Preparation method of halogenated acyl halide compound of light / deuterated isotope and high-throughput analysis method of tobacco secondary alkaloid

Abstract

The invention relates to a preparation method of light / deuterated isotope halogenated acyl halide compounds and a high-throughput analysis method of tobacco secondary alkaloids, and belongs to the field of detection of tobacco secondary alkaloids. The preparation method comprises the following steps: reacting a carboxylic acid substance, N-halogenated succinimide and thionyl chloride or phosphorus tribromide, and carrying out post-treatment to obtain the halogenated acyl halide compound. According to the method, carboxylic acid substances, N-halogenated succinimide and thionyl chloride or phosphorus tribromide serve as reaction raw materials to prepare the halogenated acyl halide compound of the light / deuterated isotope, the method is simple, efficient preparation of the halogenated acyl halide compound can be achieved, and a foundation is laid for accurate qualitation and quantification of secondary alkaloids in tobacco.

Description

Technical Field

[0001] The present invention relates to a method for preparing a light / deuterated isotope of haloacyl halide compounds and a high-throughput analysis method for tobacco secondary alkaloids, belonging to the field of detection of tobacco secondary alkaloids. Background Art

[0002] Alkaloids are nitrogen-containing compounds, mainly including nicotine, which accounts for more than 95% of tobacco alkaloids. Tobacco secondary alkaloids refer to alkaloid substances other than nicotine, mainly including nornicotine, anabasine and anatabine, and many other substances are also contained. The composition and proportion of secondary alkaloids in tobacco have an important impact on tobacco quality and are important indicators for distinguishing tobacco types and qualities. Moreover, after alkaloids enter the human body, the generated secondary alkaloid metabolites are important biomarkers for measuring the impact of tobacco on human health and distinguishing active smokers from passive smokers, and have received extensive attention in biomedical, environmental science and tobacco health research.

[0003] The content of secondary alkaloid compounds in tobacco is below the ppm level, and there are many types of secondary alkaloids. It has important practical application value and scientific significance for the species identification and accurate quantification of secondary alkaloid compounds. The analysis and testing of alkaloids in the mainstream smoke of tobacco products mainly use gas chromatography-mass spectrometry. There are many active sites on the surface of the gas chromatography flow path, and alkaloids contain two nitrogen heterocycles, which are easy to interact with the active sites, resulting in problems such as serious adsorption, signal tailing, weak response, and easy decomposition. In addition, due to the weak characteristic fragmentation ions of many secondary alkaloids in gas chromatography-mass spectrometry, it is difficult to perform qualitative analysis. Therefore, the high-throughput qualitative and quantitative analysis of secondary alkaloid compounds in tobacco is one of the difficult problems in the field of analysis and testing.

[0004] The Chinese invention patent with the authorization announcement number CN105954402B discloses a gas chromatography-flame ionization / tandem mass spectrometry detection method for alkaloids in cigarette mainstream smoke. This method uses the internal standard method for qualitative and quantitative analysis, and the internal standard is a double internal standard, namely 2-methylquinoline and 2,4'-bipyridine respectively. This method can detect the contents of 4 tobacco secondary alkaloids, namely nornicotine, myosmine, anatabine and anabasine. However, this method is not conducive to the qualitative analysis of more secondary alkaloids. In addition, the selected internal standard is not a deuterated internal standard, and it cannot effectively correct the adsorption behavior of the target in the chromatographic and mass spectrometric systems, which easily leads to problems such as inaccurate quantification. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a light / deuterated isotope of haloacyl halide compounds to solve the problems of serious adsorption, signal tailing, weak response and easy decomposition of secondary alkaloids during detection in the prior art.

[0006] The second object of the present invention is to provide a high-throughput analysis method for tobacco secondary alkaloids to solve the problems that the qualitative analysis of secondary alkaloids in the prior art is relatively difficult and the quantitative results are not accurate enough.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] A preparation method of a light / deuterated isotope of a halogenated acyl halide compound, comprising the following steps: reacting a carboxylic acid substance represented by formula I, N-halosuccinimide, with thionyl chloride or phosphorus tribromide, and obtaining a halogenated acyl halide compound through post-treatment; the structural general formula of the halogenated acyl halide compound is shown as formula II;

[0009]

[0010] In formula I and formula II, Y = H or D, n = 1 to 3, X = Cl or Br.

[0011] The preparation method of the light / deuterated isotope of the halogenated acyl halide compound of the present invention is an exploratory invention. The present invention uses a carboxylic acid substance, N-halosuccinimide, thionyl chloride or phosphorus tribromide as reaction raw materials to prepare a light / deuterated isotope of a halogenated acyl halide compound. This method is simple, can achieve the efficient preparation of halogenated acyl halide compounds, and also lays a foundation for the accurate qualitative and quantitative determination of secondary alkaloids in tobacco.

[0012] In order to further improve the reaction efficiency, preferably, the molar ratio of the carboxylic acid substance, thionyl chloride, and N-halosuccinimide is 1:(3 to 4):(2 to 3); the molar ratio of the carboxylic acid substance, phosphorus tribromide, and N-halosuccinimide is 1:(1.2 to 1.4):(1.1 to 1.3).

[0013] Preferably, the reaction is to carry out an acyl halide reaction on the carboxylic acid substance with thionyl chloride or phosphorus tribromide to obtain an acyl halide substance, and then carry out a substitution reaction on the acyl halide substance with N-chlorosuccinimide to obtain a halogenated acyl halide compound. The halogenated acyl halide compound is synthesized in two steps, and the synthesis process is mild and controllable, which is conducive to realizing industrial production.

[0014] In order to further improve the yield of the acyl halide reaction, preferably, the temperature of the acyl halide reaction is 40 to 100 °C, and the time is 30 to 300 min. More preferably, the temperature of the acyl chloride reaction is 80 to 100 °C, and the time is 30 to 90 min. The temperature of the acyl bromide reaction is 40 to 60 °C, and the time is 5 to 6 h.

[0015] In order to further improve the efficiency of the substitution reaction, preferably, the substitution reaction is a heating reflux reaction, the heating temperature is 100 to 180° C., and the reaction time is 2 to 10 hours, preferably 2 to 5 hours.

[0016] Preferably, the catalyst of the substitution reaction is concentrated hydrochloric acid or deuterated concentrated hydrochloric acid. In the presence of a catalyst, the efficiency of the substitution reaction will be accelerated, promoting the reaction to proceed in the positive direction. Further preferably, the mass fraction of the concentrated hydrochloric acid or deuterated concentrated hydrochloric acid is 37-40%.

[0017] Further preferably, the post-treatment includes distillation, and the fraction cut during distillation is determined according to the boiling point of the halogenated acyl halide compound.

[0018] More preferably, when the halogenated acyl halide compound is a chlorinated acyl chloride compound, the distillation temperature is 106-110°C; when the halogenated acyl halide compound is a brominated acyl bromide compound, the distillation temperature is 140-150°C.

[0019] The second technical solution of the present invention is:

[0020] A high-throughput analysis method for tobacco secondary alkaloids comprises: preparing light halogenated acyl halide compounds and deuterated halogenated acyl halide compounds respectively according to the above-mentioned preparation method, and then performing derivatization reaction with analytes and performing qualitative and quantitative analysis by gas chromatography-mass spectrometry.

[0021] The light / deuterated isotope halogenated acyl halide compounds of the present invention are used as derivatization reagents to convert highly active functional groups such as amino, hydroxyl and carboxyl groups in secondary alkaloids into inert groups. The product is more stable and can effectively avoid the decomposition of the secondary alkaloids at the injection port during qualitative analysis. It can better reflect the actual situation of the secondary alkaloids and enhance the signal response intensity. At the same time, the present invention realizes the dual labeling of light / heavy stable isotopes and halogen atoms. The analysis method of tobacco secondary alkaloids of the present invention can simultaneously detect 19 tobacco secondary alkaloids, achieving the effect of high-throughput analysis.

[0022] The light / deuterated isotope halogenated acyl halide compounds of the present invention are used as derivatization reagents and are also stable isotope labeled probes in qualitative and quantitative analysis. The light / heavy labeled products have the same or similar retention time, mass spectrum peak signals appear in equal proportions, and the interval between the two labeled products is a fixed value, which has high recognition and is suitable for identifying unknown substances; and the halogen elements such as Cl in the labeled products have a high recognition fixed ratio isotope mass spectrum. 35 Cl, 37 Therefore, the present invention can achieve double confirmation through the highly recognizable characteristic peak and the isotope mass spectrum of the halogen element, making the qualitative result more accurate.

[0023] Furthermore, quantitative analysis is carried out by comparing the concentrations and signal abundances of light / heavy stable isotope-labeled products. Since there is only a difference in the mass-to-charge ratio between the two labeled products, the heavy stable isotope-labeled products can effectively correct the matrix effect existing in the qualitative and quantitative analysis of light stable isotope-labeled products, and the quantitative results are more accurate.

[0024] Preferably, the secondary alkaloids include one or more combinations of 3-(2,5-dihydro-1H-pyrrol-2-yl)-pyridine, 3-(2,3-dihydro-1H-pyrrol-2-yl)-pyridine, 3-(4,5-dihydro-1H-pyrrol-2-yl)pyridine, nornicotine, nornicotineene, cotinine-N-oxide, 1,2,5,6-tetrahydro-2,3'-bipyridine, 3,4,5-tetrahydro-2,3'-bipyridine, anabasine, anabazine, N-methyl-3-pyridylbutylamine, 2-(3-pyridyl)-5-piperidone, 2-(3-pyridyl)-4-piperidone, 6-(3-pyridyl)-2-piperidone, 2-(3-pyridyl)-5-hydroxypiperidine, 3-hydroxycotinine, 2-(3-methylpyridyl)-5-hydroxypiperidine, 2-(3-methylpyridyl)-3-hydroxypiperidine, trans-1-methyl-4-carboxy-5-(3-pyridyl)-2-pyrrolidone.

[0025] The light / deuterated isotope halogenated acyl halide compounds of the present invention as derivatizing reagents and stable isotope-labeled probes for qualitative and quantitative analysis can simultaneously identify 19 secondary alkaloids, that is, the number of identified secondary alkaloids is relatively large, laying a foundation for further distinguishing tobacco types and qualities.

[0026] To improve the accuracy of qualitative analysis, preferably, the conditions of the gas chromatography include: chromatographic column: capillary chromatographic column; stationary phase: trifluoropropylmethyl polysiloxane; inlet temperature: 290-300 °C; temperature gradient: initial temperature 100-105 °C is maintained for 2 min, then raised to 160-165 °C at 10 °C / min, then raised to 175-180 °C at 2 °C / min, then raised to 260-265 °C at 10 °C / min, and then run at 280-285 °C for 10 min.

[0027] Further preferably, the conditions of the gas chromatography also include: carrier gas: helium, constant flow mode, flow rate of 1 mL / min; injection volume and split ratio: injection volume is 1 μL, pulsed splitless injection, 50 psi for 0.5 min.

[0028] Preferably, the conditions of the mass spectrometry include: EI ionization mode; electron energy of 70-75 eV; ion source temperature of 280-285 °C; quadrupole temperature of 150-170 °C; transfer line temperature of 280-290 °C; scanning mode is SIM. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a full scan chromatogram of the cigarette smoke sample in Example 4 of the present invention;

[0030] Figure 2 This is a high-resolution mass spectrum of the derivatized 3-(2,5-dihydro-1H-pyrrol-2-yl)-pyridine in Example 4 of the present invention;

[0031] Figure 3 This is a high-resolution mass spectrum of the derivatized 3,4,5-tetrahydro-2,3'-bipyridine in Example 4 of the present invention;

[0032] Figure 4 This is a high-resolution mass spectrum of the derivatized 2-(3-pyridyl)-5-piperidone in Example 4 of the present invention;

[0033] Figure 5 is a full scan chromatogram of the cigarette smoke sample in Comparative Example 1 of the present invention;

[0034] Figure 6 This is the mass spectrum of 5 μg / mL standard solution of Equisetum arvense after silylation derivatization in Comparative Example 2 of the present invention;

[0035] Figure 7 This is the mass spectrum of Pseudoargenteus arvense and co-eluting products in the flue gas sample of Comparative Example 2 of the present invention on GC-MS. DETAILED DESCRIPTION

[0036] The light / deuterated isotope halogenated acyl halide compounds of the present invention are used to derivatize the secondary alkaloids in tobacco and tobacco products respectively, and the two products after derivatization are used for the identification of the secondary alkaloids at the same time. The two products have the same or similar retention time, the mass spectrum peak signals appear in equal proportions, and the interval between the two labeled products is a fixed value, which has high recognition. Combined with the fixed proportion of halogen isotope mass spectrum, double labeling is achieved, which is convenient for quickly locating the characteristic ion fragments of the secondary alkaloids, and improves the qualitative efficiency and accuracy of the secondary alkaloids. In addition, the heavy stable isotope derivatization products of the secondary alkaloids can effectively correct the matrix effect of the light stable isotope derivatization products in qualitative and quantitative analysis, thereby improving the accuracy of the quantitative results.

[0037] The reaction process involved in the following examples is as follows:

[0038] (1) Carboxylic acid substances react with thionyl chloride to obtain acyl chloride substances, and the reaction formula is as follows:

[0039]

[0040] (2) The acyl chloride reacts with N-chlorosuccinimide, and is catalyzed by concentrated hydrochloric acid or deuterated concentrated hydrochloric acid to obtain light / deuterated isotope halogenated acyl halide compounds. The reaction formula is as follows:

[0041]

[0042] Among them, Y is H or D, and n = 1-3.

[0043] The technical solutions of the present invention will be further described below in conjunction with specific embodiments.

[0044] I. Specific examples of the preparation method of the light / deuterated isotope halogenated acyl halide compounds of the present invention are as follows:

[0045] Example 1

[0046] The preparation method of the light / deuterated isotope halogenated acyl halide compound in this example adopts the following steps:

[0047] (1) Add 200 g (1.68 mol) of thionyl chloride to a reactor equipped with a stirrer, a spherical condenser, a constant-pressure burette funnel and a thermometer. Then add 27 g (0.45 mol) of acetic acid-d4 to the constant-pressure burette funnel. Open the water inlet valve of the spherical condenser and start stirring continuously. At the same time, add acetic acid-d4 dropwise. After the addition is completed, heat up to 80 °C, age for 30 min, and cool to room temperature. During the dropping process, a large amount of bubbles are released, and the tail gas is absorbed by sodium hydroxide solution.

[0048] (2) Add 125.1 g (0.94 mol) of N-chlorosuccinimide to the solution obtained in step (1), and at the same time add 5 drops of 37% DCl solution (the solute is D2O). Heat the mixture under reflux at 160 °C for 2.5 h, and cool to room temperature.

[0049] (3) The bottom of the distillation equipment is a three-necked flask with vacuum insulation, and the column body is a glass distillation column with a size of 1800×25×1.5 mm, filled with 1.5×1.5×1.5 stainless steel triangular packing, about 50 theoretical plates. Distill the reaction product on the column. After removing the pre-fraction with a temperature t < 100 °C, continue distillation, and cut the fraction with a temperature of 106 °C < t < 110 °C, which is the synthesized deuterated acetyl chloride.

[0050] In other implementation cases, replace the raw material acetic acid-d4 in the example with acetic acid, and replace the catalyst DCl solution with concentrated hydrochloric acid, then acetyl chloride can be synthesized.

[0051] Example 2

[0052] The preparation method of the light / deuterated isotope halogenated acyl halide compound in this example is different from that in Example 1 in that: the raw material in step (1) is propionic acid-d6, and the fraction selected in step (2) is the fraction with a temperature of 115 °C < t < 120 °C. The specific steps are as follows:

[0053] (1) Add 100 g (0.84 mol) of thionyl chloride to a reactor equipped with a stirrer, a spherical condenser, a constant-pressure burette funnel, and a thermometer. Then add 18 g (0.225 mol) of propionic acid-d6 to the constant-pressure burette funnel. Open the water inlet valve of the spherical condenser and start stirring continuously. At the same time, add propionic acid-d6 dropwise. After the addition is complete, heat up to 100 °C, age for 60 min, and then cool to room temperature. During the dropwise addition, a large amount of bubbles are released, and the tail gas is absorbed by sodium hydroxide solution.

[0054] (2) Add 62.5 g (0.47 mol) of N-chlorosuccinimide to the solution obtained in step (1), and at the same time add 3 drops of 37% DCl solution (the solute is D2O). Heat the mixture under reflux at 180 °C for 2.5 h, and then cool to room temperature.

[0055] (3) The still pot of the rectification equipment is a vacuum-insulated three-necked flask, and the column body is a glass rectification column with a size of 1800×25×1.5 mm, filled with 1.5×1.5×1.5 stainless steel triangular packing, equivalent to about 50 theoretical plates. Rectify the reaction product on the column. After removing the fore fraction with a temperature of t < 110 °C, continue rectification and cut the fraction with a temperature of 115 °C < t < 120 °C, which is the synthesized deuterated propionyl chloride.

[0056] Example 3

[0057] The preparation method of bromoacetyl bromide in this example is as follows:

[0058] (1) Add 162.6 g (0.6 mol) of phosphorus tribromide to a reactor equipped with a stirrer, a spherical condenser, a constant-pressure burette funnel, and a thermometer. Then add 27 g (0.45 mol) of acetic acid-d4 to the constant-pressure burette funnel. Open the water inlet valve of the spherical condenser and start stirring continuously. At the same time, add acetic acid-d4 dropwise. After the addition is complete, heat up to 40 °C and age for 5 h. During the dropwise addition, a large amount of bubbles are released, and the tail gas is absorbed by sodium hydroxide solution.

[0059] (2) Add 89 g (0.5 mol) of N-bromosuccinimide to the solution obtained in step (1), and at the same time add 3 drops of 37% DCl solution (the solute is D2O). Heat the mixture under reflux at 100 °C for 4 h, and then cool to room temperature.

[0060] (3) The bottom of the rectification equipment is a three-necked flask with vacuum insulation, and the column body is a glass rectification column with dimensions of 1800×25×1.5 mm. It is filled with 1.5×1.5×1.5 stainless steel triangular packing, equivalent to about 50 theoretical plates. The reaction product is fed into the column for rectification. After removing the fore-fraction with a temperature t < 100 °C, rectification is continued, and the fraction with a temperature of 140 °C < t < 150 °C is cut, which is the synthesized deuterated acetyl bromide.

[0061] In other embodiments, acetic acid - d4 in the examples is replaced with acetic acid, and the catalyst is replaced from DCl solution with concentrated hydrochloric acid, then acetyl bromide can be synthesized.

[0062] II. Examples of the high-throughput analysis method of tobacco secondary alkaloids of the present invention

[0063] Example 4

[0064] For the high-throughput analysis method of tobacco secondary alkaloids in this example, deuterated acetyl chloride and acetyl chloride prepared in Example 1 are used as derivatization reagents to qualitatively analyze the secondary alkaloids in the cigarette smoke. The specific method is as follows:

[0065] (1) Collection of particulate matter in the mainstream cigarette smoke: The cigarette smoking conditions refer to the national standard GB / T16450 - 2004, that is, 1 puff is taken every 60 s, the puff volume is 35 mL, and the puff duration is 2 s. Under these conditions, 5 cigarettes are smoked, and the particulate matter in the mainstream cigarette smoke is collected with a Cambridge filter.

[0066] (2) Pretreatment of the filter: A filter with a diameter of 44 mm is placed in a 100 mL conical flask, 5 mL of 10% NaOH is added, and after standing for 10 min, 40 mL of 1% pentylamine - methyl tert-butyl ether is added, and mechanical oscillation is carried out at 220 r / min for 2 h. 4 g of anhydrous sodium sulfate is added to a 0.45 μm nylon membrane filter, and then 3 - 4 mL of the debris-free part of the upper layer of the extraction solution is added, and filtered into a chromatographic vial.

[0067] Take two 500 μL filtrates into chromatographic vials, add 20 μL of acetyl chloride and 20 μL of deuterated acetyl chloride respectively, vortex for 5 min, then mix the two solutions and perform instrumental analysis.

[0068] (3) Analysis of the sample: The sample is analyzed by gas chromatography - high-resolution mass spectrometry (GC-Orbitrap-MS). The GC analysis conditions are as follows:

[0069] Chromatographic column: Capillary chromatographic column, the stationary phase is trifluoropropylmethyl polysiloxane, with specifications of [30 m (length) × 0.25 mm (inner diameter) × 1 μm (film thickness)]; the inlet temperature should be 230 °C.

[0070] Program temperature rise: initial temperature 100℃, maintain for 2 min, increase to 160℃ at 10℃ / min, increase to 175℃ at 2℃ / min, increase to 260℃ at 10℃ / min, and then operate at 280℃ for 10 min.

[0071] Carrier gas: helium, constant flow mode, flow rate 1mL / min.

[0072] Injection volume and split ratio: injection volume was 1 μL, pulsed splitless injection, 50 psi for 0.5 min.

[0073] Orbitrap-MS analysis conditions: EI ionization mode; electron energy 70 eV; ion source temperature 230 °C; quadrupole 150 °C; transfer line temperature 280 °C; collision gas: nitrogen (99.999%); maximum target capacity: 1 × 10 6 ; Maximum injection time: 200ms; Mass accuracy: 5×10 -6 ; Solvent delay: 10 min; Acquisition mode: Full scan; Mass scan range (m / z): 40-500; Mass resolution: 60,000 half-peak width (FWHM) (m / z 200).

[0074] (4) Mass spectrum analysis: The full scan chromatogram of the cigarette smoke sample on the VF-200MS is as follows: Figure 1 As shown in Figure 2, a total of 19 alkaloids were isolated. Figure 1 Mass spectrometry is performed on each peak in to obtain a high-resolution mass spectrum. Figure 1 The high-resolution mass spectra corresponding to the 1st, 8th and 12th peaks in Figure 2 , 3 , as shown in Figure 4.

[0075] according to Figures 2 - 4 It can be seen that the three secondary alkaloids all have characteristic ions of deuterated and undeuterated acyl chloride substituents after deuteration, and the mass spectrum peak signals of the two are approximately equal, and the mass spectrum peak of the deuterated acyl chloride substituent corresponding to the same fragment ion is located after the mass spectrum peak of the undeuterated acyl chloride substituent. And the secondary alkaloids after derivatization contain Cl, and the isotope mass spectrum with a ratio of about 2:1 will appear in the high-resolution mass spectrum. 35 Cl, 37 Cl. It can be seen that the mass spectrum characteristic peaks of the secondary alkaloids after the derivatization treatment of the present invention are obvious and have high recognition.

[0076] Combining the mass spectrometry characteristic peak information with the high-resolution mass-to-charge ratio to determine the molecular composition information of each derivatized secondary alkaloid. Further, through the analysis of the secondary mass spectrometry, based on the fragmentation rules and the results of spectral library matching, the accurate structural information can be obtained. A total of 19 secondary alkaloids were identified, and the specific information is shown in Table 1. Compared with the four tobacco secondary alkaloids of nornicotine, myosmine, anabasine, and nicotine identified by the prior art, the derivatization method of the present invention can identify more tobacco alkaloids, laying a foundation for further distinguishing tobacco varieties and qualities.

[0077] Table 1 Relevant information of 19 secondary alkaloids

[0078]

[0079]

[0080]

[0081] Example 5

[0082] In this example, qualitative and quantitative analyses were performed on the known target obtained in Example 4. Specifically, GC-MS instrument was used for qualitative and quantitative analyses: Since the mass spectrometry resolution of the GC-MS instrument is low and it is not suitable for the qualitative analysis of unknown substances not covered in the mass spectrometry spectral library, but it is a conventional instrument with low maintenance cost and is suitable for the qualitative and quantitative analysis of known target substances. Therefore, for the above-mentioned identified secondary alkaloids, the GC-MS instrument was used for daily analysis. The pretreatment was basically the same as that in steps (1) and (2) of the example, except that the derivatization labeling process in (2) was as follows: Take 500 μL of the filtrate into a chromatographic vial, add 20 μL of deuterated chloroacetyl chloride, and vortex for 5 min; Take 480 μL of methyl tert-butyl ether solvent and add 20 μL of the secondary alkaloid standard solution (30 μg / mL), add chloroacetyl chloride, and vortex for 5 min; Then, the above-mentioned solutions of heavy labeling and light labeling were mixed in equal proportions and analyzed by the instrument.

[0083] The sample was analyzed by gas chromatography-mass spectrometry (GC-MS). Among them, the GC analysis conditions were the same as those in step (3), and the MS analysis conditions were: EI ionization mode; electron energy 70 eV; ion source temperature 280 °C; quadrupole 150 °C; transfer line temperature 280 °C; the scanning mode was SIM, and its parameters are shown in Table 2.

[0084] For the 19 secondary alkaloids identified in step (4), pure products of secondary alkaloids were purchased or synthesized for quantitative analysis. The standard products were respectively dissolved in methyl tert-butyl ether to prepare a stock solution of 10 mg / mL, and further diluted to obtain a working solution. The synthesized stable isotope-labeled probe was dissolved in methyl tert-butyl ether to prepare a 1 mg / mL labeled probe solution.

[0085] The stable isotope labeling strategy has the function of internal standard quantitative analysis. Taking the measured abundance ratio of light / heavy labeled products as the ordinate (y) and the concentration ratio as the abscissa (x), a standard curve is plotted. 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 product.

[0086] Among them, V is the volume of the sample solution, C 标品 is the actual concentration of the standard product.

[0087] Table 2 Retention time, quantitative and qualitative ions, and dwell time of secondary alkaloids after derivatization

[0088]

[0089]

[0090] In this example, the concentrations of each secondary alkaloid and the sensitivity of the method in this example were characterized. The results are shown in Table 3. It can be seen from Table 3 that the correlation coefficients R of the 19 compounds 2 are all above 0.999, the linear relationship is good, the detection limit is 3 - 22 ng / mL, the recovery rate range is 80.8 - 110.1%, the within-day precision is 2.2 - 7.5%, and the between-day precision is 3.3 - 9.7%. It shows that this method has high sensitivity, small result fluctuations, and is stable and reliable.

[0091] Table 3 Results of sensitivity experiment

[0092]

[0093]

[0094] In other implementation cases, the propionyl chloride compounds in Example 2 were used for derivatization experiments to qualitatively and quantitatively analyze secondary alkaloids. Based on their similar structures to acetyl chloride, experimental effects equivalent to those in this example can be achieved.

[0095] III. Comparative example

[0096] Comparative example 1

[0097] The qualitative and quantitative analysis of secondary alkaloids in the flue gas of this comparative example is different from those in Examples 4 and 5 in that: instead of using deuterated acetyl chloride and acetyl chloride as labeling probes, a conventional non-derivatization method was used, and 2-methylquinoline was used as the internal standard. The response signals of the target substances obtained are asFigure 5 As shown. Compared with the full-scan chromatogram of Example 4, it can be seen that the signal intensity of the target substance in this comparative example is weak, resulting in difficulty in qualitative analysis. Only 4 secondary alkaloids containing active hydrogen can be qualitatively determined. For the qualitatively determined secondary alkaloids, quantitative analysis is carried out. The difference from Example 5 is that the SIM parameters of the instrumental method adopt the data in Table 4, and the calculation method is: taking the ratio of the peak area of the quantitative ion in the standard working solution to the peak area of the quantitative ion of the internal standard as the ordinate, and the ratio of the concentration of each alkaloid to the concentration of the internal standard as the abscissa, draw the standard working curve y = ax + b formula (where y is the measured peak area ratio, x is the concentration ratio, a is the slope, and b is the intercept). According to this formula, C 样品 is calculated, and then based on the release amount of secondary alkaloids in each cigarette is calculated. The quantitative results are shown in Table 5.

[0098] Table 4 Retention times, quantitative and qualitative ions, and abundance ratios of secondary alkaloids and their internal standards on the chromatographic column

[0099]

[0100]

[0101] As can be seen from Table 5, the correlation coefficients R 2 of the 4 secondary alkaloids are above 0.753, the linear relationship is poor, the detection limit is 38 - 177 ng / mL, the sensitivity is low, the within-day precision is 12.5 - 17.5%, and the between-day precision is 23.3 - 39.7%, with large fluctuations, ultimately affecting the quantitative results. Table 5 Characterization data with 2-methylquinoline as the internal standard

[0102]

[0103] Comparative Example 2

[0104] The qualitative analysis of secondary alkaloids in the flue gas of this comparative example is different from that of Example 4 in that: instead of using deuterated acetyl chloride and acetyl chloride as labeling probes, the traditional silylation reagent BSTFA is used to convert the active hydrogen in secondary alkaloids into stable groups, and the specific process is the same as that of Example 4. Taking Anabasis aphylla as an example, its mass spectrum after derivation is as Figure 6 shown. Among the mass spectrum fragments with a mass-to-charge ratio exceeding 100, the characteristic fragment with the highest abundance has a mass-to-charge ratio of 156.13242, followed by the molecular ion peak 234.15521 and the low-abundance 219.35427. In the actual sample, due to interference from co-eluting substances, as Figure 7 shown, it is difficult to find its characteristic fragments and molecular ion peaks, resulting in difficulty in qualitative identification and unable to accurately identify.

Claims

1. A method for preparing a halogenated acyl halide compound of a light / deuterated isotope, characterized in that, It includes the following steps: React a carboxylic acid substance represented by Formula I, an N-halosuccinimide, and thionyl chloride or phosphorus tribromide, and obtain a halogenated acyl halide compound through post-treatment; the general structural formula of the halogenated acyl halide compound is as shown in Formula II; In Formulas I and II, Y = H or D, n = 1 - 3, and X = Cl or Br.

2. The preparation method of the light / deuterated isotope halogenated acyl halide compound according to claim 1, characterized in that, The molar ratio of the carboxylic acid substance, thionyl chloride, and N-halosuccinimide is 1:(3 - 4):(2 - 3); the molar ratio of the carboxylic acid substance, phosphorus tribromide, and N-halosuccinimide is 1:(1.2 - 1.4):(1.1 - 1.3).

3. The method for preparing the halogenated acyl halide compound of light / deuterated isotopes according to claim 2, wherein The reaction is to carry out an acyl halide reaction on the carboxylic acid substance and thionyl chloride or phosphorus tribromide to obtain an acyl halide substance, and then carry out a substitution reaction on the acyl halide substance and N-chlorosuccinimide to obtain a halogenated acyl halide compound.

4. The preparation method of the light / deuterated isotope halogenated acyl halide compound according to claim 3, characterized in that, The temperature of the acyl halide reaction is 40 - 100 °C, and the time is 30 - 300 min.

5. The preparation method of the halogenated acyl halide compound of light / deuterated isotopes according to claim 3, characterized in that, The substitution reaction is a heating reflux reaction, the heating temperature is 100 - 180 °C, and the reaction time is 2 - 10 h.

6. The preparation method of the light / deuterated isotope halogenated acyl halide compound according to any one of claims 3 to 5, characterized in that, The catalyst for the substitution reaction is concentrated hydrochloric acid or deuterated concentrated hydrochloric acid.

7. A high-throughput analysis method for tobacco secondary alkaloids, characterized in that, It includes the following steps: respectively prepare a light halogenated acyl halide compound and a deuterated halogenated acyl halide compound according to the preparation method described in any one of Claims 1 - 6, and then carry out a derivatization reaction with the analyte and perform qualitative and quantitative analysis using gas chromatography - mass spectrometry.

8. The high-throughput analysis method of tobacco secondary alkaloids according to claim 7, characterized in that, The secondary alkaloids include one or a combination of two or more of 3-(2,5-dihydro-1H-pyrrol-2-yl)-pyridine, 3-(2,3-dihydro-1H-pyrrol-2-yl)-pyridine, 3-(4,5-dihydro-1H-pyrrol-2-yl)pyridine, nornicotine, nornicotine en, norcotinine, 1,2,5,6-tetrahydro-2,3'-bipyridine, 3,4,5-tetrahydro-2,3'-bipyridine, anabasine, anabazine, N-methyl-3-pyridylbutylamine, 2-(3-pyridyl)-5-piperidone, 2-(3-pyridyl)-4-piperidone, 6-(3-pyridyl)-2-piperidone, 2-(3-pyridyl)-5-hydroxypiperidine, 3-hydroxycotinine, 2-(3-methylpyridyl)-5-hydroxydienepiperidine, 2-(3-methylpyridyl)-3-hydroxydienepiperidine, and trans-1-methyl-4-carboxy-5-(3-pyridyl)-2-pyrrolidone.

9. The high-throughput analysis method of tobacco secondary alkaloids according to claim 7 or 8, characterized in that, The conditions of the gas chromatography include: chromatographic column: capillary chromatographic column; stationary phase: trifluoropropylmethyl polysiloxane; inlet temperature: 290 - 300 °C; temperature gradient: initial temperature 100 - 105 °C for 2 min, rising at 10 °C / min to 160 - 165 °C, rising at 2 °C / min to 175 - 180 °C, rising at 10 °C / min to 260 - 265 °C, and then running at 280 - 285 °C for 10 min.

10. The high-throughput analysis method of tobacco secondary alkaloids according to claim 7 or 8, characterized in that The conditions of the mass spectrometry include: EI ionization mode; electron energy is 70 - 75 eV; ion source temperature is 280 - 285 °C; quadrupole temperature is 150 - 170 °C; transfer line temperature is 280 - 290 °C; scanning mode is SIM.

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