CN115856163B - Detection method for distinguishing radix sileris from water radix sileris - Google Patents
Detection method for distinguishing radix sileris from water radix sileris Download PDFInfo
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Abstract
The invention discloses a detection method for identifying wind prevention and water wind prevention, which comprises the following steps: (1) preparation of a sample solution; (2) Sample analysis, namely respectively collecting total ion flow diagrams of the sample solution; (3) PCA analysis and cluster analysis; (4) screening of differential markers: dividing radix Saposhnikoviae and radix Ledebouriellae into two groups by using the function of searching for characteristic compounds of MPP data processing software, screening the difference compounds among the groups according to the compound response values, and obtaining the difference markers which have good peak shape, high response value, moderate retention time and are not existing in radix Ledebouriellae by analyzing the extracted ion flow diagram of each difference compound; (5) Separating and purifying the differential markers and identifying chemical structures; (6) preparation of a reference substance solution; (7) preparing a sample solution to be tested; (8) detection and determination. The invention establishes a new method for identifying radix sileris and water radix sileris from the aspect of chemical components, and the method is sensitive, accurate and reliable and can be used for quality control of radix sileris.
Description
Technical Field
The invention relates to the technical field of wind prevention identification, in particular to a detection method for identifying wind prevention and water wind prevention.
Background
Radix Saposhnikoviae is dry root of radix Saposhnikoviae belonging to Umbelliferae, has pungent and sweet taste, and is slightly warm, and has effects of dispelling pathogenic wind, relieving exterior syndrome, eliminating dampness, relieving pain, and relieving spasm. Is mainly used for treating common cold, headache, rheumatalgia, rubella pruritus and tetanus. Radix Saposhnikoviae has long history, and is classified as superior in Shennong Ben Cao Jing (Shennong's herbal), and 10 prescriptions of "Guizhi Bai Shao Zhi Tang", tang Fang "Xiao Xue Ming Tang" and Song Fang "Xinyi san" in ancient classical prescriptions (first batch) are all available. The research shows that the pharmacological active ingredients of the radix sileris are mainly divided into coumarin, chromone, polysaccharide and volatile oil.
Radix Saposhnikoviae is a large number of medicinal materials, and has large flow rate and various production places. Wild products mainly comprise Guanfu, fangfeng and Fangbei Feng, and the provinces of inner Mongolia, heilongjiang and Gansu are taken as main production areas. The cultivated product is early cultivated and large in quantity in the Hebei area, also called Jifeng, and takes provinces of Hebei, shanxi and the like as main production areas. With the increasing market demand year by year, wild radix sileris resources are gradually depleted, and trade prices are high. The research shows that the phenomenon that the confusing products such as water-proof, sichuan-proof, cloud-proof and the like impersonate the wind-proof exists in the market, wherein the water-proof is more. The water divaricate saposhnikovia root is a dry root of the divaricate saposhnikovia root, has slightly weaker exterior-releasing effect than the divaricate saposhnikovia root, has stronger toxicity and is used with cautions in clinic.
At present, the standard of the wind-proof legal test is the Chinese pharmacopoeia of 2020 edition, and limit value regulation is made on the 6 aspects of physicochemical indexes of the sample, including the contents of characters, microscopes, thin-layer identification, moisture, total ash, cimicifuga rhizome glycoside and 5-O-methyl vitamin-amiloride. The water windproof test standard is 1991 edition Henan Chinese herbal medicine standard, and only the properties and the microscope are detected, so that the quality and the quality are difficult to control. The water windproof test is carried out according to the windproof standard, and as a result, the appearance property, microscopic characteristics and content control indexes of the water windproof meet the standard requirements, the difference between the appearance property, microscopic characteristics and content control indexes is extremely insignificant, the current standard is difficult to distinguish the authenticity of the windproof, and the error is easy to occur.
Referring to domestic and foreign documents, the method finds that only 2 documents of identification research of radix sileris and pseudo-product radix sileris based on HPLC fingerprint and identification of radix sileris medicinal material and pseudo-product based on high performance liquid fingerprint and chemometry exist so far, and the method has the following two defects that the simple analysis of the compounds contained in the radix sileris is carried out by liquid chromatography: on the one hand, the liquid chromatography is based on information of a compound collected by specific ultraviolet wavelength, and completely different fingerprints can appear when the ultraviolet wavelength is changed; on the other hand, the literature does not clearly identify specific compound components, the method is used for rapidly screening and distinguishing the authenticity of the radix sileris, the analysis of the fingerprint has the problems of long time consumption, difficult establishment and acquisition of a standard fingerprint, and the like, and is difficult to apply as a legal quality standard.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a detection method for identifying the wind prevention and the water wind prevention, which is a new method for identifying the wind prevention and the water wind prevention from the chemical composition perspective, and the method is sensitive, accurate and reliable and can be used for quality control of the wind prevention.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a detection method for identifying wind prevention and water wind prevention comprises the following steps:
(1) Preparation of test solution: respectively taking a plurality of batches of exact-source radix sileris and water radix sileris sample powder, respectively adding methanol into each sample powder, weighing, carrying out ultrasonic treatment, cooling to room temperature, weighing again, supplementing the reduced weight with methanol, shaking uniformly, centrifuging at an ultrahigh speed, and taking supernatant to obtain each sample solution;
(2) Sample analysis: respectively taking 1 mu L of the sample solution prepared in the step (1), and respectively collecting total ion flow diagrams of the sample solution by adopting a high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer;
(3) PCA analysis and cluster analysis: introducing the mass spectrum raw data obtained in the step (2) into MPP data processing software, extracting characteristic peak data, and carrying out principal component analysis and cluster analysis on the characteristic peaks;
(4) Screening of differential markers: dividing radix Saposhnikoviae and radix Ledebouriellae into two groups by using the function of searching for characteristic compounds of MPP data processing software, screening the difference compounds among the groups according to the compound response values, and obtaining the difference markers which have good peak shape, high response value, moderate retention time and are not existing in radix Ledebouriellae by analyzing the extracted ion flow diagram of each difference compound;
(5) Separation and purification of differential markers and chemical structure identification: separating and purifying high-purity differential markers from a test sample of Saponaria officinalis by silica gel column chromatography, and determining the structural formula of the differential markers as follows by mass spectrum and nuclear magnetic identification
(6) Preparation of a control solution: adding methanol into the differential markers to prepare a solution containing 2 mug of the differential markers per 1 ml;
(7) Preparing a sample solution to be tested: adding methanol into the sample powder to be measured, weighing, cooling to room temperature after ultrasonic treatment, weighing again, supplementing the reduced weight with methanol, shaking uniformly, filtering with microporous membrane, and collecting the subsequent filtrate;
(8) Detecting and judging: respectively sucking an equivalent amount of reference substance solution and a sample solution to be detected, and injecting the solution into a liquid chromatograph or a high performance liquid chromatograph-mass spectrometer for detection to obtain a liquid chromatograph or an extracted ion flow chromatograph;
if the same chromatographic peak does not appear in the liquid chromatogram of the sample solution to be detected at the position corresponding to the retention time of the chromatographic peak of the reference substance solution, judging that the sample to be detected is divaricate saposhnikovia root;
if the same chromatographic peak appears in the liquid chromatogram of the sample solution to be detected at the position corresponding to the retention time of the chromatographic peak of the reference solution, comparing ultraviolet-visible absorption spectrums of the corresponding chromatographic peak in the wavelength range of 190-400 nm by adopting a diode array detector, judging that the sample to be detected is windproof if the ultraviolet-visible absorption spectrums are different, and judging that the sample to be detected is doped with water for windproof if the ultraviolet-visible absorption spectrums are the same;
if the same chromatographic peak does not appear at the position corresponding to the retention time of the chromatographic peak of the reference substance solution in the extracted ion flow chromatography of the sample solution to be detected, judging that the sample to be detected is windproof, otherwise, judging that the sample to be detected is doped with water for windproof.
The specific steps for preparing the sample solution in the step (1) are as follows: respectively taking 1g of radix Saposhnikoviae and 1g of radix Saposhnikoviae powder, precisely weighing, respectively adding 50ml of 70v/v% methanol, weighing, performing ultrasonic treatment for 30min, cooling to room temperature, weighing, supplementing the lost weight with 70v/v% methanol, shaking, centrifuging at ultrahigh speed, and collecting supernatant.
The liquid chromatography conditions for sample analysis in step (2) are: 4.6mm by 50mm,2.7 mu mAgilent Poroshell EC-C 18 Is a chromatographic column, mobile phase: acetonitrile-0.1% formic acid solution, column temperature 30 ℃, flow rate 0.5 mL.min -1 ；
The mass spectrum condition is electrospray ionization source, the collection mode is positive ion mode, the temperature of the drying gas is 300 ℃, the flow rate of the drying gas is 5L/min, the pressure of an atomizer is 35psi, the temperature of the sheath gas is 350 ℃, the flow rate of the sheath gas is 11L/min, the capillary voltage is 3500V, the nozzle voltage is 500V, the mass scanning range is 50-1000 m/z, and the fragmentation voltage is 135V.
The specific method for PCA analysis is as follows: introducing mass spectrum raw data into MPP data processing software, and extracting data of characteristic peaks by using screening methods of peak matching, peak alignment and noise filtering; then, based on a t-test statistical algorithm with the p value smaller than 0.05, carrying out principal component analysis on the characteristic peak; and finally, sequentially compiling the statistical results of the first, second and third main component elements into an X axis, a Y axis and a Z axis, establishing a 3D structure model of 30 batches of samples, and observing the difference between wind prevention and water prevention.
The specific method for separating and purifying the differential markers comprises the following steps: taking 1kg of one batch of divaricate saposhnikovia root test sample powder, adding 95v/v% methanol 3L, soaking, carrying out ultrasonic treatment, filtering, concentrating the extracting solution under reduced pressure to dryness to obtain a tan oily extract, and separating the tan oily extract by using a silica gel column chromatography to obtain the high-purity differential marker.
The silica gel column chromatography specifically comprises the following steps: firstly, eluting with a chloroform/methanol solvent system according to a gradient of 95:5 to 50:50, and collecting a fraction Fr.A eluted by 75% of chloroform/methanol; eluting the fraction Fr.A with n-hexane/ethyl acetate solvent system according to a gradient of 75:25-30:70, and collecting 55% n-hexane/ethyl acetate eluted fraction Fr.B; and finally, purifying the fraction Fr.B by using a preparation liquid, wherein the mobile phase is acetonitrile-water with the volume ratio of 40:60, and the flow rate is 3.0mL min < -1 >, so as to obtain the high-purity differential marker.
The specific steps of the mass spectrum identification of the differential markers are as follows: adding methanol into the high-purity differential marker prepared in the step (5) to prepare a marker solution, and collecting a secondary mass spectrum fragment diagram by adopting a high performance liquid chromatography tandem four-pole time-of-flight mass spectrometer; the mass spectrum and chromatographic conditions are the same as in the step (2).
The specific preparation method of the sample solution to be detected by adopting the liquid chromatograph comprises the following steps: taking 1g of sample powder to be detected, adding 50ml of 70v/v% methanol, weighing, carrying out ultrasonic treatment for 30min, cooling to room temperature, weighing again, supplementing the weight of the sample powder to be detected with 70v/v% methanol, shaking uniformly, filtering with a 0.45 mu m microporous filter membrane, and taking a subsequent filtrate to obtain the product;
the specific preparation method of the sample solution to be detected by adopting the high performance liquid chromatography-mass spectrometer comprises the following steps: taking 0.2g of sample powder to be detected, adding 100ml of 70v/v% methanol, weighing, carrying out ultrasonic treatment for 30min, cooling to room temperature, weighing again, supplementing the lost weight with 70v/v% methanol, shaking uniformly, filtering with a 0.45 mu m microporous filter membrane, and taking the subsequent filtrate.
The method for detecting the sample solution to be detected by adopting the liquid chromatograph comprises the following steps: respectively sucking 10 μl of the reference substance solution and the sample solution to be detected, and injecting into a liquid chromatograph for measurement to obtain a liquid chromatogram;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler; the volume ratio is 70:30 acetonitrile-0.1% phosphoric acid aqueous solution as mobile phase; the detection wavelength was 222nm using a diode array detector.
The method for detecting the sample to be detected by adopting the high performance liquid chromatography-mass spectrometer comprises the following steps: respectively sucking 1 μl of the reference substance solution and 1 μl of the sample solution to be detected, and injecting into a high performance liquid chromatograph-mass spectrometer for measurement;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, and the inner diameter of a chromatographic column is 2.1mm; acetonitrile is taken as a mobile phase A, 0.1% formic acid solution is taken as a mobile phase B, and the flow rate is 0.3ml per minute;
the mass spectrum conditions are as follows: the electrospray positive ion mode, multi-reaction monitoring, and m/z 427.2- & gt 245.1 and m/z 427.2- & gt 327.1 are selected as detection ion pairs.
The beneficial effects of the invention are as follows: on one hand, the HPLC-Q-TOF-MS technology is used for carrying out statistical analysis on the radix sileris and the water radix sileris, a differential PCA model of the radix sileris and the water radix sileris is established, and the samples of different production places are subjected to cluster analysis, so that the statistical result not only effectively identifies the radix sileris and the water radix sileris, but also defines the inherent difference of wild radix sileris and cultivated radix sileris. On the other hand, the special component differential markers in the divaricate saposhnikovia root are separated and purified, and the chemical structure of the special component differential markers is identified by means of mass spectrum and nuclear magnetism, so that the special component differential markers are determined to be Xanthalin, and can be used as standard reference substances, the problem of divaricate saposhnikovia root doped in the divaricate saposhnikovia root is solved, the effective combination of the two aspects of research can rapidly and comprehensively control the divaricate saposhnikovia root quality, and the basis is provided for the supplementary test standard drafting of the divaricate saposhnikovia root.
Drawings
FIG. 1 is a total ion flow diagram of wind (S10) and water wind (S1) (A wind, B water wind);
FIG. 2 is a PCA score plot of wind versus water wind;
FIG. 3 is a cluster dendrogram of wind and water protection;
FIG. 4 is a graph of the differential compounds of Fahrenheit versus Fahrenheit;
FIG. 5 is an extract ion flow diagram of Fangfeng and Fangfeng (Fangfeng, fangfeng D);
FIG. 6 is a liquid chromatogram of a high purity differential marker;
FIG. 7 is a secondary mass spectrum of a poorly-labeled foreign object;
FIG. 8 shows the cleavage pathway of poorly-labeled foreign bodies;
FIG. 9 shows the nuclear magnetism of Xanthalin 13 C, carbon spectrogram;
FIG. 10 shows the nuclear magnetism of Xanthalin 1 H hydrogen profile;
FIG. 11 is a stereochemical structural formula of Xanthalin;
FIG. 12 is a liquid chromatography control chart of the differential marker Xanthalin of example 1 and a sample to be tested;
FIG. 13 is a liquid chromatography control chart of the differential marker Xanthalin of example 2 and a sample to be tested;
FIG. 14 is a spectrum contrast chart of the differential marker Xanthalin of example 2 and a sample to be tested;
FIG. 15 is a total ion flow and extracted ion flow diagram of the differential marker Xanthalin of example 3;
FIG. 16 is a total ion current and extracted ion flow diagram of the sample to be tested of example 3;
FIG. 17 is a total ion flow and extracted ion flow diagram of the differential marker Xanthalin of example 4;
fig. 18 is a total ion flow and extracted ion flow diagram of the sample to be tested of example 4.
Detailed Description
The invention is further described with reference to the drawings and detailed description which follow:
example 1
Instrument:
1260 high performance liquid chromatograph (Agilent Inc. of USA), 6530 quadrupole-time of flight tandem mass spectrometer (Agilent Inc. of USA), LC-20AP preparative liquid chromatograph (SHIMADZU Inc. of Japan), JNM-ECZ400S nuclear magnetic resonance (JEOL Inc. of Japan), XPE26 parts per million electronic balance (MettlerToledo Inc. of Switzerland), ZM-200 ultracentrifuge mill (Retsch Inc. of Germany), R-300 rotary evaporator (BUCHI Inc. of Switzerland), FESCO17 microcentrifuge (thermal Fisher Inc. of USA), UA22MFD digital control ultrasonic meter (Wiggens Inc. of Germany), ADVANTAGEA ultra pure water (Millipore Inc. of USA).
Reagent:
acetonitrile, formic acid (all mass spectrum grade, purchased from CNW); methanol (chromatographic grade, purchased from TEDIA); trichloromethane, methanol, n-hexane, ethyl acetate (all analytical grade, purchased from national pharmaceutical chemicals limited); deuterated chloroform (deuterated 99.8+%, for nuclear magnetic resonance, available from MERCK); column layer chromatography silica gel (purchased from MACKLIN); ultrapure water.
The detection method for identifying the wind prevention and the water wind prevention specifically comprises the following steps:
(1) Preparation of test solution: respectively taking 1g of radix Saposhnikoviae and 1g of radix Saposhnikoviae sample powder, precisely weighing, respectively adding 50ml of 70v/v% methanol, weighing, performing ultrasonic treatment for 30min, cooling to room temperature, weighing, supplementing the lost weight with 70v/v% methanol, shaking, centrifuging at ultrahigh speed, and collecting the supernatant to obtain each sample solution.
The sample radix Saposhnikoviae is obtained from the places such as inner Mongolia, heilongjiang, hebei and the like, and is divided into wild products and cultivated products, and the sample radix Saposhnikoviae is obtained from Henan and Shanxi, and is wild product. Radix Saposhnikoviae is identified according to the "Chinese pharmacopoeia" of 2020 edition and radix Ledebouriellae is identified according to the "Henan Chinese medicinal Material Standard" of 1991 edition. Specific information is shown in Table 1, wherein the reference medicinal materials are as follows: radix Saposhnikoviae (lot number: 120947-201810), and radix Saposhnikoviae (lot number: 121349-202002) were purchased from Chinese food and drug administration institute (middle inspection institute for short); wild products are given a "x" designation after the name.
Table 1 information of wind and water protection
In the step, the liquid extraction solvent is selected from methanol, 70% methanol and 30% methanol to extract the sample respectively, and as a result, the 70% methanol extraction solution has the optimal characteristic peak number, peak shape and peak area, and the obtained compound information is more comprehensive, so that 70% methanol is selected as the extraction solvent. The influence of ultrasonic time of 10, 30 and 60min on the result is compared, the content of each component is low when the ultrasonic time is 10 min, and the content of each component is basically unchanged after the ultrasonic time exceeds 30min, so that 30min is selected as the extraction time.
(2) Sample analysis: and (3) respectively taking 1 mu L of the sample solution prepared in the step (1), and respectively collecting the total ion flow diagram of the sample solution by adopting a high performance liquid chromatography-tandem four-rod time-of-flight mass spectrometer. The total ion flow diagram of radix Saposhnikoviae and radix Saposhnikoviae is shown in figure 1.
The liquid phase conditions are as follows: chromatographic column: agilentPorosill 120EC-C 18 Column (4.6 mm. Times.50 mm,2.7 μm), mobile phase: second stepNitrile-0.1% formic acid solution (gradient elution, details of which are shown in Table 2), column temperature 30 ℃, flow rate 0.5mL min -1 。
TABLE 2 gradient elution method
The mass spectrum conditions are as follows: electrospray ionization source (ESI), collection mode is positive ion mode, drying gas temperature: 300 ℃, drying gas flow rate: 5L/min, atomizer pressure: 35psi, sheath air temperature: sheath gas flow rate at 350 ℃): 11L/min, capillary voltage: 3500V, nozzle voltage: 500V, mass scan range m/z: 50-1000, fragmentation voltage: 135V.
The invention compares the analysis effects of 4 mobile phases of methanol-water, methanol-0.1% formic acid aqueous solution, acetonitrile-water and acetonitrile-0.1% formic acid aqueous solution, and as a result, when the acetonitrile-0.1% formic acid aqueous solution is used for gradient elution, the peak shape of each common peak is best, and the separation degree is higher, so that the acetonitrile-0.1% formic acid aqueous solution is selected as the mobile phase.
(3) PCA analysis and cluster analysis: introducing the mass spectrum raw data obtained in the step (2) into MPP data processing software, extracting characteristic peak data, and carrying out principal component analysis and cluster analysis on the characteristic peaks;
principal Component Analysis (PCA): the mass spectrum raw data is imported into Mass Profiler Professional (MPP) data processing software, and characteristic peak data is extracted by using screening methods of peak matching, peak alignment and noise filtering. And then carrying out principal component analysis on the characteristic peak based on a statistical algorithm of t-test (p value is smaller than 0.05). Finally, the statistical results of the first, second and third main component elements are orderly compiled into an X axis, a Y axis and a Z axis, 30 batches of 3D structural models of the samples are established, the difference between the windproof and the water windproof is observed, and the result is shown in figure 2. In general, 21 batches of windbreaks are gathered above the X axis, 9 batches of water windbreaks are gathered below the X axis, and the two batches are distinguished remarkably.
And (3) cluster analysis: and carrying out cluster analysis on characteristic peaks by using MPP software, and analyzing the influence of varieties and places of production on the composition of the compounds by observing the classification conditions of different test products. In the clustering dendrogram (figure 3), the difference between wind and water wind is quite obvious, 30 samples are classified into 2 types, S1-S9 are classified into water wind, S10-S30 are classified into wind, and the analysis result is consistent with the analysis result of PCA. Wherein 21 batches of radix sileris are classified into 2 types according to the difference of wild products and cultivated products, S10-S12 and S16-S18 are classified into one type, the wild products and the rest 15 batches are classified into one type, and the cultivated products are cultivated in family. The result of the clustering tree diagram is basically consistent with the appearance character and the information of the production place, and can be used as an effective method for identifying the authenticity of the divaricate saposhnikovia root and the production place.
(4) Screening of differential markers: the MPP software is used for searching characteristic compound functions, the divaricate saposhnikovia root and the divaricate saposhnikovia root are divided into two groups, the difference compounds among the groups are screened according to compound response values, and 2 divaricate saposhnikovia root differential markers and 6 divaricate saposhnikovia root differential markers are obtained as a result, and 18 divaricate saposhnikovia root compounds are shared, and the result is shown in figure 4. The extracted ion flow diagrams of 8 differential markers are respectively analyzed, the compound with the excimer ion peak ([ M+H ] +) M/z of 427.1762 in the divaricate saposhnikovia root is found, the peak shape is good, the response value is high, the retention time is moderate, the compound does not exist in the divaricate saposhnikovia root, and finally the compound is used as the differential marker, and the result is shown in figure 5 in detail.
(5) Separation and purification of differential markers and chemical structure identification:
(a) And (3) separating and purifying a differential marker: taking 1Kg of S2 water divaricate saposhnikovia root coarse powder, adding 95% methanol to soak for 24 hours, carrying out ultrasonic treatment for 1 hour, filtering, and concentrating the extracting solution under reduced pressure until the extracting solution is dried to obtain a tan oily extract. The separation was performed by silica gel column chromatography, first by gradient elution with chloroform/methanol solvent system (95:5.fwdarw.50:50), and collecting 75% chloroform/methanol eluted fraction fr.a. The fraction fr.a was then eluted with a gradient of n-hexane/ethyl acetate solvent system (75:25→30:70) and the 55% n-hexane/ethyl acetate eluted fraction fr.b was collected. Finally, the liquid fraction Fr.B was purified by preparative liquid chromatography, wherein the mobile phase was acetonitrile-water (40:60), the flow rate was 3.0mL min-1, and a high purity differential marker was obtained (the purity was calibrated to be about 97.3% by liquid chromatography, the detection wavelength was 222nm, and the results are shown in FIG. 6).
(b) Mass spectrometry identification of differential markers: the marker was taken out as a pure product 5.381mg, put into a 50mL measuring flask, and 70% methanol was added to prepare a marker solution. According to the condition of the step (2), the Collision Energy (CE) is set to be 20V, and a secondary mass spectrum fragment diagram is recorded, and the result is shown in FIG. 7 in detail. In the figure, m/Z427.1762 is an excimer ion peak, m/Z327.1236, 245.0814, 227.0706, 217.0859, 191.0339, 83.0499 and 55.0553 are fragment ion peaks, and the cleavage route is shown in figure 8, which accords with the coumarin compound cleavage rule reported in the literature (WANG W H, YAN P Z, YANG B.student on fragmentation patterns of coumarins in Notopterygium inchum with ultrahigh performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry [ J ]. Chin J Chin Mater Med (Chinese J.traditional Chinese medicine), 2021,46 (5): 1179-1190.).
The liquid phase conditions are as follows: chromatographic column: agilentPorosill 120EC-C 18 Column (4.6 mm. Times.50 mm,2.7 μm), mobile phase: acetonitrile-0.1% formic acid solution (gradient elution, details are shown in Table 2), column temperature 30 ℃, flow rate 0.5mL min -1 。
TABLE 2 gradient elution method
The mass spectrum conditions are as follows: electrospray ionization source (ESI), collection mode is positive ion mode, drying gas temperature: 300 ℃, drying gas flow rate: 5L/min, atomizer pressure: 35psi, sheath air temperature: sheath gas flow rate at 350 ℃): 11L/min, capillary voltage: 3500V, nozzle voltage: 500V, mass scan range m/z: 50-1000, fragmentation voltage: 135V.
(c) Nuclear magnetic identification of differential markers: the pure product marker is subjected to nuclear magnetic 1H hydrogen spectrum and 13C carbon spectrum measurement, the solvent is deuterated chloroform, the hydrogen spectrum resonance frequency is 400MHz, the carbon spectrum resonance frequency is 100MHz, and the results are shown in Table 3 in detail. Analysis of the measured chemical shifts, line intensities and coupling constants revealed substantial agreement with the document reported data for the compound Xanthalin (SHI M R, ZHAO J X, FENG Z F, et al chemical constituents from Sphallerocarpus gracilis [ J)]Nat Prod Res Dev (Natural products research and development), 2012,24 (2): 182-184. SHAO ZY, ZHANG Y H, JIANG K J, et al Chemical constituents from the roots of Sphallerocarpus gracilis[J]Nat Prod Res Dev (Natural products research and development), 2003,15 (3): 196-198.) the molecular formula of this compound was finally determined to be C 24 H 26 O 7 The structural formula is
TABLE 3 Xanthalin 1 H hydrogen spectrum 13 C carbon spectrum nuclear magnetic data
(6) Preparation of a control solution: preparation of a control solution: taking a proper amount of the differential marker Xanthalin, precisely weighing, and adding 70v/v% methanol to prepare a solution containing 20 mug of the differential marker per 1 ml.
(7) Preparing a sample solution to be tested: taking 1g of No. 1 sample powder to be detected (sieving with No. four sieve), adding 50ml of 70v/v% methanol, weighing, carrying out ultrasonic treatment for 30min, cooling to room temperature, weighing again, supplementing the lost weight with 70v/v% methanol, shaking uniformly, filtering with a 0.45 mu m microporous filter membrane, and taking the subsequent filtrate as No. 1 sample solution.
(8) Detecting and judging: respectively sucking 10 μl of the reference substance solution and 10 μl of the sample solution to be tested 1, and injecting into a liquid chromatograph for measurement to obtain a liquid chromatogram;
if the same chromatographic peak does not appear in the liquid chromatogram of the sample solution to be detected at the position corresponding to the retention time of the chromatographic peak of the reference substance solution, judging that the sample to be detected is divaricate saposhnikovia root; chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.1% phosphoric acid aqueous solution (70:30) is taken as a mobile phase; the detection wavelength was 222nm using a diode array detector. The theoretical plate number is not less than 3000 calculated according to Xanthalin peak.
If the same chromatographic peak appears in the liquid chromatogram of the sample solution to be detected at the position corresponding to the retention time of the chromatographic peak of the reference solution, comparing ultraviolet-visible absorption spectrums of the corresponding chromatographic peak in the wavelength range of 190-400 nm by adopting a diode array detector, judging that the sample to be detected is windproof if the ultraviolet-visible absorption spectrums are different, and judging that the sample to be detected is doped with water for windproof if the ultraviolet-visible absorption spectrums are the same.
As can be seen from fig. 12, the same chromatographic peak does not appear at the position corresponding to the retention time of the chromatographic peak of the control solution, and the sample to be measured is judged as saposhnikovia.
Example 2
The method of example 1 was used to identify wind and water, except that: and detecting and judging the powder of the No. 2 sample to be detected.
As can be seen from FIG. 13, in FIG. 13, the same chromatographic peak appears at the position corresponding to the retention time of the chromatographic peak of the reference solution, and as can be seen from FIG. 14, the corresponding chromatographic peak has the same ultraviolet-visible absorption spectrum in the wavelength range of 190-400 nm, and it is determined that the sample to be measured is adulterated with water for preventing wind.
Example 3
The method of example 1 was used to identify wind and water, except that: the steps (7) and (8) are changed into:
(7) Preparing a sample solution to be tested: taking 0.2g of No. 3 sample powder to be detected (sieving with No. four sieve), adding 100ml of 70v/v% methanol, weighing, carrying out ultrasonic treatment for 30min, cooling to room temperature, weighing again, supplementing the lost weight with 70v/v% methanol, shaking uniformly, filtering with a 0.45 mu m microporous filter membrane, and taking the subsequent filtrate to obtain the product.
The method for detecting the sample to be detected by adopting the high performance liquid chromatography-mass spectrometer comprises the following steps: respectively sucking 1 μl of the reference substance solution and 1 μl of the sample solution to be detected, and injecting into a high performance liquid chromatograph-mass spectrometer for measurement;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, and the inner diameter of a chromatographic column is 2.1mm; acetonitrile is taken as a mobile phase A, 0.1% formic acid solution is taken as a mobile phase B, and the flow rate is 0.3ml per minute;
(8) Detecting and judging: respectively sucking 1 μl of the reference substance solution and 1 μl of the sample solution to be detected, and injecting into a high performance liquid chromatography-mass spectrometer for measurement to obtain extracted ion flow chromatograph;
if the same chromatographic peak does not appear at the position corresponding to the retention time of the chromatographic peak of the reference substance solution in the extracted ion flow chromatography of the sample solution to be detected, judging that the sample to be detected is windproof, otherwise, judging that the sample to be detected is doped with water for windproof.
Chromatographic, mass spectrometry conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler (the inner diameter of a chromatographic column is 2.1 mm); acetonitrile as mobile phase a and 0.1% formic acid solution as mobile phase B, and the flow rate was 0.3ml per minute; electrospray positive ion mode (ESI) using a triple quadrupole mass spectrometer detector + ) Multiple Reaction Monitoring (MRM), m/z427.2 →245.1 and m/z427.2 →327.1 were chosen as detection ion pairs. The theoretical plate number is not less than 3000 calculated according to Xanthalin peak.
TABLE 5 gradient elution procedure
Comparing fig. 15 and 16, it can be seen that in the extracted ion flow chromatography of the sample solution to be tested, the same chromatographic peak does not appear at the position corresponding to the retention time of the chromatographic peak of the reference solution, and the sample to be tested is judged to be divaricate saposhnikovia root.
Example 4
The method of example 3 was used to identify wind and water, except that: and detecting and judging the powder of the sample to be detected No. 4.
Comparing fig. 17 and 18, it can be seen that in the extracted ion flow chromatograph of the sample solution to be detected, the same chromatographic peak appears at the position corresponding to the retention time of the chromatographic peak of the reference solution, and it is determined that the sample to be detected is adulterated with water for preventing wind.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (7)
1. The detection method for identifying the wind prevention and the water wind prevention is characterized by comprising the following steps of:
(1) Preparation of test solution: respectively taking a plurality of batches of exact-source radix sileris and water radix sileris sample powder, respectively adding methanol into each sample powder, weighing, carrying out ultrasonic treatment, cooling to room temperature, weighing again, supplementing the reduced weight with methanol, shaking uniformly, centrifuging at an ultrahigh speed, and taking supernatant to obtain each sample solution;
(2) Sample analysis: respectively taking 1 mu L of the sample solution prepared in the step (1), and respectively collecting total ion flow diagrams of the sample solution by adopting a high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer;
the liquid chromatography conditions for sample analysis in step (2) are: 4.6mm×50mm,2.7 μmAgilent Poroshell, EC-C 18 Is a chromatographic column, mobile phase: acetonitrile-0.1% formic acid solution, column temperature 30 ℃, flow rate 0.5 mL.min -1 The elution gradient is:
；
the mass spectrum condition is electrospray ionization source, the collection mode is positive ion mode, the temperature of the drying gas is 300 ℃, the flow rate of the drying gas is 5L/min, the pressure of an atomizer is 35psi, the temperature of the sheath gas is 350 ℃, the flow rate of the sheath gas is 11L/min, the capillary voltage is 3500V, the nozzle voltage is 500V, the mass scanning range is 50-1000 m/z, and the fragmentation voltage is 135V;
(3) PCA analysis and cluster analysis: introducing the mass spectrum raw data obtained in the step (2) into MPP data processing software, extracting characteristic peak data, and carrying out principal component analysis and cluster analysis on the characteristic peaks;
(4) Screening of differential markers: dividing radix Saposhnikoviae and radix Ledebouriellae into two groups by using the function of searching for characteristic compounds of MPP data processing software, screening the difference compounds among the groups according to the compound response values, and obtaining the difference markers which have good peak shape, high response value, moderate retention time and are not existing in radix Ledebouriellae by analyzing the extracted ion flow diagram of each difference compound;
(5) Separation and purification of differential markers and chemical structure identification: separating and purifying high-purity differential markers from a test sample of Saponaria officinalis by silica gel column chromatography, and determining the structural formula of the differential markers as follows by mass spectrum and nuclear magnetic identification
；
The specific method for separating and purifying the differential markers comprises the following steps: taking 1kg of a batch of divaricate saposhnikovia root test sample powder, adding 95v/v% methanol 3L, soaking, carrying out ultrasonic treatment, filtering, concentrating the extracting solution under reduced pressure to dryness to obtain a tan oily extract, and separating the tan oily extract by using a silica gel column chromatography, wherein the silica gel column chromatography specifically comprises: firstly, eluting with a chloroform/methanol solvent system according to a gradient of 95:5 to 50:50, and collecting a fraction Fr.A eluted by 75% of chloroform/methanol; eluting the fraction Fr.A with n-hexane/ethyl acetate solvent system according to a gradient of 75:25-30:70, and collecting 55% n-hexane/ethyl acetate eluted fraction Fr.B; finally purifying the fraction Fr.B by using a preparation liquid, wherein the mobile phase is acetonitrile-water with the volume ratio of 40:60, and the flow rate is 3.0 mL.min < -1 >, so as to obtain the high-purity differential marker;
(6) Preparation of a control solution: adding methanol into the differential markers to prepare a solution containing 2 mug of the differential markers per 1 ml;
(7) Preparing a sample solution to be tested: adding methanol into the sample powder to be measured, weighing, cooling to room temperature after ultrasonic treatment, weighing again, supplementing the reduced weight with methanol, shaking uniformly, filtering with microporous membrane, and collecting the subsequent filtrate;
(8) Detecting and judging: respectively sucking an equivalent amount of reference substance solution and a sample solution to be detected, and injecting the solution into a liquid chromatograph or a high performance liquid chromatograph-mass spectrometer for detection to obtain a liquid chromatograph or an extracted ion flow chromatograph;
if the same chromatographic peak does not appear in the liquid chromatogram of the sample solution to be detected at the position corresponding to the retention time of the chromatographic peak of the reference substance solution, judging that the sample to be detected is divaricate saposhnikovia root;
if the same chromatographic peak appears in the liquid chromatogram of the sample solution to be detected at the position corresponding to the retention time of the chromatographic peak of the reference solution, comparing ultraviolet-visible absorption spectrums of the corresponding chromatographic peak in the wavelength range of 190-400 nm by adopting a diode array detector, judging that the sample to be detected is windproof if the ultraviolet-visible absorption spectrums are different, and judging that the sample to be detected is doped with water for windproof if the ultraviolet-visible absorption spectrums are the same;
if the same chromatographic peak does not appear at the position corresponding to the retention time of the chromatographic peak of the reference substance solution in the extracted ion flow chromatography of the sample solution to be detected, judging that the sample to be detected is windproof, otherwise, judging that the sample to be detected is doped with water for windproof.
2. The method for detecting the identity of saposhnikovia divaricata and saposhnikovia divaricata according to claim 1, wherein the specific steps of preparing the test solution in the step (1) are as follows: respectively taking 1g of radix Saposhnikoviae and 1g of radix Saposhnikoviae powder, precisely weighing, respectively adding 50ml of 70v/v% methanol, weighing, performing ultrasonic treatment for 30min, cooling to room temperature, weighing, supplementing the lost weight with 70v/v% methanol, shaking, centrifuging at ultrahigh speed, and collecting supernatant.
3. The method for detecting the identity of wind and water according to claim 1, wherein the specific method of PCA analysis is: introducing mass spectrum raw data into MPP data processing software, and extracting data of characteristic peaks by using screening methods of peak matching, peak alignment and noise filtering; then, based on a t-test statistical algorithm with the p value smaller than 0.05, carrying out principal component analysis on the characteristic peak; and finally, sequentially compiling the statistical results of the first, second and third main component elements into an X axis, a Y axis and a Z axis, establishing a 3D structure model of 30 batches of samples, and observing the difference between wind prevention and water prevention.
4. The method for detecting the identification of the wind prevention and the water prevention according to claim 1, wherein the specific steps of the mass spectrum identification of the differential markers are as follows: adding methanol into the high-purity differential marker prepared in the step (5) to prepare a marker solution, and collecting a secondary mass spectrum fragment diagram by adopting a high performance liquid chromatography tandem four-pole time-of-flight mass spectrometer; the mass spectrum and chromatographic conditions are the same as in the step (2).
5. The detection method for distinguishing between radix Saposhnikoviae and radix Ledebouriellae according to claim 1, wherein the specific preparation method of the sample solution to be detected by liquid chromatograph comprises: taking 1g of sample powder to be detected, adding 50ml of 70v/v% methanol, weighing, carrying out ultrasonic treatment for 30min, cooling to room temperature, weighing again, supplementing the weight of the sample powder to be detected with 70v/v% methanol, shaking uniformly, filtering with a 0.45 mu m microporous filter membrane, and taking a subsequent filtrate to obtain the product;
the specific preparation method of the sample solution to be detected by adopting the high performance liquid chromatography-mass spectrometer comprises the following steps: taking 0.2g of sample powder to be detected, adding 100ml of 70v/v% methanol, weighing, carrying out ultrasonic treatment for 30min, cooling to room temperature, weighing again, supplementing the lost weight with 70v/v% methanol, shaking uniformly, filtering with a 0.45 mu m microporous filter membrane, and taking the subsequent filtrate.
6. The method for detecting the wind-proof and the water-proof according to claim 1, wherein the method for detecting the sample solution to be detected by using a liquid chromatograph comprises the following steps: respectively sucking 10 μl of the reference substance solution and the sample solution to be detected, and injecting into a liquid chromatograph for measurement to obtain a liquid chromatogram;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler; the volume ratio is 70:30 acetonitrile-0.1% phosphoric acid aqueous solution as mobile phase; the detection wavelength was 222nm using a diode array detector.
7. The method for detecting the wind prevention and the water prevention according to claim 1, wherein the method for detecting the sample to be detected by using a high performance liquid chromatography-mass spectrometer comprises the following steps: respectively sucking 1 μl of the reference substance solution and 1 μl of the sample solution to be detected, and injecting into a high performance liquid chromatograph-mass spectrometer for measurement;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, and the inner diameter of a chromatographic column is 2.1mm; acetonitrile is taken as a mobile phase A, 0.1% formic acid solution is taken as a mobile phase B, and the flow rate is 0.3ml per minute;
the mass spectrum conditions are as follows: the electrospray positive ion mode, multi-reaction monitoring, and m/z 427.2- & gt 245.1 and m/z 427.2- & gt 327.1 are selected as detection ion pairs.
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