Method for detecting ten tobacco alkaloids in soil

Abstract

The present application relates to the field of analytical chemistry, in particular to a method for detecting ten tobacco alkaloids in soil, which uses sodium hydroxide solution and acetonitrile to extract alkaloids in a soil sample, and uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to analyze and detect nicotine, nornicotine, myosmine, pseudotropine, anatabine, anabasine, 2,3'-bipyridine, noranatabine, N-methyl pseudotropine and nornicotine, and uses an internal standard method for quantitative analysis. The present application uses sodium hydroxide solution as an alkaloid eluent and acetonitrile as an extractant, which reduces the amount of solvent used, optimizes the operation process, uses sodium chloride to separate the organic phase and the aqueous phase, improves the sample pretreatment efficiency, uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to target analysis of 10 tobacco alkaloids, greatly improves the detection sensitivity, and has strong quantitative ability in targeted analysis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, in particular to a method for detecting ten tobacco alkaloids in soil. BACKGROUND

[0002] Tobacco alkaloids are a class of low molecular weight compounds containing nitrogen heterocyclic compounds, common tobacco alkaloids mainly include nicotine, nornicotine, myosmine, anabasine, anatabine, anatabine, anatabine, 2,3'-dipyridine, nornicotine, N-methyl anatabine, nornicotine, etc. Most of these tobacco alkaloids have strong biological toxicity, which can affect the primary metabolism of cells, gene expression, transcriptional regulation, etc. If these tobacco alkaloids are stored in the soil for a long time, they will be absorbed, transported and enriched in plant leaves and fruits by crops, which will eventually lead to potential food safety problems. Therefore, it is of great significance to establish a method for detecting tobacco alkaloids in soil.

[0003] The prior art does not have a liquid chromatography-mass spectrometry method for simultaneously detecting multiple alkaloids in soil. In addition, a small part of the gas chromatography-mass spectrometry method or the liquid chromatography-mass spectrometry method is used to detect part of the tobacco alkaloids. These methods need to perform complex pretreatment such as organic solvent extraction and ultrasonic assisted extraction on the sample. This process needs to go through multiple extraction and separation, which inevitably leads to the loss of alkaloids, making the final detection result deviate from the true value. Even the pretreatment of a sample takes several hours, which seriously restricts the detection efficiency of alkaloids.

[0004] Therefore, it is necessary to propose a rapid analysis method for tobacco alkaloids in soil, which has short time consumption, high sensitivity and can extract and detect multiple alkaloids at a time.

[0005] To solve any of the above problems, the present application is proposed. SUMMARY

[0006] In order to further improve the analysis method of tobacco alkaloids, the present application establishes an analysis method for tobacco alkaloids in soil based on sodium hydroxide solution as an alkaloid eluent, acetonitrile as an extractant, QuEChERS method as a pretreatment method for the sample to be tested, and ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection and analysis. The method has simple pretreatment, and the sample amount and solvent amount used are relatively small. The entire sample pretreatment can be completed in one hour, the steps are simple, and the sample processing time is greatly shortened and the loss of active ingredients is reduced. The multiple reaction monitoring mode is adopted, which has the advantages of high sensitivity, good reproducibility, wide linear range, etc.

[0007] The technical scheme adopted by the present application is as follows:

[0008] The method for detecting ten tobacco alkaloids in soil uses sodium hydroxide solution and acetonitrile solution to test the extraction of alkaloids in soil samples, analyzes and detects ten alkaloids including nicotine, nornicotine, myosmine, anabasine, cotinine, anatabine, 2,3'-bipyridine, noranatabine, N-methyl anabasine and anatabine in soil samples by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and quantitatively analyzes by using an internal standard method, which specifically comprises the following steps:

[0009] Step (1) sample pretreatment: precisely weigh the test soil sample, sequentially add the internal standard solution, sodium hydroxide solution and acetonitrile, seal, mix and ultrasonic for a period of time, add sodium chloride, then centrifuge, collect the upper liquid, add PSA to the upper liquid, concentrate the purified upper liquid, then add the constant volume liquid to the concentrated liquid, filter to remove PSA, and obtain the sample to be detected;

[0010] The step (1) sample pretreatment is specifically as follows: weigh 2.00g of the test soil sample and place it in a 50mL centrifuge tube, sequentially add 40μL of mixed internal standard solution of nicotine-D4 and cotinine-D3 with a concentration of 1mg / L, 10mL of 5% sodium hydroxide solution, shake, then add 10mL of acetonitrile, shake again, seal, and place it in an ultrasonic machine for ultrasonic for 30min, shake every 10min during the ultrasonic process, after the ultrasonic process, add 2g of sodium chloride to the system, centrifuge at 25℃ and 5000r / min for 5min, immediately take 7.5mL of the upper liquid to a clean centrifuge tube, add 20mg of PSA, vortex for 1min, take 5mL of the supernatant, nitrogen blow to 100μL, use 0.01% ammonia water to constant volume to 1.0mL, filter through a 0.22μm filter membrane to remove PSA, and obtain the sample to be detected;

[0011] And / or, recovery rate investigation in method verification: weigh 2.00g of blank control soil sample without alkaloids and place it in a 50mL centrifuge tube, sequentially add 40μL of mixed solution of nicotine-D4 and cotinine-D3 with a concentration of 1mg / L, 200μL of mixed external standard solution containing ten tobacco alkaloids (the concentration is determined according to the test requirement), 10mL of 5% sodium hydroxide solution, shake, then add 10mL of acetonitrile, shake again, place it in an ultrasonic machine for ultrasonic for 30min, shake every 10min, after the ultrasonic process, add 2g of sodium chloride to the system, centrifuge at 25℃ and 5000r / min for 5min, immediately take 7.5mL of the upper liquid to a clean centrifuge tube, add 20mg of PSA, vortex for 1min, take 5mL of the supernatant, nitrogen blow to 100μL, use 0.01% ammonia water to constant volume to 1.0mL, filter through a 0.22μm filter membrane to remove PSA, and obtain the sample to be detected;

[0012] and / or the investigation of the intra-day and inter-day reproducibility: 2.00 g of the test soil sample is placed in a 50 mL centrifuge tube, 40 μL of a mixed solution of nicotine-D4 and cotinine-D3 with a concentration of 1 mg / L is added, 200 μL of a mixed external standard solution containing 10 kinds of tobacco alkaloids (the concentration is determined according to the test requirements) is added, 10 mL of 5% sodium hydroxide solution is added after shaking, 10 mL of acetonitrile is added, and then shaking is performed, and the mixture is placed in an ultrasonic machine for ultrasonic treatment for 30 min, and shaking is performed every 10 min. After the ultrasonic treatment is completed, 2 g of sodium chloride is added to the system, and then the mixture is centrifuged at 25°C and 5000 r / min for 5 min. After the centrifugation is completed, 7.5 mL of the supernatant is immediately taken into a clean centrifuge tube, 20 mg of PSA is added, and then vortexing is performed for 1 min. 5 mL of the supernatant is taken, nitrogen is blown to 100 μL, 0.01% ammonia water is added to 1.0 mL, 0.22 μm filter membrane is used to remove the PSA, and then a sample to be detected is obtained.

[0013] Step (2) detection: the sample to be detected obtained in step (1) is analyzed by using a multi-reaction detection mode of an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer;

[0014] The analysis conditions of the multi-reaction detection mode of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:

[0015] ① Liquid chromatography conditions: a Waters BEH C18 chromatographic column with an inner diameter of 2.1 mm x length of 50 mm, a particle size of 1.7 μm, and a column temperature of 30°C; elution mode: gradient elution; gradient elution conditions: the flow rate is set to 0.3 mL / min; the total chromatographic running time is 10.0 min; the elution gradient is: 0.00-1 min, 85% mobile phase A + 15% mobile phase B; 1-6 min, 45% mobile phase A + 55% mobile phase B, 6-7.5 min, 10% mobile phase A + 90% mobile phase B, 7.5-10 min, 85% mobile phase A + 15% mobile phase B; the injection chamber temperature is 15°C; the injection volume is 2 μL;

[0016] Among them, the mobile phase A is 0.01% ammonia water solution, and the mobile phase B is methanol.

[0017] ② Mass spectrometry conditions: ESI source, ion source temperature is set to 550°C; ion source spray voltage is 5500V; gas curtain gas is set to 35 psi; collision activation dissociation rate is set to high; atomization gas pressure and auxiliary gas pressure are set to 50 psi;

[0018] (3) The sample is analyzed by using a multi-reaction monitoring mode; the method for optimizing the multi-reaction monitoring mode is as follows: the ion pair with the highest response is selected as the quantitative ion pair, and the ion pair with the second highest response is selected as the qualitative ion pair;

[0019] (4) The internal standard method is used for quantification, and quantitative detection analysis is carried out according to the linear quantitative curve.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The present application uses sodium hydroxide solution as an alkaloid eluent and acetonitrile as an extractant, reduces the solvent consumption, optimizes the operation process, uses sodium chloride to separate the organic phase and the aqueous phase, improves the sample pretreatment efficiency, uses ultrasonic extraction, the extraction process is simple, greatly shortens the analysis time, uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry technology to target analyze 10 kinds of tobacco alkaloids, greatly improves the detection sensitivity, the matrix effect is weak, the linear range is wide, and the target analysis quantitative capacity is strong. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the extraction ion chromatogram of 10 kinds of tobacco alkaloids DETAILED DESCRIPTION

[0023] The present application is further illustrated by the following examples, which are not limited to the present examples. The experimental methods not specified in the examples are usually carried out according to the conventional conditions and the conditions described in the manual, or the general equipment, materials, reagents, etc. used according to the conditions suggested by the manufacturer, and if not specified, they can be obtained from commercial channels.

[0024] Example 1

[0025] Evaluation of the analysis method of tobacco alkaloids in soil based on ultrasonic extraction of sodium hydroxide aqueous solution and acetonitrile, sodium chloride salting-out, and ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection.

[0026] Investigation of linearity, detection limit and quantification limit.

[0027] (1) Standard curve configuration: nicotine, nornicotine, myosmine, anabasine, cotinine, anatabine, 2,3'-dipyridyl, noranatabine, N-methyl anabasine, anhydroecgonine, nicotine-D4 and cotinine-D3 as internal standards, all of which were dissolved in acetonitrile. The concentration of the 10 alkaloids was 10 mg / mL, and the concentration of the internal standards was 100 mg / L. The 10 alkaloids and 2 internal standards were mixed to obtain a 100 mg / L alkaloid mixture: 100 μL of each of the 10 mg / mL single alkaloid solutions was mixed and diluted with acetonitrile to 10 mL, and vortexed to obtain a 100 mg / L alkaloid mixture. The 1 mg / L internal standard mixture was prepared by mixing 100 μL of each of the 100 mg / L internal standard solutions and diluting with acetonitrile to 10 mL, and vortexing. The alkaloid mixture and the internal standard mixture were diluted to obtain the different concentrations of the alkaloid mixture and the internal standard mixture. The linearity of the method was investigated by setting 7 concentration levels of the 10 alkaloid mixture. The 7 concentration levels were 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, and 5 ng / mL, respectively. 20 μL of the internal standard mixture was diluted with the above-mentioned concentrations to 1.0 mL for analysis.

[0028] (2) Liquid chromatography conditions: Waters BEH C18 column 2.1 mm x 50 mm, 1.7 μm, column temperature 30°C; mobile phase: A phase is water containing 0.01% ammonia, B phase is methanol; flow rate is set to 0.3 mL / min; total chromatography running time is 10.0 min; elution gradient is: 0.00-1 min, 85% mobile phase A + 15% mobile phase B; 1-6 min, 45% mobile phase A + 55% mobile phase B, 6-7.5 min, 10% mobile phase A + 90% mobile phase B, 7.5-10 min, 85% mobile phase A + 15% mobile phase B; injection chamber temperature is 15°C; injection volume is 2 μL;

[0029] (3) Mass spectrometry conditions: ESI source, ion source temperature is set to 550°C; ion source spray voltage 5500V; gas curtain gas is set to 35 psi; collision activation dissociation rate is set to high; nebulization gas pressure and auxiliary gas pressure are set to 50 psi;

[0030] The qualitative and quantitative ion pairs of each alkaloid were obtained by multiple reaction monitoring optimization, and the corresponding collision energy and retention time were obtained in the set method. The details are shown in Table 1. The linear range, linear correlation coefficient, detection limit and quantification limit of each alkaloid were obtained by linear analysis of the 10 alkaloid solutions at 7 concentration levels using the mass spectrometry conditions determined in Table 1. The correlation coefficient r in the table is between 0.9994 and 0.9999.

[0031] Table 11 Tobacco alkaloids multiple reaction monitoring parameter settings

[0032]

[0033] Note: "*" indicates quantitative ion

[0034] Table 2 Analysis method linearity and detection evaluation

[0035]

[0036]

[0037] The intra-day and inter-day reproducibility was investigated using actual samples with standard addition.

[0038] (1) Sample pretreatment: 2.00 g of mixed soil was weighed into a 50 mL centrifuge tube, 40 μL of mixed internal standard solution of nicotine-D4 and cotinine-D3 with a concentration of 1 mg / L was added for internal standard quantitative analysis, 10 mL of 5% sodium hydroxide solution was added to wet the soil to dissociate the alkaloids in the soil, after shaking, 10 mL of acetonitrile was added for back extraction, and then the soil was shaken again and placed in an ultrasonic machine for 30 min, and shaken every 10 min to extract the alkaloids in the soil. After taking out from the ultrasonic machine, 2 g of sodium chloride was added for salting out, and the organic phase and aqueous phase were separated, and then placed in a centrifuge at 25°C, 5000 r / min for 5 min. After taking out from the centrifuge, 7.5 mL of supernatant was immediately taken in a clean centrifuge tube for standby. 20 mg of PSA was added to the supernatant, vortexed for 1 min, and then 5 mL of nitrogen was blown to 100 μL, and then 0.01% ammonia water was added to 1.0 mL, and then 0.22 μm filter membrane was used to remove PSA, and then the solution was transferred to a brown sample bottle for detection and analysis.

[0039] (2) Standard working solution configuration: nicotine, nornicotine, myosmine, anabasine, cotinine, anatabine, 2,3'-dipyridyl, noreanatabine, N-methyl anabasine, anhydro nicotine are 10 alkaloids to be analyzed, the single standard mother liquor concentration is 10 mg / mL, nicotine-D4 and cotinine-D3 are internal standards, the mother liquor concentration is 100 mg / L, 10 kinds of alkaloids and 2 internal standards are all with pure acetonitrile as solvent. 10 kinds of alkaloid mixed standard mother liquor concentration is 100 mg / L: 100 μL of 10 mg / mL single standard alkaloid mother liquor is mixed, and acetonitrile is added to 10 mL, vortex mixed to obtain 100 mg / L of alkaloid mixed standard mother liquor; the mixed standard mother liquor concentration of nicotine-D4 and cotinine-D3 internal standard is 1 mg / L: 100 μL of 100 mg / L internal standard mother liquor is mixed, and acetonitrile is added to 10 mL, vortex mixed. The different concentrations of tobacco alkaloid mixed standard and internal standard mixed standard used thereafter are all diluted from the above two mixed standard mother liquors. Linear investigation is carried out by setting 7 concentration levels of 10 kinds of alkaloid mixed standard solutions. The 7 concentration levels are 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, respectively, 20 μL of internal standard mixed standard mother liquor is added to 1.0 mL with the above concentrations for analysis.

[0040] (3) Sample determination: the standard working solution obtained in (2) and the sample solution obtained in (1) are analyzed by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The standard working curve is prepared with the quantitative ion peak area of each alkaloid in the standard working solution as the vertical coordinate and the content of each alkaloid in the standard working solution as the horizontal coordinate; the results of (3) are substituted into the standard curve to obtain the content of alkaloids in the test solution, and the content of each alkaloid in the sample is further calculated. Five injections per day are used for the investigation of intra-day reproducibility; five injections per day for three consecutive days are used for the investigation of inter-day reproducibility; see Table 3, the RSD is below 5.5%, and the test results show that the test method has good intra-day and inter-day reproducibility.

[0041] Table 3 Evaluation of reproducibility and recovery rate of analysis method

[0042]

[0043] Investigation of recovery rate of test method: 10 ng / mL, 50 ng / mL and 400 ng / mL are selected as low, medium and high concentrations for recovery rate investigation, and blank and spiked tests are carried out, with 5 parallel samples in each group.

[0044] (1) Blank sample experiment operation: 2.00 g of mixed soil was weighed into a 50 mL centrifuge tube, 40 μL of mixed internal standard solution of nicotine-D4 and cotinine-D3 with a concentration of 1 mg / L was added for internal standard method quantification, 10 mL of 5% sodium hydroxide solution was used to wet the soil to dissociate the alkaloids in the soil, after shaking, 10 mL of acetonitrile was added for back extraction, and then shaking, ultrasonic machine was used for 30 min, and shaking was performed every 10 min to extract the alkaloids in the soil. After taking out from the ultrasonic machine, 2 g of sodium chloride was added for salting out, and the organic phase and the aqueous phase were separated, and then placed in a centrifuge at 25°C and 5000 r / min for 5 min. After taking out from the centrifuge, 7.5 mL of supernatant was immediately taken in a clean centrifuge tube for standby. 20 mg of PSA was added to the supernatant for purification, and after vortexing for 1 min, 5 mL of nitrogen was blown to 100 μL, and 0.01% ammonia water was used for constant volume to 1.0 mL. 0.22 μm filter membrane was used to remove PSA, and the solution was transferred to a brown sample bottle for detection and analysis.

[0045] (2) Sample spiking experiment operation: 2.00 g of mixed soil was weighed into a 50 mL centrifuge tube, 40 μL of mixed internal standard solution of nicotine-D4 and cotinine-D3 with a concentration of 1 mg / L was added for internal standard method quantification, 40 μL of 10 kinds of alkaloid mixed standard solution with a concentration of 1 mg / L, 200 μL of 10 kinds of alkaloid mixed standard solution with a concentration of 1 mg / L, and 160 μL of 10 kinds of alkaloid mixed standard solution with a concentration of 10 mg / L were added for low, medium and high concentration respectively, 10 mL of 5% sodium hydroxide solution was used to wet the soil to dissociate the alkaloids in the soil, after shaking, 10 mL of acetonitrile was added for back extraction, and then shaking, ultrasonic machine was used for 30 min, and shaking was performed every 10 min to extract the alkaloids in the soil. After taking out from the ultrasonic machine, 2 g of sodium chloride was added for salting out, and the organic phase and the aqueous phase were separated, and then placed in a centrifuge at 25°C and 5000 r / min for 5 min. After taking out from the centrifuge, 7.5 mL of supernatant was immediately taken in a clean centrifuge tube for standby. 20 mg of PSA was added to the supernatant for purification, and after vortexing for 1 min, 5 mL of nitrogen was blown to 100 μL, and 0.01% ammonia water was used for constant volume to 1.0 mL. 0.22 μm filter membrane was used to remove PSA, and the solution was transferred to a brown sample bottle for detection and analysis.

[0046] (3) Standard working solution preparation: nicotine, nornicotine, myosmine, anabasine, cotinine, anatabine, 2,3'-dipyridyl, norepinephrine, N-methyl anabasine, anatabine, and D4-nicotine and D3-cotinine as internal standards were analyzed. The concentration of each single standard solution was 10 mg / mL, and the concentration of each internal standard solution was 100 mg / mL. The 10 alkaloids and 2 internal standards were dissolved in acetonitrile. The concentration of the mixed standard solution of the 10 alkaloids was 100 mg / mL: 100 μL of each single standard solution was mixed and diluted with acetonitrile to 10 mL. The concentration of the mixed standard solution of D4-nicotine and D3-cotinine was 1 mg / mL: 100 μL of each internal standard solution was mixed and diluted with acetonitrile to 10 mL. The mixed standard solutions of different concentrations of tobacco alkaloids and internal standards were diluted from the above two mixed standard solutions. The linearity was investigated by setting 7 concentration levels of the mixed standard solution of the 10 alkaloids. The 7 concentration levels were 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, and 5 ng / mL, respectively. 20 μL of the mixed standard solution of the internal standards was diluted with the above-mentioned solutions to 1.0 mL for analysis.

[0047] (4) Sample determination: the standard working solution obtained in (3) and the sample solution obtained in (1) and (2) were analyzed by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The standard working curve was prepared by taking the quantitative ion peak area of each alkaloid in the standard working solution as the vertical coordinate and the content of each alkaloid in the standard working solution as the horizontal coordinate. The content of each alkaloid in the sample was calculated by substituting the results of step 3 into the standard curve.

[0048] After analysis and calculation, the recovery rates of the low, medium, and high concentrations were 82.3% to 110%, which met the analysis requirements. The above method evaluation showed that the method had high detection sensitivity, good linearity, and good reproducibility.

[0049] Example 2

[0050] Analysis of tobacco alkaloids in tobacco-growing soils from a town in Dali, Yunnan (plot 1), a town in Luquan, Yunnan (plot 2), a village in Anning, Yunnan (plot 3), and a village in Wenshan, Yunnan (plot 4)

[0051] (1) Sample pretreatment: 2.00 g of mixed soil was weighed into a 50 mL centrifuge tube, 40 μL of mixed internal standard solution of nicotine-D4 and cotinine-D3 with a concentration of 1 mg / L was added for internal standard method quantification, 10 mL of 5% sodium hydroxide solution was added to wet the soil to dissociate alkaloids in the soil, after shaking, 10 mL of acetonitrile was added for back extraction, and then the soil was shaken again and placed in an ultrasonic machine for 30 min, and the soil was shaken every 10 min to extract alkaloids in the soil. After taking out from the ultrasonic machine, 2 g of sodium chloride was added for salting out, and the organic phase and the aqueous phase were separated, and then the organic phase was placed in a centrifuge at 25°C and 5000 r / min for 5 min. After taking out from the centrifuge, 7.5 mL of supernatant was immediately taken in a clean centrifuge tube for standby. 20 mg of PSA was added to the supernatant, and after vortexing for 1 min, 5 mL of nitrogen was blown to 100 μL, and 0.01% ammonia water was added to 1.0 mL, and then 0.22 μm filter membrane was used to remove PSA, and the solution was transferred to a brown sample bottle for detection and analysis.

[0052] (2) Standard working solution configuration: nicotine, nornicotine, myosmine, anabasine, cotinine, anatabine, 2,3'-dipyridyl, nornicoumarin, N-methyl anabasine, and anatabine are 10 alkaloids to be analyzed, and the single standard stock solution has a concentration of 10 mg / mL. Nicotine-D4 and cotinine-D3 are internal standards, and the stock solution has a concentration of 100 mg / L. Ten kinds of alkaloids and two internal standards are dissolved in pure acetonitrile. The mixed standard stock solution of 10 alkaloids has a concentration of 100 mg / L: 100 μL of 10 mg / mL single standard alkaloid stock solution is mixed, and acetonitrile is added to 10 mL to obtain a mixed standard stock solution of 10 alkaloids with a concentration of 100 mg / L. The mixed standard stock solution of nicotine-D4 and cotinine-D3 has a concentration of 1 mg / L: 100 μL of 100 mg / L internal standard stock solution is mixed, and acetonitrile is added to 10 mL to obtain a mixed standard stock solution of two internal standards with a concentration of 1 mg / L. The mixed standard stock solution of tobacco alkaloids with different concentrations and the mixed standard stock solution of internal standards are diluted from the above two mixed standard stock solutions. Linear investigation is carried out by setting seven concentration levels of 10 kinds of mixed standard solutions of alkaloids. The seven concentration levels are 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, and 5 ng / mL, respectively. 20 μL of the mixed standard stock solution of internal standards is added to 1.0 mL with the above concentrations for analysis.

[0053] (3) Liquid chromatography conditions: Waters BEH C18 chromatographic column 2.1mmx50mm, 1.7um, column temperature is 30 DEG C; mobile phase: A phase is 0.01% ammonia water concentration water, B phase is acetonitrile; flow rate is set to 0.3mL / min; total chromatographic running time is 10.0min; elution gradient is: 0.00-1min, 85% mobile phase A+15% mobile phase B; 1-6min, 45% mobile phase A+55% mobile phase B, 6-7.5min, 10% mobile phase A+90% mobile phase B, 7.5-10min, 85% mobile phase A+15% mobile phase B; injection chamber temperature is 15 DEG C; injection volume is 2ul;

[0054] (4) Mass spectrometry conditions: ESI source, ion source temperature is set to 550 DEG C; ion source spray voltage 5500V; gas curtain gas is set to 35psi; collision activation dissociation rate is set to high; atomization gas pressure and auxiliary gas pressure is set to 50psi;

[0055] (5) Sample determination: the standard working solution obtained in (2), and (1) the sample solution obtained is analyzed by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The quantitative ion peak area of each alkaloid in the standard working solution is used as the ordinate, and the content of each alkaloid in the standard working solution is used as the abscissa to make a standard working curve; the results of (5) are substituted into the standard curve to obtain the content of alkaloids in the test solution, and the content of each alkaloid in the sample is further calculated.

[0056] Using the above analysis method, it is found that the tobacco alkaloid content in the soil taken from different plots is from 1.33 to 3206ng / g. As can be seen from table 4, except that N-methyl pseudotropine is not detected in all plots, the remaining nine kinds of tobacco alkaloids are detected in all plots.

[0057] Table 4: content of 10 tobacco alkaloids in soil of four tobacco growing plots

[0058]

[0059] The present application greatly shortens the sample extraction time, thereby effectively shortening the time of the entire analysis process. The method of the present application has the advantages of simple sample pretreatment, convenient extraction process, less reagent consumption, high detection sensitivity, good reproducibility and the like.

Claims

1. A method for the detection of ten tobacco alkaloids in soil, characterized in that, The method uses sodium hydroxide solution and acetonitrile to extract alkaloids in the test soil sample, detects ten alkaloids including nicotine, nornicotine, myosmine, anabasine, cotinine, anatabine, 2,3'-bipyridine, noranatabine, N-methyl anabasine and anatabine in the test soil sample by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and quantitatively analyzes by internal standard method, and specifically comprises the following steps: Step (1) sample pretreatment: precisely take the test soil sample, sequentially add the internal standard solution, sodium hydroxide solution and acetonitrile, seal, mix and ultrasonic for a period of time, add sodium chloride, then centrifuge, collect the upper liquid, add N-propyl ethylenediamine (PSA) to the upper liquid for purification, concentrate the purified upper liquid, then add constant volume liquid to the concentrated liquid, filter to remove PSA, and obtain the sample to be detected; Step (2) detection: analyze the sample to be detected obtained in step (1) by using the multiple reaction detection mode of ultra-high performance liquid chromatography-triple quadrupole mass spectrometry; The analysis conditions of the multiple reaction detection mode of ultra-high performance liquid chromatography-triple quadrupole mass spectrometry in step (2) are as follows: Liquid chromatography conditions: Waters BEH C18 chromatographic column with an inner diameter of 2.1 mm x length of 50 mm, a particle size of 1.7 μm, and a column temperature of 30 DEG C; elution mode: gradient elution; sample chamber temperature is 15 DEG C; sample volume is 2 μL; Mass spectrometry conditions: ESI source, ion source temperature is set to 550 DEG C; ion source spray voltage is 5500 V; gas curtain gas is set to 35 psi; collision activation dissociation rate is set to high; atomization gas pressure and auxiliary gas pressure are set to 50 psi; The gradient elution conditions are as follows: flow rate is set to 0.3 mL / min; total chromatographic running time is 10.0 min; elution gradient is: 0.00-1 min, 85% mobile phase A+15% mobile phase B; 1-6 min, 45% mobile phase A+55% mobile phase B, 6-7.5 min, 10% mobile phase A+90% mobile phase B, 7.5-10 min, 85% mobile phase A+15% mobile phase B; The mobile phase A is 0.01% aqueous ammonia solution, and the mobile phase B is methanol.

2. The detection method according to claim 1, characterized in that, The quantitative analysis is performed by internal standard method, and quantitative detection analysis is performed according to the linear quantitative curve.

3. The method of claim 1, wherein, The sample pretreatment of step (1) is specifically as follows: 2.00 g of the test soil sample is weighed into a 50 mL centrifuge tube, 40 μL of mixed internal standard solution of nicotine-D4 and cotinine-D3 each at a concentration of 1 mg / L is added, 10 mL of 5% sodium hydroxide solution is added after shaking, 10 mL of acetonitrile is added, and then shaking, sealing, and ultrasonic treatment in an ultrasonic machine for 30 min, with shaking every 10 min during the ultrasonic treatment, after the ultrasonic treatment, 2 g of sodium chloride is added to the system, and then centrifugation is performed at 25°C and 5000 r / min for 5 min, after the centrifugation, 7.5 mL of the supernatant is immediately taken into a clean centrifuge tube, 20 mg of PSA is added, vortexed for 1 min, 5 mL of the supernatant is taken, nitrogen blowing is performed to 100 μL, 0.01% ammonia water is added to 1.0 mL, 0.22 μm filter membrane is used to remove PSA, and a sample to be detected is obtained. And / or, recovery rate investigation in method verification: 2.00 g of blank control soil sample without alkaloids is weighed into a 50 mL centrifuge tube, 40 μL of mixed solution of nicotine-D4 and cotinine-D3 each at a concentration of 1 mg / L is added, 200 μL of mixed external standard solution containing 10 kinds of tobacco alkaloids is added, 10 mL of 5% sodium hydroxide solution is added after shaking, 10 mL of acetonitrile is added, and then shaking, ultrasonic treatment in an ultrasonic machine for 30 min, with shaking every 10 min during the ultrasonic treatment, after the ultrasonic treatment, 2 g of sodium chloride is added to the system, and then centrifugation is performed at 25°C and 5000 r / min for 5 min, after the centrifugation, 7.5 mL of the supernatant is immediately taken into a clean centrifuge tube, 20 mg of PSA is added, vortexed for 1 min, 5 mL of the supernatant is taken, 0.01% ammonia water is added to 1.0 mL, 0.22 μm filter membrane is used to remove PSA, and a sample to be detected is obtained. And / or, investigation of intra-day and inter-day reproducibility: 2.00 g of the test soil sample is weighed into a 50 mL centrifuge tube, 40 μL of mixed solution of nicotine-D4 and cotinine-D3 each at a concentration of 1 mg / L is added, 200 μL of mixed external standard solution containing 10 kinds of tobacco alkaloids is added (the concentration is determined according to the test requirement), 10 mL of 5% sodium hydroxide solution is added after shaking, 10 mL of acetonitrile is added, and then shaking, ultrasonic treatment in an ultrasonic machine for 30 min, with shaking every 10 min during the ultrasonic treatment, after the ultrasonic treatment, 2 g of sodium chloride is added to the system, and then centrifugation is performed at 25°C and 5000 r / min for 5 min, after the centrifugation, 7.5 mL of the supernatant is immediately taken into a clean centrifuge tube, 20 mg of PSA is added, vortexed for 1 min, 5 mL of the supernatant is taken, nitrogen blowing is performed to 100 μL, 0.01% ammonia water is added to 1.0 mL, 0.22 μm filter membrane is used to remove PSA, and a sample to be detected is obtained.

4. The method of claim 1, wherein In step (2), the sample is analyzed by using a multiple reaction monitoring mode; and the method for optimizing the multiple reaction monitoring mode is as follows: the ion pair with the highest response is selected as the quantitative ion pair, and the ion pair with the second highest response is selected as the qualitative ion pair.

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