CN110057927B - Detection method combining fingerprint spectrum and liquid chromatography-mass spectrometry of Qilong capsules and application of detection method - Google Patents
Detection method combining fingerprint spectrum and liquid chromatography-mass spectrometry of Qilong capsules and application of detection method Download PDFInfo
- Publication number
- CN110057927B CN110057927B CN201811621837.2A CN201811621837A CN110057927B CN 110057927 B CN110057927 B CN 110057927B CN 201811621837 A CN201811621837 A CN 201811621837A CN 110057927 B CN110057927 B CN 110057927B
- Authority
- CN
- China
- Prior art keywords
- fingerprint
- solution
- mobile phase
- capsule
- qilong
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The application belongs to the field of traditional Chinese medicine fingerprint analysis, and particularly relates to a detection method combining fingerprint analysis and liquid chromatography-mass spectrometry of Qilong capsules and application thereof. The application provides a method for establishing a fingerprint of a Qilong capsule and a method for controlling the quality of the Qilong capsule by detecting the fingerprint and astragaloside. In the application, the fingerprint is established through the high-performance liquid phase, and 15 common peaks are obtained through high-performance liquid phase separation, so that the quality condition of the Qilong capsule can be reflected integrally. The construction method is simple to operate, stable and reliable, high in precision, good in separation degree, good in stability and reproducibility of a fingerprint spectrum, large in information amount, capable of avoiding one-sidedness of determining the overall quality of the preparation due to the fact that only one or two chemical components are measured, reducing the possibility of manual treatment for reaching the quality standard, and capable of comprehensively and scientifically evaluating the quality of the Qilong capsule by performing systematic analysis on a plurality of batches of samples, so that the quality and the curative effect of the product are guaranteed.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine fingerprint analysis, and particularly relates to a method for detecting a Qilong capsule by combining fingerprint analysis and liquid chromatography-mass spectrometry of the Qilong capsule and application of the Qilong capsule as a quality control means.
Background
The Qilong capsule is a Chinese medicinal compound preparation prepared by eight medicinal materials of astragalus root, earthworm, salvia miltiorrhiza, angelica, red peony root, Szechuan lovage rhizome, safflower and peach seed through fine processing, has proved that the Qilong capsule contains a plurality of biological active substances, has good curative effects of tonifying qi, activating blood circulation, removing blood stasis and dredging collaterals, and is used for treating ischemic stroke (cerebral infarction) in the recovery period with symptoms of hemiplegia, facial distortion, slurred speech, hemianesthesia and tongue ecchymosis and petechia. In the formula, the astragalus root is used as a monarch drug to reinforce the primordial qi of the spleen and stomach, promote qi and blood circulation, remove blood stasis and remove meridian obstruction. Dang Gui is good at activating blood and has the action of resolving stasis without damaging blood. Is used as a ministerial drug. Salvia miltiorrhiza, as a ministerial drug, passes through blood vessels, removes stasis and relieves pain. The hemlock parsley, the red paeony root, the peach seed and the safflower are adjuvant drugs for assisting the angelica in promoting blood circulation and removing blood stasis and the earthworm in clearing and activating the channels and collaterals.
Thrombosis and embolism are the basis of cerebral infarction, so the ideal method is to restore the normal blood flow of ischemic brain tissue before necrosis occurs, and the brain tissue obtains early reperfusion of the cerebral blood flow, thereby relieving the degree of ischemia and limiting the damage of nerve cells and functions thereof. Huang Qi is sweet and warm, entering spleen and lung meridians, and good at tonifying spleen and lung qi. The radix salviae miltiorrhizae is added into the astragalus-dragon capsule to enhance the effects of tonifying qi, promoting the production of body fluid, tonifying qi, generating blood and the like, and can obviously improve the symptoms of thrombus, embolism and the like. The Qilong capsule has the effects of benefiting qi and nourishing blood, activating blood circulation to dissipate blood stasis and treating both principal and secondary aspects of diseases, and is mainly used for qi deficiency and blood stasis in the meridian recovery period of ischemic stroke (cerebral infarction).
The traditional Chinese medicine and the compound thereof are applied and researched under the guidance of the theory of traditional Chinese medicine, the components are complex, the part exerting the drug effect is not a single component but a group curative effect, so any single component cannot reflect the whole curative effect of the traditional Chinese medicine and the compound thereof. Therefore, it is necessary to perform comprehensive analysis on the traditional Chinese medicine and the compound thereof macroscopically. The traditional Chinese medicine fingerprint comprehensively reflects the quality information of the traditional Chinese medicine and the compound thereof by various spectrum, wave spectrum and chromatographic techniques, and is an internationally recognized important means for controlling the quality of the traditional Chinese medicine and the compound thereof at present.
The traditional Chinese medicine fingerprint spectrum technology reflects the holistic concept of the traditional Chinese medicine theoretical system and carries out scientific analysis on the chemical components in the traditional Chinese medicine and the compound thereof. In recent years, researches on fingerprint technology and pharmacodynamics are becoming mature. The traditional Chinese medicine fingerprint spectrum technology has an indispensable effect in the field of traditional Chinese medicine quality control and the aspect of pharmacodynamic research. The astragalus-dragon capsule is used as an addition and subtraction method of yang-tonifying five-returning decoction, and the research on the fingerprint spectrum establishment method of the yang-tonifying five-returning decoction in the prior art is relatively mature. However, the ingredients and the dosage of the Qilong capsule are not completely the same as those of the Yang tonifying and Yang restoring decoction, and the ingredients of the Qilong capsule are changed after the steps of alcohol extraction, ultrafiltration and the like are carried out in the preparation process. Aiming at the quality control research of the Qilong capsule, no report is found yet, and the provision of the fingerprint spectrum establishment method of the Qilong capsule has important significance.
Disclosure of Invention
Aiming at the technical problems, the application provides a method for establishing a fingerprint of a Qilong capsule. The research on the analysis of the effective components in the astragalus-dragon capsule in the field is blank, and the components of the compound in the capsule are complex and not completely clear. Based on the current research, the mixed standard substance capable of reflecting the overall quality condition of the capsule is screened out, and the five components of paeoniflorin, ferulic acid, tanshinone IIA, salvianolic acid B and hydroxysafflor flavin A have high pharmacological activity and rich content, and can well indicate the overall quality change condition of the capsule when being used as the mixed standard substance. In addition, the research of the application provides a corresponding liquid chromatography analysis method aiming at the analysis of the astragalus-dragon capsule, and the liquid phase condition can separate 15 common peaks, which is beneficial to reflecting the quality change condition of the astragalus-dragon capsule on the whole.
In addition, the application also provides a quality control method of the astragalus-dragon capsules, the astragalus-dragon capsules of a plurality of batches are measured by the fingerprint spectrum established by the method, and the quality condition of the astragalus-dragon capsules is evaluated by investigating the similarity of the spectrogram and the content of the astragaloside in the astragalus-dragon capsules. As known in the art, the astragaloside IV is taken as the main effective component of the monarch drug radix astragali in the formula, and only has absorption at the ultraviolet end, so that the detection interference is high, and the sensitivity is low. The quality control method of the application provides a liquid chromatography-mass spectrometry method for analyzing the astragalus membranaceus-dragon capsule, and provides a method for detecting the content of astragaloside by high performance liquid separation-link mass spectrometry aiming at the technical problem that astragaloside is not suitable for ultraviolet detection.
In order to achieve the above technical effects, the present application provides the following technical solutions:
in a first aspect of the application, a fingerprint spectrum establishment method of a Qilong capsule is provided, and the method takes paeoniflorin, ferulic acid, tanshinone IIA, salvianolic acid B and hydroxysafflorin A as mixed standard substances.
Preferably, the fingerprint establishing method comprises the following steps:
(1) preparation of a test solution: taking astragalus-dragon capsule powder, adding methanol, carrying out ultrasonic extraction for a period of time, centrifuging and taking supernatant to obtain a solution A; adding water into the residue, heating in water bath for a period of time, centrifuging to obtain supernatant to obtain solution B, and mixing solution A and solution B to obtain test solution;
(2) preparation of a reference solution: collecting constant weight reference substances of ferulic acid, paeoniflorin, salvianolic acid B, tanshinone IIA, and hydroxysafflor yellow A, respectively adding methanol to obtain reference substance solutions;
(3) and (3) determination: respectively and precisely measuring a test solution and a reference solution, injecting the test solution and the reference solution into a high performance liquid chromatograph for separation, and performing gradient elution to obtain a mobile phase A: 5mM ammonium acetate, 0.1% acetic acid in water, mobile phase B: acetonitrile; analyzing and processing the chromatogram obtained by the high performance liquid chromatography by fingerprint software to obtain the Qilong capsule fingerprint.
Preferably, a certain amount of the astragalus membranaceus-dragon capsule powder is weighed in the step (1), added with methanol, subjected to ultrasonic treatment for 0.8-1.2h, centrifuged at 3500-4500r/min for 8-12min, and centrifuged to obtain a supernatant solution A, wherein the astragalus membranaceus-dragon capsule powder: the ratio of methanol was (180-220) mg: (4-6) mL. Adding triple distilled water into the residue, uniformly mixing, performing water bath at 65-75 ℃ for 1.8-2.2h, and centrifuging at 3500-. Mixing the solution A and the solution B according to the following ratio (4-6): (6-8), adding a mobile phase for diluting by 4-6 times, and filtering by an organic filter membrane to obtain a test solution.
Preferably, the preparation method of the reference solution in the step (2) is as follows: precisely weighing 0.0100g of ferulic acid standard, 0.0099g of paeoniflorin standard, 0.0101g of tanshinone IIA standard, 0.0102g of salvianolic acid B standard and 0.0099g of hydroxysafflor A standard, placing in a 10mL volumetric flask, adding methanol to dissolve and dilute to scale, and respectively preparing standard solutions.
Preferably, the column used in the step (3) is CAPCELL PAK C18 (2.0X 150mm, 5 μm, Shiseido, Japan); the flow rate of the mobile phase is 0.3 ml/min; the column temperature was 35 ℃; the detection wavelength was 275nm, and the amount of sample was 20. mu.L.
Preferably, the gradient elution mode in the step (3) is as follows: 0-25min, mobile phase B10-25%; 25-30min, mobile phase B25-39%; 30-35min, mobile phase B39-42%; 35-40min, 42-50%; 40-45min, mobile phase B50-55%; 45-60min, and mobile phase B55-90%.
Wherein: 1) investigation of test solution preparation method: the test compares the effect of adding mobile phase dilution factor on the detection effect of the sample solution, such as 20-fold, 10-fold, 5-fold and 2-fold dilution, and the result is that the detection effect is better when the initial mobile phase (5mM ammonium acetate, 0.1% acetic acid in water: acetonitrile: 9: 1) is diluted 5-fold.
2) Selection and optimization of chromatographic conditions: the effect of different salt solutions (ammonium acetate and phosphoric acid) and ammonium acetate buffer ratios (5mM and 10mM) and acetate buffer ratios (0.1%, 0.2% and 0.3%) on the chromatographic separation in the mobile phase was examined. The test result shows that: when the proportion of the ammonium acetate buffer solution in the mobile phase A is 5mM, the chromatographic separation effect is good, the base line is stable, the peak shape is symmetrical, the separation degree is good, and the stability of the retention time of the chromatographic peak can be obviously improved after 0.1% acetic acid is added, so that the mobile phase is determined as follows: phase A (5mM ammonium acetate, 0.1% acetic acid in water) -phase B (acetonitrile). After the elution proportion of the mobile phase at different time is adjusted, the retention time of each chromatographic peak is moderate, the base line is stable, the drift is not easy, the separation degree of the chromatogram is improved, the tailing phenomenon of the chromatogram is effectively avoided, and the detection and the analysis of the chromatographic fingerprint are facilitated.
3) Selection of detection wavelength: comparing and analyzing chromatograms of a photodiode array detector (PDAD) under various wavelengths scanned at 190-400 nm, setting 8 wavelengths for simultaneous detection (including 203nm,210nm,230nm,260nm,275nm and 300nm), wherein the detection result shows that: the chromatogram detected at 275nm has the advantages of stable base line, more chromatographic peaks, large information amount, good separation effect of each chromatographic peak and large peak area. Therefore, 275nm was selected as the detection wavelength. Meanwhile, chromatograms at other wavelengths are examined according to the requirements of the trial standard, and the result shows that the chromatographic fingerprint of the astragalus-dragon capsule at different wavelengths has better similarity.
4) Selection of analysis time: the chromatogram for 2h was recorded when the elution time of the fingerprint was chosen. The result shows that no obvious chromatographic peak appears after 55min, and in order to take care of the difference of the batch samples, the characteristic peaks of all the batch samples can be detected, so 60min is selected as the analysis time.
5) Selection of column temperature: the influence of four different column temperatures (e.g. 25 ℃, 30 ℃, 35 ℃ and 40 ℃) on the fingerprint detection result is tested. The result shows that when the column temperature is 25 ℃, the retention time of chromatographic peaks is appropriate, the base line is stable, the separation degree of each chromatographic peak is good, and the peak shapes are symmetrical, so the column temperature is selected to be 25 ℃.
6) Selection of flow rate: the effect of four flow rates (0.3mL/min,0.4mL/min and 0.5mL/min) on the fingerprint detection results was tested. The results show that: when the flow rate is 0.3mL/min, the separation effect is optimal, the retention time of each chromatographic peak is proper, the separation degree is good, the base line is stable, and the peak shapes are symmetrical, so that the flow rate is selected to be 0.3 mL/min.
The fingerprint of the astragalus-dragon capsule obtained by the construction method of the fingerprint of the astragalus-dragon capsule has 15 common peaks.
The second aspect of the invention provides a quality control method of astragalus-dragon capsules, which comprises the following steps: taking multiple batches of Qilong capsules to obtain the Qilong capsule sample maps according to the fingerprint map method, and comparing the similarity of the maps of the Qilong capsules to be detected with the reference sample maps, wherein the similarity calculation method is a correlation coefficient method.
Preferably, the method for establishing the reference sample fingerprint in the quality control method comprises the following steps: and calculating the similarity between each sample spectrum and other sample spectrums, selecting the sample with the maximum sum of the similarities with other samples as a reference sample, and taking the fingerprint spectrum as the fingerprint spectrum of the reference sample.
In a third aspect of the invention, another quality control method of the astragalus-dragon capsule is provided, and the quality control method detects the content of the astragaloside in the astragalus-dragon capsule through LC-MS.
Compared with UV, the LC-MS/MS system has the characteristic of high detection sensitivity and the characteristic of high precision compared with an evaporation light detector, so that the quality control method can provide a reference method for detecting astragaloside IV with higher sensitivity and high precision for pharmacopoeia.
Preferably, the astragalus-dragon capsule to be detected is taken, the astragaloside IV map and the astragaloside IV standard map in the astragalus-dragon capsule are obtained by a liquid chromatography-mass spectrometry method, and the content of the astragaloside IV in the astragalus-dragon capsule is determined according to an external standard one-point method.
Preferably, the liquid chromatography conditions in the quality control method are as follows: the column was CAPCELL PAKC18 (2.0X 150mm, 5 μm, Shiseido, Japan); mobile phase: phase a-aqueous solution containing 5mM ammonium acetate, 0.1% acetic acid, phase B-acetonitrile; and (3) an elution mode: during the gradient elution, the changes of mobile phase a and mobile phase B are: 60-60% of B for 0-3 min; 60% -90% of B in 3-3.1 min; 90-90% of B in 3.1-6.0 min; 90-60% of B in 6.0-6.1 min; 60-60% of B in 6.1-10.0 min. The flow rate of the mobile phase is 0.350 ml/min; the column temperature is 30 ℃; the amount of the sample was 10. mu.L.
Preferably, the mass spectrometry conditions in the mass control method are: an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: a positive ion; detecting ion pairs: astragaloside m/z: 785.5/587.3; ion source voltage: 5000V; ion source temperature: at 450 ℃; air curtain air: 10 psi; atomizing: 45 psi; auxiliary gas: 55 psi; cluster-splitting voltage: 55V; collision induced dissociation voltage: 24V.
The invention has the beneficial effects that:
(1) the present invention adopts a water solution-acetonitrile system containing 5mM ammonium acetate and 0.1% acetic acid, and adopts a gradient elution method to establish the fingerprint of the Qilong capsule, and the method has the advantages of simple operation, stability, reliability, high precision, good separation degree, good stability and reproducibility of the fingerprint, and large information amount.
(2) Because the fingerprint is not used for measuring the accurate content of a certain component, but is used for fully reflecting the information of chemical components, the invention selects the wavelength of 275nm for measurement, the number of peaks is more, the reflected information is more complete, the absorption value of each peak is good, and the baseline is stable.
(3) The invention adopts the fingerprint of the astragalus-dragon capsule as the quality control means of the astragalus-dragon capsule, thereby not only avoiding the one-sidedness of judging the whole quality of the preparation by only measuring one or two chemical components, but also reducing the possibility of manual treatment for reaching the quality standard, and more comprehensively and scientifically evaluating the quality of the astragalus-dragon capsule by carrying out systematic analysis on a plurality of batches of samples, thereby ensuring the quality and the curative effect of the product.
(4) The invention adopts the reference sample fingerprint to compare the fingerprints, avoids the influence of each batch of samples on the comparison fingerprint, enhances the quality distinguishing capability of different batches of samples, has better performance than the comparison fingerprint, and more accords with the actual condition of the judgment result.
(5) The invention adopts the high performance liquid chromatography and mass spectrometry combined technology to determine the content of the active ingredient astragaloside in the astragalus-dragon capsule, and effectively solves the defects of weak ultraviolet absorption and difficult detection of the astragaloside. Can be used as quality control and evaluation means of QILONG Capsule.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 shows HPLC finger print and control finger print of each batch of QILONG capsule;
FIG. 2 is a finger print of astragaloside IV reference substance mixture;
FIG. 3 is a LC-MS spectrum of a control solution of astragaloside IV;
FIG. 4 is a LC-MS spectrum of astragaloside IV in QILONG Capsule.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced by the background art, a stable and reliable method for controlling the quality of the astragalus-dragon capsule in the prior art is not available, and in order to solve the technical problems, the application provides a method for establishing the fingerprint of the astragalus-dragon capsule.
Example 1: construction of fingerprint and methodological verification of Qilong capsule
(1) Preparation of a test solution: precisely weighing 200mg of dry astragalus-dragon capsule powder to be detected, placing a sample in a 10mL centrifuge tube, adding 5mL of methanol, performing ultrasonic treatment for 1h, centrifuging at the rotating speed of 4000r/min for 10min, and taking 4.5mL of supernatant to obtain a fat-soluble methanol extracting solution. Adding 5mL of triple distilled water into the residue, mixing, centrifuging at 70 deg.C water bath for 2h at 4000r/min for 10min, taking out about 4.5mL of water-soluble extract, mixing the two at a ratio of 5:7, and diluting with mobile phase by 5 times. Filtering with 0.22 μm organic filter membrane to obtain 5 times diluted sample solution.
(2) Preparation of control solutions:
precisely weighing 0.0100g of ferulic acid standard, placing in a 10mL volumetric flask, adding methanol for dissolving, and diluting to scale to obtain ferulic acid solution with concentration of 1.002 mg/mL;
accurately weighing 0.0099g of paeoniflorin standard, placing in a 10mL volumetric flask, adding methanol for dissolving, and diluting to scale to obtain a paeoniflorin solution with a concentration of 0.998 mg/mL;
accurately weighing 0.0101g tanshinone IIA standard, placing in a 10mL volumetric flask, adding methanol to dissolve and diluting to scale to obtain tanshinone IIA solution with concentration of 1.012 mg/mL;
accurately weighing 0.0102g of salvianolic acid B standard, placing in a 10mL volumetric flask, adding methanol for dissolving, and diluting to scale to obtain salvianolic acid B solution with concentration of 1.023 mg/mL;
0.0099g of hydroxysafflor flavin A standard substance was precisely weighed, placed in a 10mL volumetric flask, dissolved in methanol and diluted to the mark to obtain a hydroxysafflor flavin A solution at a concentration of 0.995 mg/mL.
(3) And (3) determination: respectively and precisely absorbing the test solution and the reference solution, injecting the test solution and the reference solution into a high performance liquid chromatograph for measurement, recording a chromatogram, and carrying out methodology investigation.
The chromatographic conditions are as follows: the column was CAPCELL PAK C18 (2.0X 150mm, 5 μm, Shiseido, Japan); mobile phase: phase a-aqueous solution containing 5mM ammonium acetate, 0.1% acetic acid, phase B-acetonitrile; and (3) an elution mode: gradient elution; the flow rate of the mobile phase is 0.3 ml/min; the column temperature was 35 ℃; the detection wavelength was 275nm, and the amount of sample was 20. mu.L. The theoretical plate number is not less than 5000 according to paeoniflorin chromatographic peak, the separation degree from other main chromatographic peaks is greater than 1.0, and all components are detected within 60 min. Wherein, the mobile phase linear gradient is shown in table 1:
TABLE 1 chromatographic mobile phase gradient elution conditions
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 25 | 75 | 25 |
| 30 | 61 | 39 |
| 35 | 58 | 42 |
| 40 | 50 | 50 |
| 45 | 45 | 55 |
| 60 | 10 | 90 |
The methodology investigation mainly comprises stability, repeatability and precision investigation, and the investigation method and the result are as follows:
① stability experiment samples of the same batch of Qilong capsules are taken, after pretreatment, under optimized chromatographic conditions, sample injection analysis is carried out for 0h, 2h, 4h, 8h, 12h, 16h and 24h respectively, chromatogram is recorded, the result is shown in table 2. as can be seen from table 2, relative retention time of each main chromatographic peak and relative peak area ratio have no obvious change, RSD is 0.14% -0.93% and 1.8% -5.0% respectively, which indicates that the components of the test solution are stable within 24 h.
② precision experiment takes the same batch of Qilong capsule sample, after pretreatment, under the optimized chromatographic condition, continuously measures for 6 times, records chromatogram, the result is shown in Table 2, the relative retention time of each main chromatogram peak and the relative peak area ratio have no obvious change, the RSD is 0.35-2.3% and 0.38-4.59%, the RSD is less than 5.0%, and the precision of the instrument is good.
③ repeatability experiment takes 5 parts of the same batch of Qilong capsule samples, after pretreatment, under the optimized chromatographic condition, the samples are respectively injected and analyzed, chromatogram is recorded, the result is shown in Table 2, the relative retention time of each main chromatographic peak and the relative peak area ratio have no obvious change, RSD is 0.68-2.77% and 1.04-4.07%, RSD is less than 5.0%, which shows that the repeatability of the experiment method is better.
TABLE 2 fingerprint chromatogram methodological verification results of Qilong capsules
(4) Processing the obtained chromatogram with fingerprint software to obtain the Qilong capsule fingerprint. Wherein, the fingerprints of different batches of QILONG Capsule samples and reference substances are shown in figure 1, and the fingerprint of the reference substance is shown in figure 2.
Compared with the fingerprint of the Qilong capsule in figure 1, the 5 components which need to be identified and quantified are well separated, the peak shapes of chromatographic peaks are symmetrical, and the base line is stable. The retention time of hydroxysafflor yellow A, paeoniflorin, ferulic acid, salvianolic acid B and tanshinone IIA is respectively as follows: 4.37, 11.96, 12.88, 16.51 and 55.96 min.
Example 2: quality control of QILONG Capsule by using fingerprint
The quality control of the Qilong capsule is carried out by utilizing the fingerprint, and the specific method comprises the following steps:
(1) constructing the fingerprint of the Qilong capsule to be detected: taking 11 batches of astragalus-dragon capsule samples to be detected, and constructing the fingerprint of the astragalus-dragon capsule to be detected according to the method in the embodiment 1;
(2) matching HPLC fingerprint chromatogram peaks of 11 batches of Qilong capsule samples by adopting software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system. S1 (sample No. 1) was selected as the reference, and the resulting control fingerprint was R. The results of the similarity of the fingerprints of the samples in each batch to the control fingerprints and between the samples are shown in table 3.
Similarity value of fingerprint of astragalus-dragon capsules of 311 batches (correlation coefficient method)
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | S11 | S12 | S13 | S14 | S15 | R | |
| S1 | 1 | 0.951 | 0.957 | 0.996 | 0.955 | 0.957 | 0.961 | 0.996 | 0.962 | 0.96 | 0.996 | 0.959 | 0.953 | 0.954 | 0.99 | 0.981 |
| S2 | 0.951 | 1 | 0.993 | 0.961 | 0.992 | 0.991 | 0.992 | 0.964 | 0.992 | 0.993 | 0.962 | 0.989 | 0.965 | 0.964 | 0.964 | 0.989 |
| S3 | 0.957 | 0.993 | 1 | 0.964 | 0.999 | 0.996 | 0.999 | 0.97 | 0.999 | 0.999 | 0.97 | 0.997 | 0.967 | 0.964 | 0.963 | 0.994 |
| S4 | 0.996 | 0.961 | 0.964 | 1 | 0.963 | 0.962 | 0.967 | 0.995 | 0.968 | 0.966 | 0.995 | 0.963 | 0.961 | 0.962 | 0.992 | 0.985 |
| S5 | 0.955 | 0.992 | 0.999 | 0.963 | 1 | 0.993 | 0.999 | 0.966 | 0.998 | 0.998 | 0.968 | 0.994 | 0.964 | 0.96 | 0.96 | 0.992 |
| S6 | 0.957 | 0.991 | 0.996 | 0.962 | 0.993 | 1 | 0.996 | 0.97 | 0.997 | 0.997 | 0.968 | 0.998 | 0.962 | 0.964 | 0.962 | 0.992 |
| S7 | 0.961 | 0.992 | 0.999 | 0.967 | 0.999 | 0.996 | 1 | 0.973 | 0.999 | 0.999 | 0.974 | 0.998 | 0.968 | 0.965 | 0.965 | 0.995 |
| S8 | 0.996 | 0.964 | 0.97 | 0.995 | 0.966 | 0.97 | 0.973 | 1 | 0.973 | 0.972 | 0.998 | 0.972 | 0.965 | 0.966 | 0.993 | 0.989 |
| S9 | 0.962 | 0.992 | 0.999 | 0.968 | 0.998 | 0.997 | 0.999 | 0.973 | 1 | 0.999 | 0.973 | 0.998 | 0.968 | 0.967 | 0.966 | 0.995 |
| S10 | 0.96 | 0.993 | 0.999 | 0.966 | 0.998 | 0.997 | 0.999 | 0.972 | 0.999 | 1 | 0.972 | 0.997 | 0.967 | 0.965 | 0.965 | 0.994 |
| S11 | 0.996 | 0.962 | 0.97 | 0.995 | 0.968 | 0.968 | 0.974 | 0.998 | 0.973 | 0.972 | 1 | 0.972 | 0.965 | 0.963 | 0.991 | 0.989 |
| S12 | 0.959 | 0.989 | 0.997 | 0.963 | 0.994 | 0.998 | 0.998 | 0.972 | 0.998 | 0.997 | 0.972 | 1 | 0.963 | 0.963 | 0.962 | 0.993 |
| S13 | 0.953 | 0.965 | 0.967 | 0.961 | 0.964 | 0.962 | 0.968 | 0.965 | 0.968 | 0.967 | 0.965 | 0.963 | 1 | 0.996 | 0.97 | 0.98 |
| S14 | 0.954 | 0.964 | 0.964 | 0.962 | 0.96 | 0.964 | 0.965 | 0.966 | 0.967 | 0.965 | 0.963 | 0.963 | 0.996 | 1 | 0.97 | 0.979 |
| S15 | 0.99 | 0.964 | 0.963 | 0.992 | 0.96 | 0.962 | 0.965 | 0.993 | 0.966 | 0.965 | 0.991 | 0.962 | 0.97 | 0.97 | 1 | 0.985 |
| R | 0.981 | 0.989 | 0.994 | 0.985 | 0.992 | 0.992 | 0.995 | 0.989 | 0.995 | 0.994 | 0.989 | 0.993 | 0.98 | 0.979 | 0.985 | 1 |
As can be seen from table 3, the similarity between each batch of samples and R is above 0.900, which indicates that the chemical compositions of each batch of samples of the astragalus dragon capsule are relatively similar. The reference fingerprint is a fingerprint generated by fixing the retention time of the common peak according to the characteristics of 15 batches of samples. And comparing the similarity of the fingerprint of the astragalus-dragon capsule to be detected with the fingerprint of the reference sample to evaluate the quality of the astragalus-dragon capsule to be detected. In each batch of samples, the peak calibrated after sample injection according to the standard comprises the known components: hydroxysafflor yellow A, paeoniflorin, ferulic acid, salvianolic acid B and tanshinone IIA.
Example 3: quality control of QILONG Capsule by LC-MS/MS
The astragaloside IV in the Qilong capsule as the monarch drug can not be absorbed under the ultraviolet wavelength of 275nm because of weak ultraviolet absorption, so the invention provides a method for detecting the active ingredient astragaloside IV in the finished product of the Qilong capsule by using the technology of combining high performance liquid chromatography and mass spectrum.
Taking 10mg of astragaloside IV standard substance, taking a mobile phase according to a mobile phase ratio, and performing gradient dilution to obtain a standard solution: 25ng/mL, 50ng/mL, 75ng/mL, 100ng/mL, 200 ng/mL. Taking the astragalus-dragon capsule extract solution, and diluting by 400 times to obtain the astragalus-dragon capsule test solution. And respectively injecting the standard solution and the test solution into a high performance liquid chromatography-mass spectrometer. The content of the astragaloside in the astragalus-dragon capsule measured by the external standard one-point method is as follows: the content of astragaloside in the astragalus-dragon capsule is measured according to a standard curve as follows: 160ng/mL to obtain 200mg of astragalus-dragon capsule containing 0.64mg of astragaloside.
Setting mass spectrum conditions: an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: a positive ion; detecting ion pairs: astragaloside m/z: 785.5/587.3; ion source voltage: 5000V; ion source temperature: at 450 ℃; air curtain air: 10 psi; atomizing: 45 psi; auxiliary gas: 55 psi; cluster-splitting voltage: 55V; collision induced dissociation voltage: 24V; chromatographic conditions are as follows: the column was CAPCELL PAKC18 (2.0X 150mm, 5 μm, Shiseido, Japan); mobile phase: phase a-aqueous solution containing 5mM ammonium acetate, 0.1% acetic acid, phase B-acetonitrile; and (3) an elution mode: during the gradient elution, the changes of mobile phase a and mobile phase B are: 60-60% of B for 0-3 min; 60% -90% of B in 3-3.1 min; 90-90% of B in 3.1-6.0 min; 90-60% of B in 6.0-6.1 min; 60-60% of B in 6.1-10.0 min. The flow rate of the mobile phase is 0.350 ml/min; the column temperature is 30 ℃; the amount of the sample was 10. mu.L. The liquid phase mass spectrum of astragaloside IV standard product is shown in figure 3.
The methodology investigation mainly comprises the linear, stability and precision investigation, and the investigation method and the result are as follows:
① stability experiment takes the same concentration astragaloside standard solution, after pretreatment, under the optimized chromatographic condition, the solution is injected and analyzed for 0h, 2h, 4h, 8h, 12h, 16h and 24h respectively, mass spectrogram is recorded, the result is shown in table 4.48 h, the RSD value of the peak area is higher than 2%, which indicates that the stability of astragaloside is good.
TABLE 4 stability test results of astragaloside IV
② precision experiment takes 3 concentrations of astragaloside IV standard solution 50ng/mL, 100ng/mL, 200ng/mL, after pretreatment, under optimized chromatographic condition, continuously measuring for 4 times, recording mass spectrogram, RSD values of three concentrations of astragaloside IV reference substance peak areas are 1.84%, 1.27%, 1.54%, RSD values are less than 2%, indicating that the instrument precision is good.
③ Linear experiment using prepared astragaloside IV standard solution 25ng/mL, 50ng/mL, 75ng/mL, 100ng/mL, 200ng/mL, pretreating, performing sample injection analysis under optimized chromatographic condition, recording mass spectrum, and obtaining astragaloside IV linear equation of y 55x-307 (regression coefficient R)20.9490) proves that the linear relation of astragaloside in the Mongolian astragalus is good in the range of 25-200 ng/mL, and the standard curve of the astragaloside control is shown in Table 5.
TABLE 5 Peak areas at different concentrations of astragaloside IV control
The technology of combining high performance liquid chromatography and mass spectrometry is used for detecting the Chinese medicinal herbs, the method has the advantages of high throughput screening, high sensitivity and the like, the mass spectrometry is used as a common detector, the performance is superior to that of an ultraviolet detector, and the judgment result is more in line with the actual situation. The quality of the finished product of the Qilong capsule can be judged according to the similarity of the fingerprint and the content of the active ingredient astragaloside in the liquid.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific examples and comparative examples.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (4)
1. A quality control method of Qilong capsules is characterized by comprising the following steps: taking multiple batches of astragalus-dragon capsules to obtain astragalus-dragon capsule sample maps according to a fingerprint map establishing method, and carrying out similarity comparison on the maps of the astragalus-dragon capsules to be detected and reference sample maps, wherein a similarity calculation method is a correlation coefficient method;
the fingerprint spectrum establishing method comprises the following steps:
(1) preparation of a test solution: taking astragalus-dragon capsule powder, adding methanol, carrying out ultrasonic extraction for a period of time, centrifuging and taking supernatant to obtain a solution A; adding water into the residue, heating in water bath for a period of time, centrifuging to obtain supernatant to obtain solution B, and mixing solution A and solution B to obtain test solution;
(2) preparation of a reference solution: collecting constant weight reference substances of ferulic acid, paeoniflorin, salvianolic acid B, tanshinone IIA, and hydroxysafflor yellow A, respectively adding methanol to obtain reference substance solutions;
(3) and (3) determination: respectively and precisely measuring a test solution and a reference solution, injecting the test solution and the reference solution into a high performance liquid chromatograph for separation, wherein the high performance liquid chromatograph has the separation conditions that: CAPCELL PAK C18 for chromatographic column, 2.0 × 150mm, 5 μm in specification, SHISEIDO corporation, Japan; the flow rate of the mobile phase is 0.3 ml/min; the column temperature was 35 ℃; the detection wavelength is 275nm, and the sample injection amount is 20 mu L; gradient elution was used, mobile phase a: 5mM ammonium acetate, 0.1% acetic acid in water, mobile phase B: acetonitrile; the gradient elution mode is as follows: at 0-25min, mobile phase B accounts for 10% → 25%; at 25-30min, mobile phase B accounts for 25% → 39%; at 30-35min, mobile phase B accounts for 39% → 42%; at 35-40min, mobile phase B accounts for 42% → 50%; at 40-45min, mobile phase B accounts for 50% → 55%; when the time is 45-60min, the mobile phase B accounts for 55% → 90%; analyzing and processing the chromatogram obtained by the high performance liquid chromatography by fingerprint software to obtain the Qilong capsule fingerprint.
2. The quality control method according to claim 1, wherein the reference sample map is created by: and calculating the similarity between each sample spectrum and other sample spectrums, selecting the sample with the maximum sum of the similarities with other samples as a reference sample, and taking the fingerprint spectrum as the reference sample spectrum.
3. The quality control method as claimed in claim 1, wherein the fingerprint spectrum establishment method comprises the steps of (1) weighing a certain amount of the stilbene dragon capsule powder, adding methanol, performing ultrasonic treatment for 0.8-1.2h, centrifuging at 3500-4500r/min for 8-12min, and obtaining the supernatant solution A after centrifuging, wherein the stilbene dragon capsule powder: the mixture ratio of the methanol is 180-220 mg: 4-6 mL; adding triple distilled water into the residue, uniformly mixing, and centrifuging for 8-12min in a water bath at 65-75 ℃ for 1.8-2.2h and at 3500 r/min of 4500r/min to obtain a supernatant solution B; mixing the solution A and the solution B according to a ratio of 4-6:6-8, adding a mobile phase to dilute by 4-6 times, and filtering by an organic filter membrane to obtain a test sample solution.
4. The quality control method according to claim 1, wherein the reference solution in the step (2) of the fingerprint spectrum establishing method is prepared by the following method: precisely weighing 0.0100g of ferulic acid standard, 0.0099g of paeoniflorin standard, 0.0101g of tanshinone IIA standard, 0.0102g of salvianolic acid B standard and 0.0099g of hydroxysafflor A standard, placing in a 10mL volumetric flask, adding methanol to dissolve and dilute to scale, and respectively preparing standard solutions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811621837.2A CN110057927B (en) | 2018-12-28 | 2018-12-28 | Detection method combining fingerprint spectrum and liquid chromatography-mass spectrometry of Qilong capsules and application of detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811621837.2A CN110057927B (en) | 2018-12-28 | 2018-12-28 | Detection method combining fingerprint spectrum and liquid chromatography-mass spectrometry of Qilong capsules and application of detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110057927A CN110057927A (en) | 2019-07-26 |
| CN110057927B true CN110057927B (en) | 2020-04-21 |
Family
ID=67315379
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811621837.2A Active CN110057927B (en) | 2018-12-28 | 2018-12-28 | Detection method combining fingerprint spectrum and liquid chromatography-mass spectrometry of Qilong capsules and application of detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110057927B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113075325B (en) * | 2021-03-30 | 2022-12-20 | 贵州医科大学 | Method for simultaneously measuring contents of 8 index components in cynanchum wilfordii |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102183590A (en) * | 2011-01-31 | 2011-09-14 | 山东沃华医药科技股份有限公司 | Measuring method of Xinkeshu preparation finger-print |
| CN104849364A (en) * | 2015-05-05 | 2015-08-19 | 山东大学 | Canzhiling oral solution fingerprint map building method, fingerprint map and application thereof |
-
2018
- 2018-12-28 CN CN201811621837.2A patent/CN110057927B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102183590A (en) * | 2011-01-31 | 2011-09-14 | 山东沃华医药科技股份有限公司 | Measuring method of Xinkeshu preparation finger-print |
| CN104849364A (en) * | 2015-05-05 | 2015-08-19 | 山东大学 | Canzhiling oral solution fingerprint map building method, fingerprint map and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| HPLC-DAD 法测定脑血栓片中苦杏仁苷、羟基红花黄色素 A、芍药苷、阿魏酸、丹酚酸 B 和丹参酮ⅡA;周军等;《现代药物与临床》;20150331;第30卷(第3期);第262-266页 * |
| HPLC-ESI-MSn法测定黄芪药材中黄芪甲苷;李晶等;《中成药》;20110430;第33卷(第4期);第720-722页 * |
| 芪骨胶囊 HPLC 指纹图谱研究;范兴等;《中草药》;20140531;第45卷(第9期);全文 * |
| 芪龙胶囊的质量标准研究;叶素艳等;《科技信息》;20120630(第18期);第375-377页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110057927A (en) | 2019-07-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yan et al. | Advancement in the chemical analysis of Paeoniae Radix (Shaoyao) | |
| CN109307721B (en) | Detection method for determining content of effective components in leech capsule by HPLC-QQQ/MS method | |
| CN111999423B (en) | Establishment method and application of neck pain granule fingerprint spectrum | |
| CN110412155B (en) | Detection method of HPLC (high performance liquid chromatography) characteristic spectrum of Erdi decoction | |
| CN114152686A (en) | Fingerprint construction method and application of traditional Chinese medicine compound containing cinnamon | |
| CN114184704B (en) | Construction method and application of UPLC fingerprint of Xiaoyao pill | |
| CN110031564B (en) | Quality detection method of natural plant anticoccidial feed additive based on HPLC fingerprint | |
| Qi et al. | HPLC fingerprint and LC-TOF-MS analysis on extract from roots of Gentiana macrophylla | |
| CN106226435B (en) | A kind of fingerprint atlas detection method of Yixinshu tablet saponin component | |
| CN110057927B (en) | Detection method combining fingerprint spectrum and liquid chromatography-mass spectrometry of Qilong capsules and application of detection method | |
| CN110568108B (en) | Multi-component content determination method of Ganfule preparation | |
| CN112684036A (en) | Fingerprint spectrum determination method of kidney-tonifying capsules containing leeches and application of fingerprint spectrum determination method | |
| CN102133333A (en) | Quality control method for shenmai injection mass spectrum finger prints | |
| CN108469473B (en) | Method for detecting metabolite of honeysuckle in rat serum by using RRLC-DAD-ESI-Q-TOF-MS | |
| CN114113403B (en) | Method for determining liquid chromatography-mass spectrometry (LC-MS) of Hedan tablets | |
| CN110412154B (en) | Quantitative determination method for multi-component liquid quality of Qilong capsule and application thereof | |
| CN108205024B (en) | Fingerprint quality control method for ficus microcarpa dry extract | |
| CN113759011B (en) | Method for establishing characteristic spectrum of starwort root and preparation thereof | |
| CN106918670B (en) | A kind of quality determining method of pharmaceutical composition | |
| Liu et al. | Simultaneous determination of twelve bioactive constituents in Buyang Huanwu decoction by HPLC‐DAD‐ELSD and HPLC‐TOF/MS | |
| CN114088823A (en) | Method for measuring content of multi-index components in Tongtian oral liquid | |
| CN108107130B (en) | Method for measuring fingerprint spectrum of Shenzhiling preparation | |
| CN111551662A (en) | Quality detection method of angelica sinensis blood-enriching soup | |
| CN111189952A (en) | Method for measuring special content of verbena and controlling quality of verbena | |
| CN112147243B (en) | Detection method for simultaneously determining multiple active ingredients in lung-clearing medicament |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |