CN107941927B - Method for determining lobetyolin content by UPLC/Q-TOF-MS - Google Patents
Method for determining lobetyolin content by UPLC/Q-TOF-MS Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicine pharmacokinetics, and particularly relates to a method for determining spleen tonifying formula and codonopsis pilosula alkynoside content in codonopsis pilosula extract by using UPLC/Q-TOF-MS, which comprises the following steps: 1) preparing a spleen-tonifying prescription or a codonopsis pilosula extracting solution; 2) separating and detecting lobetyolin by adopting a high performance liquid chromatography-mass spectrometer; 3) plotting different concentrations of lobetyolin and corresponding response peak areas to obtain a standard curve of lobetyolin, and calculating the content of lobetyolin of the sample to be measured by comparing the peak areas of the sample to be measured with the standard curve. The invention establishes a method for detecting the content of lobetyolin in the traditional Chinese medicine codonopsis pilosula and the spleen-tonifying formula by applying a liquid chromatography-mass spectrometry technology. The method is rapid, sensitive and stable, and provides a new reference method for determining the content of lobetyolin.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine pharmacokinetics, and particularly relates to a method for determining lobetyolin content by UPLC/Q-TOF-MS.
Background
Pharmacokinetics (PK) of traditional Chinese medicine is an emerging subject which applies the principle of dynamics to study the dynamic change rules of active ingredients, effective parts, single medicine and compound in vivo absorption (Abosorpton), Distribution (Distribution), Metabolism (Metabolism), Excretion (Excretion) and toxicity (ADME/Tox) of traditional Chinese medicine and the relationship between the amount, the effect and the time effect in vivo, and is quantitatively described by mathematical functions. The Chinese medicinal composition plays an important role in clarifying the substance basis of the efficacy of Chinese medicaments, disclosing the scientific connotation of the Chinese medicaments, and researching the creation, the improvement of the dosage form and the compatibility mechanism of the components of the prescription of the Chinese medicaments, and becomes an inseparable important component in the modern research chain of the Chinese medicaments, thereby becoming a hotspot of the pharmacokinetic research of China. The blood concentration method is a classical method for pharmacokinetic research and is the most common and accurate determination method for calculating pharmacokinetics, so accurate determination of the blood concentration is a crucial link for traditional Chinese medicine pharmacokinetic research. LC-MS is widely applied in the research of traditional Chinese medicine pharmacokinetics.
Wuxiaxia and the like start from alkaloids of the coptis detoxification decoction, establish a method for measuring the blood concentrations of jatrorrhizine, palmatine and small fruit alkaloid by combining (LC-MS/MS) technology, are successfully applied to the pharmacokinetics research of the 3 components in the body of a normal rat of the coptis detoxification decoction, and provide a referential analysis method for the pharmacokinetics research of the traditional Chinese medicine compound prescription. The method for determining tanshinone IIA in plasma of beagle dogs is established by taking diazepam as an internal standard and adopting an HPLC-MS/MS technology, the minimum quantitative concentration of the method is 1ng/ml, the method can completely meet the experimental requirements, the sample treatment and determination process is simple and convenient, and the method is suitable for the research of pharmacokinetics in beagle dogs. LI and the like establish a rapid, specific and sensitive pharmacokinetic parameter after UPLC-MS/MS (ultra performance liquid chromatography-mass spectrometry/mass spectrometry) technology is used for analyzing the oral administration of the phlorizin and the phloretin (drynaria extract) of the rat, and the result shows that the method meets the analysis standard of biological medicines under the guidance of FDA (food and drug administration), and the pharmacokinetic research of the phlorizin and the active metabolite phlorizin thereof can provide a reasonable reference for the clinical application of the drynaria. Chenning and the like establish a method for measuring the content of astragaloside in the plasma of rats by SPE-HPLC-MS, and study the pharmacokinetics and tissue distribution of astragaloside in the bodies of rats. Yujuan Li applied LC-MS/MS to quantitatively analyze the content of atractylenolide I in rat plasma and research the pharmacokinetics of the atractylenolide I. The method is characterized in that the method applies UPLC-MS to simultaneously detect 6 components of calycosin glucoside, formononetin, calycosin, formononetin, astragaloside IV and astragaloside II in blood plasma of rats after astragalus mongholicus is orally taken for extraction at night, and pharmacokinetic research is carried out on each component. The content of 6 active ingredients in 7 Chinese patent medicine preparations based on the three-yellow diarrhea center decoction is researched by applying UPLC-MS/MS technology by Mei-Ling Hou and the like. And compares the pharmacokinetics process of the rhein monomer after the rat gavage traditional Chinese medicine monomer rhein, the traditional Chinese medicine rhubarb and the traditional Chinese medicine compound Sanhuang heart-purging decoction. Research shows that the absorption rate of rhein in the Chinese herbal compound and the single Chinese herbal medicine is higher than that of rhein monomer, so that the conclusion that the three-yellow heart-fire-purging decoction compound is superior to the single Chinese herbal medicine rhein and the single Chinese herbal medicine rhein in the aspect of rhein absorption rate is obtained. The traditional Chinese medicine has more impurities, the concentration or activity of a detected object is extremely low, the amount of samples for analysis is small, especially in the continuous determination process, completely same samples are difficult to obtain again, in addition, the workload is large, because the traditional Chinese medicine contains various chemical components, and the content of each chemical component is low, a large amount of data processing and analysis are needed, certain difficulties are brought to the analysis, the development of the traditional Chinese medicine is also severely limited, the liquid chromatography-mass spectrometry separation efficiency is high, the sensitivity is high, the selectivity is high, the interference of the factors is eliminated, and the development of the traditional Chinese medicine pharmacology is promoted.
The Gegenqinlian decoction is a traditional compound prescription for treating gastrointestinal diseases. Zhang Y and the like adopt a liquid chromatography-mass spectrometry technology to establish an effective method for simultaneously detecting puerarin and daidzein. The method compares the pharmacokinetic differences of puerarin and daidzein in the Gegenqinlian decoction and the Gegen extract. The results show that compared with the pueraria decoction, the puerarin and the daidzein in the pueraria qinlian decoction are absorbed more efficiently, and the elimination rate is reduced. Provides scientific basis for disclosing the compatibility rule of the Gegenqinlian decoction.
The Codonopsis pilosula is a traditional and rare Chinese medicinal material in China, and the Codonopsis pilosula collected in the pharmacopoeia of the people's republic of China 2010 is derived from 3 varieties of platycodiaceae plants, namely dried roots of Codonopsis pilosula Codonopsis pliosula (Franch) Nannf, Codonopsis pilosula Codonopsis pilosula Nannf. The codonopsis pilosula is sweet in taste and neutral in nature, enters spleen and lung meridians, and has the effects of tonifying middle-jiao and Qi, promoting fluid production and harmonizing stomach. The traditional Chinese medicine composition is mainly used for treating symptoms such as weakness of spleen and stomach, deficiency of middle-jiao, deficiency of spleen-qi, fluid impairment due to heat disease and the like in clinic. The chemical components in the radix codonopsitis comprise sterols, glycosides, alkaloids, nitrogenous components, volatile oil components, triterpenes and other components, and a plurality of inorganic elements and amino acids which are necessary for human bodies. Modern pharmacological research shows that the codonopsis pilosula has various effects of regulating blood sugar, promoting hematopoietic function, reducing blood pressure, resisting anoxia, resisting fatigue, enhancing the immunity of the organism, regulating gastric contraction, resisting ulcer and the like. The polyacetylene compound in radix Codonopsis, radix Codonopsis alkynoside, is a water soluble component separated from radix Codonopsis. The lobetyolin has good protection effect on gastric mucosa injury caused by ethanol, conforms to the traditional efficacy of codonopsis pilosula for tonifying spleen and stomach, and is one of active ingredients of codonopsis pilosula for protecting gastric mucosa. Can be used as index component for quality control of radix Codonopsis.
The spleen-tonifying prescription has the main effects of tonifying spleen and qi, and the codonopsis pilosula is a commonly used spleen-tonifying medicament in clinic. The lobetyolin is a polyacetylene compound separated from codonopsis pilosula, and is used as an index component for quality control of codonopsis pilosula in the Chinese pharmacopoeia (part one) of the 2010 edition. At present, HPLC-UV and RP-HPLC methods are mostly adopted at home and abroad to measure the content of lobetyolin. The methods need complicated and complicated pretreatment of the codonopsis pilosula, and cause difficulty in measuring the content of specific components because the codonopsis pilosula contains various chemical components. The liquid chromatography-mass spectrometry technology has high separation efficiency, high sensitivity and high selectivity, eliminates the interference of the factors and provides an excellent technical means for the determination of the lobetyolin.
Disclosure of Invention
In order to solve the problems, the invention provides a method for measuring the content of lobetyolin in a spleen tonifying formula and a codonopsis pilosula extract by UPLC/Q-TOF-MS.
The method for determining the spleen tonifying formula and the content of lobetyolin in the codonopsis pilosula extract by using UPLC/Q-TOF-MS comprises the following steps: 1) preparing a spleen-tonifying prescription or a codonopsis pilosula extracting solution; 2) separating and detecting lobetyolin by adopting a high performance liquid chromatography-mass spectrometer; 3) plotting different concentrations of lobetyolin and corresponding response peak areas to obtain a standard curve of lobetyolin, and calculating the content of lobetyolin of the sample to be measured by comparing the peak areas of the sample to be measured with the standard curve.
The spleen tonifying formula extract is prepared by the following specific steps: weighing radix astragali, radix codonopsitis and rhizoma atractylodis macrocephalae according to the weight ratio of 1:1:1, and mixing the raw materials in a proportion of 1: 10, adding 50% ethanol into the mixture, performing reflux extraction for 2 times, performing reflux extraction for 2 hours each time, concentrating to a constant volume of 200mL, precisely sucking 100 mu L of the extracting solution into a 50mL volumetric flask, performing ultrasonic dissolution by using 50% methanol water, cooling, fixing the volume to a scale, precisely sucking a proper amount of the extracting solution into an inlet EP tube, centrifuging at 12000r/min for 20min, and taking a proper amount of supernatant to obtain an upper sample bottle.
Wherein, the specific steps for preparing the codonopsis pilosula extract are as follows: weighing radix codonopsis, and mixing the raw materials in a proportion of 1: 10, adding 50 percent ethanol into the mixture, and performing reflux extraction for 2 times, wherein each time is 2 hours; concentrating to 200mL, precisely sucking 100 μ L of the extractive solution into a 50mL volumetric flask, ultrasonically dissolving with 50% methanol water, cooling, precisely sucking to desired volume, centrifuging at 12000r/min for 20min, and taking appropriate amount of supernatant to sample bottle.
Wherein, the chromatographic conditions are as follows:
a chromatographic column: waters BEH C18 column (2.1X 50mm,1.7 μm); column temperature: 40 ℃; mobile phase A: methanol, B: an aqueous solution containing 0.1% formic acid water; gradient elution: 0-1.5min, 60% -80% A; 80-60% for 1.5-2 min; balancing samples for 3 min; analysis time: 2 minutes; the flow rate is 0.2 ml/min; the sample injection amount is 5 mu L;
mass spectrum conditions:
electrospray ion source (ESI); capillary voltage: 2500V; ion source temperature: 100 ℃; atomization temperature: 100 ℃; taper hole gas velocity: 50L/h; atomizing gas speed: 800L/h; detection mode: a positive ion; detecting m/z: 50-1200.
Common extraction methods of lobetyolin include cold soaking, decoction, ethanol reflux and ultrasonic extraction. The difference of the lobetyolin content in the codonopsis pilosula obtained by different extraction processes is investigated in the prior article. The method adopts a method of condensing, refluxing and extracting for 2 hours by 50 percent ethanol for two times by combining the existing conditions of a laboratory.
Under the condition of high performance liquid chromatography, the content of the lobetyolin is detected by an elution mode of taking acetonitrile as an organic phase and pure water as an inorganic phase at equal degrees. The invention adopts acetonitrile in the pre-test: water (78:22, 75: 25); methanol: water (78:22, 75: 25); isocratic elution is carried out, the peak shape of the lobetyolin obtained as a result is not good, several subsequent tests show that the lobetyolin obtained by using a gradient elution mode and methanol as an organic phase has good peak shape, and 0.1% of formic acid is added into a pure water inorganic phase, so that the separation is better. Gradient elution was performed using methanol and 0.1% formic acid as mobile phase. The response values of the lobetyolin in the positive ion mode and the negative ion mode are compared, and the response value in the positive ion mode is superior to the response value in the negative ion mode, so that data are acquired in the positive ion mode.
The sensitivity of the LC-MS technology is extremely high, so that the sample processing requirements are strict. The traditional Chinese medicine components are complex, and in order to avoid blocking of a chromatographic column by insoluble substances in the traditional Chinese medicine, a common sample processing method comprises a microfiltration membrane impurity removal method or an ultrahigh-speed centrifugal precipitation impurity removal method. Because the sample to be detected is treated by the mobile phase (methanol water) before sample introduction, and the mobile phase is prevented from dissolving substances in the filter membrane to generate interference on experimental results, the pretreatment of the sample before sample introduction for research adopts an ultra-high speed centrifugation method to remove impurities in the sample.
The invention establishes a method for detecting the content of lobetyolin in the traditional Chinese medicine codonopsis pilosula and the spleen-tonifying formula by applying a liquid chromatography-mass spectrometry technology. The method is rapid, sensitive and stable, and provides a new reference method for determining the content of lobetyolin.
Drawings
FIG. 1 shows the lobetyolin standard curve.
FIG. 2 shows the mass spectrogram of radix Codonopsis alkyne glycoside standard, radix Codonopsis extract, and spleen invigorating formula.
Fig. 3 shows a total ion flow diagram in the spleen invigorating formulation positive ion mode.
Fig. 4 shows a total ion flow diagram in the spleen invigorating formulation negative ion mode.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
1. An experimental instrument:
a traditional Chinese medicine crusher: model 103, renian, perpetual calendar pharmaceutical machinery ltd;
intelligent ultrasonic cleaner: model DL-360B, Shanghai letters instruments Inc.;
electric heating constant temperature water bath: model HWS24, shanghai-constant technology ltd;
one tenth electronic balance: model JJ2000, double jie test instrument factory, ever-maturing city;
one in ten thousand electronic balance BSA124S, Saedodes scientific instruments (Beijing) Inc.;
the special ultra-pure water machine for the laboratory: arium 611 type, Sartorius, Germany
A high-speed refrigerated centrifuge: 3-30K, Sigma Germany
Rotary evaporator System R-215, Switzerland B m CHI Co
Mass spectrometry: xevo G2-S Q-TOF, Waters corporation, USA
Ultra-high performance liquid chromatograph: waters corporation, USA.
2. Reagent
Radix Codonopsis, radix astragali, and rhizoma Atractylodis Macrocephalae were purchased from Guangdong Tiancheng Chinese medicinal decoction pieces, Inc. The company provides a report on the examination of each drug.
Codonopsis pilosula alkyne glycoside standard (batch No. D-026-130528, Shanghai Sheng traditional Chinese medicine chemical Co., Ltd.)
Both methanol and acetonitrile are chromatographically pure (Merck, German)
Formic acid Sigma ethanol chromatography grade.
EXAMPLE 1 preparation of Codonopsis pilosula extract
Weighing radix codonopsis, and mixing the raw materials in a proportion of 1: 10, adding 50 percent ethanol into the mixture, and performing reflux extraction for 2 times, wherein each time is 2 hours; concentrating to 200mL, precisely sucking 100 μ L of the extractive solution into a 50mL volumetric flask, ultrasonically dissolving with 50% methanol water, cooling, precisely sucking to desired volume, centrifuging at 12000r/min for 20min, and taking appropriate amount of supernatant to sample bottle.
Example 2 preparation of spleen invigorating formulation extract
Weighing radix astragali, radix codonopsitis and rhizoma atractylodis macrocephalae according to the weight ratio of 1:1:1, and mixing the raw materials in a proportion of 1: 10, adding 50% ethanol into the mixture, performing reflux extraction for 2 times, performing reflux extraction for 2 hours each time, concentrating to a constant volume of 200mL, precisely sucking 100 mu L of the extracting solution into a 50mL volumetric flask, performing ultrasonic dissolution by using 50% methanol water, cooling, fixing the volume to a scale, precisely sucking a proper amount of the extracting solution into an inlet EP tube, centrifuging at 12000r/min for 20min, and taking a proper amount of supernatant to obtain an upper sample bottle.
Example 3 preparation of a Standard Curve
Precisely sucking 25, 50, 100, 400, 500, 800 and 1000 mu L of standard mother liquor into a 10mL volumetric flask (marked by a reference mark), and making into standard solutions of 0.0625 mu g/mL, 0.125 mu g/mL, 0.25 mu g/mL, 1 mu g/mL, 1.24 mu g/mL, 2 mu g/mL and 2.5 mu g/mL by using 50% methanol solution to fix the volume to the scale. And respectively taking a proper amount of the mixture to an inlet EP pipe. Centrifuging at 12000r/min for 20min, and taking the supernatant to an upper sample bottle. And (4) measuring under the liquid condition, recording the peak area, and obtaining a standard curve by taking the concentration as an abscissa and the peak area as an ordinate.
The standard solution is gradually diluted and measured, and the amount of the standard is taken as a quantification limit and a detection limit when the signal-to-noise ratio S/N is 10 and the signal-to-noise ratio S/N is 3.
Precisely preparing 6 parts of radix codonopsis alkynoside standard substance with the same concentration, operating according to the operation method, recording the peak area of each part of radix codonopsis alkynoside, and calculating the RSD value.
A known sample was prepared, and 3 parts of each standard, 0.25. mu.g/mL, 1.24. mu.g/mL, 2.5. mu.g/mL, were added in parallel, and the recovery formula (measured amount-added amount)/sample weight X100% was determined under the same conditions as described above.
And (4) conclusion:
1. the concentration of the codonopsis pilosula alkynoside standard substance is taken as an abscissa (x axis), and the peak area corresponding to each concentration is taken as an ordinate (y axis). Masslynx 4.1Software was used to plot a codonopsis pilosula alkynin standard curve and to derive a regression equation (FIG. 1). The correlation coefficient was 0.999503, and the linear relationship was good in the range of 0.0625 to 2.5. mu.g/mL. See figure 1, lobetyolin standard curve.
2. The quantitative limit and the detection limit of the lobetyolin are respectively 10ng/mL and 5ng/mL of the amount of each standard substance when the signal to noise ratio S/N is 10 and S/N is 3.
3. Precision of Codonopsis tangynoside
4. Recovery rate of lobetyolin
| Serial number | Sample size (μ g) | Addition amount (μ g) | Measured quantity (μ g) | The recovery rate is high |
| 1 | 1 | 0.125 | 1.09±0.02 | 96.9 |
| 2 | 1 | 0.62 | 1.56±0.06 | 96.0 |
| 3 | 1 | 1.25 | 2.13±0.04 | 94.7 |
Example 4 screening of the Mobile phase
Elution was carried out by the following method
1) A: acetonitrile, B: isocratic elution with water (78:22, 75: 25);
2) a: methanol, B: isocratic elution with water (78:22, 75: 25);
3) a: methanol B: gradient elution with 0.1% aqueous formic acid.
And (4) conclusion: a: methanol, B: an aqueous solution containing 0.1% formic acid water; gradient elution: 0-1.5min, 60% -80% A; 80-60% for 1.5-2 min; balancing samples for 3 min; analysis time: 2 minutes; the flow rate is 0.2 ml/min; the sample injection amount is 5 mu L; the lobetyolin has good separation and peak shape, and the retention time is 0.87 min. See figure 2, cortex spectrogram of radix Codonopsis alkyne glycoside standard, radix Codonopsis extract, and spleen invigorating formula.
EXAMPLE 5 setting of column temperature of chromatography column
The test investigates the influence of the column temperature of 35 ℃ and 40 ℃ on the separation of the compounds in the compound, the column pressure fluctuation degree of the chromatographic column of the compound at the normal temperature and the column temperature of 35 ℃ is larger than that of 40 ℃, and the separation degree of each compound is lower than that of 40 ℃, so the column temperature of the chromatographic column is set to be 40 ℃.
EXAMPLE 6 screening of the extraction conditions of the formulations
Respectively using pure water, 30%, 50%, 70% and 95% ethanol as solvents, and carrying out condensation reflux extraction for 2 hours for two times. And (4) inspecting the extracted part by using a liquid chromatography-mass spectrometry technology.
And (4) conclusion: the 50% ethanol extract is the most abundant in substances in the total ion flow diagram under positive and negative modes, so 50% ethanol is selected as the extraction solvent. See fig. 3 and 4, the total ion flow diagram of the codonopsis pilosula alkyne glycoside spleen tonifying prescription in the positive ion mode.
Example 7 test for determining the content of lobetyolin in Codonopsis pilosula extract and spleen invigorating formula with optimized System
Precisely sucking 100 mu L of spleen tonifying formula and codonopsis pilosula extracting solution respectively into 50mL volumetric flasks, ultrasonically dissolving with 50% methanol, cooling, and fixing the volume to the scale. Precisely sucking a proper amount of the supernatant into an inlet EP tube, centrifuging at 12000r/min at an ultra-high speed for 20min, taking the supernatant into an upper sample bottle, paralleling 6 samples, and measuring under the same liquid quality condition.
TABLE 2 radix Codonopsis extractive solution and radix Codonopsis alkyne glycoside content (μ g/mL) in spleen invigorating formula
Note: codonopsis pilosula extracting liquid ii spleen invigorating prescription
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. A method for determining the content of lobetyolin in a spleen tonifying formula and a codonopsis pilosula extract by UPLC/Q-TOF-MS is characterized by comprising the following steps: 1) preparing a spleen tonifying formula or a codonopsis pilosula extracting solution, extracting the spleen tonifying formula or the codonopsis pilosula for 2 hours by adopting 50% ethanol in a condensing reflux manner, and extracting twice, wherein the spleen tonifying formula is prepared from astragalus, codonopsis pilosula and bighead atractylodes rhizome in a weight ratio of 1:1: 1; 2) separating and detecting lobetyolin by adopting a high performance liquid chromatography-mass spectrometer; 3) plotting different concentrations of lobetyolin and corresponding response peak areas to obtain a standard curve of lobetyolin, and calculating the content of lobetyolin of a sample to be detected by comparing the peak area of the sample to be detected with the standard curve, wherein the chromatographic conditions are as follows:
a chromatographic column: a 2.1X 50mm,1.7 μm waters BEH C18 column; column temperature: 40 ℃; mobile phase A: methanol, B: an aqueous solution containing 0.1% formic acid water; gradient elution: 0-1.5min, 60% -80% A; 80-60% for 1.5-2 min; balancing samples for 3 min; analysis time: 2 minutes; the flow rate is 0.2 ml/min; the sample injection amount is 5 mu L;
mass spectrum conditions:
electrospray ion source (ESI); capillary voltage: 2500V; ion source temperature: 100 ℃; atomization temperature: 100 ℃; taper hole gas velocity: 50L/h; atomizing gas speed: 800L/h; detection mode: a positive ion; detecting m/z: 50-1200.
2. The method for determining the content of lobetyolin in the spleen-invigorating formula and the codonopsis pilosula extract according to claim 1, wherein the method for preparing the spleen-invigorating formula extract comprises the following specific steps: weighing radix astragali, radix codonopsitis and rhizoma atractylodis macrocephalae according to the weight ratio of 1:1:1, and mixing the raw materials in a proportion of 1: 10, adding 50% ethanol into the mixture, performing reflux extraction for 2 times, performing reflux extraction for 2 hours each time, concentrating to a constant volume of 200mL, precisely sucking 100 mu L of the extracting solution into a 50mL volumetric flask, performing ultrasonic dissolution by using 50% methanol water, cooling, fixing the volume to a scale, precisely sucking a proper amount of the extracting solution into an inlet EP tube, centrifuging at 12000r/min for 20min, and taking a proper amount of supernatant to obtain an upper sample bottle.
3. The method for determining the content of lobetyolin in the spleen invigorating formula and the codonopsis pilosula extract according to claim 1, wherein the preparation of the codonopsis pilosula extract comprises the following specific steps: weighing radix codonopsis, and mixing the raw materials in a proportion of 1: 10, adding 50 percent ethanol into the mixture, and performing reflux extraction for 2 times, wherein each time is 2 hours; concentrating to 200mL, precisely sucking 100 μ L of the extractive solution into a 50mL volumetric flask, ultrasonically dissolving with 50% methanol water, cooling, precisely sucking to desired volume, centrifuging at 12000r/min for 20min, and taking appropriate amount of supernatant to sample bottle.
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