A liquid chromatography-tandem mass spectrometry analysis method for distinguishing natural nicotine from synthetic nicotine
Through liquid chromatography-tandem mass spectrometry analysis combined with stoichiometry, using isotope mode and OPLS-DA model, the problem of difficulty in identifying natural and synthetic nicotine in the existing technology is solved, and the accurate identification and supervision of nicotine sources is achieved, and the simplification of simplification Operational procedures and reduce costs.
Patent Information
- Application Number
- CN202211541700.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-03
- Publication Date
- 2024-12-20
- Estimated Expiration
- 2042-12-03
AI Technical Summary
The prior art is difficult to accurately identify high-purity natural (S)-(-)-nicotine and synthetic (S)-(-)-nicotine. The commonly used methods are complex and costly, so it is impossible to effectively identify natural and synthetic nicotine. Source.
The liquid chromatography-tandem mass spectrometry analysis method was used to distinguish the source of nicotine through isotope mode, and the identification model was constructed in combination with stoichiometric analysis. Nicotine was extracted using ultrapure water, and [M+H]+ and [M+H+]+ 1]+ peak abundance and area ratio, as well as isotope abundance ratio of precursor ions, to verify the source of nicotine, screen the main variables that affect the difference, and construct an OPLS-DA evaluation model.
The accurate identification of natural and synthetic nicotine is achieved, with simple and fast operation, and the model prediction accuracy reaches 97.87%. It does not require complex sample pretreatment processes, and can accurately identify synthetic tobacco products on the market.
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Figure CN115993411B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to a liquid chromatography-tandem mass spectrometry analysis method for identifying natural nicotine and synthetic nicotine. Background Art
[0002] Nicotine is an important chemical component of tobacco products, and its content is closely related to the quality and taste of tobacco. Natural nicotine (>99% (S)-(-)-nicotine) mainly comes from tobacco plants, such as flue-cured tobacco and cigarettes. With the development of technology, new tobacco products containing synthetic nicotine have become a substitute for traditional tobacco products. Synthetic nicotine is different from natural nicotine and contains equal amounts of two stereoisomers (R, S-(±)-nicotine), which are mainly prepared from chemical raw materials. Since natural nicotine does not come from tobacco plants, this poses a great challenge to the supervision and management of tobacco products by the Food and Drug Administration. Therefore, there is an urgent need to develop a reliable method to identify the sources of natural and synthetic nicotine.
[0003] At present, liquid chromatography-mass spectrometry based on chiral columns is a commonly used method for identifying nicotine. However, this technology cannot distinguish high-purity natural (S)-(-)-nicotine from synthetic (S)-(-)-nicotine. In recent years, stable isotope ratio technology has played an important role in the identification of synthetic and naturally extracted taurine, caffeine, and ephedrine. In addition, Hu et al. studied the differences between natural and synthetic nicotine based on the site-specific peak intensity ratio analysis technology of hydrogen nuclear magnetic spectroscopy (Liu B, Anal Bioanal Chem, 2019, 411 (24): 6427-6434). However, these two methods require the operator to have high professional knowledge, and the experimental process is complicated and costly.
[0004] Chemometric statistical analysis is very important for the interpretation of data and the construction of classification models for samples of unknown origin. Typical chemometric data analysis includes unsupervised principal component analysis and hierarchical cluster analysis; as well as supervised linear discriminant analysis, partial least squares discriminant analysis, orthogonal partial least squares discriminant analysis, etc. Compared with PLS-DA, OPLS-DA can filter out noise that is irrelevant to the classification signal and improve the analytical ability and effectiveness of the model. This study developed a tandem mass spectrometry technology based on isotope pattern peaks and combined it with chemometric analysis to identify the sources of natural and synthetic nicotine, so as to achieve accurate identification, labeling and supervision of tobacco products containing synthetic nicotine on the market. Summary of the invention
[0005] The purpose of the present invention is to establish a liquid chromatography-tandem mass spectrometry analysis method for distinguishing natural nicotine from synthetic nicotine based on the above-mentioned deficiencies in the prior art. It is the first time that the isotope pattern of tandem mass spectrometry is used to distinguish and identify the source of nicotine. It has the characteristics of simple and rapid operation and no need for quantification.
[0006] The objective of the present invention is achieved through the following technical solutions:
[0007] A liquid chromatography-tandem mass spectrometry analysis method for identifying natural nicotine and synthetic nicotine, firstly, natural nicotine in tobacco is extracted with ultrapure water, and synthetic nicotine is directly diluted with ultrapure water; then, the sample is loaded, and nicotine is separated from other impurities in the extract by liquid chromatography, and liquid chromatography-tandem mass spectrometry is used for detection, wherein: by comparing the [M+H] + and [M+H+1] + Peak abundance and area ratio, and [M+H] + and [M+H+1] + The isotope abundance ratio of the fragment peak of the parent ion is used to verify the source or naturalness of nicotine; by combining the chemometric method, an evaluation model for the identification of natural and synthetic nicotine is further constructed, and the main variables affecting the difference between the two are screened.
[0008] The present invention specifically comprises the following steps:
[0009] (1) Nicotine extraction: natural nicotine in tobacco is extracted using ultrapure aqueous solution, and synthetic nicotine is diluted; the specific method is as follows:
[0010] Natural nicotine: Accurately weigh 0.01g (accurate to 0.0001g) of natural tobacco products, add 50mL of ultrapure water, extract by ultrasonic at 30℃ for 20min, let stand overnight, take an appropriate amount of the extract and filter it through a 0.22μm water filter membrane;
[0011] Synthetic nicotine: Pure nicotine was diluted with water to 20 μg / mL.
[0012] (2) using ultra-high performance liquid chromatography to separate nicotine from other impurities in the extract;
[0013] Liquid chromatography conditions were as follows: mobile phase A: 10 mM ammonium formate, pH 3.5 aqueous solution; mobile phase B: acetonitrile. Flow rate: 0.4 mL / min, elution mode: 70% B isocratic elution for 7 min. Chromatographic column: ACQUITY UPLC BEH HILIC (2.1×150 mm, 1.7 μm, Waters, USA), column temperature was 40°C. Injection volume was 2 μL.
[0014] (3) Comparison of [M+H] of nicotine by mass spectrometry + and [M+H+1] + Peak abundance and area ratio, and [M+H] + and [M+H+1] +The isotope abundance ratio of the peak as the fragment peak of the parent ion is used to verify the source or naturalness of nicotine;
[0015] The mass spectrometry conditions were as follows: the ion source was an electrospray ionization (ESI) ion source, data were collected in positive ion mode, and the collision gas was argon; the capillary voltage, the nebulizer gas pressure, and the drying gas temperature were -4.5 kV, 1.8 bar, and 220°C, respectively; the mass spectrometry internal standard was sodium formate solution, and the m / z range was 50-1300 Th; subsequently, nicotine m / z 163 and 164 were subjected to MS / MS fragmentation analysis, and the collision energy (CID) in the secondary fragmentation was 10 eV; the fragment ions of m / z 163 were 84 and 130, and the fragment ions of m / z 164 were 85 and 131.
[0016] (4) An OPLS-DA evaluation model for the identification of natural and synthetic nicotine was constructed by combining chemometric methods, and the main variables affecting the differences between the two were screened;
[0017] The OPLS-DA sample classification model was used for quantitative statistical analysis. From the VIP values, it can be seen that variables 164-85 / 84 and 164-85 / (84+85) can represent natural nicotine samples, while variables 163-131 / 131 and 163-131 / (130+131) represent synthetic nicotine samples. The overall prediction accuracy of the model was 97.87%; the prediction accuracy of natural nicotine was 95.45%, and the prediction accuracy of synthetic nicotine samples was 100%.
[0018] The method of the present invention does not require complicated sample pre-treatment processes such as complex nicotine extraction and purification. By developing a tandem mass spectrometry technology based on isotope pattern peaks and combining chemometric analysis methods to identify the sources of natural and synthetic nicotine, accurate identification, labeling and supervision of tobacco products containing synthetic nicotine on the market can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Figure 2 shows the MS / MS spectra of protonated nicotine parent ions at m / z 163 and m / z 164.
[0020] Figure 2 (A) Score plot and (B) Load plot of the OPLS-DA model for natural and synthetic nicotine identification. The dots in the figure represent natural nicotine samples, and the squares represent synthetic nicotine samples.
[0021] Figure 3 VIP values for 12 variables for identification of natural and synthetic nicotine.
[0022] Figure 4 .Natural and synthetic nature of 5 types of nicotine. DETAILED DESCRIPTION
[0023] The present invention is further described by the following specific implementation:
[0024] 1. Instruments and Reagents
[0025] All mass spectrometry data in this experiment were completed on an impact II quadrupole time-of-flight mass spectrometer (Bruker, Germany), which was equipped with an Ultimate 3000 UHPLC liquid chromatograph (Thermo Fisher, USA).
[0026] Ammonium formate (purity ≥ 99.99%) and chromatography-grade formic acid (purity ≥ 98.0%) were purchased from Sigma-Aldrich, USA; chromatography-grade acetonitrile was purchased from Merck, Germany. Experimental water was prepared by Milli-Q ultrapure water preparation system (Millipore, USA).
[0027] Natural nicotine was extracted from different types of tobacco products: 4 traditional cigarette samples, 4 heated cigarette samples and 2 electronic cigarette samples. Seven types of synthetic nicotine, including R-nicotine, R,S-(±)-nicotine and 5 different synthetic route nicotine samples purchased from TRC, were used as synthetic nicotine. Model construction was performed.
[0028] 2. Sample Pretreatment
[0029] Accurately weigh 0.01 g of cigarette tobacco or 5 ul of e-cigarette liquid and add it to 50 mL of water. Perform ultrasonic extraction at 30°C for 20 min, let it stand overnight, and filter the extract through a 0.22 μm water filter membrane.
[0030] Synthetic nicotine was diluted to 20 μg / mL using ultrapure water.
[0031] 3. Instrumental analysis conditions
[0032] Chromatographic conditions: aqueous solution containing 10 mM ammonium formate, pH 3.5 as mobile phase A; pure acetonitrile as mobile phase B. Elution method: flow rate 0.4 mL / min, isocratic elution at 70% B for 7 min. Chromatographic column: ACQUITY UPLC BEHHILIC (2.1×150 mm, 1.7 μm, Waters, USA), column temperature: 40°C. Injection volume 2 μL.
[0033] Mass spectrometry conditions: The ion source is an electrospray ionization (ESI) ion source, data acquisition is performed in positive ion mode, and the collision gas is argon. The capillary voltage, nebulizer gas pressure, and drying gas temperature are -4.5 kV, 1.8 bar, and 220 ° C, respectively. The mass spectrometer internal standard is sodium formate solution, and the m / z range is 50-1300 Th. Subsequently, MS / MS fragmentation analysis was performed on nicotine m / z 163 and 164, respectively, and the collision energy (CID) in the secondary fragmentation was 10 eV.
[0034] 4. Data Processing
[0035] All raw mass spectrometry data were analyzed using Data Analysis 4.0 software (Bruker, Germany). The abundance ratio and peak area ratio of m / z 163 and 164 in the MS mass spectrum were extracted. Subsequently, the abundance ratios of m / z 84, 85, 130, and 131 in the MS / MS mass spectrum with parent ions of 163 and 164 were extracted respectively. The collected data were imported into SPSS 23.0 (IBM, USA) and SIMCA 14.0 (Umetrics, Sweden) for univariate and multivariate analysis.
[0036] 5. Experimental Results
[0037] First, OPLS-DA model was constructed using 10 natural nicotine and 7 synthetic nicotine. The model score and loading diagrams are shown in Figure 2 As shown in Figure 2, the VIP values of the 12 variables for the identification of natural and synthetic nicotine are as follows: Figure 3 The abundance ratio and peak area ratio are shown in Table 1. The accuracy of natural nicotine and synthetic nicotine obtained by the model is 100%. In order to further verify the predictability of the discriminant model, 200 model permutation cross-tests were performed. The R of the permutation test 2 =0.203, Q 2 =-0.719, indicating that the model has no overfitting phenomenon (R 2 <0.5, Q 2 <0), which was statistically significant.
[0038] Table 1. Isotope abundance ratios of fragment peaks of natural nicotine and synthetic nicotine samples under the conditions of MS mass spectra, MS / MS mass spectra of m / z 163, and MS / MS mass spectra of m / z 164, respectively.
[0039]
[0040]
[0041] Implementation case: Identification of the naturalness of 5 types of nicotine
[0042] Sources of nicotine used in this experiment: 3 natural samples: TN1, TN2, TN3 and 2 synthetic samples: TS1, TS2 were tested. Natural nicotine was extracted from different types of tobacco products: 1 traditional cigarette sample, 1 heated cigarette sample and 1 electronic cigarette sample. R-nicotine and R, S-(±)-nicotine purchased from TRC were used as synthetic nicotine for testing. The sample pretreatment method and instrument analysis conditions are all referred to the specific implementation plan. According to the above model, the 8 parameters with VIP values greater than 1 (FD164 / 163, FD164 / (163+164), MJ164 / 163, MJ164 / (163+164), 163-131 / 130, 163-131 / (130+131), 164-85 / 84, 164-85 / (84+85)) were tested and analyzed, and the analysis results are shown in Table 2. The above established model was used to analyze the five nicotine samples. Figure 4 As shown, the three samples in the upper left corner are natural nicotine, and the two samples in the lower right corner are synthetic nicotine. This proves that this method can accurately identify natural and synthetic nicotine.
[0043] Table 2. Isotope abundance ratios / area ratios of fragmentation peaks under eight parameters of five samples under MS mass spectra, MS / MS mass spectra of m / z 163, and MS / MS mass spectra of m / z 164.
[0044]
Claims
1. A liquid chromatography-tandem mass spectrometry analysis method for identifying natural nicotine and synthetic nicotine, firstly using ultrapure water to extract natural nicotine from tobacco, and directly diluting synthetic nicotine with ultrapure water; then loading the sample, using liquid chromatography to separate nicotine from other impurities in the extract, and using liquid chromatography-tandem mass spectrometry for detection, characterized in that: By comparing the [M+H] + and [M+H+1] + Peak abundance and area ratio, and [M+H] + and [M+H+1] + The isotope abundance ratio of the fragment peak of the parent ion is used to verify the source or naturalness of nicotine; an evaluation model for the identification of natural and synthetic nicotine is further constructed by combining chemometric methods, and the main variables affecting the difference between the two are screened; Extract MS mass spectra m / z The abundance ratio and peak area ratio of 163 and 164 are respectively extracted from the MS / MS mass spectra of the parent ions 163 and 164. m / z is the abundance ratio of 84, 85, 130 and 131; variables 164-85 / 84 and 164-85 / (84+85) may represent natural nicotine samples, while variables 163-131 / 131 and 163-131 / (130+131) represent synthetic nicotine samples.
2. The liquid chromatography-tandem mass spectrometry analysis method for distinguishing natural nicotine from synthetic nicotine according to claim 1, characterized in that: The specific steps are: (1) Nicotine extraction: Use ultrapure aqueous solution to extract natural nicotine from tobacco and dilute synthetic nicotine; (2) Using ultra-high performance liquid chromatography to separate nicotine from other impurities in the extract; (3) Comparison of [M+H] of nicotine by mass spectrometry + and [M+H+1] + Peak abundance and area ratio, and [M+H] + and [M+H+1] + The isotope abundance ratio of the peak as the fragment peak of the parent ion is used to verify the source or naturalness of nicotine; (4) An OPLS-DA evaluation model for the identification of natural and synthetic nicotine was constructed by combining chemometric methods, and the main variables affecting the differences between the two were screened.
3. The liquid chromatography-tandem mass spectrometry analysis method for distinguishing natural nicotine from synthetic nicotine according to claim 2, characterized in that: The nicotine extraction treatment method in step (1) is specifically as follows: Natural nicotine: Accurately weigh 0.01 g of natural tobacco products, add 50 mL of ultrapure water, extract by ultrasonic at 30°C for 20 min, let stand overnight, take an appropriate amount of the extract and filter through a 0.22 μm water filter; Synthetic nicotine: Pure nicotine was diluted with water to 20 μg / mL.
4. The liquid chromatography-tandem mass spectrometry analysis method for distinguishing natural nicotine from synthetic nicotine according to claim 2, characterized in that: The liquid chromatography conditions in step (2) are: Mobile phase A was an aqueous solution containing 10 mM ammonium formate, pH 3.5; pure acetonitrile was used as mobile phase B; the flow rate was 0.4 mL / min, and the isocratic elution was performed at 70% B for 7 min; the chromatographic column was ACQUITY UPLC BEH HILIC, size 2.1 × 150 mm, 1.7 μm, and the column temperature was 40 ˚C; the injection volume was 2 μL.
5. The liquid chromatography-tandem mass spectrometry analysis method for distinguishing natural nicotine from synthetic nicotine according to claim 2, characterized in that: The mass spectrometry conditions in step (3) are: The ion source was an electrospray ion source, data were collected in positive ion mode, and the collision gas was argon; the capillary voltage, nebulizer gas pressure, and drying gas temperature were -4.5 kV, 1.8 bar, and 220°C, respectively; the mass spectrometer internal standard was sodium formate solution. m / z The range was 50-1300 Th; then, nicotine m / z MS / MS fragmentation analysis was performed for 163 and 164, and the collision energy in the secondary fragmentation was 10 eV; m / z The fragment ions of 163 are 84 and 130. m / z The fragment ions of 164 are 85 and 131.
6. The liquid chromatography-tandem mass spectrometry analysis method for distinguishing natural nicotine from synthetic nicotine according to claim 2, characterized in that: The model in step (4) is as follows: The OPLS-DA sample classification model is used for quantitative statistical analysis. From the VIP value, it can be seen that variables 164-85 / 84 and 164-85 / (84+85) can represent natural nicotine samples, while variables 163-131 / 131 and 163-131 / (130+131) represent synthetic nicotine samples.