CN111983109B - Chiral analysis method of alkaloid in tobacco or tobacco product - Google Patents
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Abstract
The invention relates to a chiral analysis method of alkaloid in tobacco or tobacco products, which comprises the following steps: (1) weighing tobacco or tobacco products, adding the internal standard solution and the ammonium acetate aqueous solution, oscillating and mixing uniformly, carrying out ultrasonic extraction, filtering the filtrate, adding methanol, oscillating, and centrifuging to obtain a supernatant; (2) taking the supernatant obtained in the step (1) to perform high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis; (3) and calculating the content of the target object in the sample. The method realizes the simultaneous chiral analysis of nicotine, cotinine, anatabine, nornicotine and N-methylanabasine.
Description
Technical Field
The invention belongs to the technical field of tobacco chemical analysis, and particularly relates to a chiral analysis method of alkaloid in tobacco or tobacco products.
Background
The tobacco alkaloid refers to a class of organic compounds (except proteins, titanium compounds, amino acids, ammonia, amino sugar, nucleotide and nucleic acid) containing nitrogen heterocycles and existing in tobacco, and generally has alkalinity and shows physiological activity. Alkaloids have a special position in tobacco and products thereof and are important quality factors of tobacco. Most of the tobacco alkaloids have a chiral center, so most of the tobacco alkaloids have a pair of enantiomers, mainly including nicotine, cotinine, anatabine, nornicotine, N-methyl anabasine and the like. The metabolic mechanism and the biological activity of the enantiomer generally have larger difference, so that the establishment of the chiral separation method with high flux, high sensitivity and high selectivity for the tobacco alkaloid has important significance for metabolism and accumulation of the alkaloid, smoking and health research.
At present, chiral separation methods of nicotine mainly include a Liquid Chromatography (LC) method, a Supercritical Fluid Chromatography (SFC) method, a Gas Chromatography (GC) method, a Capillary Electrophoresis (CE) method and the like, and research on other tobacco alkaloids is relatively few. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) has the advantages of high speed, sensitivity, good reproducibility, high flux and the like, and has wide application prospect in chiral separation of alkaloids, but related researches are few. For example, in 2017, Hellinghausen et al (Drug test. anal.2017,9 (6)), 944-948. select a NicoShell chromatographic column to establish a method for measuring the enantiomer ratio of nicotine in products such as smokeless tobacco by liquid chromatography, but the research only carries out chiral separation on nicotine and does not research other alkaloids. In 2018, the team based on a liquid chromatography-mass spectrometry combined method (LC-MS, Talanta 2018,181,132-141.), the NicoShell chromatographic column is used for realizing chiral separation of nicotine, anatabine, nornicotine, N-methyl anabasine and the like, the TeicoSall chromatographic column is used for realizing chiral separation of cotinine, however, the method does not simultaneously realize chiral separation of 5 enantiomers, has poor separation effect on cotinine (does not realize baseline separation), in addition, the LC-MS method uses single quadrupole for qualitative and quantitative determination, such as application to complex tobacco matrix samples, the accuracy of target object analysis is not enough, and the method has non-ideal separation effect on target objects, and is easy to generate false positive results. In 2019, Ji et al (heiyon.2019, 5(5), e01719.) have established an ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining the enantiomeric ratios of nicotine, nornicotine, anatabine and the like in tobacco, but the method does not realize chiral separation of cotinine, N-methylanabasine and the like, and the chiral separation of nicotine, nornicotine and anatabine respectively needs two sets of different analysis systems, so that the process is complicated and the analysis efficiency is low.
Disclosure of Invention
The invention aims to establish a chiral analysis method of alkaloid in tobacco or tobacco products based on the technical defects, and the method uses HPLC-MS/MS technology to realize simultaneous chiral separation of nicotine, cotinine, anatabine, nornicotine and N-methylanabasine for the first time and realize baseline chiral separation of cotinine for the first time, has the advantages of high flux, good separation degree, simple and convenient operation, high sensitivity, good recovery rate and repeatability and the like, and is suitable for chiral analysis of main alkaloid in tobacco and tobacco products.
The purpose of the invention is realized by the following technical scheme:
a method of chiral analysis of an alkaloid in tobacco or a tobacco product, the method comprising the steps of:
(1) weighing tobacco or tobacco products, adding the internal standard solution and the ammonium acetate aqueous solution, oscillating and mixing uniformly, carrying out ultrasonic extraction, filtering the filtrate, adding methanol, oscillating, and centrifuging to obtain a supernatant;
(2) taking the supernatant obtained in the step (1) to perform high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis;
(3) and calculating the content of the target object in the sample.
Preferably, in step (1), the internal standard solution is a methanol solution of Mycoplanin-d 4 with a concentration of 8 μ g/mL.
Preferably, in the step (1), the concentration of the ammonium acetate aqueous solution is 0.05mol/L to 0.2mol/L, preferably 0.1 mol/L.
Preferably, in the step (1), the time of the ultrasonic extraction is 30-60 min, preferably 30 min.
Preferably, in the step (1), the speed of the centrifugation is 10000-15000 rpm.
Preferably, in the step (2), the chromatographic conditions of the high performance liquid chromatography-tandem mass spectrometry comprise: the chromatographic column is a Chiralpak IG-3 chromatographic column (250X 4.6mm X3 μm); the column temperature is 20 ℃; the mobile phase is 0.2 percent of ammonium formate/methanol solution by mass fraction.
Preferably, in the step (2), the chromatographic conditions of the high performance liquid chromatography-tandem mass spectrometry further include: the gradient elution procedure was: the initial flow rate is 0.4mL/min, the flow rate is kept for 12min, the flow rate is increased to 0.8mL/min when the flow rate is 13min, the flow rate is kept for 17min, and the total operation time is 30 min; the injection volume was 5. mu.L.
Preferably, in step (2), the mass spectrometry conditions are: an ion source: an electrospray ion source; an ionization mode: ESI (+); detection mode: an MRM mode; ion source Temperature (TEM): 550 ℃; electrospray voltage (IS): 5500V; collision gas (CAD): 4.8X 104Pa; air curtain gas (CUR): 2.8X 105Pa; ion source gas flow (GS 1): 3.8X 105Pa; ion source gas flow (GS 2): 4.1X 105Pa; inlet voltage (EP): 10V; emission voltage (CXP): 10V.
Preferably, in step (2), the MRM mass spectrometric detection parameters of the target compound and the internal standard are as follows:
note: "+" indicates the quantitative ion;
preferably, in step (3), the content of the target in the sample is quantitatively calculated by using an internal standard curve method, wherein the internal standard curve method comprises the following steps: preparing a series of standard working solutions containing a target object, adding an internal standard (Mysmine-d 4), and making a standard working curve by taking the quantitative ion peak area ratio of the target object to the internal standard object in each standard working solution as a vertical coordinate and the content of the target object in each standard working solution as a horizontal coordinate; and (3) substituting the analysis result obtained in the step (2) into the standard working curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
Preferably, the concentration ranges of each target in the series of standard working solutions are as follows: nicotine is 0.20-20 mu g/mL, cotinine is 2-500 ng/mL, nornicotine is 10-1000 ng/mL, anatabine is 2-5000 ng/mL, and N-methyl anabasine is 2-500 ng/mL.
Preferably, the alkaloid is nicotine, cotinine, nornicotine, anatabine and N-methylanabasine.
Preferably, the tobacco or tobacco product comprises a sample of tobacco leaves, such as flue-cured tobacco, burley tobacco, oriental tobacco, cigar, maryland tobacco and the like, smokeless tobacco products, cigars, heat-not-burn cigarettes, tobacco sheets, cut filler.
In a specific embodiment, the method comprises the steps of:
(1) weighing 50-200 mg of sample into a 15mL centrifuge tube, adding 1mL of internal standard solution (8 mug/mL of Mesamine-d 4 methanol solution) and 9mL of 0.1mol/L ammonium acetate aqueous solution, oscillating and mixing uniformly, performing ultrasonic extraction for 30-60 min, filtering the extract liquor through a 0.22 mu m filter membrane, taking 100-1000 mu L of the extract liquor into the centrifuge tube, adding 5-10 times of methanol in volume of the extract liquor, performing vortex oscillation for 2-5 min, centrifuging at 10000-15000 rpm for 5-10 min, and taking supernatant for later use;
(2) and (2) taking the supernatant obtained in the step (1) to perform high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, wherein the analysis conditions are as follows:
chromatographic conditions are as follows: the chromatographic column is a Chiralpak IG-3 chromatographic column (250X 4.6mm X3 μm) and the column temperature is 20 ℃; the mobile phase is ammonium formate/methanol solution with the mass fraction of 0.2%, and the gradient elution procedure is as follows: the initial flow rate is 0.4mL/min, the flow rate is kept for 12min, the flow rate is increased to 0.8mL/min when the flow rate is 13min, the flow rate is kept for 17min, and the total operation time is 30 min; the sample injection volume is 5 mu L;
mass spectrum conditions: an ion source: an electrospray ion source; an ionization mode: ESI (+); detection mode: an MRM mode; ion source Temperature (TEM): 550 ℃; electrospray voltage (IS): 5500V; collision gas (CAD): 4.8X 104Pa; air curtain gas (CUR): 2.8X 105Pa; ion source gas flow (GS 1): 3.8X 105Pa; ion source gas flow (GS 2): 4.1X 105Pa；Inlet voltage (EP): 10V; emission voltage (CXP): 10V; the MRM mass spectrometric detection parameters of the target compound and the internal standard are as follows:
note: "" indicates a quantitative ion.
(3) Quantitatively calculating the content of the target object in the sample by adopting an internal standard curve method; wherein, the internal standard curve method is as follows: preparing a series of standard working solutions containing a target, adding an internal standard (Mysmine-d 4), and making a standard working curve by taking the quantitative ion peak area ratio of the target to the internal standard in each standard working solution as a vertical coordinate and the content of the target in each standard working solution as a horizontal coordinate; and (3) substituting the analysis result of the step (2) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
Compared with the prior art, the invention has at least the following beneficial technical effects:
the invention is based on HPLC-MS/MS technology, inspects the selection of chromatographic column, the composition of mobile phase, the flow rate of mobile phase, the temperature of column, and the like, establishes a chiral analysis method of main alkaloid in tobacco or tobacco products, realizes the simultaneous chiral analysis of nicotine, cotinine, anatabine, nornicotine and N-methylanabasine, realizes the baseline chiral separation of nicotine, cotinine and N-methylanabasine, has the advantages of high flux, good separation degree, simple and convenient operation, high sensitivity, good recovery rate and repeatability, and the like, and is suitable for the chiral analysis of main alkaloid in tobacco and tobacco products.
Drawings
FIG. 1 is a comparison graph of the separation effect of different chromatographic columns (taking nicotine and cotinine as an example), wherein FIGS. 1A and 1B are chromatograms of NicoShell chromatographic columns for separating enantiomers of nicotine and cotinine, respectively; FIGS. 1C and 1D are chromatograms of a TeicoSchell column for separating the enantiomers of nicotine and cotinine, respectively; FIGS. 1E and 1F are chromatograms of chiral chromatographic columns for Chiralpak IG-3 separation of enantiomers of nicotine and cotinine, respectively;
FIG. 2 is a diagram showing the separation effect of different mobile phase compositions, wherein the mobile phase in FIG. 2A is acetonitrile, the mobile phase in FIG. 2B is a formic acid-methanol solution containing 0.1% volume fraction, and the mobile phase in FIG. 2C is an ammonium acetate-methanol solution containing 0.2% mass fraction;
FIG. 3 is a graph showing the separation effect of different mobile phases, wherein the flow rate of the mobile phase in FIG. 3A is 0.7mL/min, and the flow rate of the mobile phase in FIG. 3B is 0.4 mL/min;
FIG. 4 is a graph showing the separation effect at a column temperature of 40 ℃;
FIG. 5 is a Multiple Reaction Monitoring (MRM) chromatogram of a target and an internal standard in a standard working solution.
Detailed Description
The technical solution of the present invention will be further described below with reference to specific embodiments.
Example 1: chiral analysis method of alkaloid in tobacco or tobacco product
(1) Instruments and reagents
The instrument comprises the following steps: the liquid chromatography-tandem mass spectrometer (HPLC-MS/MS) system consists of an American Agilent 1200 high performance liquid chromatograph (comprising a G1367D autosampler, a G1312B binary solvent pump and a G1316B column incubator) and an AB SCIEX 5500 triple quadrupole mass spectrometer (provided with an ESI ion source), and data acquisition and processing Software is analysis 1.5.1 Software; ultrasonic apparatus (YM-100S, Union); an electronic balance (AE163, Mettler, Switzerland, feeling: 0.0001 g); talboys digital display multi-tube vortex mixer; centrifuge (3-30K, SIGMA).
Reagent consumables: (R, S) -nicotine, S-nicotine, R-nicotine, (R, S) -anatabine, S-anatabine, R-anatabine, (R, S) -nornicotine, S-nornicotine, R-nornicotine, (R, S) -cotinine, S-cotinine, R-cotinine, (R, S) -N-methylanabasine, Masmine-d 4 are all purchased from TRC, methanol (DUKSAN, chromatographic grade), ammonium formate (Acros, purity > 99%), and the ultrapure water used is obtained from Milli-Q system (Milford, MA, USA).
(2) Working conditions of the apparatus
Chromatographic conditions are as follows: the chromatographic column is a Chiralpak IG-3 chromatographic column (250X 4.6mm X3 μm) and the column temperature is 20 ℃; the mobile phase is ammonium formate/methanol solution with the mass fraction of 0.2%, and the gradient elution procedure is as follows: the initial flow rate is 0.4mL/min, the flow rate is kept for 12min, the flow rate is increased to 0.8mL/min when the flow rate is 13min, the flow rate is kept for 17min, and the total operation time is 30 min; the sample injection volume is 5 mu L;
mass spectrum conditions: an ion source: an electrospray ion source; an ionization mode: ESI (+); detection mode: an MRM mode; ion source Temperature (TEM): 550 ℃; electrospray voltage (IS): 5500V; collision gas (CAD): 4.8X 104Pa; air curtain gas (CUR): 2.8X 105Pa; ion source gas flow (GS 1): 38X 105Pa; ion source gas flow (GS 2): 41X 105Pa; inlet voltage (EP): 10V; emission voltage (CXP): 10V; the mass spectrometric parameters of the target compound and the internal standard MRM are as follows:
note: "+" indicates the quantitative ion.
(3) Sample pretreatment
Weighing 100mg of sample into a 15mL plastic centrifuge tube, adding 1mL of internal standard solution and 9mL of ammonium acetate aqueous solution with the concentration of 0.1mol/L, oscillating and mixing uniformly, and performing ultrasonic extraction for 30 min. Filtering the extract with 0.22 μm filter membrane, placing 200 μ L in 2mL centrifuge tube, adding 1400 μ L methanol, vortex oscillating for 3min, centrifuging at 11000rpm for 5min, and transferring the supernatant to chromatographic analysis bottle for detection.
(4) Standard working solution preparation
Preparing an internal standard solution: 50mg of the myosmine-d 4 is accurately weighed in a 50mL brown volumetric flask, diluted by methanol to a constant volume to a scale, and the internal standard stock solution is obtained. And diluting the internal standard stock solution by 125 times with methanol to obtain an internal standard solution.
Preparation of first-grade standard stock solution
Preparing a first-level nicotine standard stock solution: 100.0mg of (R, S) -nicotine was weighed out accurately and placed in a 10mL brown volumetric flask and diluted to volume with methanol. The solution should be stored at 4-8 deg.C in the dark.
Preparing a primary secondary alkaloid standard stock solution: respectively and accurately weighing 10.0mg of (R, S) -anatabine, (R, S) -nornicotine, (R, S) -cotinine and (R, S) -N-methyl anabasine, placing in a 10mL brown volumetric flask, diluting with methanol and fixing the volume to the scale. The solution should be stored at 4-8 deg.C in the dark.
Preparation of second-level standard stock solution
Preparing a secondary nicotine standard stock solution: accurately transferring 1.0mL of first-level nicotine standard stock solution, placing the first-level nicotine standard stock solution in a 10mL brown volumetric flask, and diluting with methanol to a constant volume to be calibrated. The solution should be stored at 4-8 deg.C in the dark.
Preparing a secondary alkaloid standard stock solution: accurately transferring 0.1mL of primary secondary alkaloid standard stock solution, placing the stock solution in a 10mL brown volumetric flask, diluting with methanol and fixing the volume to the scale. The solution should be stored at 4-8 deg.C in the dark.
Preparation of standard working solution
Preparation of nicotine standard working solution: accurately transferring 2 mu L, 5 mu L, 10 mu L, 20 mu L, 50 mu L, 100 mu L and 200 mu L of secondary nicotine standard stock solutions into different 10mL brown volumetric flasks respectively, then accurately adding 125 mu L of internal standard solution respectively, diluting with methanol to constant volume to scale, and obtaining 7 series of standard solutions with different concentrations.
Preparation of standard working solution of secondary alkaloid: accurately transferring 2 mu L, 5 mu L, 10 mu L, 20 mu L, 50 mu L and 100 mu L of secondary 10 kinds of secondary alkaloid standard stock solutions and 2 mu L, 5 mu L, 10 mu L, 20 mu L, 50 mu L and 100 mu L of primary 10 kinds of secondary alkaloid standard stock solutions into different 10mL brown volumetric flasks, accurately adding 125 mu L of internal standard solutions respectively, diluting with methanol to a constant volume to scale, and obtaining 12 series of standard solutions with different concentrations.
(5) Sample assay
And (3) respectively carrying out HPLC-MS/MS analysis on the standard working solution obtained in the step (4) and the sample solution obtained in the step (3), wherein the target substance and the internal standard Multiple Reaction Monitoring (MRM) chromatogram thereof in the standard working solution are shown in figure 5. Taking the quantitative ion peak area ratio of the target substance and the internal standard substance in each standard working solution as a vertical coordinate, and taking the content of the target substance in each standard working solution as a horizontal coordinate to prepare a standard working curve; and (4) substituting the analysis result of the step (3) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
(6) Method verification
According to the lowest-level standard working solution, the detection limit of the method is calculated according to 3 times of signal-to-noise ratio, the standard addition recovery rate is calculated according to 3 addition levels of low, medium and high, 5 parallel samples are added at each level, and the test precision is calculated according to the parallel test result. The linear range, linear coefficient, detection limit, average recovery rate by adding standard, and average precision of the method are shown in table 1.
TABLE 1 Linear Range, Linear coefficient, detection Limit, average spiked recovery and average precision for each target①
Note:①the response of each target enantiomer is the same, so the standard addition recovery rate and the like are calculated by S configuration;②nicotine concentration units are μ g/mL, other alkaloids are ng/mL.
(7) Analysis of actual samples
According to the above measurement method, 5 kinds of tobacco and tobacco product samples were selected, and the contents of the target substances were measured as shown in Table 2 (nicotine unit: mg/g, other alkaloid unit: μ g/g):
TABLE 2 results for enantiomeric contents of target in typical samples
Example 2: selection experiment of chromatographic column
Considering that nicotine and cotinine have the greatest difficulty in achieving chiral separation simultaneously, NicoShell (100 mm. times.4.6 mm. times.2.7 μm) and TeicoShell (150 mm. times.4.6 mm. times.2.7 μm) from AZYP and Chiralpak IG-3 chiral chromatography column (250 mm. times.4.6 mm. times.3 μm) from Dacello were selected as subjects. The detection method is as shown in example 1.
The research finds that:
first, NicoShell was able to achieve baseline separation of nicotine enantiomers (separation degree R2.22, shown in fig. 1A) but less effective separation of cotinine enantiomers (separation degree R0.88, shown in fig. 1B) according to the column recommendation mobile phase (methanol-ammonium formate solution). Further optimizing the mobile phase, such as gradient elution by matching the water phase with the organic phase, adjusting the temperature of the column incubator, the flow rate of the mobile phase and the like, has no obvious improvement on the separation effect of the target object.
②, according to the column recommendation mobile phase (100% methanol), nicotine enantiomer did not remain on teicosell column, shown in figure 1C, cotinine enantiomer separation effect was not ideal (separation degree R is 0.56, shown in figure 1D), by optimizing the chromatographic conditions did not significantly improve the separation effect.
③ Chiralpak IG-3 chiral chromatographic column, nicotine enantiomer can be completely separated basically (separation degree R ═ 1.40, shown in fig. 1E), cotinine enantiomer can be completely separated also (separation degree R >10, shown in fig. 1F).
Therefore, the finally determined chiral analytical column is a Chiralpak IG-3 chiral chromatographic column.
Example 3: selection experiment of mobile phase
In order to examine the influence of the flow on the detection method, several mobile phases were selected for the study, water, acetonitrile, formic acid/methanol with a volume fraction of 0.1%, ammonium acetate/methanol with a mass fraction of 0.2%, and ammonium formate/methanol with a mass fraction of 0.2%. The rest of the process, except for the mobile phase, is referred to example 1.
As a result, it was found that:
firstly, the chiral separation effect of the target substance is affected by using water or acetonitrile as the mobile phase, and the chiral separation of nornicotine, anatabine and N-methylanabasine cannot be realized (taking acetonitrile as an example, shown in FIG. 2A).
② adding formic acid (containing 0.1 percent of formic acid-methanol solution by volume fraction shown in figure 2B) or ammonium acetate (containing 0.2 percent of ammonium acetate-methanol solution by mass fraction shown in figure 2C) into the mobile phase methanol, the peak shape of the target is poor and the chiral separation of nornicotine, anatabine and N-methylanabasine can not be realized.
③ 0.2 percent of ammonium formate-methanol solution, and obtains more ideal effect.
The mobile phase composition finally determined was therefore a 0.2% mass fraction ammonium formate/methanol solution.
Example 4: selection experiment of mobile phase flow rate
In order to examine the influence of the flow rate of the mobile phase on the detection method, the flow rate of the mobile phase is set to be 0.7 mL/min; 0.4 mL/min; and an initial flow rate of 0.4mL/min for 12min, followed by a 13min flow rate ramp to 0.8mL/min for 17 min. The rest of the process is referred to example 1, except for the flow rate of the mobile phase.
As a result, it was found that:
under the high flow rate experiment condition of 0.7mL/min, chiral base line separation of nicotine can not be realized (shown in figure 3A).
② under the low flow rate experiment condition of 0.4mL/min, the chiral base line separation of nicotine can be realized, but the R-cotinine peak time is late (RT is 39.23min) (shown in figure 3B), the whole analysis time is longer.
And thirdly, the initial flow rate is 0.4mL/min, the flow rate is kept for 12min, the flow rate is increased to 0.8mL/min when the flow rate is 13min, the gradient flow rate mode of 17min is kept, and the separation degree and the analysis time are considered.
Therefore, the scheme (c) is finally determined.
Example 5: column temperature selection experiment
The inventors also examined the effect of different column temperatures on the detection method. The column temperatures were respectively: at 40 deg.C and 20 deg.C. The rest of the process is referred to example 1.
As a result, it was found that:
when the column temperature is set to 40 ℃, the tail of nicotine reduction peak type is more serious (shown in figure 4), and the tail phenomenon is improved by reducing the column temperature.
The column temperature was finally determined to be 20 ℃.
Claims (6)
1. A method of chiral analysis of an alkaloid in tobacco or a tobacco product, the method comprising the steps of:
(1) weighing 50-200 mg of sample into a 15mL centrifuge tube, adding 1mL of internal standard solution and 9mL of ammonium acetate aqueous solution with the concentration of 0.1mol/L, oscillating and mixing uniformly, performing ultrasonic extraction for 30-60 min, filtering the extract liquid through a 0.22 mu m filter membrane, taking 100-1000 mu L of the extract liquid into the centrifuge tube, adding 5-10 times of methanol in volume of the extract liquid, performing vortex oscillation for 2-5 min, centrifuging at 10000-15000 rpm for 5-10 min, and taking supernatant for later use;
(2) taking the supernatant obtained in the step (1) to perform high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, wherein the analysis conditions are as follows:
chromatographic conditions are as follows: the chromatographic column is a Chiralpak IG-3 chromatographic column with the length multiplied by the inner diameter multiplied by the particle size of 250 multiplied by 4.6mm multiplied by 3 mu m, and the temperature of the chromatographic column is 20 ℃; the mobile phase is ammonium formate/methanol solution with the mass fraction of 0.2%, and the gradient elution procedure is as follows: the initial flow rate is 0.4mL/min, the flow rate is kept for 12min, the flow rate is increased to 0.8mL/min when the flow rate is 13min, the flow rate is kept for 17min, and the total operation time is 30 min; the sample injection volume is 5 mu L;
mass spectrum conditions: an ion source: an electrospray ion source; an ionization mode: ESI +; detection mode: an MRM mode; ion source Temperature (TEM): 550 ℃; electrospray voltage (IS): 5500V; collision gas (CAD): 4.8X 104Pa; air curtain gas (CUR): 2.8X 105Pa; ion source gas flow GS 1: 3.8X 105Pa; ion source gas flow GS 2: 4.1X 105Pa; inlet voltage (EP): 10V; emission voltage (CXP): 10V;
the MRM mass spectrometric detection parameters of the target compound and internal standard are as follows:
(3) quantitatively calculating the content of the target object in the sample by adopting an internal standard curve method; wherein, the internal standard curve method is as follows: preparing a series of standard working solutions containing a target object, adding an internal standard Maistine-d 4, and making a standard working curve by taking the peak area ratio of the target object to the internal standard object in each standard working solution as a vertical coordinate and the content of the target object in each standard working solution as a horizontal coordinate; and (3) substituting the analysis result of the step (2) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
2. The method according to claim 1, wherein in step (1), the internal standard solution is a methanol solution of myosmine-d 4 with a concentration of 8 μ g/mL.
3. The method according to claim 1, wherein in step (1), the time of the ultrasonic extraction is 30 min.
4. The method of any one of claims 1 to 3, wherein the concentration ranges of each target in the series of standard working solutions are as follows: 0.20-20 mu g/mL of nicotine, 2-500 ng/mL of cotinine, 10-1000 ng/mL of nornicotine, 2-5000 ng/mL of anatabine and 2-500 ng/mL of N-methyladamicine.
5. A method according to any one of claims 1 to 3, wherein the tobacco or tobacco product comprises a tobacco sample, a smokeless tobacco product, a cigar, a heat-not-burn cigarette, a tobacco sheet, a cut filler.
6. The method of claim 5, wherein the tobacco sample is selected from the group consisting of flue-cured tobacco, burley tobacco, oriental tobacco, and maryland tobacco.
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| CN117388406A (en) * | 2023-11-03 | 2024-01-12 | 东莞市鸿馥生物科技有限公司 | Determination method for chiral resolution of 1-methyl-2 alpha- (6-methyl-3-pyridyl) pyrrolidine |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100100154A (en) * | 2009-03-05 | 2010-09-15 | 한국과학기술연구원 | Simultaneous quantitaive analysis method for tobacco elements and metabolites thereof in human urine |
| CN106908540A (en) * | 2017-03-10 | 2017-06-30 | 国家烟草质量监督检验中心 | One grow tobacco and tobacco product in nicotine chiral analysis high performance liquid chromatography |
| CN108535389A (en) * | 2018-07-13 | 2018-09-14 | 国家烟草质量监督检验中心 | One grows tobacco the separation of middle NNN optical isomers and the conjunction phase chromatographic tandem mass spectrometry method that measures |
| CN111257490A (en) * | 2020-02-17 | 2020-06-09 | 沈阳农业大学 | Method for simultaneously detecting contents of 13 substances in tobacco leaves |
-
2020
- 2020-09-03 CN CN202010914235.7A patent/CN111983109B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100100154A (en) * | 2009-03-05 | 2010-09-15 | 한국과학기술연구원 | Simultaneous quantitaive analysis method for tobacco elements and metabolites thereof in human urine |
| CN106908540A (en) * | 2017-03-10 | 2017-06-30 | 国家烟草质量监督检验中心 | One grow tobacco and tobacco product in nicotine chiral analysis high performance liquid chromatography |
| CN108535389A (en) * | 2018-07-13 | 2018-09-14 | 国家烟草质量监督检验中心 | One grows tobacco the separation of middle NNN optical isomers and the conjunction phase chromatographic tandem mass spectrometry method that measures |
| CN111257490A (en) * | 2020-02-17 | 2020-06-09 | 沈阳农业大学 | Method for simultaneously detecting contents of 13 substances in tobacco leaves |
Non-Patent Citations (3)
| Title |
|---|
| Xinyu Liu 等.Analysis of nicotine and nicotine-related compounds in electronic cigarette liquids and aerosols by liquid chromatography-tandem mass spectrometry.《Beiträge zur Tabakforschung International/Contributions to Tobacco Research》.2017,第27卷(第7期), * |
| 手性色谱柱-HPLC-DAD法分离分析烟草与烟草制品中烟碱旋光异构体;余晶晶 等;《轻工学报》;20180930;第33卷(第05期);44-52 * |
| 烟碱手性异构体的分离分析;杨强 等;《云南化工》;20200131;第47卷(第01期);65-67,70 * |
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