CN111198236A - Method for identifying honeysuckle and lonicera confusa by utilizing high performance liquid chromatography and liquid chromatography-mass spectrometry - Google Patents
Method for identifying honeysuckle and lonicera confusa by utilizing high performance liquid chromatography and liquid chromatography-mass spectrometry Download PDFInfo
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Abstract
The invention belongs to the technical field of identification of source varieties of traditional Chinese medicinal materials, and relates to a method for identifying honeysuckle and lonicera confusa by utilizing high performance liquid chromatography and liquid chromatography-mass spectrometry. By adopting the method, when the test sample is one or two of honeysuckle or lonicera confusa, the weight percentages of the strychnic acid and the chlorogenic acid in the dry test sample are detected by high performance liquid chromatography, and the ratio of the weight percentages of the chlorogenic acid and the strychnic acid is calculated; detecting the weight percentage of the teasel saponin B in the dry sample by LC-MS; the honeysuckle is identified by utilizing the weight percentage ratio of chlorogenic acid to the strychnic acid and the weight percentage of the teasel saponin B. The method is not limited by the properties of the test product, can be used for identifying the single medicinal material or the mixture of the honeysuckle and the lonicera confusa, and can also be used for identifying the single medicinal material or the mixture of the honeysuckle and the lonicera confusa in Chinese patent medicine, so that the identification accuracy is effectively improved, and the basis for identifying the truth is provided for the production, circulation and use processes of the medicine.
Description
Technical Field
The invention belongs to the technical field of identification of source varieties of traditional Chinese medicinal materials, relates to a method for identifying honeysuckle and lonicera confusa, and particularly relates to a method for identifying the honeysuckle and the lonicera confusa by utilizing high performance liquid chromatography and liquid chromatography-mass spectrometry.
Background
Honeysuckle and lonicera confusa are listed as two medicinal materials from the pharmacopoeia of the people's republic of China 2005 edition. Because the species of the original plants of the lonicera confusa and the lonicera confusa are similar, the appearance and the shape of the medicinal materials are similar and difficult to distinguish, the honeysuckle and the lonicera confusa in the market are unclear in source and disordered in variety, because the lonicera confusa honeysuckle phenomenon frequently exists in the market due to large yield and low price, but the substance basis of the lonicera confusa honeysuckle is greatly different, so that the curative effect of the medicinal materials and the preparation thereof is directly influenced, more seriously, the lonicera confusa has more saponin content than the honeysuckle, and the saponin can cause the hemolysis of the injection, and the life safety of a user is directly harmed.
The existing methods for identifying the varieties of the honeysuckle and the lonicera confusa have some defects. For example, the traditional appearance and shape identification depends on experience and has large subjectivity, and particularly, the mixed product or the crushing state of the two products in the market cannot be identified; the DNA molecular identification method has complex technology, ten thousand-level experiment environment, high requirement, high cost, long inspection period and poor repeatability caused by a plurality of influencing factors. Pharmacological methods have high verification cost, long cycle and large errors caused by a plurality of influence factors, and although the methods can distinguish closely related varieties to a certain extent, the methods cannot evaluate the quality of the drug effect. Especially, the above methods cannot identify the adulteration phenomenon in the mixture in a crushed state or the Chinese patent medicine.
Patent CN 103173532B (a method for identifying honeysuckle and lonicera confusa and application thereof) relates to a method for identifying honeysuckle and lonicera confusa and application thereof. The method comprises PCR amplification of ITS2 nucleotide sequence, including 1) extraction of sample DNA; 2) PCR amplifying a fragment containing the ITS2 sequence of ribosomal DNA; 3) splicing the amplified products to obtain a complete ITS2 gene spacer region; 4) an NJ tree of ITS2 sequences was constructed for the samples. Patent CN 104419754 a (a method for rapidly identifying the authenticity of honeysuckle) discloses a method for identifying honeysuckle and common counterfeit lonicera confusa thereof, which is to analyze the marker nucleic acid sites of honeysuckle and lonicera confusa by using a high resolution dissolution curve technique. The method confirms the type of the marker nucleic acid locus by carrying out DNA extraction, PCR amplification and HRM analysis on a sample of the honeysuckle to be tested, and further identifies the authenticity of the honeysuckle. The method belongs to a DNA molecule identification method, requires PCR amplification, and has complex technology and high cost.
The patent CN 105277721B (a method for identifying lonicera confusa and lonicera confusa raw materials of a preparation for treating stomatitis) discloses an identification method for identifying lonicera confusa and lonicera confusa, which comprises the following steps of taking lonicera confusa or a preparation containing lonicera confusa raw materials as a control, constructing an acute oral inflammation model, and identifying and distinguishing lonicera confusa or a compound preparation thereof by comparing the influence of a test sample and a control standard on the expression levels of inflammatory factors TNF- α, IL-6, IL-8 and IL-10 in cells of the acute oral inflammation model.
Although the prior art reports that a single index of the content ratio of chlorogenic acid to mucronate or the content of the teasel saponin B is taken as an identification basis, the application of the method has the premise that a character observation sample is a single medicinal material decoction piece, and the method is limited to the identification of mixed medicinal materials or medicinal materials of which the characters cannot be observed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for identifying honeysuckle and lonicera confusa by using high performance liquid chromatography and liquid chromatography-mass spectrometry. The method takes the weight percentage ratio of chlorogenic acid to semen strychni Pulveratum and the weight percentage of dipsacus asperoides saponin B as indexes, and adopts high performance liquid chromatography and LC-MS to detect the weight percentage ratio of chlorogenic acid to semen strychni Pulveratum and the weight percentage of dipsacus asperoides saponin B in the honeysuckle and lonicera confusa dry products, so as to identify the honeysuckle and the lonicera confusa.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for identifying flos Lonicerae and flos Lonicerae by high performance liquid chromatography and LC-MS comprises detecting the weight percentage of strychnic acid and chlorogenic acid in dry sample by high performance liquid chromatography when the sample is one or two of flos Lonicerae or flos Lonicerae, and calculating the ratio of chlorogenic acid to strychnic acid; detecting the weight percentage of the teasel saponin B in the dry sample by liquid chromatography-mass spectrometry. The honeysuckle and the lonicera confusa are identified by utilizing the weight percentage ratio of chlorogenic acid to the strychnic acid and the weight percentage of the dipsacoside B.
And when the weight percentage ratio of the chlorogenic acid to the strychnic acid in the dry sample is less than 2 and the weight percentage of the teasel saponin B is 0, judging the honeysuckle.
The method for detecting the content of chlorogenic acid and mucronate comprises the following steps: injecting the reference substance solution and the test solution into a high performance liquid chromatograph, measuring peak areas at the wavelength of 210-360 nm, calculating by adopting an external standard method or a standard curve method to obtain the weight percentage of chlorogenic acid and secoisolariciresinol diglucoside in the test solution, and then calculating the ratio of the chlorogenic acid to the secoisolariciresinol diglucoside in the test solution; the detection method of the content of the dipsacoside B comprises the following steps: and (3) injecting the reference substance solution and the test sample solution into a liquid chromatograph-mass spectrometer, measuring the area of the characteristic ions by liquid phase separation and mass spectrum, and calculating by adopting an external standard method or a standard curve method to obtain the weight percentage of the dipsacoside B in the test sample.
Preferably, (1) the measurement conditions of the high performance liquid chromatography are: column C18, 4.6mm X250 mm, 5 μm; the wavelength of the secoisolariciresinol is 240nm, and the wavelength of the chlorogenic acid is 324 nm; taking acetonitrile as a mobile phase A, taking an acid water solution with the pH value of 2.5 as a mobile phase B, and adopting gradient elution, wherein the mobile phase A is 0-20 min, and the volume percentage is 5% → 15%; 20-30 min, 15% → 28% by volume; the balance is mobile phase B.
(2) The determination conditions of the LC-MS are as follows: liquid phase partial chromatography column: c18 chromatographic column, 10mm × 2.1mm, 1.7 μm; column temperature: 45 ℃; flow rate: 0.3 mL/min; sample introduction volume: 1 mu L of the solution; mobile phase: taking a 0.1% formic acid (containing 10mmol/L ammonium formate) solution as a mobile phase A, taking acetonitrile as a mobile phase B, and adopting gradient elution, wherein the mobile phase A is 0-3 min, 92% by volume, 3-5 min, 92% by volume, → 35%, 5-8 min, 35% by volume, → 15%, 8-10 min, and 15% by volume; the rest is mobile phase B; mass spectrometry ion source mode: an Electrospray (ESI) ion source; scanning mode: selective ion detection (SRM); ionization voltage: 3500V; temperature of the atomization chamber: 200 ℃; sheath gas: high-purity nitrogen gas, 35L/min; auxiliary gas: high-purity nitrogen gas, 10L/min; ion transfer tube temperature: 290 ℃; collision gas: high-purity argon gas; the anion is adopted to collect the teasel saponin B, the mass-to-charge ratio of the teasel saponin B is 1075.0, the mass-to-charge ratio of the mother ion is 749.2, and the collision energy is 40 eV.
The acid is phosphoric acid or formic acid or acetic acid or phosphate.
Further, the preparation method of the test solution in the method for detecting the content of chlorogenic acid and mucronate in (1) comprises the following steps: taking 0.25g of test sample powder, precisely weighing, placing in a conical flask with a plug, precisely adding 10-50mL of solvent, carrying out ultrasonic treatment for 15-60 minutes, shaking up, and filtering to obtain the product.
(2) The preparation method of the test solution in the detection method of the content of the dipsacoside B comprises the following steps: pulverizing the sample into fine powder, precisely weighing about 0.25g, placing into 100mL conical flask with plug, precisely adding 85% methanol 20-50mL, precisely weighing, ultrasonically extracting at room temperature for 30min, cooling, precisely weighing, adding 85% methanol to make up the weight, filtering, and filtering with 0.22 μm filter membrane to obtain the final product. The 85% methanol is a mixed solution of methanol and ultrapure water, and the volume percentage of the methanol is 85%.
The power of the ultrasound is 250W, and the frequency is 35 kHz.
The solvent is water or 0-50% ethanol solution or 0-50% methanol solution or a mixture thereof, wherein the 0-50% ethanol/methanol solution is a mixed solution of ethanol/methanol and ultrapure water, and the percentage by volume is percentage.
The invention has the beneficial effects that:
(1) according to the invention, the high performance liquid chromatography is used for detecting the weight percentage of chlorogenic acid and the secoisolariciresinol diglucoside in the dry sample to be tested and calculating the ratio of the chlorogenic acid to the secoisolariciresinol diglucoside, the liquid chromatography-mass spectrometry is used for detecting the weight percentage of the dipsacus asperoides saponin B, and the two indexes of the ratio of the chlorogenic acid to the secoisolariciresinol diglucoside and the weight percentage of the dipsacus asperoides saponin B are adopted for identifying the sample to be tested, so that.
(2) The method is used for identifying the honeysuckle and lonicera confusa mixed product by taking the content ratio of chlorogenic acid to secoisolariciresinol diglucoside and the content of dipsacus asperoides saponin B as evaluation indexes without observing whether a sample is a single medicinal material decoction piece by observing the characters firstly, and achieves the purpose of identifying aiming at the condition that a lot of crushed or partially crushed mixed medicinal materials, medicinal materials in Chinese patent medicines and the like cannot observe the characters by naked eyes in the market.
(3) In the invention, the weight percentage ratio of the chlorogenic acid to the secoisolariciresinol diglucoside is strong in regularity, and is easy to observe and judge by being converted into an integer; the liquid chromatography-mass spectrometry combination has stronger specificity, higher precision, higher sensitivity and higher accuracy, and provides a basis for identifying the truth of the medicine in the production, circulation and use processes.
Drawings
FIG. 1 is a chlorogenic acid control map;
FIG. 2 is a spectrum of chlorogenic acid of flos Lonicerae of Henan of China;
FIG. 3 is a spectrum of chlorogenic acid of flos Lonicerae of Hebei;
FIG. 4 is a spectrum of chlorogenic acid of flos Lonicerae of different batches;
FIG. 5 is a graph of chlorogenic acid of Lonicera confusa in different batches;
FIG. 6 is a spectrum of a strychnic acid reference;
FIG. 7 is the acid spectrum of the nux vomica of different batches of honeysuckle in Henan;
FIG. 8 is the acid spectrum of the nux vomica of different batches of Lonicera japonica Thunb in Hebei;
FIG. 9 is the acid spectrum of the Lonicera Japonica flos of different batches in Shandong;
FIG. 10 is the acid spectrum of the nux vomica of different batches of lonicera confusa;
FIG. 11 is the map of the control;
FIG. 12 is the spectrum of the saponin B of different batches of the honeysuckle teasel roots in Henan;
FIG. 13 is the spectrum of the saponin B of different batches of Lonicera japonica Thunb in Hebei;
FIG. 14 shows the spectrum of saponin B of different batches of Lonicera japonica Thunb;
FIG. 15 shows the spectrum of the saponins B of Hibiscus Sabdariffa Hirsonia in different batches.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.
Example 1 establishing a detection method
1. Apparatus and materials
1.1 instruments
TSQ-TUANTOM liquid chromatograph/mass spectrometer (THERMO FISH Co., Ltd.), electrospray ion source (ESI) mass spectrometer, waters C18 chromatography column (2.1X 10mm, 1.7 μm);
agilent 1260 quaternary HPLC, Agilent G4212B diode array detector, Agilent Eclipse XDB-C18Chromatography column (250 mm. times.4.6 mm, 5 μm);
KH500B ultrasonic cleaner (GRAD, kunshan), bright color D24UV ultrapure water system (millipore china).
1.2 materials and reagents
Acetonitrile (Fisher Scientific) is in chromatographic grade, water is in first-grade ultrapure water, and other reagents such as absolute ethyl alcohol (COMIO) are in analytical grade; formic acid and ammonium formate are in mass spectrum level.
The seconux vomica acid reference substance is purchased from Shanghai Shidande Standard technical service Co., Ltd, and has the following batch number: 60077-46-5;
the chlorogenic acid reference substance is purchased from China institute for testing biological products of drugs, and has a batch number: 110753-200413.
The teasel root saponin B reference substance is purchased from China pharmaceutical biological products institute, and the batch number is as follows: 111685-200401.
2. Test method
2.1 chromatographic conditions
(1) The determination conditions of the high performance liquid chromatography are as follows: column C18, 4.6mm X250 mm, 5 μm; wavelength 240, 324 nm; using acetonitrile as a mobile phase A, using an acid water solution with the pH value of 1.5-4 as a mobile phase B, and adopting gradient elution, wherein the mobile phase A is 0-20 min, and the volume percentage is 5% → 15%; 20-30 min, 15% → 28% by volume; the balance is mobile phase B.
(2) The determination conditions of the LC-MS are as follows: liquid phase partial chromatography column: c18 chromatographic column, 10mm × 2.1mm, 1.7 μm; column temperature: 45 ℃; flow rate: 0.3 mL/min; sample introduction volume: 1 mu L of the solution; mobile phase: taking a 0.1% formic acid (containing 10mmol/L ammonium formate) solution as a mobile phase A, taking acetonitrile as a mobile phase B, and adopting gradient elution, wherein the mobile phase A is 0-3 min, 92% by volume, 3-5 min, 92% by volume, → 35%, 5-8 min, 35% by volume, → 15%, 8-10 min, and 15% by volume; the balance is mobile phase B. Mass spectrometry ion source mode: an Electrospray (ESI) ion source; scanning mode: selective ion detection (SRM); ionization voltage: 3500V; temperature of the atomization chamber: 200 ℃; sheath gas: high-purity nitrogen gas, 35L/min; auxiliary gas: high-purity nitrogen gas, 10L/min; ion transfer tube temperature: 290 ℃; collision gas: high-purity argon gas; the anion is adopted to collect the teasel saponin B, the mass-to-charge ratio of the teasel saponin B is 1075.0, the mass-to-charge ratio of the mother ion is 749.2, and the collision energy is 40 eV.
2.2 preparation of control solutions
2.2.1 preparation of Dipsacaceae saponin B reference substance
Accurately weighing 0.00558g of the dipsacoside reference substance, placing in a 5mL volumetric flask, adding 85% methanol to dilute to constant volume to scale, shaking up, and making into a dipsacoside reference substance stock solution.
Precisely measuring 100 μ L of radix Dipsaci saponin B reference substance stock solution, placing in 10mL volumetric flask, adding 85% methanol to dilute to scale, and shaking to obtain radix Dipsaci saponin B reference substance solution I.
Precisely sucking 2ml to 10ml of the teasel saponin B reference substance solution I, adding 85% methanol to dilute to scale, and shaking up to obtain a teasel saponin B reference substance solution II.
The 85% methanol is a mixed solution of methanol and ultrapure water, and the volume percentage of the methanol is 85%.
2.2.2 preparation of chlorogenic acid reference solution
Precisely weighing appropriate amount of chlorogenic acid reference substance, placing in brown measuring flask, and adding 50% methanol to obtain solution containing 40 μ g per lml (preservation at below 10 deg.C). The 50% methanol is a mixed solution of methanol and ultrapure water, and the volume percentage of the methanol is 50%.
2.2.3 preparation of a control solution of secoStrychnos acid
Precisely weighing secoisolariciresinol diglucoside reference substance, and adding 20% ethanol to obtain 0.25mg/mL solution, i.e. secoisolariciresinol diglucoside reference substance solution. The 20% ethanol is a mixed solution of ethanol and ultrapure water, and the volume percentage of the ethanol is 20%.
2.3 preparation of test solutions
2.3.1 preparation of Dipsacaceae saponin B test solution
Taking about 0.25g of test sample powder (passing through a No. four sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 85% methanol, precisely weighing, ultrasonically extracting (with the power of 250W and the frequency of 35kHz) for 30min, cooling, supplementing the weight with 85% methanol, shaking up, filtering, and filtering with a 0.22 mu m filter membrane to obtain the final product.
The 85% methanol is a mixed solution of methanol and ultrapure water, and the volume percentage of the methanol is 85%.
2.3.2 preparation of test solution of secoisolariciresinol
Taking about 0.25g of test sample powder (passing through a No. four sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 20% ethanol, carrying out ultrasonic treatment (power 250W and frequency 35kHz) for 30 minutes, cooling, supplementing the weight with 20% ethanol, shaking up, and filtering to obtain the product. The 20% ethanol is a mixed solution of ethanol and ultrapure water, and the volume percentage of the ethanol is 20%.
2.3.3 preparation of chlorogenic acid test solution
Taking about 0.25g of test sample powder (passing through a No. four sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 50% methanol, carrying out ultrasonic treatment (power 250W and frequency 35kHz) for 30 minutes, cooling, supplementing the weight with 50% methanol, shaking up, and filtering to obtain the product. The 50% methanol is a mixed solution of methanol and ultrapure water, and the volume percentage of the methanol is 50%.
2.4 Studies of Dipsacus asperoides saponin B methodology
2.4.1 Linear relationship
Precisely absorbing 1, 2, 5, 10, 20 and 25 mu L of the teasel saponin B reference substance solution II, respectively performing machine analysis, recording peak areas, performing linear regression by taking the peak area Y of a standard sample as a vertical coordinate and the sample injection mass X (C, ng) as a horizontal coordinate, and calculating a regression equation and a correlation coefficient. The linear equation: Y2890.27X +1954.28 with a correlation coefficient of 0.9949. The linear range is 2.232-55.800 ng.
2.4.2 instrumental precision investigation
Sampling the Dipsacusin B reference solution I continuously for 6 times under 2.1 chromatography, recording peak area, and calculating RSD, the results are shown in Table 1.
TABLE 1 Instrument precision investigation
2.4.3 stability test in days
Sampling the extract of Lonicera macranthoides produced in Hunan under the chromatographic condition of 2.1 under LF-M-2 for 0, 4, 8, 12, 16 and 20h, recording peak areas, and calculating RSD, wherein the results are shown in Table 2.
TABLE 2 stability survey in days
2.4.4 repeatability
Taking the same batch of samples (3 Guizhou grey felt-1), taking 6 parts in parallel, preparing the test solution according to the requirements of the preparation item of the 2.3.1 dipsacoside test solution, precisely absorbing the test solution and a dipsacoside reference solution I, injecting the test solution and the dipsacoside reference solution I into a liquid chromatograph, recording the peak area, and calculating the content. The results are shown in Table 3:
TABLE 3 repeatability test investigation
The results show that: RSD 3.80%, the repeatability experiment was good.
2.4.5 spiking recovery Studies
Taking the same sample (Hebei 3-4), taking six parts in parallel, each part being about 0.25g, precisely weighing, placing in a conical flask with a plug, precisely adding 24.5mL of 85% methanol, respectively adding 0.5mL of dipsacus asperoides saponin B reference substance stock solution, precisely weighing, ultrasonically extracting for 30min, cooling, precisely weighing, supplementing the lost weight with 85% methanol, filtering, taking the subsequent filtrate, filtering with a 0.22 μm filter membrane, performing sample injection analysis according to the above conditions, recording the peak area, and calculating the recovery rate, wherein the results are shown in Table 4.
The 85% methanol is a mixed solution of methanol and ultrapure water, and the volume percentage of the methanol is 85%.
TABLE 4 recovery rates of asperosaponin B
2.5 Breast Strychnos acid methodology study
2.5.1 Linear investigation
Precisely sucking 1, 5, 10, 15, 20 and 25 μ l of the seconux vomica acid control solution (0.226mg/ml), respectively injecting into a liquid chromatograph, and recording peak area. Linear regression was performed on the sample injection mass (m, μ g) with peak area (a), the regression equation being: a is 1213C-12, and the correlation coefficient R is 1.00, which shows that the strychnic acid has good linear relationship between 0.2264 μ g and 5.66 μ g. The results are shown in Table 5.
TABLE 5 Linear relationship examination
2.5.2 precision of the Instrument
Precisely sucking 10 μ L of the seconux vomica acid reference substance solution (0.233mg/ml), injecting into a liquid chromatograph, recording peak area, calculating instrument precision, and finding out the following results in Table 6:
TABLE 6 Instrument precision investigation
The results show that: the mean peak area 2945.2, RSD 0.88%, instrument precision was good.
2.5.3 repeatability
Taking the same batch of samples (honeysuckle SD1-1 produced by Shandong), taking 6 parts in parallel, preparing the test solution under the preparation item of the 2.3.2-item strychninic acid test solution, precisely absorbing 10 mu L of each of the test solution and the 2.2.3-item strychninic acid reference solution, injecting into a liquid chromatograph, recording peak area, and calculating content. The results are shown in Table 7:
TABLE 7 repeatability test investigation
The results show that: RSD 1.83%, the repeatability of the experiment was good.
2.6 chlorogenic acid methodology Studies
2.6.1 Linear investigation
Precisely sucking 4, 8, 12, 16, 20 and 40 μ l of chlorogenic acid reference solution (0.1088mg/ml), respectively injecting into liquid chromatograph, and recording peak area. Linear regression was performed on the sample injection mass (m, μ g) with peak area (a), the regression equation being: a is 2110.0C-3.6, and the correlation coefficient R is 0.99991, so that the chlorogenic acid is in a good linear relation between 0.4352 mu g and 4.352 mu g.
The results are shown in Table 8.
TABLE 8 Linear relationship examination
2.6.2 precision of the Instrument
Precisely absorbing 10 μ L of chlorogenic acid reference solution (0.1088mg/ml), injecting into liquid chromatograph, recording peak area, calculating instrument precision, and finding out the result shown in Table 9:
TABLE 9 Instrument precision investigation
The results show that: the mean peak area 2299.2, RSD 0.43%, instrument precision was good.
2.6.3 repeatability
Taking the same batch of samples (flos Lonicerae SD1-1 produced by Shandong), taking 6 parts in parallel, preparing the sample solution according to the preparation item of the chlorogenic acid sample solution of 2.3.3, precisely absorbing 10 μ L of each of the sample solution and the chlorogenic acid reference solution, injecting into a liquid chromatograph, recording peak area, and calculating content. The results are shown in Table 10:
TABLE 10 repeatability test study
The results show that: RSD 1.73%, the repeatability of the experiment was good.
2.7 sample determination
Respectively taking 0.25g of honeysuckle or lonicera confusa sample, and carrying out sample injection analysis according to the conditions.
2.7.1 the content of the asperosaponin B in the honeysuckle of different producing areas and different batches
The major producing areas of honeysuckle are Hebei province, Henan province and Shandong province, different batches of honeysuckle in each area are respectively taken as test samples to be detected according to the detection conditions of the method, and the obtained detection results are shown in Table 11.
TABLE 11 Himalayan teasel root saponin B content in Lonicera japonica Thunb of Hebei province, Henan province and Shandong province
2.7.2 the content of the saponin B of the teasel roots in the lonicera confusa of different producing areas and different batches
The major production areas of lonicera confusa are Guizhou province, Hunan province, Guangxi province, Guangdong province and Sichuan province, different batches of lonicera confusa in different production areas are respectively taken as test products to be detected according to the detection conditions of the method, and the obtained detection results are shown in Table 12.
TABLE 12 detection results of Lonicera confusa in different provinces
The results show that:
as can be seen from tables 11-12, (1) the asperosaponin B content in flos Lonicerae is high except for individual batches, and the asperosaponin B content in flos Lonicerae is zero except for 0.03 batch produced by Hebei. Wherein the content of the asperosaponin B in the lonicera confusa in 12 Sichuan and lonicera confusa in 22 Sichuan is low and nearly zero.
(2) Most of the lonicera confusa has high content of the dipsacoside, so when the lonicera confusa is used for the honeysuckle injection, the honeysuckle and the lonicera confusa are necessary to be identified by technical means.
Example 2 verification experiment
1. The contents of strychnic acid and chlorogenic acid in different production places and batches of honeysuckle
Each batch of honeysuckle in the north Hebei province, the south Henan province and the Shandong province is taken as a test sample to be detected according to the detection conditions of the method in the example 1, and the obtained detection results are shown in table 13.
TABLE 13 detection results of Lonicera japonica Thunb in Hebei province, Henan province and Shandong province
2. The content of strychnos acid and chlorogenic acid in different production places and different batches of lonicera confusa
Various batches of lonicera confusa in Guizhou province, Hunan province, Guangxi province, Guangdong province and Sichuan province are taken as test samples and detected according to the detection conditions of the method in the example 1, and the detection results are shown in Table 14.
TABLE 14 detection results of Lonicera confusa in different provinces
3. Results and conclusions
As can be seen from tables 13-14, the content ratios of chlorogenic acid and secoisolariciresinol diglucoside in (1) flos lonicerae are both greater than 2, and the content ratios of chlorogenic acid and secoisolariciresinol diglucoside in flos lonicerae are both less than 2. (2) The content ratio of chlorogenic acid to mucronate in the honeysuckle flower, Guangdong south China honeysuckle (three batches) is larger than 2 but the value is not large.
In the analysis market, the honeysuckle is far higher than the lonicera confusa (11 months in 2019, the wholesale price of honeysuckle is 360 yuan/kg, and the wholesale price of lonicera confusa is 78 yuan/kg), so the phenomenon that lonicera confusa is sold in the market is common, but the situation that the lonicera confusa is sold is not found, the curative effect is only improved even if the situation exists, and the potential safety hazard is avoided, so the method is mainly used for preventing the lonicera confusa from being sold.
If the lonicera confusa of three batches of 12 Sichuan lonicera confusa or 22 Sichuan lonicera confusa (Shandong Baoli) is used as the honeysuckle or is mixed into the honeysuckle, only the asperosaponin B is used as an evaluation index, and the content of the asperosaponin B is very low and close to zero, so that certain misjudgment exists, but the ratio is far higher than 2 when the content ratio of chlorogenic acid to the strychnine acid is measured in an auxiliary manner, so that the identification purpose is achieved.
If honeysuckle is faked or mixed with honeysuckle in three batches of lonicera confusa, the ratio is close to 2 only by adopting the content ratio of chlorogenic acid to strychnic acid for identification, and if the amount of the faked honeysuckle is less or even lower than 2, certain misjudgment exists, and the asperosaponin B is assisted to serve as an evaluation index, so that the identification purpose is achieved.
If other lonicera confusa (except 12 Sichuan lonicera confusa or 22 Sichuan lonicera confusa (Shandong Baoli and three batches of Guangdong south honeysuckle) is used, the content ratios of the teasel saponin B, the chlorogenic acid and the strychnic acid are greatly different from the honeysuckle, so the identification purpose is easily achieved.
In order to better illustrate that the judgment rule of the invention is applicable to the mixture of honeysuckle and lonicera confusa in different proportions, the mixture of honeysuckle and lonicera confusa in different proportions is taken as a research object, the content of each component and the content ratio of chlorogenic acid to secoisolariciresinol diglucoside are respectively taken, and the result is analyzed, the experiment is as follows:
example 3 determination of mixed sample (honeysuckle and lonicera confusa mixed at a mass ratio of 1:1)
Preparation of a mixed sample: respectively taking flos Lonicerae and flos Lonicerae of equal weight according to Table 15, pulverizing, mixing well, determining as sample according to determination method of chlorogenic acid, semen Strychni Pulveratum acid, and radix Dipsaci saponin B in example 1, and calculating content ratio of chlorogenic acid and semen Strychni Pulveratum acid and radix Dipsaci saponin B content, the result is shown in Table 16.
TABLE 15 honeysuckle and Lonicera confusa mixed sample composition table (quality ratio 1:1)
| Number of mixed sample | Name of honeysuckle | Name of Lonicera confusa variety | Composition mass ratio |
| 1 | First batch of Henan, first-class | 3 Guizhou grey felt-1 | 1：1 |
| 2 | First batch of Henan, first-class | 14 flos Lonicerae of Longhui mountain of Hunan province | 1：1 |
| 3 | First batch of Henan, first-class | 29 sample honeysuckle purchased from san Guangxi province | 1：1 |
| 4 | First batch of Henan, first-class | Guangdong south honeysuckle 1 | 1：1 |
| 5 | First batch of Henan, first-class | 12 Sichuan mountain honeysuckle flower | 1：1 |
| 6 | Shandong SD1-1 | 3 Guizhou grey felt-1 | 1：1 |
| 7 | Shandong SD1-1 | 14 flos Lonicerae of Longhui mountain of Hunan province | 1：1 |
| 8 | Shandong SD1-1 | 29 sample honeysuckle purchased from san Guangxi province | 1：1 |
| 9 | Shandong SD1-1 | Guangdong south honeysuckle 1 | 1：1 |
| 10 | Shandong SD1-1 | 12 Sichuan mountain honeysuckle flower | 1：1 |
| 11 | Hebei 2-2(4) | 3 Guizhou grey felt-1 | 1：1 |
| 12 | Hebei 2-2(4) | 14 flos Lonicerae of Longhui mountain of Hunan province | 1：1 |
| 13 | Hebei 2-2(4) | 29 sample honeysuckle purchased from san Guangxi province | 1：1 |
| 14 | Hebei 2-2(4) | Guangdong south honeysuckle 1 | 1：1 |
| 15 | Hebei 2-2(4) | 12 Sichuan mountain honeysuckle flower | 1：1 |
TABLE 16 weight ratio of each component and chlorogenic acid to secoisolariciresinol diglucoside when flos Lonicerae and flos Lonicerae are mixed at 1:1
The results show that:
(1) in this example: in the No. 4 mixed sample 'Henan Ying first batch-Guangdong south China Lonicera 1', the content ratio of chlorogenic acid to strychnic acid is 1.57 and is less than 2, but the content of the teasel root saponin B is 15.3mg/g and is obviously more than 0, and the product can be judged to be non-honeysuckle. On the contrary, the content of the asperosaponin B in the mixed sample No. 5, namely the first batch of Yinhua 12 Sichuan mountain in Henan, is 0.09mg/g and is close to 0, and misjudgment can be caused under the conditions of low precision or low operation recovery rate, but the content ratio of the chlorogenic acid to the secoisolariciresinol is 3.20 and is more than 2, so that the product can be judged to be non-honeysuckle.
(2) Compared with the method that only one of the ratio of the content of chlorogenic acid to the content of the secoisolariciresinol diglucoside and the content of the dipsacoside serving as evaluation indexes is used for identifying the honeysuckle and the lonicera confusa mixture, the identification accuracy can be effectively improved, and the mixtures in the example can be distinguished.
Example 4 determination of mixed sample (honeysuckle and lonicera confusa in a mass ratio of 1:4)
Preparation of a mixed sample: respectively pulverizing flos Lonicerae and flos Lonicerae in equal weight according to Table 17, mixing, determining as sample according to determination method of chlorogenic acid, semen Strychni Pulveratum acid, and radix Dipsaci saponin B in example 1, and calculating content ratio of chlorogenic acid and semen Strychni Pulveratum acid and content of radix Dipsaci saponin B, the results are shown in Table 18
TABLE 17 honeysuckle and Lonicera confusa mixed sample composition table (quality ratio 1:4)
| Number of mixed sample | Name of honeysuckle | Name of Lonicera confusa variety | Composition by weight ratio |
| 1 | First batch of Henan, first-class | 3 Guizhou grey felt-1 | 1:4 |
| 2 | First batch of Henan, first-class | 14 flos Lonicerae of Longhui mountain of Hunan province | 1:4 |
| 3 | First batch of Henan, first-class | 29 sample honeysuckle purchased from san Guangxi province | 1:4 |
| 4 | First batch of Henan, first-class | Guangdong south honeysuckle 1 | 1:4 |
| 5 | First batch of Henan, first-class | 12 Sichuan mountain honeysuckle flower | 1:4 |
| 6 | Shandong SD1-1 | 3 Guizhou grey felt-1 | 1:4 |
| 7 | Shandong SD1-1 | 14 flos Lonicerae of Longhui mountain of Hunan province | 1:4 |
| 8 | Shandong SD1-1 | 29 sample honeysuckle purchased from san Guangxi province | 1:4 |
| 9 | Shandong SD1-1 | Guangdong south honeysuckle 1 | 1:4 |
| 10 | Shandong SD1-1 | 12 Sichuan mountain honeysuckle flower | 1:4 |
| 11 | Hebei 2-2(4) | 3 Guizhou grey felt-1 | 1:4 |
| 12 | Hebei 2-2(4) | 14 flos Lonicerae of Longhui mountain of Hunan province | 1:4 |
| 13 | Hebei 2-2(4) | 29 sample honeysuckle purchased from san Guangxi province | 1:4 |
| 14 | Hebei 2-2(4) | Guangdong south honeysuckle 1 | 1:4 |
| 15 | Hebei 2-2(4) | 12 Sichuan mountain honeysuckle flower | 1:4 |
TABLE 18 weight ratio of flos Lonicerae and flos Lonicerae at 1:4 and content ratio of chlorogenic acid and semen Strychni Pulveratum acid
The results show that: (1) in this example: the content ratio of chlorogenic acid to secoisolariciresinol diglucoside in the No. 14 mixed sample 'Hebei 2-2(4) -Guangdong south Lonicera japonica Thunb 1' is 1.63 and less than 2, but the content of the dipsacus asperoides saponin B is 25.11mg/g and obviously more than 0, and the product can be judged to be non-honeysuckle. On the contrary, the content of the asperosaponin B in the mixed sample No. 5, namely the first batch of Yinhua 12 Sichuan mountain in Henan, is 0.24mg/g, which is close to 0, and the misjudgment can be caused under the conditions of low precision or low operation recovery rate, but the content ratio of the chlorogenic acid to the secoisolariciresinol is 6.51, which is more than 2, so that the product can be judged to be non-honeysuckle.
(2) Compared with the method that only one of the ratio of the content of chlorogenic acid to the content of the secoisolariciresinol diglucoside and the content of the dipsacoside serving as evaluation indexes is used for identifying the honeysuckle and the lonicera confusa mixture, the identification accuracy can be effectively improved, and the mixtures in the example can be distinguished.
Example 5 determination of Mixed sample (honeysuckle and Lonicera confusa at a mass ratio of 4:1)
Preparation of a mixed sample: respectively taking flos Lonicerae and flos Lonicerae of equal weight according to Table 19, pulverizing, mixing well, determining as sample according to determination method of chlorogenic acid, semen Strychni Pulveratum acid, and radix Dipsaci saponin B in example 1, and calculating content ratio of chlorogenic acid and semen Strychni Pulveratum acid and radix Dipsaci saponin B content. The results are shown in Table 20.
TABLE 19 honeysuckle and Lonicera confusa mixed sample composition table (quality ratio 4:1)
| Number of mixed sample | Name of honeysuckle | Name of Lonicera confusa variety | Composition by weight ratio |
| 1 | First batch of Henan, first-class | 3 Guizhou grey felt-1 | 4:1 |
| 2 | First batch of Henan, first-class | 14 flos Lonicerae of Longhui mountain of Hunan province | 4:1 |
| 3 | First batch of Henan, first-class | 29 sample honeysuckle purchased from san Guangxi province | 4:1 |
| 4 | First batch of Henan, first-class | Guangdong south honeysuckle 1 | 4:1 |
| 5 | First batch of Henan, first-class | 12 Sichuan mountain honeysuckle flower | 4:1 |
| 6 | Shandong SD1-1 | 3 Guizhou grey felt-1 | 4:1 |
| 7 | Shandong SD1-1 | 14 flos Lonicerae of Longhui mountain of Hunan province | 4:1 |
| 8 | Shandong SD1-1 | 29 sample No. 29Yingsanguangxi mountain honeysuckle flower | 4:1 |
| 9 | Shandong SD1-1 | Guangdong south honeysuckle 1 | 4:1 |
| 10 | Shandong SD1-1 | 12 Sichuan mountain honeysuckle flower | 4:1 |
| 11 | Hebei 2-2(4) | 3 Guizhou grey felt-1 | 4:1 |
| 12 | Hebei 2-2(4) | 14 flos Lonicerae of Longhui mountain of Hunan province | 4:1 |
| 13 | Hebei 2-2(4) | 29 sample honeysuckle purchased from san Guangxi province | 4:1 |
| 14 | Hebei 2-2(4) | Guangdong south honeysuckle 1 | 4:1 |
| 15 | Hebei 2-2(4) | 12 Sichuan mountain silverweedFlower (A. B. A | 4:1 |
TABLE 20 weight ratio of the components and the ratio of chlorogenic acid to secoisolaricid acid when flos Lonicerae and flos Lonicerae are mixed at 4:1
The results show that: (1) in this example: the content ratio of chlorogenic acid to secoisolariciresinol diglucoside in the No. 14 mixed sample 'Hebei 2-2(4) -Guangdong south Lonicera japonica Thunb 1' is 1.02 and less than 2, but the content of the dipsacus asperoides saponin B is 8.43mg/g and obviously more than 0, and the product can be judged to be non-honeysuckle. On the contrary, the content of the asperosaponin B in the 15 # mixed sample, Hebei 2-2(4) -12 Sichuan lonicera japonica, is 0.08mg/g, which is close to 0, and may cause misjudgment under the conditions of low precision or low operation recovery rate, but the content ratio of the chlorogenic acid to the strychnic acid is 2.55, which is more than 2, and the product can be judged to be non-honeysuckle.
(2) Compared with the method that only one of the ratio of the content of chlorogenic acid to the content of the secoisolariciresinol diglucoside and the content of the dipsacoside serving as evaluation indexes is used for identifying the honeysuckle and the lonicera confusa mixture, the identification accuracy can be effectively improved, and the mixtures in the example can be distinguished.
By combining the embodiments 3, 4 and 5, the honeysuckle and the lonicera confusa are respectively selected and mixed according to different weight proportions, and by adopting the method disclosed by the invention, the honeysuckle and lonicera confusa mixture can be effectively identified by taking the content ratio of the chlorogenic acid to the strychnic acid as an evaluation index, and the content of the dipsacoside B as an evaluation index, and whether a sample is a single medicinal material decoction piece or not is not observed by observing characters in the experimental process. For the adulteration identification of the lonicera confusa honeysuckle in the Chinese patent medicine, the content ratio of chlorogenic acid to strychnic acid and the content of the dipsacus asperoides B in the input medicinal materials can be converted by checking the added amount and carrying out weight conversion, and the identification purpose is achieved by utilizing the judgment key points. The aim of identifying the condition that a plurality of crushed or partially crushed mixed medicinal materials, medicinal materials in Chinese patent medicine and the like in the market cannot be observed by naked eyes.
Claims (9)
1. The method for identifying the honeysuckle and the lonicera confusa by utilizing the high performance liquid chromatography and the LC-MS is characterized in that when the test sample is one or two of the honeysuckle or the lonicera confusa, the weight percentages of the strychnic acid and the chlorogenic acid in the dry test sample are detected by the high performance liquid chromatography, and the ratio of the weight percentages of the chlorogenic acid and the strychnic acid is calculated; detecting the weight percentage of the teasel saponin B in the dry sample by LC-MS; the ratio of the weight percentage of chlorogenic acid to the weight percentage of the strychnic acid and the weight percentage of the teasel root saponin B are utilized to identify whether the honeysuckle is mixed with the lonicera confusa or not.
2. The method as claimed in claim 1, wherein the honeysuckle is determined as honeysuckle when the ratio of the weight percentage of chlorogenic acid to the weight percentage of secoisolariciresinol diglucoside in the dry sample is less than 2 and the weight percentage of the asperosaponin B is 0.
3. The method for identifying honeysuckle and lonicera confusa according to claim 1 or 2, wherein the method for detecting the content of chlorogenic acid and secoisolariciresinol diglucoside comprises the following steps: injecting the reference substance solution and the test solution into a high performance liquid chromatograph, measuring peak areas at the wavelength of 210-360 nm, calculating by adopting an external standard method or a standard curve method to obtain the weight percentage of chlorogenic acid and secoisolariciresinol diglucoside in the test solution, and then calculating the ratio of the chlorogenic acid to the secoisolariciresinol diglucoside in the test solution; the detection method of the content of the dipsacoside B comprises the following steps: and (3) injecting the reference substance solution and the test sample solution into a liquid chromatograph-mass spectrometer, measuring the area of the characteristic ions by liquid phase separation and mass spectrum, and calculating by adopting an external standard method or a standard curve method to obtain the weight percentage of the dipsacoside B in the test sample.
4. The method of claim 3, wherein the identification of honeysuckle and lonicera confusa is carried out,
(1) the determination conditions of the high performance liquid chromatography are as follows: column C18, 4.6mm X250 mm, 5 μm; wavelength 240-324 nm; taking acetonitrile as a mobile phase A, taking an acid water solution with the pH value of 1.5-4 as a mobile phase B, and adopting gradient elution, wherein the mobile phase A is 0-20 min, and the volume percentage is 5% → 15%; 20-30 min, 15% → 28% by volume; the rest is mobile phase B;
(2) the determination conditions of the LC-MS are as follows: liquid phase partial chromatography column: c18 chromatographic column, 10mm × 2.1mm, 1.7 μm; column temperature: 45 ℃; flow rate: 0.3 mL/min; sample introduction volume: 1 mu L of the solution; mobile phase: taking a 0.1% formic acid (containing 10mmol/L ammonium formate) solution as a mobile phase A, taking acetonitrile as a mobile phase B, and adopting gradient elution, wherein the mobile phase A is 0-3 min, 92% by volume, 3-5 min, 92% by volume, → 35%, 5-8 min, 35% by volume, → 15%, 8-10 min, and 15% by volume; the rest is mobile phase B; mass spectrometry ion source mode: an electrospray ion source; scanning mode: selecting ions for detection; ionization voltage: 3500V; temperature of the atomization chamber: 200 ℃; sheath gas: high-purity nitrogen gas, 35L/min; auxiliary gas: high-purity nitrogen gas, 10L/min; ion transfer tube temperature: 290 ℃; collision gas: high-purity argon gas; the anion is adopted to collect the teasel saponin B, the mass-to-charge ratio of the teasel saponin B is 1075.0, the mass-to-charge ratio of the mother ion is 749.2, and the collision energy is 40 eV.
5. The method as claimed in claim 4, wherein the acid is phosphoric acid, formic acid, acetic acid or phosphate.
6. The method of claim 4, wherein the detection wavelength of the strychnos nux-vomica acid is 240nm, and the detection wavelength of the chlorogenic acid is 324 nm.
7. The method of claim 3, wherein the identification of honeysuckle and lonicera confusa is carried out,
(1) the preparation method of the test solution in the detection method of the content of chlorogenic acid and secoisolariciresinol diglucoside comprises the following steps: precisely weighing 0.05-0.25g of test sample powder, placing into a conical flask with a plug, precisely adding 10-50mL of solvent, performing ultrasonic treatment for 15-60min, shaking up, and filtering to obtain the final product.
(2) The preparation method of the test solution in the detection method of the content of the dipsacoside B comprises the following steps: pulverizing the sample into fine powder, precisely weighing about 0.1-0.5g, placing into a conical flask with a plug, precisely adding 20-50mL of 85% methanol, precisely weighing, performing ultrasonic extraction at room temperature for 15-60min, cooling, precisely weighing, adding 85% methanol to make up the weight, filtering, and filtering with 0.22 μm filter membrane to obtain the final product.
8. The method as claimed in claim 7, wherein the power of the ultrasonic wave is 250W and the frequency is 35 kHz.
9. The method of claim 7, wherein the solvent is water or 0-50% ethanol solution or 0-50% methanol solution or mixture thereof.
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