CN114460209A - Method for measuring contents of seven alkaloids in fresh tobacco leaves - Google Patents
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- CN114460209A CN114460209A CN202210104650.5A CN202210104650A CN114460209A CN 114460209 A CN114460209 A CN 114460209A CN 202210104650 A CN202210104650 A CN 202210104650A CN 114460209 A CN114460209 A CN 114460209A
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- 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
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- 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
- G01N30/08—Preparation using an enricher
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G—PHYSICS
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- 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
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Abstract
The invention discloses a method for measuring the content of seven alkaloids in fresh tobacco leaves, which comprises the following steps: preparing standard solutions of seven alkaloids; processing a sample: weighing a certain amount of freeze-dried fresh tobacco powder, adding ammonia water and dichloromethane for extraction and separation to obtain supernatant; thirdly, high performance liquid chromatography-tandem mass spectrometry; fourthly, calculating: obtaining working curves and correlation coefficients of various alkaloids by adopting a weighted regression method; calculating to obtain the content of seven alkaloids in the fresh tobacco leaves. The method of the invention has simple and convenient sample treatment and accurate measurement result.
Description
Technical Field
The invention belongs to the technical field of tobacco, and particularly relates to a method for rapidly and accurately measuring the content of seven alkaloids in fresh tobacco leaves by using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS).
Background
The tobacco alkaloid is an important chemical component of tobacco and smoke, the composition and the content of the tobacco alkaloid are important factors influencing the quality of tobacco leaves, and the tobacco alkaloid directly influences the strength, the irritation, the taste and the safety of tobacco products. Among the tobacco alkaloids, nicotine is the most important and accounts for more than 95% of the total amount of the tobacco alkaloids; the second is nornicotine, anatabine, anabasine (anabasine) and the like, and the rest is trace alkaloid. In recent years, new cigarette production processes and novel tobacco products have brought forward special requirements on nicotine content, and important biological breeding projects started by the national tobacco agency include nicotine regulation and control research. Therefore, the accurate and rapid analysis of the tobacco alkaloid is an important link for the promotion of the matching project.
The analytical methods of tobacco alkaloids are many, and capillary electrophoresis and gas chromatography are mainly adopted in the early stage, and liquid chromatography is also adopted for analysis. Because the chromatographic method has not ideal analytical sensitivity to trace alkaloids and is easily interfered by impurities, the current chromatographic-mass spectrometry method still dominates. The chromatography-mass spectrometry mainly comprises a gas chromatography-single quadrupole mass spectrometry combined method, a full two-dimensional gas chromatography-flight time mass spectrometry combined method, a multidimensional gas chromatography-mass spectrometry combined method and the like. The industry standard YC/T-2018 recommends using gas chromatography-single quadrupole mass spectrometry as a conventional detection method of tobacco alkaloid and gas chromatography-tandem mass spectrometry as a confirmation method of trace alkaloid. However, the method recommended by the standard YC/T-2018 has the following problems: the sensitivity of the gas chromatography-single quadrupole mass spectrometry to the trace alkaloid is not ideal enough and is easily interfered by impurities; the nicotine in the alkaloid is overloaded due to high content by using gas chromatography-tandem mass spectrometry detection, and the quantitative analysis is difficult. YC/T-2018 recommends that gas chromatography-single quadrupole mass spectrometry and gas chromatography-tandem mass spectrometry are combined for use, two sets of equipment are required to be configured, and sample introduction and analysis are respectively carried out, so that the method is complicated.
The present invention has been made to solve the above problems.
Disclosure of Invention
The method adopts ammonia water for infiltration, then dichloromethane is used for extracting alkaloid in fresh tobacco leaves, N-propyl-ethylenediamine (PSA) adsorbent is used for purification, and UPLC-MS-MS is used for establishing a method for rapidly analyzing the content of seven alkaloids in the fresh tobacco leaves. The method of the invention has the advantages of more convenient and accurate sample treatment and analysis method. Compared with the liquid chromatography method in the prior art, the method has lower detection limit on trace alkaloid and can better prevent false positive. Provides technical support for material screening and alkaloid metabolism research in alkaloid regulation and control research.
The technical scheme of the invention is as follows:
a method for rapidly and accurately measuring the contents of seven alkaloids in fresh tobacco leaves comprises the following steps:
preparing standard solutions of seven alkaloids;
processing a sample: weighing a certain amount of freeze-dried fresh tobacco powder, adding ammonia water and dichloromethane, extracting and separating to obtain supernatant;
thirdly, ultra-high performance liquid chromatography-tandem mass spectrometry;
fourthly, calculating: obtaining working curves and correlation coefficients of various alkaloids by adopting a weighted regression method; calculating to obtain the content of seven alkaloids in the fresh tobacco leaves.
Preferably, the seven alkaloids in step (i) are nicotine (nicotinine), neonicotinine (anabasine), anatabine (anarabine), nornicotine (nornicotine), 2,3 '-dipyridine (2, 3' -dipyridine), cotinine (cotinine), and myosamine (myostatin); the preparation method of the seven alkaloid standard solutions comprises the following steps: respectively weighing 0.1g of nicotine and 0.01g of each of six other alkaloids to the accurate value of 0.0001 g; then respectively putting the mixed solution into a 100mL volumetric flask, and diluting to the constant volume by using methanol to prepare standard solutions with the nicotine concentration of 1000 mug/mL and the six other alkaloids concentrations of 100 mug/mL.
Preferably, the sample processing in step (ii) comprises the following steps: accurately weighing 50mg of freeze-dried tobacco powder into a 15mL centrifuge tube; adding 2mL of 6% ammonia water, oscillating for 2min, standing and soaking for 10 min; adding 10mL of dichloromethane, and performing ultrasonic treatment for 20 min; centrifuging at 4000r/min for 5min, and taking the lower organic phase into a purification tube containing the matrix dispersed solid phase extraction adsorbent; vortex and oscillate at 2500r/m for 2min, and then centrifuge at 10000r/m for 5 min; taking 0.5mL of supernatant to a chromatographic sampling bottle, blowing nitrogen to be nearly dry, adding 1mL of methanol, performing ultrasonic redissolution, and analyzing.
Preferably, the matrix dispersed solid phase extraction adsorbent is N-propyl-ethylenediamine.
Preferably, the analysis conditions of the step three ultra performance liquid chromatography-tandem mass spectrometry are as follows:
a chromatographic column: waters Acquity BEH C18(2.1 × 100mm, 1.7 μm), or Waters Acquity CSH C18(2.1 × 100mm, 1.7 μm), or Waters Acquity HSS T3(2.1 × 100mm, 1.7 μm); column temperature: 30 ℃; sample introduction amount: 2 mu L of the solution; mobile phase: a is ultrapure water containing 0.2% ammonia water, B is methanol containing 0.2% ammonia water; elution procedure: 0-5 min, 5% -50% B; 5-6.5 min, 50% B; 6.5-7 min, 50% -5% B; 7-10 min, 5% B; flow rate: 0.3 mL/min;
mass spectrum scanning mode: electrospray positive ion mode; the monitoring mode is as follows: monitoring multiple reactions; spraying voltage: 4500V; atomizing gas pressure: 750L/h; auxiliary gas pressure: 50L/h; ion source temperature: at 150 ℃.
Preferably, the column is a Waters Acquity BEH C18 (2.1X 100mm, 1.7 μm).
Preferably, the monitoring ion pair optimization mode is as follows: respectively injecting 1.0 mu g/mL of single standard solution of seven alkaloids into a mass spectrum at the flow rate of 10 mu L/min by adopting a flow injection mode, carrying out secondary mass spectrum scanning by taking a molecular ion peak of each alkaloid as a parent ion in an electrospray positive ion mode, selecting two fragment ions with higher abundance as characteristic ions, taking the fragment ions with the maximum strength as a quantitative ion pair, and taking the other pair as an auxiliary qualitative ion pair; then, mass spectrum parameters such as the voltage of a taper hole, collision energy and the like of the quantitative ion pair and the qualitative ion pair of each alkaloid are optimized, so that the ion pair strength of the quantitative ions and the qualitative ions reaches the maximum; thereby obtaining the optimal mass spectrum detection parameters of each alkaloid.
Preferably, 163.1/116.9 is used as the nicotine-quantifying ion pair. Because the nicotine content is far higher than other 6 kinds of trace alkaloids, in order to take account of the mass spectrum response intensity of other alkaloids, the quantitative ion pair of nicotine does not adopt 163.1/130.1 with the best response, but adopts 163.1/116.9, and the collision energy does not need to be optimal, thereby expanding the linear range of nicotine.
The invention has the following beneficial effects:
1. according to the sample treatment method, 0.2% ammonia water is respectively added into the water phase, the organic phase and the two phases. The retention time of the alkaloid eluted by the three is approximate, but the peak area is sequentially two-phase ammonia water addition, aqueous phase ammonia water addition and organic phase ammonia water addition; for precise quantitative elution, 0.2% ammonia was added to both phases. In order to keep consistency with the extract, methanol was used as the organic phase for subsequent analysis.
2. The ultra-high performance liquid chromatography-tandem mass spectrometry for analyzing the tobacco alkaloid has short analysis time and is simpler and more convenient than a gas chromatography-tandem mass spectrometry in instrument use and maintenance. Compared with the method recommended by the standard YC/T-2018, the method disclosed by the invention can be used for quickly, accurately and simply measuring the contents of seven alkaloids including nicotine, neonicotinoid, anatabine, nornicotine, myosmine, cotinine and 2, 3' -bipyridine in fresh tobacco leaves.
3. In the prior art, alkaloid in tobacco leaves is extracted, and generally, alkali liquor is added to dissociate the bound alkaloid in the tobacco leaves, and then solvent is used for extraction. The invention discovers that when methanol, ethanol or pure water is used for extraction, more pigment impurities can be extracted, and in addition, the sugar content of tobacco is high, the extract liquor can also extract higher sugar content, so that a serious matrix effect is caused; however, the pigment and the saccharide cannot be extracted by using dichloromethane, and the dichloromethane is easy to be replaced by methanol for redissolution by nitrogen blowing, so that the method adopts ammonia water infiltration and dichloromethane extraction modes to extract the alkaloid. Meanwhile, the method uses a matrix dispersed solid phase extraction adsorbent N-propyl-ethylenediamine (PSA) to reduce the influence of matrix effect on analysis; the matrix dispersed solid phase extraction adsorbent is used for purifying the extracting solution, so that impurities such as pigment, organic acid and the like can be effectively removed, and the interference of matrix effect is effectively reduced.
4. The method provided by the invention is used for measuring the mixed standard solution of seven tobacco alkaloids with different concentrations, and three sample injections are carried out for average correction at each concentration, so that the result is that the linearity of six trace alkaloids (other six alkaloids except nicotine) is good within the range of 0.01-1 mu g/mL, and the linearity of nicotine is good within the range of 10-100 mu g/mL; the detection limit of nicotine is 0.45mg/g, and the quantitative limit is 1.5 mg/g; the detection limit of other six trace alkaloids is 0.15-0.6 μ g/g, and the quantitative limit is 0.5-2 μ g/g. Therefore, the method of the invention has better recovery rate and precision for seven kinds of tobacco alkaloids, and can meet the quantitative analysis requirement of the seven kinds of alkaloids in fresh tobacco leaves
5. According to the method, the alkaloid standard solution is added to the fresh tobacco leaf samples at 3 levels to carry out recovery tests, and six parallel tests are repeatedly carried out; the recovery rate of the seven alkaloids is between 86.5 and 102 percent, and the relative standard deviation is 2.2 to 5.4 percent. According to the calculation of 3 times of the minimum detection concentration relative standard deviation, the quality of a pre-treated sample, the extraction volume, the dilution multiple and the like, the method has better detection limit and quantitative limit on the hepta-alkaloid.
Drawings
FIG. 1 is a MRM chromatogram of seven tobacco alkaloids eluted with methanol from column BEH C18.
FIG. 2 is a MRM chromatogram of seven tobacco alkaloids eluted from chromatographic column BEH C18 acetonitrile.
FIG. 3 is a MRM chromatogram of seven tobacco alkaloids eluted from a chromatographic column CSH C18 acetonitrile.
The reference signs are: 1. cotinine; 2. reducing nicotine; 3. anatabine; 4. 1, the product is Maisamine; 5. a neonicotinoid; 6. 2, 3' -bipyridine; 7. nicotine.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
Materials and reagents
Seven alkaloid standards are purchased from the canadian TRC company and include 7 alkaloids, such as nicotine (nicotinine), neonicotinine (anabase), anatabine (anabase), nornicotine (nornicotin), 2,3 '-bipyridine (2, 3' -bipyridine), cotinine (cotinine), and myosmine (myostatin), all with a purity of greater than 99.0%.
Chromatographically pure methanol, acetonitrile, dichloromethane were purchased from Fisher, usa; high-grade pure formic acid and ammonium formate, which are purchased from Dikma company; analytically pure ammonia, purchased from Xiong chemical Co., Ltd.
Purification tube containing matrix dispersed solid phase extraction adsorbent: the purge tube contained 25mg of N-propyl-ethylenediamine (PSA) powder; PSA is a product of Agilent corporation, USA.
Apparatus and device
Waters Xevo ultra performance liquid chromatography-tandem mass spectrometer (Waters, USA), Talboys multi-tube vortex oscillator (Troemner, USA), Eppendorf5804 desktop centrifuge (Eppendorf, Germany), JP-080 ultrasonic cleaner (40kHz, Shenzhen Jie cleaning devices, Ltd.).
Examples
The method for determining the content of the 7 alkaloids in the fresh tobacco leaves comprises the following steps:
step 1, preparing a standard solution: respectively weighing 0.1g of nicotine standard substance and 0.01g to 100mL of six other trace alkaloid standard substances in a volumetric flask, and accurately weighing to 0.0001 g; methanol is used for constant volume, and a single standard stock solution with the nicotine concentration of 1000 mug/mL and the six other trace alkaloids concentration of 100 mug/mL is prepared.
Step 3, high performance liquid chromatography-tandem mass spectrometry analysis: the analysis conditions were as follows: a chromatographic column: waters Acquity BEH C18 (2.1X 100mm, 1.7 μm); column temperature: 30 ℃; sample introduction amount: 2 mu L of the solution; mobile phase: a is ultrapure water containing 0.2 percent of ammonia water (V/V), and B is methanol containing 0.2 percent of ammonia water (V/V); elution procedure: 0-5 min, 5% -50% B; 5-6.5 min, 50% B; 6.5-7 min, 50% -5% B; 7-10 min, 5% B; flow rate: 0.3 mL/min.
The results were: the organic phase is poor in chromatographic separation no matter methanol or acetonitrile is used, and the nicotine and the neonicotine cannot be separated by chromatography; after the mobile phase is added with 0.1 percent formic acid or 10mmol/L ammonium formate, the nicotine and the neonicotine still can not realize chromatographic separation, which is inconsistent with the conclusion of the prior art; after ammonia water is added into the two phases, both the chromatographic peak shape and the separation degree are improved, the nicotine and the neonicotinoid realize chromatographic separation, and because the nicotine and the neonicotinoid have the same parent ions (163.1m/e), the same daughter ions can be generated, even if different quantitative ion pairs are manually selected, the potential interference still exists, and the quantitative accuracy is influenced. Chromatographic separation must therefore be achieved for accurate quantification. Respectively adding 0.2% ammonia water into the water phase, the organic phase and the two phases, wherein the retention time of the alkaloid eluted by the three phases is approximate, but the peak area size is that the ammonia water is added into the two phases, the ammonia water is added into the water phase, and the ammonia water is added into the organic phase. Therefore, 0.2% ammonia was added to both phases during elution.
The results of the comparison of the elution effects of methanol and acetonitrile show that the chromatographic separation effect using acetonitrile at a flow rate of 0.15ml/min is equivalent to that of methanol at a flow rate of 0.30ml/min under the same elution procedure, and in order to keep the same with the extract, the subsequent analysis all uses methanol as an organic phase.
The same specifications of the Waters Acquity BEH C18, CSH C18 and HSS T3 chromatographic columns are compared, and only the BEH C18 and CSH C18 chromatographic columns meet the chromatographic separation requirements. The order of the peaks of neonicotinoids and 2, 3' -bipyridine was changed (see FIGS. 1-3). This example employs a more general purpose Waters Acquity BEH C18 chromatography column.
Mass spectrum scanning mode: electrospray positive ion mode; the monitoring mode is as follows: multiple reaction monitoring, the monitoring parameters of 7 alkaloids are shown in table 1; spraying voltage: 4500V; atomizing gas pressure: 750L/h; auxiliary gas pressure: 50L/h; ion source temperature: at 150 ℃. The optimization mode of monitoring ion pairs is as follows: respectively injecting 1.0 mu g/mL of single standard solution of seven alkaloids into a mass spectrum at the flow rate of 10 mu L/min by adopting a flow injection mode, performing secondary mass spectrum scanning by taking the molecular ion peak of each alkaloid as a parent ion in an electrospray positive ion mode, selecting two fragment ions with higher abundance as characteristic ions, taking the maximum strength as a quantitative ion pair, and taking the other pair as an auxiliary qualitative ion pair; and then optimizing mass spectrum parameters such as the voltage of a taper hole, collision energy and the like of the quantitative ion pair and the qualitative ion pair of each alkaloid to enable the ion pair strength of the quantitative ion and the qualitative ion to be maximum, thereby obtaining the optimal mass spectrum detection parameter of each alkaloid, wherein the parameters are shown in table 1.
Table 1 alkaloid species and assay parameters
Step 4, calculating: preparing mixed standard solutions of seven tobacco alkaloids with different concentrations, measuring the series of mixed standard working solutions, carrying out sample injection for 3 times at each concentration, and carrying out average correction; and obtaining working curves and correlation coefficients of various alkaloids by adopting a weighted regression method. The six trace alkaloids have good linearity in the range of 0.01-1 mug/mL, and the nicotine has good linearity in the range of 10-100 mug/mL.
The recovery test was performed by adding alkaloid standard solution at 3 levels, and the recovery, precision and quantitation limits of the method of the present invention are shown in table 2, which was repeated 6 times. As shown in Table 2, the recovery rate of the seven alkaloids is 86.5% -102%, and the relative standard deviation is 2.2% -5.4%. According to the calculation of 3 times of the relative standard deviation of the minimum detection concentration, the quality of a pre-treated sample, the extraction volume, the dilution multiple and the like, the detection limit of the method to nicotine is 0.45mg/g, and the quantification limit is 1.5 mg/g; the detection limit of six trace alkaloids is 0.15-0.6 mug/g, and the quantitative limit is 0.5-2 mug/g. The method has better recovery rate and precision for seven alkaloids, and can meet the quantitative analysis requirement of the seven alkaloids in the fresh tobacco leaves.
TABLE 2 recovery, precision and limits of quantitation of the process of the invention
In the embodiment, 20 fresh tobacco leaf samples are analyzed, the nicotine content ranges from 15 mg/g to 30mg/g, the nornicotine content ranges from 55.4 μ g/g to 104.9 μ g/g, the neonicotine content ranges from 36.4 μ g/g to 110.2 μ g/g, the anabasine content ranges from 121.9 μ g/g to 191.2 μ g/g, the myosmine content ranges from 5.0 μ g/g to 18.4 μ g/g, the cotinine content ranges from 4.3 μ g/g to 12.8 μ g/g, and the 2, 3' -bipyridine content ranges from 9.2 μ g/g to 20.8 μ g/g.
Therefore, compared with the method recommended by the standard YC/T-2018, the determination method disclosed by the invention has the advantages that the measurement result is accurate, the sample treatment is simple, and the recovery rate and the precision of the alkaloid, particularly the trace alkaloid, are higher.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A method for measuring the content of seven alkaloids in fresh tobacco leaves is characterized by comprising the following steps:
preparing standard solutions of seven alkaloids;
processing a sample: weighing a certain amount of freeze-dried fresh tobacco powder, adding ammonia water and dichloromethane, extracting and separating to obtain supernatant;
thirdly, high performance liquid chromatography-tandem mass spectrometry;
fourthly, calculating: obtaining working curves and correlation coefficients of various alkaloids by adopting a weighted regression method; calculating to obtain the content of seven alkaloids in the fresh tobacco leaves.
2. The method according to claim 1, wherein the seven alkaloids in step (r) are nicotine, neonicotinoid, anatabine, nornicotine, 2, 3' -bipyridine, cotinine and myosmine, respectively; the preparation method of the seven alkaloid standard solutions comprises the following steps: respectively weighing 0.1g of nicotine and 0.01g of each of six other alkaloids to the accurate value of 0.0001 g; putting into 100mL volumetric flasks respectively, and diluting to constant volume with methanol to obtain standard solutions with nicotine concentration of 1000 μ g/mL and six other alkaloids concentrations of 100 μ g/mL.
3. The method of claim 1, wherein the step of processing the sample in step (ii) is: accurately weighing 50mg of freeze-dried tobacco powder into a 15mL centrifuge tube; adding 2mL of 6% ammonia water, oscillating for 2min, standing and soaking for 10 min; adding 10mL of dichloromethane, and performing ultrasonic treatment for 20 min; centrifuging at 4000r/min for 5min, and taking the lower organic phase into a purification tube containing the matrix dispersed solid phase extraction adsorbent; vortex and oscillate at 2500r/m for 2min, and then centrifuge at 10000r/m for 5 min; taking 0.5mL of supernatant to a chromatographic sampling bottle, blowing nitrogen to be nearly dry, adding 1mL of methanol, performing ultrasonic redissolution, and analyzing.
4. The method of claim 3, wherein the matrix dispersed solid phase extraction adsorbent is N-propyl-ethylenediamine.
5. The method of claim 1, wherein the analysis conditions of the hplc-tandem mass spectrometry of step (c) are:
a chromatographic column: waters Acquity BEH C18(2.1 × 100mm, 1.7 μm), or Waters Acquity CSH C18(2.1 × 100mm, 1.7 μm), or Waters Acquity HSS T3(2.1 × 100mm, 1.7 μm); column temperature: 30 ℃; sample introduction amount: 2 mu L of the solution; mobile phase: a is ultrapure water containing 0.2% ammonia water, B is methanol containing 0.2% ammonia water; elution procedure: 0-5 min, 5% -50% B; 5-6.5 min, 50% B; 6.5-7 min, 50% -5% B; 7-10 min, 5% B; flow rate: 0.3 mL/min;
mass spectrometry scan mode: electrospray positive ion mode; the monitoring mode is as follows: monitoring multiple reactions; spraying voltage: 4500V; atomizing gas pressure: 750L/h; auxiliary gas pressure: 50L/h; ion source temperature: at 150 ℃.
6. The method of claim 5, wherein the chromatography column is a Waters Acquity BEH C18(2.1 x 100mm, 1.7 μm).
7. The method of claim 5, wherein the optimization of monitoring ion pairs is: respectively injecting 1.0 mu g/mL of single standard solution of seven alkaloids into a mass spectrum at the flow rate of 10 mu L/min by adopting a flow injection mode, carrying out secondary mass spectrum scanning by taking a molecular ion peak of each alkaloid as a parent ion in an electrospray positive ion mode, selecting two fragment ions with higher abundance as characteristic ions, taking the fragment ions with the maximum strength as a quantitative ion pair, and taking the other pair as an auxiliary qualitative ion pair; and then optimizing the cone hole voltage and collision energy of the quantitative ion pair and the qualitative ion pair of each alkaloid, so that the ion pair strength of the quantitative ion and the qualitative ion is maximized.
8. The method of claim 7, wherein 163.1/116.9 is used as the nicotine-quantifying ion pair.
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