Method for detecting ten tobacco alkaloids in soil

Abstract

The invention relates to the field of analytical chemistry, in particular to a method for detecting ten tobacco alkaloids in soil, which comprises the following steps: extracting alkaloids in a soil sample by using a sodium hydroxide solution and acetonitrile; ten alkaloids such as nicotine, nornicotine, myosmine, anabasine, cotinine, diene nicotine, 2, 3 '-bipyridine, nordiene nicotine, N-methyl anabasine and neonicotinine are analyzed and detected through ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and quantitative analysis is carried out by adopting an internal standard method. According to the method, a sodium hydroxide solution is used as an alkaloid dissolving agent, acetonitrile is used as an extraction agent, the solvent dosage is reduced, the operation process is optimized, sodium chloride is used for layering an organic phase and a water phase, and the sample pretreatment efficiency is improved; 10 tobacco alkaloids are subjected to targeted analysis by adopting an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry technology, so that the detection sensitivity is greatly improved, and the targeted analysis and quantification capability is high.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry, and in particular to a method for detecting ten tobacco alkaloids in soil. Background Art

[0002] Tobacco alkaloids are a class of low-molecular compounds containing nitrogen heterocycles. Common tobacco alkaloids include nicotine, nornicotine, mysmine, anabasine, anabasine, cotinine, dienicotine, 2,3'-bipyridine, nornicotine, N-methyl anabasine, and anatabine. Most of these tobacco alkaloids have strong biological toxicity and can affect the primary metabolism, gene expression, and transcriptional regulation of cells. If these tobacco alkaloids are stored in the soil for a long time, they will be absorbed and transported by crops and enriched in plant leaves and fruits, eventually bringing potential food safety problems. Based on this, it is of great significance to establish a detection method for tobacco alkaloids in soil.

[0003] The prior art does not have a liquid chromatography-mass spectrometry method to simultaneously detect multiple alkaloids in soil. In addition, a small number of methods use gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry to detect some alkaloids in tobacco. These methods require complex pre-treatment of samples such as organic solvent extraction and ultrasound-assisted extraction. The process requires multiple extraction and separation, which inevitably leads to alkaloid loss, causing the final test results to deviate from the true value. It may even take several hours to complete the pre-treatment of a sample, which seriously restricts the detection efficiency of alkaloids.

[0004] Therefore, it is necessary to propose a rapid analysis method for tobacco alkaloids in soil that is time-saving, highly sensitive, and can extract and detect multiple alkaloids in a single time.

[0005] The present invention is proposed to solve any of the above problems. Summary of the invention

[0006] In order to further improve the analysis method of tobacco alkaloids, the present invention establishes an analysis method of tobacco alkaloids in soil based on sodium hydroxide solution as an alkaloid dissolving agent, acetonitrile as an extractant, QuEChERS method as a pretreatment method of a 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 both small. The entire sample pretreatment can be completed within one hour, the steps are simple, the sample processing time is greatly shortened, and the loss of effective components is reduced; the multiple reaction monitoring mode is adopted, and the method has the advantages of high sensitivity, good reproducibility, wide linear range, etc.

[0007] The technical solution adopted by the present invention is as follows:

[0008] The method for detecting ten tobacco alkaloids in soil comprises the following steps: extracting alkaloids from test soil samples using sodium hydroxide solution and acetonitrile solution, detecting nicotine, nornicotine, myosamine, anabasine, cotinine, nicotine diene, 2,3'-bipyridine, nornicotine, N-methyl anabasine and anatabine by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and quantitatively analyzing by internal standard method. The method comprises the following steps:

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

[0010] The sample pretreatment in step (1) is specifically as follows: 2.00 g of the test soil sample is weighed and placed in a 50 mL centrifuge tube, 40 μL of a mixed internal standard solution of nicotine-D4 and cotinine-D3 with a concentration of 1 mg / L and 10 mL of a 5% sodium hydroxide solution are added in sequence, the mixture is shaken well, 10 mL of acetonitrile is added, the mixture is shaken well again, sealed and placed in an ultrasonic machine for 30 min, the mixture is shaken well every 10 min, 2 g of sodium chloride is added to the system after the ultrasonication is completed, and the mixture is centrifuged at 25° C. and 5000 r / min for 5 min. After the centrifugation is completed, 7.5 mL of the upper layer liquid 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 is blown to 100 μL, the volume is fixed to 1.0 mL with 0.01% ammonia water, and the mixture is filtered through a 0.22 μm filter membrane to remove PSA, thereby obtaining a sample to be tested;

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

[0012] And / or investigation of intra-day and inter-day reproducibility: weigh 2.00g of the test soil sample and place it in a 50mL centrifuge tube, add 40μL of a mixed solution of nicotine-D4 and cotinine-D3 with a concentration of 1mg / L, 200μL of a mixed external standard solution containing 10 tobacco alkaloids (the concentration is determined according to the test needs), 10mL of 5% sodium hydroxide solution, shake well, add 10mL of acetonitrile, shake well again, place it in an ultrasonic machine for 30min, shake well every 10min, after the ultrasound is completed, add 2g of sodium chloride to the system, use a centrifuge to centrifuge for 5min at 25℃ and 5000r / min, after the centrifugation is completed, immediately take 7.5mL of the upper layer liquid into a clean centrifuge tube, add 20mg PSA, vortex for 1min, take 5mL of the supernatant and blow it to 100μL with nitrogen, make up to 1.0mL with 0.01% ammonia water, pass through a 0.22μm filter membrane to remove PSA, and obtain the sample to be tested.

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

[0014] The analysis conditions of the multiple reaction detection mode of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry are:

[0015] ① Liquid chromatography conditions: Waters BEH C18 chromatographic column inner diameter 2.1mm × length 50mm, particle size 1.7μm, column temperature 30℃; elution mode: gradient elution; gradient elution conditions: flow rate set to 0.3mL / min; total chromatographic running time 10.0min; elution gradient: 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 15℃; injection volume 2μL;

[0016] The mobile phase A is 0.01% ammonia aqueous solution, and the mobile phase B is methanol.

[0017] ②Mass spectrometry conditions: ESI source, ion source temperature set to 550℃; ion source spray voltage 5500V; curtain gas set to 35psi; collision activated dissociation rate set to high; nebulizer gas pressure and auxiliary gas pressure set to 50psi;

[0018] (3) Analyzing the samples in a multiple reaction monitoring mode; optimizing the multiple reaction monitoring mode method to select the ion pair with the highest response as the quantitative ion pair and the ion pair with the second highest response as the qualitative ion pair;

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

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

[0021] The invention adopts sodium hydroxide solution as an alkaloid dissolving agent and acetonitrile as an extracting agent, thereby reducing the amount of solvent used, optimizing the operation process, and adopting sodium chloride to separate the organic phase and the aqueous phase, thereby improving the sample pretreatment efficiency; adopts ultrasonic extraction, the extraction process is simple, and the analysis time is greatly shortened; the method adopts ultra-high performance liquid chromatography-triple quadrupole mass spectrometry coupling technology to target and analyze 10 tobacco alkaloids, thereby greatly improving the detection sensitivity, having a weak matrix effect, a wide linear range, and a strong quantitative ability of targeted analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the extracted ion chromatogram of 10 tobacco alkaloids DETAILED DESCRIPTION

[0023] The present invention is further described below by way of examples, which are not limited to the examples. Experimental methods without specific conditions specified in the examples are generally carried out under conventional conditions and conditions described in the manual, or under conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all commercially available unless otherwise specified.

[0024] Example 1

[0025] Evaluation of analytical methods for tobacco alkaloids in soil based on ultrasonic extraction with aqueous sodium hydroxide and acetonitrile, salting out with sodium chloride, and detection by ultra-performance liquid chromatography-triple quadrupole mass spectrometry.

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

[0027] (1) Standard curve configuration: Nicotine, nornicotine, mysamine, anabasine, cotinine, nicotine diene, 2,3'-bipyridine, nornicotine, N-methyl anabasine, and anatabine were the 10 alkaloids to be analyzed, and the single standard stock solution concentration was 10 mg / mL. Nicotine-D4 and cotinine-D3 were used as internal standards, and the stock solution concentration was 100 mg / L. The 10 alkaloids and the two internal standards were all prepared in pure acetonitrile as solvent. The concentration of the 10 alkaloid mixed standard stock solution is 100 mg / L: 100 μL of each 10 mg / mL single standard alkaloid stock solution is mixed, and the volume is adjusted to 10 mL with acetonitrile, and vortexed to obtain a 100 mg / L alkaloid mixed standard stock solution; the concentration of the mixed standard stock solution of nicotine-D4 and cotinine-D32 internal standards is 1 mg / L: 100 μL of each 100 mg / L internal standard stock solution is mixed, and the volume is adjusted to 10 mL with acetonitrile, and vortexed. After that, the different concentrations of tobacco alkaloid mixed standards and internal standard mixed standards used are all diluted from the above two mixed standard stock solutions. Linearity investigation was carried out by setting 7 concentration levels of 10 alkaloid mixed standard solutions. The 7 concentration levels are 500ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 20ng / mL, 10ng / mL, and 5ng / mL. Take 20μL of the internal standard mixed standard stock solution and dilute it to 1.0mL with the above concentrations for analysis.

[0028] (2) Liquid chromatography conditions: Waters BEH C18 chromatographic column 2.1 mm × 50 mm, 1.7 μm, column temperature 30°C; mobile phase: phase A is an aqueous solution containing 0.01% ammonia, phase B is methanol; flow rate is set to 0.3 mL / min; total chromatographic running time is 10.0 min; elution gradient: 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 set to 550 °C; ion source spray voltage 5500 V; curtain gas set to 35 psi; collision-activated dissociation rate set to high; nebulizer gas pressure and auxiliary gas pressure set to 50 psi;

[0030] The qualitative and quantitative ion pairs of each alkaloid were optimized by multiple reaction monitoring, and the corresponding collision energy and retention time obtained in the set method are shown in Table 1 for details. The linear analysis of the method was performed on 10 alkaloid solutions at 7 concentration levels using the mass spectrometry conditions determined in Table 1, and the linear range, linear correlation coefficient, detection limit and quantification limit of each alkaloid were shown in Table 2. The correlation coefficient r in the table is between 0.9994 and 0.9999.

[0031] Table 110 tobacco alkaloids MRM parameter settings

[0032]

[0033] Note: “*” indicates quantitative ion

[0034] Table 2 Evaluation of the linearity and detection of analytical methods

[0035]

[0036]

[0037] The intra-day and inter-day reproducibility were investigated by spiking actual samples.

[0038] (1) Sample pretreatment: Weigh 2.00 g of mixed soil and place it in a 50 mL centrifuge tube. Add 40 μL of a mixed internal standard solution of nicotine-D4 and cotinine-D3 at a concentration of 1 mg / L for internal standard quantification. Moisten the soil with 10 mL of 5% sodium hydroxide solution to dissociate the alkaloids in the soil. After shaking, add 10 mL of acetonitrile for back extraction. Shake again and place it in an ultrasonic machine for 30 min. Shake once every 10 min to fully extract the alkaloids in the soil. After taking it out of the ultrasonic machine, add 2 g of sodium chloride for salting out to separate the organic phase and the aqueous phase. Then place it in a centrifuge and centrifuge it at 25°C and 5000 r / min for 5 min. After taking it out of the centrifuge, immediately take 7.5 mL of the upper layer liquid and place it in a clean centrifuge tube for later use. The supernatant was purified by adding 20 mg PSA, vortexed for 1 min, and 5 mL of nitrogen was blown to 100 μL, and the volume was made up to 1.0 mL with 0.01% ammonia water, filtered through a 0.22 μm filter membrane to remove PSA, and the solution was transferred to a brown injection bottle for detection and analysis.

[0039] (2) Standard working solution preparation: Nicotine, nornicotine, mysamine, anabasine, cotinine, nicotine diene, 2,3'-bipyridine, nornicotine diene, N-methyl anabasine, and anatabine were the 10 alkaloids to be analyzed. The single standard stock solution concentration was 10 mg / mL. Nicotine-D4 and cotinine-D3 were used as internal standards. The stock solution concentration was 100 mg / L. The 10 alkaloids and the two internal standards were all prepared in pure acetonitrile. The concentration of the 10 alkaloid mixed standard stock solution is 100 mg / L: 100 μL of each 10 mg / mL single standard alkaloid stock solution is mixed, and the volume is adjusted to 10 mL with acetonitrile, and vortexed to obtain a 100 mg / L alkaloid mixed standard stock solution; the concentration of the mixed standard stock solution of nicotine-D4 and cotinine-D32 internal standards is 1 mg / L: 100 μL of each 100 mg / L internal standard stock solution is mixed, and the volume is adjusted to 10 mL with acetonitrile, and vortexed. After that, the different concentrations of tobacco alkaloid mixed standards and internal standard mixed standards used are all diluted from the above two mixed standard stock solutions. Linearity investigation was carried out by setting 7 concentration levels of 10 alkaloid mixed standard solutions. The 7 concentration levels are 500ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 20ng / mL, 10ng / mL, and 5ng / mL. Take 20μL of the internal standard mixed standard stock solution and dilute it to 1.0mL with the above concentrations for analysis.

[0040] (3) Sample determination: The standard working solution obtained in (2) and the sample solution obtained in (1) were subjected to ultra-high performance liquid chromatography-triple quadrupole mass spectrometry analysis. A standard working curve was prepared with the quantitative ion peak area of ​​each alkaloid in the standard working solution as the ordinate and the content of each alkaloid in the standard working solution as the abscissa; the result of (3) was substituted into the standard curve to obtain the content of alkaloids in the solution to be tested, and then the content of each alkaloid in the sample was further calculated. Five injections were made every day for intra-day reproducibility; five injections were made every day for three consecutive days for inter-day reproducibility; as shown in Table 3, the RSDs were all below 5.5%. The test results showed that the test method had good intra-day and inter-day reproducibility.

[0041] Table 3 Evaluation of reproducibility and recovery of analytical methods

[0042]

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

[0044] (1) Operation of blank sample experiment: Weigh 2.00 g of mixed soil and place it in a 50 mL centrifuge tube, add 40 μL of a mixed internal standard solution of nicotine-D4 and cotinine-D3 with a concentration of 1 mg / L for internal standard method quantification, moisten the soil with 10 mL of 5% sodium hydroxide solution to dissociate the alkaloids in the soil, shake well, add 10 mL of acetonitrile for back extraction, shake well again, place it in an ultrasonic machine for 30 min, shake well every 10 min to fully extract the alkaloids in the soil, take it out from the ultrasonic machine, add 2 g of sodium chloride for salting out, separate the organic phase and the aqueous phase, and then put it in a centrifuge and centrifuge it at 25°C and 5000 r / min for 5 min. After taking it out from the centrifuge, immediately take 7.5 mL of the upper layer liquid and put it in a clean centrifuge tube for later use. The supernatant was purified by adding 20 mg PSA, vortexed for 1 min, and 5 mL of nitrogen was blown to 100 μL, and the volume was made up to 1.0 mL with 0.01% ammonia water, filtered through a 0.22 μm filter membrane to remove PSA, and the solution was transferred to a brown injection bottle for detection and analysis.

[0045] (2) Sample spike experiment operation: Weigh 2.00 g of mixed soil and place it in a 50 mL centrifuge tube, and add 40 μL of a mixed internal standard solution of nicotine-D4 and cotinine-D3 with a concentration of 1 mg / L for internal standard quantification. The low, medium, and high concentrations are 40 μL of a mixed standard solution of 10 alkaloids with a concentration of 1 mg / L, 200 μL of a mixed standard solution of 10 alkaloids with a concentration of 1 mg / L, 160 μL of a mixed standard solution of 10 alkaloids with a concentration of 10 mg / L, and 10 mL 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 stripping, and then shaken. The soil was ultrasonicated for 30 minutes, and the alkaloids in the soil were fully extracted every 10 minutes. After being taken out of the ultrasonic machine, 2 g of sodium chloride was added for salting out to separate the organic phase and the aqueous phase. The soil was then placed in a centrifuge and centrifuged at 25°C and 5000 r / min for 5 minutes. After being taken out of the centrifuge, 7.5 mL of the upper layer of liquid was immediately taken into a clean centrifuge tube for later use. 20 mg of PSA was added to the supernatant for purification. After vortexing for 1 minute, 5 mL of nitrogen was taken to 100 μL, and the volume was adjusted to 1.0 mL with 0.01% ammonia water. The solution was filtered through a 0.22 μm filter membrane to remove PSA, and the solution was transferred to a brown injection bottle for detection and analysis.

[0046] (3) Standard working solution preparation: Nicotine, nornicotine, mysamine, anabasine, cotinine, nicotine diene, 2,3'-bipyridine, nornicotine diene, N-methyl anabasine, and anatabine were the 10 alkaloids to be analyzed. The single standard stock solution concentration was 10 mg / mL. Nicotine-D4 and cotinine-D3 were used as internal standards. The stock solution concentration was 100 mg / L. The 10 alkaloids and the two internal standards were all prepared in pure acetonitrile. The concentration of the 10 alkaloid mixed standard stock solution is 100 mg / L: 100 μL of each 10 mg / mL single standard alkaloid stock solution is mixed, and the volume is adjusted to 10 mL with acetonitrile, and vortexed to obtain a 100 mg / L alkaloid mixed standard stock solution; the concentration of the mixed standard stock solution of nicotine-D4 and cotinine-D32 internal standards is 1 mg / L: 100 μL of each 100 mg / L internal standard stock solution is mixed, and the volume is adjusted to 10 mL with acetonitrile, and vortexed. After that, the different concentrations of tobacco alkaloid mixed standards and internal standard mixed standards used are all diluted from the above two mixed standard stock solutions. Linearity investigation was carried out by setting 7 concentration levels of 10 alkaloid mixed standard solutions. The 7 concentration levels are 500ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 20ng / mL, 10ng / mL, and 5ng / mL. Take 20μL of the internal standard mixed standard stock solution and dilute it to 1.0mL with the above concentrations for analysis.

[0047] (4) Sample determination: The standard working solution obtained in (3) and the sample solutions obtained in (1) and (2) are respectively subjected to ultra-high performance liquid chromatography-triple quadrupole mass spectrometry analysis. A standard working curve is prepared with the quantitative ion peak area of ​​each alkaloid in the standard working solution as the ordinate and the content of each alkaloid in the standard working solution as the abscissa; the result of step 3 is substituted into the standard curve to obtain the content of the alkaloid in the solution to be tested, and then the content of each alkaloid in the sample is further calculated.

[0048] After analysis and calculation, the spiked recoveries of low, medium and high concentrations are shown in Table 3, ranging from 82.3% to 110%, which can meet the analysis requirements. The above method evaluation shows that this method has high detection sensitivity, good linearity and reproducibility.

[0049] Example 2

[0050] Analysis of tobacco alkaloids in tobacco-growing soils from four different tobacco-growing soils: 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: Weigh 2.00 g of mixed soil and place it in a 50 mL centrifuge tube. Add 40 μL of a mixed internal standard solution of nicotine-D4 and cotinine-D3 at a concentration of 1 mg / L for internal standard quantification. Wet the soil with 10 mL of 5% sodium hydroxide solution to dissociate the alkaloids in the soil. After shaking, add 10 mL of acetonitrile for back extraction. Shake again and place it in an ultrasonic machine for 30 min. Shake once every 10 min to fully extract the alkaloids in the soil. After taking it out of the ultrasonic machine, add 2 g of sodium chloride for salting out to separate the organic phase and the aqueous phase. Then place it in a centrifuge and centrifuge it at 25°C and 5000 r / min for 5 min. After taking it out of the centrifuge, immediately take 7.5 mL of the upper layer liquid and place it in a clean centrifuge tube for later use. The supernatant was purified by adding 20 mg PSA, vortexed for 1 min, and 5 mL of nitrogen was blown to 100 μL, and the volume was made up to 1.0 mL with 0.01% ammonia water, filtered through a 0.22 μm filter membrane to remove PSA, and the solution was transferred to a brown injection bottle for detection and analysis.

[0052] (2) Standard working solution preparation: Nicotine, nornicotine, mysamine, anabasine, cotinine, nicotine diene, 2,3'-bipyridine, nornicotine diene, N-methyl anabasine, and anatabine were the 10 alkaloids to be analyzed. The single standard stock solution concentration was 10 mg / mL. Nicotine-D4 and cotinine-D3 were used as internal standards. The stock solution concentration was 100 mg / L. The 10 alkaloids and the two internal standards were all prepared in pure acetonitrile. The concentration of the 10 alkaloid mixed standard stock solution is 100 mg / L: 100 μL of each 10 mg / mL single standard alkaloid stock solution is mixed, and the volume is adjusted to 10 mL with acetonitrile, and vortexed to obtain a 100 mg / L alkaloid mixed standard stock solution; the concentration of the mixed standard stock solution of nicotine-D4 and cotinine-D32 internal standards is 1 mg / L: 100 μL of each 100 mg / L internal standard stock solution is mixed, and the volume is adjusted to 10 mL with acetonitrile, and vortexed. After that, the different concentrations of tobacco alkaloid mixed standards and internal standard mixed standards used are all diluted from the above two mixed standard stock solutions. Linearity investigation was carried out by setting 7 concentration levels of 10 alkaloid mixed standard solutions. The 7 concentration levels are 500ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 20ng / mL, 10ng / mL, and 5ng / mL. Take 20μL of the internal standard mixed standard stock solution and dilute it to 1.0mL with the above concentrations for analysis.

[0053] (3) Liquid chromatography conditions: Waters BEH C18 column 2.1 mm × 50 mm, 1.7 μm, column temperature 30°C; mobile phase: phase A is water containing 0.01% ammonia, phase B is acetonitrile; flow rate is set to 0.3 mL / min; total chromatographic run time is 10.0 min; elution gradient: 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 15°C; injection volume 2 μL;

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

[0055] (5) Sample determination: The standard working solution obtained in (2) and the sample solution obtained in (1) are respectively subjected to ultra-high performance liquid chromatography-triple quadrupole mass spectrometry analysis. A standard working curve is prepared with the quantitative ion peak area of ​​each alkaloid in the standard working solution as the ordinate and the content of each alkaloid in the standard working solution as the abscissa; the result of (5) is substituted into the standard curve to obtain the content of the alkaloid in the solution to be tested, and then the content of each alkaloid in the sample is further calculated.

[0056] Using the above analysis method, it was found that the tobacco alkaloid content in the soil taken from different plots ranged from 1.33 to 3206 ng / g. As shown in Table 4, except for N-methyl arbutin, which was not detected in all plots, the other 9 tobacco alkaloids were detected in all plots.

[0057] Table 4 Contents of 10 tobacco alkaloids in soils of four tobacco-growing plots

[0058]

[0059] This invention greatly shortens the sample extraction time, thereby effectively shortening the time of the entire analysis process. The method of the invention has the advantages of simple sample pretreatment, convenient extraction process, small amount of reagents, high detection sensitivity, good reproducibility, etc.

Claims

1. The method for detecting ten tobacco alkaloids in soil is characterized by: The method uses sodium hydroxide solution and acetonitrile to extract alkaloids in the test soil sample, and detects ten alkaloids, including nicotine, nornicotine, myosamine, anabasine, cotinine, dienicotine, 2,3'-bipyridine, nornicotine, N-methyl anabasine and anatabine, by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and quantitatively analyzes the alkaloids by an internal standard method, which specifically includes the following steps: Step (1) Sample pretreatment: accurately weigh the test soil sample, add internal standard solution, sodium hydroxide solution, and acetonitrile in sequence, seal, mix, and ultrasonicate for a period of time, add sodium chloride, and then centrifuge to collect the upper layer liquid, add N-propylethylenediamine (PSA) to the upper layer liquid for purification, concentrate the purified upper layer liquid, and then add a constant volume solution to the concentrated liquid to make it constant volume, filter to remove PSA, and obtain a sample to be tested; Step (2) detection: using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry in multiple reaction detection mode to analyze the sample to be detected obtained in step (1).

2. The detection method according to claim 1, characterized in that: The quantitative analysis is performed by using an internal standard method and performing quantitative detection and analysis according to a linear quantitative curve.

3. The detection method according to claim 1, characterized in that: The sample pretreatment in step (1) is specifically as follows: 2.00 g of the test soil sample is weighed and placed in a 50 mL centrifuge tube, 40 μL of a mixed internal standard solution of nicotine-D4 and cotinine-D3 with a concentration of 1 mg / L and 10 mL of a 5% sodium hydroxide solution are added in sequence, the mixture is shaken well, 10 mL of acetonitrile is added, the mixture is shaken well again, sealed and placed in an ultrasonic machine for 30 min, the mixture is shaken well every 10 min, 2 g of sodium chloride is added to the system after the ultrasonication is completed, and the mixture is centrifuged at 25° C. and 5000 r / min for 5 min. After the centrifugation is completed, 7.5 mL of the upper layer liquid 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 is blown to 100 μL, the volume is fixed to 1.0 mL with 0.01% ammonia water, and the mixture is filtered through a 0.22 μm filter membrane to remove PSA, thereby obtaining a sample to be tested; And / or, recovery rate investigation in method validation: weigh 2.00g of blank control soil sample without alkaloids and place it in a 50mL centrifuge tube, add 40μL of a mixed solution of nicotine-D4 and cotinine-D3 with a concentration of 1mg / L each, 200μL of a mixed external standard solution containing 10 tobacco alkaloids (the concentration is determined according to the test needs), 10mL of 5% sodium hydroxide solution, shake well, add 10mL of acetonitrile, shake well again, place it in an ultrasonic machine for 30min, shake well every 10min, after the end of the ultrasound, add 2g of sodium chloride to the system, use a centrifuge to centrifuge for 5min at 25℃ and 5000r / min, after the centrifugation, immediately take 7.5mL of the upper layer liquid into a clean centrifuge tube, add 20mg PSA, vortex for 1min, take 5mL of the supernatant, dilute to 1.0mL with 0.01% ammonia water, pass through a 0.22μm filter membrane to remove PSA, and obtain a sample to be tested; And / or investigation of intra-day and inter-day reproducibility: weigh 2.00g of the test soil sample and place it in a 50mL centrifuge tube, add 40μL of a mixed solution of nicotine-D4 and cotinine-D3 with a concentration of 1mg / L, 200μL of a mixed external standard solution containing 10 tobacco alkaloids (the concentration is determined according to the test needs), 10mL of 5% sodium hydroxide solution, shake well, add 10mL of acetonitrile, shake well again, place it in an ultrasonic machine for 30min, shake well every 10min, after the ultrasound is completed, add 2g of sodium chloride to the system, use a centrifuge to centrifuge for 5min at 25℃ and 5000r / min, after the centrifugation is completed, immediately take 7.5mL of the upper layer liquid into a clean centrifuge tube, add 20mg PSA, vortex for 1min, take 5mL of the supernatant and blow it to 100μL with nitrogen, make up to 1.0mL with 0.01% ammonia water, pass through a 0.22μm filter membrane to remove PSA, and obtain the sample to be tested.

4. The detection method according to claim 1, characterized in that: In the step (2), the sample is analyzed in multiple reaction monitoring mode; the method of optimizing the multiple reaction monitoring mode is to select the ion pair with the highest response as the quantitative ion pair and the ion pair with the second highest response as the qualitative ion pair.

5. The detection method according to claim 1, wherein the analysis conditions of the multiple reaction detection mode of ultra-high performance liquid chromatography-triple quadrupole mass spectrometry in step (2) are: Liquid chromatography conditions: Waters BEH C18 column, inner diameter 2.1 mm × length 50 mm, particle size 1.7 μm, column temperature 30°C; elution mode: gradient elution; injection chamber temperature 15°C; injection volume 2 μL; Mass spectrometry conditions: ESI source, ion source temperature set to 550°C; ion source spray voltage 5500V; curtain gas set to 35psi; collision activated dissociation rate set to high; nebulizer gas pressure and auxiliary gas pressure set to 50psi.

6. According to the detection method of claim 5, it is characterized in that the gradient elution conditions are: the flow rate is set to 0.3mL / min; the total chromatographic running time is 10.0min; the 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.

7. The detection method according to claim 6, wherein the mobile phase A is a 0.01% ammonia aqueous solution and the mobile phase B is methanol.

Images