark, which belongs to the original Chinese wolfberry.
To sum UP, the method (1) extracts cortex lycii radicis by using a methanol-acetic acid mixed solution, and injects the cortex lycii radicis into a liquid chromatograph, the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, methanol as a mobile phase A, and a trifluoroacetic acid aqueous solution as a mobile phase B to perform gradient elution, so as to establish a UP L C characteristic map of the cortex lycii radicis medicinal material, and simultaneously, UP L C-MS-MS is adopted to confirm basic research of related substances, so as to calibrate 2 characteristic peaks.
(2) The method utilizes the UP L C characteristic spectrum of the cortex lycii radicis medicinal material, and can effectively identify different primordia and the pseudo-product cortex periplocae of the cortex lycii radicis medicinal material by calculating the relative retention time and the relative peak area of each characteristic peak of the chromatogram of the test sample.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method for constructing a characteristic map of cortex lycii radicis UP L C is characterized by comprising the following steps:
(1) respectively extracting cortex Lycii reference material, cortex Lycii B reference substance, and kukoamine A reference substance with solvent to obtain reference substance solution;
(2) extracting cortex Lycii powder with methanol-acetic acid mixed solution to obtain sample solution;
(3) injecting preset amounts of reference substance solution and test solution into a liquid chromatograph, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, methanol as a mobile phase A and trifluoroacetic acid aqueous solution as a mobile phase B for gradient elution to establish a cortex lycii radicis UP L C characteristic map.
2. The method for constructing a characteristic map of cortex lycii radicis UP L C according to claim 1, wherein the step (1) comprises:
taking cortex lycii radicis as a reference medicinal material, precisely weighing, placing into a volumetric flask with a plug, adding a mixed solution of 50% methanol and 0.5% acetic acid, carrying out ultrasonic treatment, cooling, adding a mixed solution of 50% methanol and 0.5% acetic acid, centrifuging, filtering, and taking a subsequent filtrate as a reference medicinal material solution;
accurately weighing cortex Lycii B reference substance and kukoamine reference substance, dissolving in methanol, and making into mixed solution containing 80-100 μ g of kukoamine per 1ml as reference substance solution.
3. The method for constructing a characteristic map of cortex lycii radicis UP L C according to claim 2, wherein the sampling amount of the cortex lycii radicis control drug is 0.5-1g, the volume of the mixed solution of 50% methanol-0.5% acetic acid is 20-30ml first, and the volume of the mixed solution of 50% methanol-0.5% acetic acid is 5-10ml after ultrasonic treatment;
in the mixed solution of 50% methanol and 0.5% acetic acid, the dosage ratio of 50% methanol to 0.5% acetic acid is (1-2) to (1-2);
the power of the ultrasonic treatment is 400-600W, and the frequency is 30-50 KHz.
4. The method for constructing a characteristic map of cortex lycii radicis UP L C according to claim 1, wherein the step (2) comprises:
taking cortex lycii radicis powder, sieving, precisely weighing, placing into a centrifuge tube, adding a mixed solution of 50% methanol and 0.5% acetic acid, carrying out ultrasonic treatment and centrifugation, taking supernatant, transferring into a measuring flask, repeatedly extracting for 2-3 times, washing residues with a proper amount of mixed solution of 50% methanol and 0.5% acetic acid, combining extracting solutions, adding a mixed solution of 50% methanol and 0.5% acetic acid, filtering, and taking a subsequent filtrate to obtain a sample solution.
5. The method for constructing characteristic map of cortex lycii radicis UP L C according to claim 4, wherein in the mixed solution of 50% methanol and 0.5% acetic acid, the ratio of 50% methanol to 0.5% acetic acid is (0.5-1.5): 1;
the power of the ultrasonic treatment is 400-600W, and the frequency is 30-50 KHz.
6. The method for constructing a characteristic map of cortex Lycii UP L C as claimed in claim 1, wherein in step (3), octadecylsilane chemically bonded silica is used as filler, the column length is 100mm, the inner diameter is 2.1mm, the particle size is 1.7 μm, the column temperature is 40-42 ℃, the flow rate is 0.25ml per minute, and the detection wavelength is 278-;
the mobile phase B is trifluoroacetic acid aqueous solution with the concentration of 0.05-0.3%.
7. The method for constructing a characteristic map of cortex lycii radicis UP L C according to claim 1 or 6, wherein the gradient elution is carried out according to the following procedure:
0-5 min, wherein the mobile phase A is 8% → 14%, and the mobile phase B is 92% → 86%;
5-14 min, wherein the mobile phase A is from 14% → 25%, and the mobile phase B is from 86% → 75%;
14-23 min, wherein the content of mobile phase A is 25% → 50%, and the content of mobile phase B is 75% → 50%;
23-26 min, wherein the mobile phase A is 50% → 90%, and the mobile phase B is 50% → 10%;
26-26.1 min, wherein the mobile phase A is 90% → 8%, and the mobile phase B is 10% → 92%;
26.1-30 min, 8% of mobile phase A and 92% of mobile phase B.
8. The method of claim 1, wherein the characteristic map of Gotu-Qi UP L C comprises 7 characteristic peaks, wherein the peak corresponding to kukotin reference is S peak, and the relative retention time of each characteristic peak to S peak is within + -10% of the predetermined value, and the predetermined value comprises 0.473 for Peak 1, 0.833 for Peak 2, 1.067 for Peak 4, 1.508 for Peak 5, 1.781 for Peak 6, and 1.996 for Peak 7.
9. A detection method for identifying different primordia and pseudo-product cortex periplocae of cortex lycii radicis medicinal materials is characterized by comprising the following steps:
(1) respectively extracting cortex Lycii reference material, cortex Lycii B reference substance, and kukoamine A reference substance with solvent to obtain reference substance solution;
(2) extracting cortex Lycii powder with methanol-acetic acid mixed solution to obtain sample solution;
(3) injecting a preset amount of reference substance solution into a liquid chromatograph, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, methanol as a mobile phase A and trifluoroacetic acid aqueous solution as a mobile phase B for gradient elution to establish a cortex lycii radicis UP L C characteristic map;
(4) injecting a preset amount of a test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filler, methanol as a mobile phase A and a trifluoroacetic acid aqueous solution as a mobile phase B by the liquid chromatograph to obtain a test chromatogram;
(5) comparing the chromatogram of the test sample with the characteristic spectrum of the UP L C of the cortex lycii radicis, and judging different primordia and counterfeit products of the cortex lycii radicis medicinal material according to the relative retention time of each characteristic peak of the chromatogram of the test sample and/or the relative peak area of each characteristic peak.
10. The detecting method for identifying different primordia of cortex lycii radicis medicinal material and its pseudo-product cortex periplocae as claimed in claim 9, wherein the chromatogram of the sample shows 7 characteristic peaks corresponding to 7 characteristic peaks in the characteristic map of UP L C of cortex lycii radicis, wherein the peak corresponding to the lycium barbarum reference is S peak, the relative retention time of each characteristic peak and S peak of the chromatogram of the sample is calculated, and the relative retention time should be within ± 10% of the specified values, the specified values include peak 1 of 0.473, peak 2 of 0.833, peak 4 of 1.067, peak 5 of 1.508, peak 6 of 1.781 and peak 7 of 1.996;
calculating relative peak areas of the peak 2, the peak 5 and the S peak, and judging according to the relative peak areas and a specified range, wherein the specified range is as follows: peak 2 should not exceed 0.050 and peak 5 should not exceed 0.297;
if the peak 2 and the peak 5 are in the specified range, judging the Chinese wolfberry root-bark, wherein the Chinese wolfberry root-bark belongs to the original Chinese wolfberry root-bark;
if the peak 2 is not in the specified range and the peak 5 is in the specified range, the Chinese wolfberry is judged as the cortex lycii radicis, and the original root of the Chinese wolfberry belongs to Ningxia Chinese wolfberry;
and if the peak 5 is more than 0.800, judging the fake product of the cortex periplocae.
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