Preparation method of light / deuterated N, N-alkyl halogenated aniline acyl chloride compound and stable isotope labeling analysis method of tobacco secondary alkaloid

Abstract

The invention relates to a preparation method of a light / deuterated N, N-alkyl halogenated aniline acyl chloride compound and a stable isotope labeling analysis method of tobacco secondary alkaloids, and belongs to the field of detection of tobacco secondary alkaloids. The preparation method comprises the following steps: carrying out reaction on aniline, light / heavy monoiodoalkane, 1, 3-dihalo-5, 5-dimethyl hydantoin and trichloromethyl carbonate, so as to prepare the light / deuterated N, N-alkyl halogenated aniline acyl chloride compound. The reaction is simple, convenient to operate and suitable for large-scale production and utilization, the prepared light / deuterated N, N-alkyl halogenated aniline acyl chloride compound can be used as a derivatization reagent, reactive hydrogen such as amino, hydroxyl and carboxyl in a target object can be converted into inert groups, meanwhile, stable isotope tag groups and halogen atoms are introduced, and therefore the light / deuterated N, N-alkyl halogenated aniline acyl chloride compound is obtained. Double labeling is achieved, the mass spectrum signal recognition degree is remarkably enhanced, and qualitative and quantitative efficiency and accuracy are improved.

Description

Technical Field

[0001] The present invention relates to a method for preparing a light / deuterated N,N-alkylhaloaniline acyl chloride compound and a method for stable isotope labeling analysis of tobacco secondary alkaloids, belonging to the field of detection of tobacco secondary alkaloids. Background Art

[0002] Tobacco secondary alkaloid compounds refer to alkaloid substances in tobacco other than nicotine. Many are metabolites produced during baking, production, fermentation, storage, etc. of nicotine, such as cotinine, hydroxycotinine, nicotine glycosides, etc. The accurate analysis of these tobacco secondary alkaloid compounds is of great significance for evaluating the safety of tobacco products, studying the dynamic process changes of secondary alkaloid substances, and formulating relevant regulatory standards.

[0003] Currently reported analysis methods for tobacco secondary alkaloids mainly include: gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, high performance liquid chromatography, ion chromatography, capillary electrophoresis, and supercritical fluid chromatography, etc. Among them, liquid chromatography-mass spectrometry is one of the commonly used methods for analyzing secondary alkaloids in tobacco products. At present, due to the low recognition of response signals of secondary alkaloid substances in liquid mass spectrometry, the weak characteristic of mass spectrometry fragmentation product ions, and the difficulty in qualitative analysis, the types of identified substances are few, and it is impossible to comprehensively and accurately understand the composition and content of tobacco secondary alkaloid compounds. In addition, due to the matrix effect in liquid chromatography-mass spectrometry, stable isotope internal standards need to be used for quantitative detection, and the existing stable isotope internal standards are expensive, resulting in too high test costs and making large-scale detection and research difficult to carry out.

[0004] In summary, there is an urgent practical need and important application value to develop an analysis method for tobacco secondary alkaloids with low cost, accurate qualitative analysis, and capable of realizing high-throughput determination. Summary of the Invention

[0005] The first object of the present invention is to provide a method for preparing a light / deuterated N,N-alkylhaloaniline acyl chloride compound to solve the problem in the prior art of lacking a labeling reagent capable of accurately determining the composition and content of secondary alkaloids in tobacco.

[0006] The second object of the present invention is to provide a method for stable isotope labeling analysis of tobacco secondary alkaloids to solve the problems in the prior art that it is difficult to qualitatively analyze tobacco secondary alkaloids by the internal standard method, the types of identified secondary alkaloids are few, and the accuracy of quantitative analysis results is not high.

[0007] To achieve the above objects, the technical solution of the present invention is as follows:

[0008] A method for preparing a light / deuterated N,N-alkyl halogenated aniline acyl chloride compound, comprising the following steps: reacting aniline, light / heavy iodoalkane, 1,3-dihalo-5,5-dimethylhydantoin, and trichloromethyl carbonate to obtain the light / deuterated N,N-alkyl halogenated aniline acyl chloride compound; the general structural formula is shown in Formula I:

[0009]

[0010] In Formula I, X = Cl or Br, Y = H or D, and RY3 = methyl, ethyl, deuterated methyl or deuterated ethyl.

[0011] The method for preparing the light / deuterated N,N-alkyl halogenated aniline acyl chloride compound of the present invention is an exploratory invention. The method for preparing the light / deuterated N,N-alkyl halogenated aniline acyl chloride compound of the present invention has easily available and inexpensive reaction raw materials, a simple reaction and convenient operation, and is suitable for large-scale production and utilization. It not only improves the preparation efficiency of the light / deuterated N,N-alkyl halogenated aniline acyl chloride compound, but also reduces the production cost of such compounds.

[0012] The light N,N-alkyl halogenated aniline acyl chloride compound and deuterated N,N-alkyl halogenated aniline acyl chloride compound of the present invention can be used as derivatization reagents, which can convert active hydrogens such as amino, hydroxyl and carboxyl groups in the target substance into inert groups, and at the same time introduce stable isotope label groups and halogen atoms to achieve dual labeling, significantly enhancing the mass spectrometry signal recognition, and improving the efficiency and accuracy of qualitative and quantitative analysis. Moreover, the light / deuterated N,N-alkyl halogenated aniline acyl chloride compound of the present invention is used for derivatizing the target substance for qualitative and quantitative analysis, with lower cost.

[0013] To further improve the reaction efficiency and reduce the waste of raw materials, preferably, the molar ratio of aniline, light / heavy iodoalkane, 1,3-dihalo-5,5-dimethylhydantoin, and trichloromethyl carbonate is (1.5 - 1.8):(4.1 - 4.4):1:(0.4 - 0.7).

[0014] Preferably, aniline reacts with light / heavy iodoalkane and an acid-binding agent in a first substitution reaction to obtain an N,N-alkyl aniline substance. The N,N-alkyl aniline substance reacts with 1,3-dihalo-5,5-dimethylhydantoin in a second substitution reaction to obtain a 2-halo-N,N-alkyl aniline substance. The 2-halo-N,N-alkyl aniline substance reacts with trichloromethyl carbonate in an acylation reaction to obtain the light / deuterated N,N-alkyl halogenated aniline acyl chloride compound.

[0015] Preferably, the temperature of the first substitution reaction is 60-100 °C and the time is 12-24 h. At this temperature, aniline reacts with light / heavy monoiodoalkane faster and more completely, and the reaction time can ensure complete reaction. Also, the end of the reaction can be determined by observing the amount of reaction raw materials. More preferably, it is 60 °C.

[0016] Preferably, the second substitution reaction is carried out at room temperature and the time is 10-24 h. The reaction conditions are mild and easy to control. More preferably, it is 14-24 h.

[0017] To further improve the reaction rate, preferably, the temperature of the acyl chlorination reaction is 50-100 °C and the time is 2-5 h. More preferably, it is 65 °C.

[0018] Preferably, the acid-binding agent for the first substitution reaction is potassium carbonate.

[0019] To further ensure that the reaction proceeds in the forward reaction direction, preferably, the catalyst for the second substitution reaction is one or a combination of two or more of diisopropyl ammonium halide salts, pyrrolidine ammonium halide salts, and piperidine ammonium halide salts. More preferably, it is diisopropyl ammonium halide salt.

[0020] The second technical solution of the present invention is as follows:

[0021] A method for analyzing stable isotope labeling of tobacco secondary alkaloids, comprising the following steps: reacting a light N,N-alkylhaloaniline acyl chloride compound and a deuterated N,N-alkylhaloaniline acyl chloride compound with an analyte respectively, and then performing qualitative and quantitative analysis by liquid chromatography-mass spectrometry; the light / deuterated N,N-alkylhaloaniline acyl chloride compound is prepared according to the above method.

[0022] The light N,N-alkylhaloaniline acyl chloride compound and the deuterated N,N-alkylhaloaniline acyl chloride compound of the present invention, as derivatization reagents and stable isotope labeling probes for qualitative and quantitative analysis, the light / heavy two labeled products have highly similar behaviors in chromatography and mass spectrometry, have the same or similar retention times in the chromatogram, and the signal ratio of the mass spectrometry peaks in the mass spectrum is close to the concentration ratio, the mass-to-charge ratio difference is a fixed value, and the characteristic peaks are highly recognizable, which is suitable for identifying unknown substances. And the two labeled products contain halogen elements, such as Cl or Br, and have two highly recognizable natural isotope mass spectrometry peaks in the mass spectrum, such as 35 Cl, 37 Cl and 79 Br, 81 Br. Therefore, the identification result can achieve double confirmation of high recognition of characteristic peaks and natural isotope mass spectrometry peaks of halogens, and the qualitative analysis is more accurate.

[0023] In addition, during quantitative analysis, since the chemical properties of the light / heavy labeled products are highly similar, the heavy labeled products can usually effectively correct the severe matrix effects of the light labeled products in liquid chromatography, especially in mass spectrometry. Therefore, the accuracy of the quantitative results can be significantly improved.

[0024] The light / deuterated N,N-alkylhaloaniline acyl chloride compounds of the present invention as derivatization reagents for secondary alkaloids and stable isotope labeled probes for qualitative and quantitative analysis can significantly increase the number of qualitative substances and the accuracy of quantification, and achieve high-throughput determination of secondary alkaloids. At the same time, the cost of qualitative and quantitative analysis of secondary alkaloids is reduced.

[0025] Preferably, the secondary alkaloids include one or more combinations of nornicotine enol, nornicotine, anabasine, norcotinine, anabasin, 2-(3-pyridyl)-5-piperidone, 3-hydroxycotinine, isohydroxycotinine, diene nicotine glycoside, anabasine glycoside, nicotine-N-β-glucuronide, anabasin glycoside, cotinine glycoside, 3-hydroxycotinine glycoside. The analytical method of the present invention has identified 14 secondary alkaloids, laying a foundation for further research on the quality and characteristics of tobacco and tobacco products.

[0026] Preferably, the conditions of the liquid chromatography include: the stationary phase of the chromatographic column is C18, the column temperature is 45-48 °C, the mobile phase A is ammonium acetate, the mobile phase B is acetonitrile, the flow rate is 0.4-0.6 mL / min, the injection volume is 1-1.2 μL, and the temperature of the autosampler is 10-12 °C. Further preferably, the concentration of the ammonium acetate is 10 mM and the pH is 10.

[0027] Preferably, the liquid chromatography uses gradient elution, and the gradient elution program is: 0 min, 95% A, 5% B; 3 min, 45% A, 55% B; 3.1 min, 10% A, 90% B; 4 min, 10% A, 90% B; 4.5 min, 95% A, 5% B; 5.5 min, 95% A, 5% B.

[0028] Preferably, the conditions of the mass spectrometry are: electrospray ionization (ESI) source, positive (ESI+) ionization mode; the ion source temperature is 550-553 °C; the spray voltage in the ESI+ mode is 5500-5505 V; multiple reaction monitoring (MRM) mode. Description of the Drawings

[0029] Figure 1 It is the HPLC-Orbitrap-MS chromatogram of the light / heavy N,N-methylchloroaniline acyl chloride derivatization products of the sample extract in Example 3 of the present invention;

[0030] Figure 2High-resolution mass spectrometry of light / heavy derivatization products of norcotinine in Example 3 of the present invention on HPLC-Orbitrap;

[0031] Figure 3 High-resolution mass spectrometry of light / heavy derivatization products of 2-(3-pyridyl)-5-piperidone in Example 3 of the present invention on HPLC-Orbitrap;

[0032] Figure 4 High-resolution mass spectrometry of light / heavy derivatization products of isonorcotinine in Example 3 of the present invention on HPLC-Orbitrap;

[0033] Figure 5 HPLC-Orbitrap-MS chromatogram of light / heavy acetyl chloride of the sample extract in Comparative Example 2 of the present invention;

[0034] Figure 6 HPLC-Orbitrap-MS chromatogram of light / heavy acetyl chloride of norcotinine in Comparative Example 2 of the present invention. Detailed implementation manners

[0035] The reaction processes involved in the following Example 1 are as follows:

[0036] (1) Aniline reacts with deuterated iodomethane to obtain deuterated N,N-methylaniline substances, and the reaction formula is as follows:

[0037]

[0038] (2) The deuterated N,N-methylaniline substances react with 1,3-dichloro-5,5-dimethylhydantoin under the catalysis of diisopropylammonium chloride salt to obtain deuterated 2-chloro-N,N-methylaniline substances, and the reaction formula is as follows:

[0039]

[0040] (3) The deuterated 2-chloro-N,N-methylaniline substances react with triphosgene to obtain deuterated N,N-methylchloroaniline acyl chloride compounds, and the reaction formula is as follows:

[0041]

[0042] The technical solutions of the present invention will be further described below in conjunction with the specific implementation manners.

[0043] I. Specific examples of the preparation method of the light / deuterated N,N-alkylhaloaniline acyl chloride compound of the present invention are as follows:

[0044] Example 1

[0045] The light / deuterated N,N-alkyl halogenated aniline acyl chloride compound of this embodiment is a light / heavy N,N-methyl chlorinated aniline acyl chloride compound, and its structural formula is as follows:

[0046]

[0047] Among them, Y is H or D.

[0048] The preparation method of the light / deuterated N,N-alkyl halogenated aniline acyl chloride compound of this embodiment includes the following steps:

[0049] (1) Add 5 L of acetonitrile, 140 g (1.5 mol) of aniline, 700 g of deuterated iodomethane (4.1 mol), and 900 g of potassium carbonate to a reactor equipped with a stirrer, a spherical condenser, and a thermometer. Before adding these reagents, ensure that the reactor is clean and dry. The acetonitrile used needs to be anhydrous treated, and the purity of aniline, deuterated iodomethane, and potassium carbonate should also meet the experimental requirements. Open the upper water valve of the spherical condenser and start stirring continuously. Heat up to 60 °C, after reacting for 16 h, cool down to 0 °C, quench with saturated sodium chloride aqueous solution, and then perform extraction, brine washing, and concentration. After concentration, purify by column chromatography.

[0050] Among them, during ethyl acetate extraction, transfer the reaction solution to a separatory funnel, add about 200 mL of ethyl acetate for extraction each time, and repeat the extraction operation 3 - 4 times to ensure that the product is fully transferred to the organic phase. Then wash with saturated sodium chloride aqueous solution, transfer the combined organic phase to a round-bottom flask of a rotary evaporator, set the temperature at 40 °C, and maintain the vacuum at about 0.08 MPa. When it is observed that the volume of the solution significantly decreases and a small amount of viscous substance appears, the temperature can be raised to 50 °C to continue concentration until most of the solvent is evaporated.

[0051] For column chromatography purification, make a slurry of 50 g of silica gel (100 - 200 mesh) with 50 mL of n-hexane, and slowly pour it into the column while gently tapping the chromatography column on the side to make the silica gel settle evenly and form a tight stationary phase. Flush the chromatography column with n-hexane to ensure that the stationary phase is packed tightly and without bubbles. After the stationary phase is balanced, dissolve the concentrated sample with a small amount of ethyl acetate, slowly add it to the top of the column, and then use a solution of n-hexane and ethyl acetate mixed in a ratio of 5:1 as the mobile phase for elution, and collect the eluate containing the target product. Finally, concentrate the collected eluate again, remove the solvent, and perform structural identification and purity analysis of the product by mass spectrometry (MS), with the purity exceeding 90%.

[0052] (2) Take 170 g (1.2 mol) of the product from step (1) and add it to 1 L of toluene. Then add 197 g (1 mol) of 1,3-dichloro-5,5-dimethylhydantoin and 15 g of diisopropylammonium chloride salt to the toluene solution, and stir the reaction at room temperature for 14 h. Then remove the impurities and toluene solvent after the reaction. The specific operation is as follows:

[0053] Add saturated brine to the toluene phase, shake it, and let it stand for liquid separation, and then perform liquid separation again. Saturated brine can reduce the solubility of the product in water and further remove the possibly remaining water-soluble impurities. Repeat the above saturated brine washing step 2 - 3 times to ensure that the impurities are fully removed.

[0054] Add 500 g of anhydrous sodium sulfate to the toluene phase, shake it, and let it stand for 12 h to remove the remaining water in the toluene phase. Then, at a temperature of 60 °C and a vacuum degree maintained at about 0.08 MPa, rotary evaporate and concentrate to remove the toluene solvent.

[0055] (3) Add 500 mL of chlorobenzene and 120 g (0.4 mol) of trichloromethyl carbonate at room temperature, start stirring, slowly heat up to 100 °C, and dropwise add 500 mL of a chlorobenzene solution containing 175 g of the product from the above step (2) (1 mol) in about 30 min. After the dropping is completed, lower the temperature to 65 °C to improve the stability of the target product and prevent the product from further reacting. At this temperature, react for 2 - 3 h, and then cool the reaction solution. At a temperature of 60 °C and a vacuum degree maintained at about 0.08 MPa, perform vacuum fractional distillation to obtain the final product deuterated N,N-methylchloroaniline acyl chloride.

[0056] In other implementation cases, replace the deuterated deuterated iodomethane in step (1) with iodomethane to prepare N,N-methylchloroaniline acyl chloride.

[0057] Example 2

[0058] The light / deuterated N,N-alkylhaloaniline acyl chloride compound of this example is a light / heavy N,N-methylbromoaniline acyl chloride compound, and its structural formula is as follows:

[0059]

[0060] Among them, Y is H or D.

[0061] The preparation method of the light / deuterated N,N-alkylhaloaniline acyl chloride compound of this example is different from that of Example 1 in that 1,3-dibromo-5,5-dimethylhydantoin is used instead of 1,3-dichloro-5,5-dimethylhydantoin in step (2), and the addition amount is 285 g (1 mol); the specific steps are as follows:

[0062] (1) Add 5 L of acetonitrile, 140 g (1.5 mol) of aniline, 700 g of deuterated iodomethane (4.1 mol), and 900 g of potassium carbonate to a reactor equipped with a stirrer, a spherical condenser, and a thermometer. Open the water inlet valve of the spherical condenser, start continuous stirring, heat up to 60 °C, react for 16 h, then cool down to 0 °C, quench with saturated sodium chloride aqueous solution, extract with ethyl acetate, wash with brine, and concentrate and purify by column chromatography after concentration.

[0063] (2) Take 170 g (1.2 mol) of the product from step (1) and add it to 1 L of toluene. Then add 285 g (1 mol) of 1,3-dibromo-5,5-dimethylhydantoin and 15 g of diisopropylammonium chloride salt to the toluene solution, and stir at room temperature for 14 h.

[0064] (3) Add 500 mL of chlorobenzene and 120 g (0.4 mol) of trichloromethyl carbonate at room temperature, start stirring, slowly heat up to 100 °C, and add 500 mL of a chlorobenzene solution containing 175 g of the above product (1 mol) dropwise in about 30 min. After the addition, react at 65 °C for 2 - 3 h, then cool the reaction solution, and fractionate under reduced pressure to obtain the final product deuterated N,N-methyl bromoaniline acyl chloride.

[0065] In other embodiments, N,N-methyl bromoaniline acyl chloride can be prepared by replacing deuterated iodomethane in step (1) with iodomethane.

[0066] II. Examples of the method for analyzing stable isotope labeling of tobacco secondary alkaloids of the present invention

[0067] Example 3

[0068] For the method for analyzing stable isotope labeling of tobacco secondary alkaloids in this example, the light / heavy N,N-methyl chloroaniline acyl chloride of Example 1 is used as a derivatization reagent and for stable isotope labeling to qualitatively and quantitatively analyze tobacco secondary alkaloids. The specific steps are as follows:

[0069] (1) Pretreatment of the sample: Weigh 1 g of the fermented tobacco leaf sample into a 50 mL centrifuge tube, add 3 mL of 10% NaOH, let it stand for 30 min, then add 20 mL of 1% pentylamine-methyl tert-butyl ether, and vortex at the maximum speed for 15 min; add 3 - 4 mL of the extract to a filter with a 0.45 μm nylon membrane and filter it into a chromatographic vial. Take two 500 μL filtrates into the chromatographic vial, add 20 μL of N,N-methyl chloroaniline acyl chloride and 20 μL of deuterated N,N-methyl chloroaniline acyl chloride respectively, vortex for 5 min, then mix the two solutions and conduct instrumental analysis.

[0070] (2) Analysis of the sample: The sample was analyzed by liquid chromatography-high resolution mass spectrometry (HPLC-Orbitrap-MS). The HPLC analysis conditions are as follows:

[0071] —— Chromatographic column: The stationary phase is made of C18 material, with a specification of [2.1 mm × 100 mm × 1.7 μm];

[0072] —— The column temperature is 45 °C;

[0073] —— Mobile phase A: 10 mM ammonium acetate, pH 10; Mobile phase B: acetonitrile;

[0074] —— The flow rate is 0.4 mL / min;

[0075] —— Injection volume and split ratio: The injection volume is 1 μL, and the temperature of the autosampler is 10 °C;

[0076] Among them, the gradient change of the mobile phase is shown in Table 1:

[0077] Table 1 Gradient change of the mobile phase

[0078]

[0079] (3) Positive ion mode; spray voltage 3 kV; capillary temperature 325 °C; heater temperature 350 °C; sheath gas pressure 4 MPa; auxiliary gas pressure 1 MPa; scanning mode is FullMS / dd-MS 2 ; mass scanning range m / z 80 - 1100; full scan of the first stage (Full MS): resolution 70000 FWHM, maximum capacity of C-trap (AGC target) 1×10 6 , maximum injection time of C-trap 200 ms; data-dependent second-stage product ion scanning (dd-MS 2 ) : resolution 17500 FWHM, maximum capacity of C-trap 2×10 5 , maximum injection time of C-trap 100 ms; number of cycles is 5; normalized collision energy 20, 40, 60 eV; dynamic exclusion time 8 s.

[0080] Data-dependent acquisition (DIA): mass scanning range m / z 50 - 950; resolution 35000; automatic gain 2×10 5; The maximum ion implantation time is auto; the MSX count is 1; the normalized collision energies are 20, 40, and 60 eV; the acquisition is performed in 2 acquisition windows. The first acquisition window: the mass scan range is m / z 50 - 450, the isolation window is m / z 52.0, and the first-stage scan mass range is divided into 8 segments: m / z 50 - 100, 100 - 150, 150 - 200, 200 - 250, 250 - 300, 300 - 350, 350 - 400, 400 - 450; the second acquisition window: the mass scan range is m / z 450 - 950, the isolation window is m / z 104.0, and the first-stage scan mass range is divided into 5 segments: m / z 450 - 550, 550 - 650, 650 - 750, 750 - 850, 850 - 950. Mass spectrum analysis and qualitative results.

[0081] Among them, the HPLC-Orbitrap-MS chromatogram of the light / heavy N,N-methylchloroaniline acyl chloride derivatives of the sample extract is as Figure 1 shown. It can be clearly observed from this figure that compared with Figure 5 , the chromatographic peak signals in this chromatogram are extremely significant. This indicates that under the experimental conditions of this experiment, the mass spectrometry response of the light / heavy N,N-methylchloroaniline acyl chloride derivatives in the sample extract is strong, and the chromatography is well separated, which can provide strong data support for subsequent qualitative and quantitative analysis.

[0082] Next, taking the detected secondary alkaloids nornicotine, 2-(3-pyridyl)-5-piperidone, and norhydrocotinine as examples, the process of qualitative analysis is introduced:

[0083] The high-resolution mass spectra of the light / heavy derivatives of the alkaloids nornicotine, 2-(3-pyridyl)-5-piperidone, and norhydrocotinine are as Figures 2 - 4 shown. The primary mass spectrum is basically the parent ion. After one active hydrogen in the secondary alkaloid is replaced by light / heavy acyl chloride, both deuterated and non-deuterated characteristic ions exist in the mass spectrum, and the deuterated and non-deuterated mass spectral peak signals of the same characteristic ion are approximately in equal proportion. The intensity of the isotope peak containing Cl 37 of the same characteristic ion is about 1 / 3 of that of Cl 35 , and the mass number difference is 2 m / z. Screening is carried out according to this principle, and combined with the specific value of the high-resolution mass-to-charge ratio, the molecular composition information is obtained. Further, through the analysis of the secondary mass spectrum, the structural information is obtained. A total of 14 secondary alkaloids are identified, and the specific information is shown in Table 2. Compared with the 7 alkaloids of nornicotine, anabasin, anabasine, nornicotineene, nicotine glycoside, cotinine glycoside, and hydroxycotinine glycoside reported in the current cigarette smoke, the number of identified ones has increased significantly.

[0084] Table 2 Information on secondary alkaloids identified by qualitative analysis

[0085]

[0086]

[0087] (4) Quantitative analysis: For the 14 compounds identified in step (3), purchase or synthesize the corresponding standards for quantitative analysis. Dissolve the standards in methyl tert-butyl ether respectively to prepare a stock solution of 10 mg / mL, and further dilute to obtain the working solution. Dissolve the synthesized stable isotope-labeled probe in methyl tert-butyl ether to prepare a 500 mg / L labeled probe solution.

[0088] The pretreatment conditions are similar to those in step (1), except that the standard solution is lightly labeled and the sample solution is heavily labeled. Then, mix the two in equal volumes. After passing through the membrane, perform instrumental analysis. The instrument uses a liquid chromatography-triple quadrupole mass spectrometer (HPLC-MS) to analyze the sample. The HPLC analysis conditions are the same as those in step (3), and the MS conditions are as follows: electrospray ionization (ESI) source, positive (ESI+) ionization mode; ion source temperature 550 °C; spray voltage 5500 V in ESI+ mode; multiple reaction monitoring (MRM) mode, and its parameters are shown in Table 3.

[0089] The stable isotope labeling strategy has the function of internal standard quantitative analysis. Taking the measured abundance ratio of the light / heavy labeled products as the ordinate (y) and the concentration ratio as the abscissa (x), plot the standard curve. The 5 concentration ratios are 1:3, 1:1, 3:1, 6:1, and 9:1 respectively, and the formula y = ax + b can be plotted. Among them, y is the measured abundance ratio, x is the concentration ratio, a is the slope, and b is the intercept. According to this formula, the concentration ratio can be calculated based on the measured abundance ratio in the sample, and the content of alkaloids can be calculated based on the actual concentration of the standard.

[0090] Among them, V is the volume of the sample solution, C 标品 is the actual concentration of the standard.

[0091] Table 3 Retention time of alkaloids after derivatization on the liquid chromatography column and characteristic ion pairs of the mass spectrum

[0092]

[0093] Use methodological investigation to systematically characterize the working curve, detection limit, recovery rate, and precision of the method in this example. The results are shown in Table 4. According to Table 4, the correlation coefficient R of the 14 compounds 2All are above 0.999, showing a good linear relationship. The limit of quantification is 2 - 5.2 ng / mL, the recovery rate ranges from 76.1 - 120%, the intra-day precision is 2.5 - 7.5%, and the inter-day precision is 3.4 - 9.5%. This indicates that the method has high sensitivity, small result fluctuations, and is stable and reliable.

[0094] Table 4 Characterization data

[0095]

[0096]

[0097] In other implementation cases, the light / heavy N,N-methyl bromoaniline acyl chloride compound of Example 2 is used for qualitative and quantitative analysis of secondary alkaloids. Based on its structure similar to that of the N,N-methyl chloroaniline acyl chloride compound, experimental effects equivalent to those of this example can be achieved.

[0098] III. Comparative example

[0099] Comparative example 1

[0100] For the qualitative and quantitative analysis of secondary alkaloids in the fermented tobacco of this comparative example, the difference from Example 3 is that traditional 4-dimethylaminobenzoyl chloride is used for derivatization treatment. However, this derivatization reagent has no stable isotope label, so 2-methylquinoline is selected as the internal standard. The quantitative and characterization results are shown in Table 5. According to Table 5, this method can only identify 4 secondary alkaloids, namely nornicotineene, nornicotine, anabasine, and anatabine. Moreover, the linearity of this quantitative analysis is poor, the sensitivity is low, the fluctuations are large, and the deviation between the quantitative result and the actual value is relatively large.

[0101] The following is the specific qualitative and quantitative process:

[0102] The pretreatment is similar to that of Example 3, except that 100 μL of 2-methylquinoline internal standard (concentration: 1 mg / mL) is added before extraction. After the extraction solution is filtered, 1 mL of the sample extraction solution is taken and 50 μL of 4-dimethylaminobenzoyl chloride derivatization reagent is added, and the reaction is carried out at 60 °C for 30 minutes. At the same time, 200 μL of triethylamine is added as an acid-binding agent to promote the derivatization reaction.

[0103] The instruments used for qualitative and quantitative analysis are the same as those in Example 3. Quantitative analysis is carried out by the internal standard method of 2-methylquinoline: First, a series of standard solutions of secondary alkaloids with different concentrations are prepared, an equal amount of internal standard working solution is added, after derivatization reaction, it is injected into the instrument for analysis, and the peak areas of the secondary alkaloid derivatives and the internal standard in each standard solution are recorded. Taking the ratio of the concentration of the secondary alkaloid standard solution to the concentration of the internal standard as the abscissa, and the ratio of the peak area or peak height of the secondary alkaloid derivative to the peak area or peak height of the internal standard as the ordinate, a standard curve is plotted. Linear regression analysis is carried out using appropriate data analysis software to obtain the standard curve equation and the correlation coefficient. The peak areas or peak heights of the secondary alkaloid derivatives and the internal standard in the sample solution are substituted into the standard curve equation to calculate the concentration of the secondary alkaloids in the sample solution.

[0104] Table 5 Quantitative and characterization data

[0105]

[0106] Comparative Example 2

[0107] For the qualitative and quantitative analysis of secondary alkaloids in the fermented tobacco of this comparative example, the difference from Example 3 is that: traditional light / heavy acetyl chloride is used for isotope labeling, and the specific process is the same as that in Example 3. After the labeling probe is labeled, due to the absence of a permanently charged group, the mass spectrometry response is as Figure 5 , 6 shown. Compared with Figure 1 , it can be seen that the product after labeling with traditional light / heavy acetyl chloride as an isotope labeling probe has no permanently charged group in its molecular structure. During the ionization process, compared with some substances that themselves carry a permanent charge, its ionization efficiency is low, and the number of ions generated is small. This leads to relatively weak signal intensity during mass spectrometry detection, and the sensitivity is not significantly enhanced, resulting in no response and non-detection of many low-content target substances in the mass spectrometry; moreover, due to the weak signal, in a complex sample system, it is more likely to be interfered by impurities in the co-eluting peaks, such as cotinine derivatives, and the characteristic ions are mainly the molecular ion 204.15678 and the fragment ion 78.04219. These characteristic ions cannot be significantly distinguished from the impurities, resulting in difficult qualitative identification and inability to accurately identify.

Claims

1. A method for preparing a light / deuterated N,N-alkyl aniline acyl chloride compound, characterized in that, Comprising the following steps: React aniline, light / heavy monoiodoalkane, 1,3-dihalo-5,5-dimethylhydantoin, and trichloromethyl carbonate to obtain a light / deuterated N,N-alkylhaloaniline acyl chloride compound; the general structural formula thereof is as shown in Formula I: In Formula I, X = Cl or Br, Y = H or D, RY3 = methyl, ethyl, deuterated methyl, or deuterated ethyl.

2. The preparation method of the light / deuterated N,N-alkyl aniline acyl chloride compound according to claim 1, characterized in that, The molar ratio of the aniline, light / heavy monoiodoalkane, 1,3-dihalo-5,5-dimethylhydantoin, and trichloromethyl carbonate is (1.5 - 1.8):(4.1 - 4.4):1:(0.4 - 0.7).

3. The preparation method of the light / deuterated N,N-alkyl aniline acyl chloride compound according to claim 2, characterized in that, The aniline reacts with the light / heavy monoiodoalkane and an acid-binding agent in a first substitution reaction to obtain an N,N-alkylaniline substance. The N,N-alkylaniline substance undergoes a second substitution reaction with 1,3-dihalo-5,5-dimethylhydantoin to obtain a 2-halo-N,N-alkylaniline substance. The 2-halo-N,N-alkylaniline substance undergoes an acyl chlorination reaction with trichloromethyl carbonate to obtain a light / deuterated N,N-alkylhaloaniline acyl chloride compound.

4. The method for preparing a light / deuterated N,N-alkyl aniline acyl chloride compound according to claim 3, characterized in that, The temperature of the first substitution reaction is 60 - 100°C, and the time is 12 - 24 h.

5. The preparation method of the light / deuterated N,N-alkyl aniline acyl chloride compound according to claim 3, characterized in that, The second substitution reaction is carried out at room temperature, and the time is 10 - 24 h.

6. The preparation method of the light / deuterated N,N-alkyl aniline acyl chloride compound according to claim 3, characterized in that, The temperature of the acyl chlorination reaction is 50 - 100°C, and the time is 2 - 5 h.

7. The method for preparing a light / deuterated N,N-alkyl aniline acyl chloride compound according to claim 3 or 4, characterized in that, The acid-binding agent for the first substitution reaction is potassium carbonate.

8. The method for preparing a light / deuterated N,N-alkyl aniline acyl chloride compound according to claim 3 or 5, characterized in that, The catalyst for the second substitution reaction is one or a combination of two or more of diisopropyl ammonium halide salts, pyrrolidinium ammonium halide salts, and piperidinium ammonium halide salts.

9. A method for the analysis of stable isotope-labeled tobacco secondary alkaloids, characterized in that, Comprising the following steps: Respectively derivatize the light N,N-alkylhaloaniline acyl chloride compound and the deuterated N,N-alkylhaloaniline acyl chloride compound with the analyte, and then perform qualitative and quantitative analysis using liquid chromatography-mass spectrometry; the light / deuterated N,N-alkylhaloaniline acyl chloride compound is prepared according to the preparation method of the light / deuterated N,N-alkylhaloaniline acyl chloride compound described in any one of claims 1 - 8.

10. The method for stable isotope labeling analysis of tobacco secondary alkaloids according to claim 9, characterized in that, The secondary alkaloids include one or a combination of two or more of nornicotine enamine, nornicotine, anabasine, norcotinine, anabasin, 2-(3-pyridyl)-5-piperidone, 3-hydroxycotinine, iso-hydroxycotinine, diene nicotine glycoside, anabasine glycoside, nicotine-N-β-glucuronide, anabasin glycoside, cotinine glycoside, and 3-hydroxycotinine glycoside.

11. The method for analyzing stable isotope-labeled tobacco secondary alkaloids according to claim 9 or 10, characterized in that, The conditions of the liquid chromatography include: the stationary phase of the chromatographic column is C18, the column temperature is 45 - 48°C, mobile phase A is ammonium acetate, mobile phase B is acetonitrile, the flow rate is 0.4 - 0.6 mL / min, the injection volume is 1 - 1.2 μL, and the temperature of the autoinjector is 10 - 12°C.

12. The method for analyzing the stable isotope labeling of tobacco secondary alkaloids according to claim 11, characterized in that, The liquid chromatography uses gradient elution, and the gradient elution program is: 0 min, 95% A, 5% B; 3 min, 45% A, 55% B; 3.1 min, 10% A, 90% B; 4 min, 10% A, 90% B; 4.5 min, 95% A, 5% B; 5.5 min, 95% A, 5% B.

13. The method for analyzing stable isotope labeling of tobacco secondary alkaloids according to claim 9 or 10, characterized in that, The conditions of the mass spectrometry are as follows: electrospray ionization (ESI) source, positive (ESI+) ionization mode; ion source temperature 550 - 553 °C; spray voltage in ESI+ mode 5500 - 5505 V; multiple reaction monitoring (MRM) mode.

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