CN110187015B - Method for detecting content of higenamine in traditional Chinese medicine, condiment and externally applied medicine - Google Patents
Method for detecting content of higenamine in traditional Chinese medicine, condiment and externally applied medicine Download PDFInfo
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Abstract
The application discloses a detection method of the content of higenamine in traditional Chinese medicines, seasonings and externally applied medicines, which comprises the following steps: step 1, preparing a sample to be detected; step 2, preparing an extraction solution and preparing an internal standard solution; step 3, preparing a sample purifying agent; step 4, preparing a solution to be detected by using the sample to be detected, the extraction solution, the internal standard solution and the sample purifying agent; and 5, injecting the solution to be detected into a liquid chromatogram-tandem mass spectrometer for detection, and recording a chromatogram, wherein the liquid chromatogram conditions are as follows: chromatography column (
3 μm,3mm × 100 mm); flow rate: 0.40 mL/min; column temperature: 30 ℃; sample introduction amount: 5 mu L of the solution; the mobile phase is as follows: phase A: ammonium acetate aqueous solution, phase B: methanol solution, and the tandem mass spectrometry detection conditions are as follows: in a multiple reaction monitoring mode, qualitative ion pairs of higenamine are 272.0/255.0, 272.0/161.0 and 272.0/107.0, and quantitative ion pairs are 272.0/107.0. Through the technical scheme in the application, the simplicity and the batch detection capability of the higenamine detection method are improved, and the high-sensitivity detection of the higenamine in the traditional Chinese medicine, the condiment and the externally applied medicine is realized.
Description
Technical Field
The application relates to the technical field of drug analysis, in particular to a detection method of higenamine content in traditional Chinese medicines, seasonings and externally applied medicines.
Background
Higenamine is a medicinal component existing in many natural plants, has the effects of stimulating beta-adrenergic receptors and relaxing blood vessels, and can be clinically used as a cardiotonic. Since 2017, higenamine is classified as an S3 (beta-2 agonist) class banned substance by WADA (International anti-excitant organization). The higenamine mainly exists in Nandina domestica, radix Aconiti lateralis Preparata, herba asari, Annona squamosa, and flos Nelumbinis, which are the components of Chinese medicinal materials or daily diet flavoring agent. Therefore, athletes are likely to take higenamine in daily diet to cause positive detection of stimulant detection. In addition, since higenamine exists in various traditional Chinese medicines and Tibetan medicines, higenamine can be contained in a plurality of external medicines. In order to avoid the phenomenon that athletes mistakenly take higenamine due to daily diet, use of externally applied medicines and the like, a high-sensitivity and reliable higenamine detection method aiming at traditional Chinese medicines, seasonings and externally applied medicines possibly involved in daily diet and treatment is required to be established.
Because the matrixes of the traditional Chinese medicine, the seasoning and the externally applied medicine are complex and various, and the existing content of the higenamine in the sample is generally low, a proper sample pretreatment technology needs to be established to extract the higenamine in the sample. In addition, due to the need of screening higenamine in traditional Chinese medicines, seasonings and externally applied medicines in large scale and large scale, a sample pretreatment technology is also required to have batch processing performance. The existing higenamine detection method cannot well meet the requirement of detecting higenamine.
Disclosure of Invention
The purpose of this application lies in: the simplicity, the reliability and the batch detection capability of the higenamine detection method are improved, and the high-sensitivity detection of the higenamine in the traditional Chinese medicine, the condiment and the externally applied medicine is realized.
The technical scheme of the application is as follows: provides a method for detecting the content of higenamine in traditional Chinese medicines, seasonings and externally applied medicines, which comprises the following steps: step 1, preparing a sample to be detected; step 2, preparing an extraction solution and preparing an internal standard solution; step 3, preparing a sample purifying agent; step 4, preparing a solution to be detected by using the sample to be detected, the extraction solution, the internal standard solution and the sample purifying agent; and 5, injecting the solution to be detected into a liquid chromatogram-tandem mass spectrometer for detection, and recording a chromatogram, wherein the chromatogram condition is as follows: chromatography column (
3 μm,3mm × 100 mm); flow rate: 0.40 mL/min; column temperature: 30 ℃; sample introduction amount: 5 mu L of the solution; the mobile phase is as follows: phase A: ammonium acetate aqueous solution, phase B: methanol solution. The mass spectrum conditions are as follows: in a multiple reaction monitoring mode, qualitative ion pairs of higenamine are 272.0/255.0, 272.0/161.0 and 272.0/107.0, and quantitative ion pairs are 272.0/107.0.
In any of the above technical solutions, further, the aqueous ammonium acetate solution is a 0.5mmol/L aqueous ammonium acetate solution containing 0.1% formic acid, and the methanol solution is a methanol solution containing 0.1% formic acid.
In any of the above technical solutions, further, the ionization mode of the liquid chromatography-tandem mass spectrometer is: ion source ESI +; spray voltage (IS): 5500V; the detection mode is as follows: monitoring multiple reactions; qualitative ion pairs of higenamine are 272.0/255.0, 272.0/161.0 and 272.0/107.0, quantitative ion pair is 272.0/107.0, clustering removing voltage (DP) is 40V, and collision gas energy (CE) is 20 eV, 25 eV and 30eV respectively; the internal standard detected ion pair (m/z) was 304.2/135.3.
In any of the above embodiments, further, the extraction solution is an ethanol solution containing 0.5% (v/v) formic acid.
In any of the above technical solutions, further, the ethanol solution is an unhydrated ethanol solution.
In any of the above technical solutions, further, the preparing an internal standard solution specifically includes: weighing 10.0mg of fenoterol standard, dissolving with methanol, diluting to a constant volume of 10mL, marking the solution after constant volume as an internal standard solution, injecting into a brown volumetric flask, and storing in a dark environment at 4 ℃.
In any one of the above technical solutions, further, in step 4, the method specifically includes: step 41, weighing 2.0g of the homogenized sample to be detected and placing the weighed sample into a 15mL centrifuge tube by using an analytical balance with the accuracy of 0.001 g; step 42, adding 5mL of extraction solution and 50 mu L of internal standard solution into a centrifugal tube, putting the centrifugal tube into a vortex mixer, and carrying out vortex mixing, wherein the extraction solution is an ethanol solution which is not hydrated and contains 0.5% formic acid, and the internal standard solution is fenoterol methanol solution with the concentration of 1.0 mu g/mL; 43, placing the centrifugal tube on an oscillator to vibrate for 30min, then placing the centrifugal tube on a centrifugal machine with the preset rotating speed of 8000r/min to centrifuge, and extracting 1mL of first supernatant into an EP tube after centrifuging for 10 min; step 44, adding a sample purifying agent into the EP pipe, placing the EP pipe in a vortex mixer for vortex mixing for 1min, then placing the EP pipe on a centrifugal machine for centrifugal operation, and after centrifuging for 5min, extracting a second supernatant in the EP pipe; and step 45, filtering the second supernatant by using a 0.22-micron microfiltration membrane, and recording the filtered second supernatant as the solution to be detected.
In any one of the above technical solutions, further, when the solution to be detected is injected into the liquid chromatography-tandem mass spectrometer for detection, the method includes a gradient elution process, where the gradient elution process is as follows:
the beneficial effect of this application is: the extraction solution and the internal standard solution are added into a sample to be detected, vortex mixing and oscillation operation are carried out, higenamine in the sample to be detected is extracted, then supernatant containing the higenamine is extracted, a sample purifying agent is added, so that interference of other substances in the sample to be detected on a higenamine detection result is reduced, the higenamine content in the sample to be detected is detected through a liquid chromatography-tandem mass spectrometer, the simplicity, the reliability and the batch detection capability of the higenamine detection method are improved, and the high-sensitivity detection of the higenamine in traditional Chinese medicines, seasonings and externally-applied medicines is realized.
According to the application, through a large number of data comparison tests, unhydrated ethanol solution containing 0.5% (v/v) formic acid is used as extraction solution, so that the extraction efficiency of higenamine in a sample to be detected is improved, 50mg of N-propyl ethylenediamine, 50mg of carbon 18 and 7.5mg of graphitized carbon black are used as sample purifying agents, and the supernatant of the sample to be detected is purified, so that the influence of residues on higenamine content detection is reduced, and the accuracy and reliability of higenamine content joint detection are improved.
Drawings
The advantages of the above and/or additional aspects of the present application will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic flow chart of a method for detecting higenamine content in Chinese herbs and spices according to one embodiment of the present application;
FIG. 2 is a schematic diagram of the chemical structure of higenamine according to one embodiment of the present application;
FIG. 3 is a graph comparing the extraction efficiency of higenamine with aqueous hydrochloric acid, methanol and ethanol according to one embodiment of the present application;
FIG. 4 is a graph comparing the extraction efficiency of ethanol with different formic acid content to higenamine according to one embodiment of the present application;
FIG. 5 is a graph comparing the extraction efficiency of higenamine with different water content according to one embodiment of the present application.
Detailed Description
In order that the above objects, features and advantages of the present application can be more clearly understood, the present application will be described in further detail with reference to the accompanying drawings and detailed description. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, however, the present application may be practiced in other ways than those described herein, and therefore the scope of the present application is not limited by the specific embodiments disclosed below.
As shown in fig. 1, the present embodiment provides a method for detecting the content of higenamine in traditional Chinese medicine, flavoring and external application medicine, which comprises:
step 1, preparing a sample to be detected;
specifically, when the sample to be measured is a solid sample, 500g of the solid sample (such as traditional Chinese medicine and seasoning) can be selected, the solid sample is crushed by a crusher, if the components are not uniform after crushing, the crushed powder is ground by a porcelain mortar, the powder is filtered by a screen after being uniformly ground, and the sample powder after filtering large particles is recorded as the sample to be measured. When the sample to be tested is a powdery sample (such as internal medicine powder of externally applied medicine), the sample is directly filtered by a screen, and the filtered powder is recorded as the sample to be tested.
Selecting higenamine with the purity of 99.0 percent as a higenamine standard substance, weighing 10.0mg of the higenamine standard substance, dissolving the higenamine standard substance by using methanol and fixing the volume to 10mL, placing the solution with the fixed volume into a brown volumetric flask, storing the solution in a dark environment at-18 ℃, wherein the concentration of the solution is 1.0mg/mL, and recording the solution as the higenamine standard solution.
The sample preparation process also comprises the preparation of a calibration sample, wherein the preparation of the calibration sample comprises the steps of preparing a blank sample, such as dried rice, into powder, adding a higenamine standard solution into the blank sample, controlling the content of higenamine in the blank sample to be 20 microgram/kg, and calibrating the blank sample so as to be used as a simulation sample to carry out a condition optimization experiment, so that the detection result of the detection method in the embodiment is detected, and the accuracy of the detection method in the embodiment is improved.
Step 2, preparing an extraction solution and preparing an internal standard solution;
further, the extraction solution was an ethanol solution containing 0.5% (v/v) formic acid.
Preferably, the ethanol solution is a non-hydrated ethanol solution.
Specifically, higenamine is a benzylisoquinoline alkaloid existing in natural plants, the chemical structural formula of the higenamine is shown in fig. 2, the LogP value of the higenamine is 2.23, the pKa of the higenamine is 9.72, and the higenamine is an alkaloid with stronger polarity, so that the higenamine is suitable for being extracted by a (acid-containing) polar solution.
Firstly, 5mL of hydrochloric acid-containing aqueous solution, methanol and ethanol are selected as extraction solutions for investigation, experimental data statistics shows that the extraction efficiency of the three solutions on higenamine is shown in figure 3, and data analysis shows that the extraction efficiency of ethanol on higenamine is high, so that ethanol is selected as a solvent.
Secondly, the applicant has noted that a small formic acid doping can significantly increase the extraction efficiency, whereas the extraction efficiency of formic acid alone is inferior to that of acetic acid alone. Specifically, 5mL of ethanol containing no formic acid and 0.2% (v/v), 0.5% (v/v) and 1.0% (v/v) of formic acid is used as the extraction solution, experimental data statistics shows that the extraction efficiency of higenamine by ethanol under different formic acid contents is shown in FIG. 4, and by data analysis, the applicant finds that when the formic acid concentration in ethanol reaches 0.2% (v/v), the increase of the formic acid concentration has no influence on the extraction efficiency of higenamine, so that the ethanol solution with the formic acid concentration of 0.2-0.5% (v/v) can be used as the extraction solution of higenamine.
Finally, the applicant noticed that the polarity of the extraction solution greatly affects the extraction efficiency of higenamine when the actual sample is processed, in other words, the water content in the sample is likely to affect the extraction efficiency of higenamine, and it is necessary to optimize the effect. Taking a dry rice sample as a blank matrix, adding unequal amounts of distilled water into the sample to simulate actual samples with different water contents, adopting 5mL of ethanol solution containing 0.5% (v/v) formic acid to extract higenamine, and investigating the extraction efficiency of the higenamine, wherein the result is shown in figure 5 through experimental data statistics, so that the increase of the water content in the sample can cause the reduction of the extraction effect of the higenamine.
In summary, an ethanol solution with a formic acid concentration of 0.5% (v/v) was prepared as an extraction solution of higenamine using formic acid as a solute and unhydrated ethanol as a solvent.
Further, preparing an internal standard solution specifically comprises: weighing 10.0mg of fenoterol standard, dissolving with methanol, diluting to a constant volume of 10mL, marking the solution after constant volume as an internal standard solution, injecting into a brown volumetric flask, and storing in a dark environment at 4 ℃.
Step 3, preparing a sample purifying agent;
further, the sample purifying agent was a mixture of 50mg of N-propylethylenediamine, 50mg of carbon 18, and 7.5mg of graphitized carbon black.
Specifically, in order to reduce the influence of other substances in a sample to be detected on the detection of the content of higenamine, a mixture of three adsorbents, namely N-Propylethylenediamine (PSA), carbon 18(C18) and Graphitized Carbon Black (GCB) is selected as a sample purifying agent, the recovery rate of the higenamine is set as a detection index, the usage amount of the three adsorbents is detected by adopting a three-level three-factor orthogonal test, the three-level three-factor orthogonal test conditions are shown in table 1, and the corresponding detection results are shown in table 2.
TABLE 1
TABLE 2
Combining the data in tables 1 and 2, it can be seen that the effect of the three adsorbents on the recovery of higenamine is PSA > C18 ≈ GCB, wherein the higher the amount of PSA used, the lower the recovery of higenamine, while the addition of C18 and GCB has little effect on the recovery of higenamine. Through experimental observation, the color of the sample can be obviously lightened along with the increase of the added amount of GCB, so that in the embodiment, the sample purifying agent is a mixture of 50mg of N-propyl ethylene diamine, 50mg of carbon 18 and 7.5mg of graphitized carbon black.
Step 4, preparing a solution to be detected by using the sample to be detected, the extraction solution, the internal standard solution and the sample purifying agent;
further, step 4 specifically includes:
step 41, weighing 2.0g of the homogenized sample to be detected and placing the weighed sample into a 15mL centrifuge tube by using an analytical balance with the accuracy of 0.001 g;
step 42, adding 5mL of the extraction solution and 50 μ L of the internal standard solution into a centrifuge tube, placing the centrifuge tube into a vortex mixer, performing vortex mixing,
wherein the extraction solution is an unhydrated ethanol solution containing 0.5% formic acid, and the internal standard solution is a fenoterol methanol solution with the concentration of 1.0 mug/mL;
43, placing the centrifugal tube on an oscillator to vibrate for 30min, then placing the centrifugal tube on a centrifugal machine with the preset rotating speed of 8000r/min to centrifuge, and extracting 1mL of first supernatant into an EP tube after centrifuging for 10 min;
step 44, adding a sample purifying agent into the EP pipe, placing the EP pipe in a vortex mixer for vortex mixing for 1min, then placing the EP pipe on a centrifugal machine for centrifugal operation, and after centrifuging for 5min, extracting a second supernatant in the EP pipe;
and step 45, filtering the second supernatant by using a 0.22-micron microfiltration membrane, and recording the filtered second supernatant as the solution to be detected.
Specifically, in the present embodiment, in order to verify the influence of the length of the oscillation time on the extraction efficiency of higenamine, three different sets of oscillation times were set, which were 20min, 30min, 45min, and 60min in this order. The grouping experiment result shows that the oscillation time does not have obvious influence on the extraction of the higenamine, so that the sample to be detected can be fully oscillated on the premise of ensuring the experiment efficiency by selecting 30min as the oscillation time of the sample to be detected on the oscillator.
More specifically, in the existing detection method for higenamine, a sample to be detected is usually soaked so as to extract the higenamine from the sample to be detected. In this embodiment, in order to verify the comprehensiveness of the detection method, a soaking experiment for the sample to be detected is added, and before the sample to be detected is put into an oscillator for oscillation, three sets of comparison experiments are set: soaking, soaking for 0.5h and soaking for 1h to verify the extraction efficiency of higenamine in the sample to be detected by using the extraction solution in the embodiment. The data statistics result shows that when the extraction solution in the embodiment is used for extracting higenamine in a sample to be detected, whether the sample to be detected is soaked or not and the soaking time is short or long, and the extraction of the higenamine is not influenced, so that the sample to be detected is not soaked in a subsequent detection experiment and is directly extracted, a large amount of time is saved, and the method for detecting the higenamine content in the embodiment has the advantages of simplicity and batch detection capability.
And 5, injecting the solution to be detected into a liquid chromatogram-tandem mass spectrometer for detection, and recording a chromatogram, wherein the chromatographic conditions of the liquid chromatogram-tandem mass spectrometer are as follows:
chromatography column (
3μm,3mm×100mm)；
Flow rate: 0.40 mL/min;
column temperature: 30 ℃;
sample introduction amount: 5 mu L of the solution;
the mobile phase is as follows: phase A: ammonium acetate aqueous solution, phase B: methanol solution.
Further, the aqueous ammonium acetate solution was a 0.5mmol/L aqueous ammonium acetate solution containing 0.1% formic acid, and the methanol solution was a methanol solution containing 0.1% formic acid.
Further, the detection mode of the liquid chromatography-tandem mass spectrometer is as follows:
an ion source: ESI +;
spray voltage (IS): 5500V;
the detection mode is as follows: monitoring multiple reactions;
qualitative ion pairs of higenamine are 272.0/255.0, 272.0/161.0 and 272.0/107.0, quantitative ion pair is 272.0/107.0, clustering removing voltage (DP) is 40V, and collision gas energy (CE) is 20 eV, 25 eV and 30eV respectively;
the internal standard detected ion pair (m/z) was 304.2/135.3.
Further, when the solution to be detected is injected into the liquid chromatogram-tandem mass spectrometer for detection, a gradient elution process is included, and the gradient elution process is as follows:
further, the detection method of the liquid chromatography-tandem mass spectrometer is configured to:
the detection limit is 0.03ng/mL, the linear range is 0.10-100ng/mL, and the linear equation y is 0.3078x-0.0024(R2 is 0.9933). The precision of the method was 4.33% (c: 0.50ng/mL, n: 7).
Specifically, in this embodiment, an shimadzu LC-20ADXR type liquid chromatography-tandem mass spectrometer is used as the liquid chromatography-tandem mass spectrometer for higenamine, the liquid chromatography-tandem mass spectrometer is configured according to the above parameters, the higenamine content in 13 traditional Chinese medicines, 4 flavorings and 1 Tibetan medicine plaster is detected, and the detected samples are as follows: dried lotus leaves, tuckahoe, astragalus, angelica, fructus gardeniae, almond, pseudo-ginseng, ligusticum wallichii, white paeony root, dried lotus seeds, Chinese yam, polygonatum, Chinese yam, hot pepper, fennel, pepper, cassia bark and a Tibetan medicine bone pain plaster sold in the market. Wherein, the content of higenamine can be detected from dried lotus leaf, dried lotus seed, Chinese yam, fragrant solomonseal rhizome, Chinese yam, pepper, cassia bark and the Tibetan medicine plaster sold in the market for treating ostealgia, and is shown in table 3.
TABLE 3
More specifically, in order to further verify the accuracy of the higenamine content detection method in this embodiment, two actual samples, i.e., zanthoxylum bungeanum and cinnamomum cassia bark, were labeled (adding higenamine standard solution), and the experimental detection data are shown in table 4.
TABLE 4
The verification tests show that the addition standard recovery rate of higenamine of two actual samples is 92.6-109.8%, and the method for detecting the higenamine content in the embodiment has the advantages of good accuracy, high sensitivity and batch detection capability. The method has important significance, and can be proved to meet the high requirement of practical application in the detection of practical samples.
The technical scheme of the application is explained in detail in the above with reference to the accompanying drawings, and the application provides a detection method of the content of higenamine in traditional Chinese medicines, seasonings and externally applied medicines, which comprises the following steps: step 1, preparing a sample to be detected; step 2, preparing an extraction solution and preparing an internal standard solution; step 3, preparing a sample purifying agent; step 4, preparing a solution to be detected by using the sample to be detected, the extraction solution, the internal standard solution and the sample purifying agent; step 5, injecting the solution to be detected into the solutionDetecting by using a liquid chromatography-tandem mass spectrometer, and recording a chromatogram, wherein the chromatographic conditions of the liquid chromatography-tandem mass spectrometer are as follows: chromatography column (
3 μm,3mm × 100 mm); flow rate: 0.40 mL/min; column temperature: 30 ℃; sample introduction amount: 5 mu L of the solution; the mobile phase is as follows: phase A: ammonium acetate aqueous solution, phase B: methanol solution, and the tandem mass spectrometry detection conditions are as follows: in a multiple reaction monitoring mode, qualitative ion pairs of higenamine are 272.0/255.0, 272.0/161.0 and 272.0/107.0, and quantitative ion pairs are 272.0/107.0. Through the technical scheme in the application, the simplicity, the reliability and the batch detection capability of the higenamine detection method are improved, and the high-sensitivity detection of higenamine in traditional Chinese medicines, seasonings and externally applied medicines is realized.
The steps in the present application may be sequentially adjusted, combined, and subtracted according to actual requirements.
The units in the device can be merged, divided and deleted according to actual requirements.
Although the present application has been disclosed in detail with reference to the accompanying drawings, it is to be understood that such description is merely illustrative and not restrictive of the application of the present application. The scope of the present application is defined by the appended claims and may include various modifications, adaptations, and equivalents of the invention without departing from the scope and spirit of the application.
Claims (3)
1. A sample pretreatment method for detecting the content of higenamine in traditional Chinese medicines, seasonings and externally applied medicines is characterized by comprising the following steps:
step 1, preparing a sample to be detected; wherein, the detected samples are as follows: dried lotus leaves, poria cocos, astragalus membranaceus, angelica sinensis, fructus gardeniae, almonds, pseudo-ginseng, ligusticum wallichii, radix paeoniae alba, dried lotus seeds, Chinese yam, radix polygonati officinalis, Chinese yam, hot peppers, fennel, pepper, cinnamon and a commercially available Tibetan medicine ostalgia plaster;
step 2, preparing an extraction solution and an internal standard preparation solution, wherein the extraction solution is an ethanol solution containing 0.5% v/v formic acid, and the ethanol solution is an absolute ethanol solution;
step 3, preparing a sample purifying agent; wherein the sample purifying agent is a mixture of 50mg of N-propyl ethylenediamine, 50mg of carbon 18 and 7.5mg of graphitized carbon black;
and 4, preparing a solution to be detected by using the sample to be detected, the extraction solution, the internal standard solution and the sample purifying agent.
2. The sample pretreatment method for detecting the higenamine content in the traditional Chinese medicine, the seasoning and the externally applied medicine according to claim 1, wherein the preparation of the internal standard solution specifically comprises the following steps:
weighing 10.0mg of fenoterol standard, dissolving with methanol, diluting to a constant volume of 10mL, marking the solution with constant volume as the internal standard solution, injecting into a brown volumetric flask, and storing in a dark environment at 4 ℃.
3. The sample pretreatment method for detecting the content of higenamine in traditional Chinese medicines, condiments and externally applied medicines according to claim 1, wherein the step 4 specifically comprises the following steps:
step 41, weighing 2.0g of the homogenized sample to be detected and placing the weighed sample into a 15mL centrifuge tube by using an analytical balance with the accuracy of 0.001 g;
step 42, adding 5mL of the extraction solution and 50 μ L of the internal standard solution into the centrifuge tube, placing the centrifuge tube into a vortex mixer, performing vortex mixing,
wherein the extraction solution is an ethanol solution containing 0.5% formic acid, the ethanol solution is an absolute ethanol solution, and the internal standard solution is a fenoterol methanol solution with the concentration of 1.0 mug/mL;
step 43, placing the centrifugal tube on an oscillator to vibrate for 30min, then placing the centrifugal tube on a centrifuge with a preset rotating speed of 8000r/min to centrifuge, and after centrifuging for 10min, extracting 1mL of first supernatant into an EP tube;
step 44, adding the sample purifying agent into the EP pipe, placing the EP pipe into the vortex mixer for vortex mixing for 1min, then placing the EP pipe on the centrifuge for centrifugal operation, and after centrifuging for 5min, extracting a second supernatant in the EP pipe;
and step 45, filtering the second supernatant by using a 0.22-micron microporous filter membrane, and recording the filtered second supernatant as the solution to be detected.
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