Method for determination of nicotine salt content in tobacco and tobacco products

Abstract

The present invention relates to a method for determining the nicotine salt content in tobacco and tobacco products, the method comprising the following steps: (1) weighing tobacco into a centrifuge tube, adding an internal standard solution and an aqueous solution, and standing for later use; (2) adding methanol to the tobacco solution obtained in step (1), ultrasonically extracting, and setting for later use; (3) subjecting the extract obtained in step (2) to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis; and (4) quantitatively calculating the content of a target substance in the sample using an internal standard standard curve method.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and particularly relates to a method for determining the nicotine salt content in tobacco and tobacco products. Background Art

[0002] Nicotine accounts for over 95% of the total tobacco alkaloids in tobacco. Most of it forms salts with organic acids and exists as nicotine salts. Nicotine salts in tobacco and tobacco products mainly include nicotine malate, citrate, and benzoate, and their content is closely related to tobacco quality (Tobacco Science and Technology, 2003, (6): 6-10.). At present, the main methods for testing nicotine in tobacco and tobacco products are gas chromatography (YC / T 246-2008), gas chromatography-mass spectrometry (YC / T 559-2018), and liquid chromatography-tandem mass spectrometry (Journal of Chromatography A1999,852(2):451-463). The main methods for testing organic acids are gas chromatography (YC / T 288-2009), liquid chromatography (Acta Tobacco Sinica 2007,(1):11-14), and liquid chromatography-tandem mass spectrometry (Jiangxi Journal of Agricultural Sciences 2018,30(6):75-79). However, there is no method for simultaneously determining the content of nicotine salts (nicotine and organic acids) in tobacco and tobacco products. The gas chromatography pretreatment in organic acid testing requires derivatization, which is cumbersome and prone to false positive results in liquid chromatography. However, the liquid chromatography-tandem mass spectrometry pretreatment is simple and does not require derivatization. Summary of the Invention

[0003] The present invention aims to fill a gap in the prior art by establishing a method for determining the content of nicotine salts in tobacco and tobacco products. The method uses LC-MS / MS technology to simultaneously detect nicotine, benzoic acid, salicylic acid, pyruvic acid, citric acid, malic acid, tartaric acid, sorbic acid, and succinic acid in nicotine salts. The method has the advantages of simple operation, high throughput, and high resolution, and is suitable for the analysis and determination of organic acids and nicotine in tobacco and tobacco products.

[0004] The object of the present invention is achieved through the following technical solutions:

[0005] A method for determining the nicotine salt content in tobacco and tobacco products, the method comprising the following steps:

[0006] (1) Weigh tobacco into a centrifuge tube, add internal standard solution and aqueous solution, and let stand for later use;

[0007] (2) adding methanol to the tobacco solution obtained in step (1), performing ultrasonic extraction, and setting aside;

[0008] (3) subjecting the extract obtained in step (2) to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis;

[0009] (4) The internal standard curve method was used to quantitatively calculate the content of the target substance in the sample.

[0010] Preferably, in step (1), the internal standard solution is a methanol solution of benzoic acid-d5 and nicotine-d3, with concentrations of 500 μg / mL and 400 μg / mL.

[0011] Preferably, in step (1), the aqueous solution is pure water, formic acid aqueous solution, acetic acid aqueous solution, preferably acetic acid aqueous solution.

[0012] Preferably, in step (1), when the aqueous solution is a formic acid aqueous solution or an acetic acid aqueous solution, the concentration of the solution is 0.2-1% (volume ratio), preferably 0.2% (volume ratio).

[0013] Preferably, in step (1), when the aqueous solution is pure water, the volume is 5 to 15 mL, preferably 5 mL.

[0014] Preferably, in step (2), the volume of methanol is 10 to 20 mL, preferably 20 mL.

[0015] Preferably, in step (3), the chromatographic column used for the liquid chromatography-tandem mass spectrometry analysis is selected from Phenomenex Kinetex HILIC (4.6×150mm, 2.6μm), Phenomenex Luna C18 (2) (4.6×150mm, 3μm), Waters Xbridge BEH Amide (2.1×100mm, 2.5μm) and Thermo SCIENTIFIC Acclaim Trinity P1 (3×100mm, 3μm) chromatographic columns, preferably Waters Xbridge BEH Amide (2.1×100mm, 2.5μm) chromatographic column.

[0016] Preferably, in step (3), the mobile phase used in the liquid chromatography-tandem mass spectrometry analysis is selected from a 5 mmol / L ammonium acetate aqueous solution and an acetonitrile / water (V:V=95 / 5) solution containing 5 mmol / L ammonium acetate, a 5 mmol / L ammonium acetate aqueous solution (pH 9.0) and an acetonitrile / water (V:V=95 / 5, pH 9.0) solution containing 5 mmol / L ammonium acetate, preferably a 5 mmol / L ammonium acetate aqueous solution (pH 9.0) and an acetonitrile / water (V:V=95 / 5, pH 9.0) solution containing 5 mmol / L ammonium acetate.

[0017] Preferably, in step (3), the chromatographic conditions of the liquid chromatography-tandem mass spectrometry analysis further include: a column temperature of 40°C; a gradient elution program: an initial flow rate of 0.6 mL / min, a flow rate reduced to 0.4 mL / min at 0.5 min, a flow rate increased to 0.6 mL / min at 7.2 min, maintained for 1.8 min, and a total running time of 9 min; and an injection volume of 1 μL.

[0018] Preferably, in step (3), the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry analysis include: ion source: electrospray ion source; ionization mode: ESI (+); detection mode: MRM mode; cone temperature (Cone Temperature): 130°C; electrospray voltage (Spray Voltage): 4000V (+), 3500V (-); cone gas flow (Cone Gas Flow): 20psi; heated probe temperature (Heated Probe Temperature): 350°C; probe gas flow (Probe Gas Flow): 50psi; nebulizer gas flow (Nebulizer Gas Flow): 55psi.

[0019] Preferably, in step (3), the MRM mass spectrometry detection parameters of the target compound and the internal standard are as follows:

[0020]

[0021] Preferably, in step (4), the internal standard standard curve method is as follows: a series of standard working solutions containing the target substance are prepared, internal standards (benzoic acid-d5 and nicotine-d3) are added, and a standard working curve is prepared with the quantitative ion peak area ratio of the target substance to the internal standard in each standard working solution as the vertical coordinate and the content of the target substance in each standard working solution as the horizontal coordinate; the analysis result of step (3) is substituted into the standard curve to obtain the content of the target substance in the solution to be tested, and then the content of each target substance in the sample is further calculated.

[0022] Preferably, in step (4), the concentration range of each target substance in the series of standard working solutions is: nicotine is 0.10-100 μg / mL, citric acid is 10-100 μg / mL, tartaric acid is 5-100 μg / mL, pyruvic acid is 1-100 μg / mL, and other organic acids are 0.1-100 μg / mL.

[0023] Preferably, the nicotine salt includes a salt formed by nicotine and benzoic acid, salicylic acid, pyruvic acid, succinic acid, sorbic acid, malic acid, tartaric acid, or citric acid.

[0024] In a specific embodiment, the method comprises the following steps:

[0025] (1) Weigh tobacco into a centrifuge tube, add an internal standard solution and a 0.2% (volume ratio) acetic acid aqueous solution, and let it stand for later use;

[0026] (2) Methanol is added to the tobacco solution obtained in step (1), and ultrasonic extraction is performed for later use.

[0027] (3) subjecting the extract obtained in step (2) to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis;

[0028] Chromatographic conditions: The chromatographic column was a Waters XBridge BEH Amide column (2.1×100 mm, 2.5 μm), and the column temperature was 40°C. The mobile phase consisted of 5 mmol / L ammonium acetate solution (pH 9.0) and acetonitrile / water (V:V=95 / 5, pH 9.0) containing 5 mmol / L ammonium acetate. The gradient elution program was as follows: initial flow rate 0.6 mL / min, the flow rate decreased to 0.4 mL / min at 0.5 min, and the flow rate increased to 0.6 mL / min at 7.2 min and maintained for 1.8 min. The total run time was 9 min. The injection volume was 1 μL.

[0029] Mass spectrometry conditions: ion source: electrospray ionization source; ionization mode: ESI (+); detection mode: MRM mode; cone temperature (Cone Temperature): 130 ° C; electrospray voltage (Spray Voltage): 4000 V (+), 3500 V (-); cone gas flow (Cone Gas Flow): 20 psi; heated probe temperature (Heated Probe Temperature): 350 ° C; probe gas flow (Probe Gas Flow): 50 psi; nebulizer gas flow (Nebulizer Gas Flow): 55 psi.

[0030] The MRM mass spectrometry parameters for the target compound and internal standard are as follows:

[0031]

[0032]

[0033] Note: “*” indicates quantification ion.

[0034] (4) The internal standard curve method was used to quantitatively calculate the content of the target substance in the sample.

[0035] The internal standard standard curve method comprises the following steps: preparing a series of standard working solutions containing the target substance, adding internal standards (benzoic acid-d5 and nicotine-d3), and using the quantitative ion peak area ratio of the target substance to the internal standard in each standard working solution as the ordinate, and using the content of the target substance in each standard working solution as the abscissa to prepare a standard working curve; substituting the analysis result of step (3) into the standard curve to obtain the content of the target substance in the solution to be tested, and then further calculating the content of each target substance in the sample;

[0036] The concentration ranges of the target substances in the series of standard working solutions are: nicotine is 0.10-100 μg / mL, citric acid is 10-100 μg / mL, tartaric acid is 5-100 μg / mL, pyruvic acid is 1-100 μg / mL, and other organic acids are 0.1-100 μg / mL.

[0037] Compared with the prior art, the present invention has at least the following beneficial technical effects:

[0038] The present invention is based on LC-MS / MS technology, investigates the chromatographic column, mobile phase composition, mobile phase flow rate and mass spectrometry conditions, and establishes a method for analyzing non-volatile organic acids and nicotine in electronic cigarette liquid. The method realizes the simultaneous analysis of benzoic acid, salicylic acid, pyruvic acid, succinic acid, sorbic acid, malic acid, tartaric acid, citric acid and nicotine. The method has the advantages of high throughput, good separation, simple operation, good recovery rate and repeatability, and is suitable for the analysis of non-volatile organic acids and nicotine in electronic cigarette liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the MRM chromatogram of the target compound by the liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis method in Example 1. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0041] Example 1: A method for detecting organic acid nicotine salts in tobacco

[0042] (1) Instruments and reagents

[0043] Instruments: The liquid chromatography-tandem mass spectrometry (LC-MS / MS) system consisted of an Agilent 1290 ultra-high-performance liquid chromatograph (including a G7129B autosampler, a G7120A quaternary solvent pump, and a G1316B column oven) and a Bruker EVOQ triple quadrupole mass spectrometer (with an ESI ion source). The data acquisition and processing software was HyStar Software. The system also included an ultrasonicator (YM-100S, Yumeng); an electronic balance (AE163, Mettler, Switzerland, sensitivity: 0.0001 g); and a MultiVortex multi-sample vortex mixer (Detelogy).

[0044] Reagents and consumables: Benzoic acid, salicylic acid, succinic acid, sorbic acid, citric acid, tartaric acid, malic acid, pyruvic acid, and nicotine were purchased from Alfa Aesar with a purity greater than 88%. Benzoic acid-d5 and nicotine-d3 were purchased from Macklin with a purity greater than 98%. Ultrapure water, methanol (chromatographic grade), acetic acid (chromatographic grade), and ammonium acetate (purity greater than 99%) were used. Ultrapure water was obtained using a Milli-Q system (Milford, MA, USA).

[0045] (2) Instrument working conditions

[0046] This experiment used an Agilent 1290 ultra-high performance liquid chromatograph with a Waters XBridge BEHAmide column (2.1 × 100 mm, 2.5 μm); mobile phase A: 5 mmol / L ammonium acetate solution (pH 9.0); mobile phase B: acetonitrile / water (V:V = 95 / 5, pH 9.0) containing 5 mmol / L ammonium acetate; injection volume: 1 μL; column temperature: 40°C. The gradient elution program is shown in the following table:

[0047] Time (min) A(％) B(％) Flow rate (μL / min) 0.00 0 100 600 0.50 0 100 400 7.00 50 50 400 7.20 0 100 600 9.00 0 100 600

[0048] Mass spectrometry conditions: Cone Temperature: 130°C; Spray Voltage: 4000 V (+), 3500 V (-); Cone Gas Flow: 20 psi; Heated Probe Temperature: 350°C; Probe Gas Flow: 50 psi; Nebulizer Gas Flow: 55 psi.

[0049] The MRM mass spectrometry parameters for the target compound and internal standard are as follows:

[0050]

[0051]

[0052] The multiple reaction monitoring (MRM) chromatogram of the target compound in the liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis method is as follows: Figure 1 shown.

[0053] (3) Sample pretreatment

[0054] Weigh 50 mg of tobacco sample into a 50 mL centrifuge tube, add 500 μL of internal standard (benzoic acid-d5 at a concentration of 500 μg / mL and nicotine-d3 at a concentration of 400 μg / mL), add 5 mL of 0.2% aqueous acetic acid, and let stand for 30 minutes. Add 20 mL of methanol and ultrasonically extract for 30 minutes. Transfer 1 mL of the extract to a chromatographic vial for analysis.

[0055] (4) Preparation of standard working solution

[0056] ① Preparation of internal standard solution: Accurately weigh 50 mg of benzoic acid-d5 and 40 mg of nicotine-d3 into a 10 mL brown volumetric flask. Add methanol to the volume to prepare internal standard stock solutions at concentrations of approximately 5 mg / mL and 4 mg / mL, respectively. Nicotine-d3 is used for the determination of nicotine, and benzoic acid-d5 is used for the determination of benzoic acid, salicylic acid, succinic acid, sorbic acid, tartaric acid, malic acid, citric acid, and pyruvic acid.

[0057] ② Preparation of primary standard stock solution

[0058] Accurately weigh 2.0 g each of benzoic acid, salicylic acid, succinic acid, sorbic acid, citric acid, tartaric acid, malic acid, pyruvic acid, and nicotine and place them in a 25 mL brown volumetric flask. Add methanol to the mark to prepare a standard stock solution with a concentration of approximately 80 mg / mL. Store this solution at 4°C–8°C in the dark.

[0059] ③. Preparation of secondary standard stock solution

[0060] Accurately pipette a certain volume of benzoic acid, salicylic acid, succinic acid, sorbic acid, citric acid, tartaric acid, pyruvic acid, malic acid, and nicotine stock solutions into a 50 mL brown volumetric flask. Add methanol to the mark to prepare a mixed secondary standard stock solution with a concentration of 5 mg / mL.

[0061] ④. Preparation of standard working solution

[0062] Accurately pipette the mixed secondary standard stock solution and internal standard solution, and use methanol to prepare standard working solutions with final concentrations of 0.1, 1, 5, 10, 20, 40, 50, 80, and 100 μg / mL, respectively. The final concentration of benzoic acid-d5 in the internal standard is 10 μg / mL, and the final concentration of nicotine-d3 is 8 μg / mL.

[0063] (5) Sample measurement

[0064] The standard curve solution obtained in step (4) and the sample solution obtained in step (3) were subjected to HPLC-MS / MS analysis, and the multiple reaction monitoring (MRM) chromatograms of the target substance and its internal standard in the standard working solution are shown in the attached figure. A standard working curve was prepared with the quantitative ion peak area ratio of the target substance to the internal standard substance in each standard working solution as the ordinate and the content of the target substance in each standard working solution as the ordinate; the analysis result of step (3) was substituted into the standard curve to obtain the content of the target substance in the test solution, and the content of each target substance in the sample was further calculated.

[0065] (6) Method validation

[0066] The detection limit of this method was calculated based on a signal-to-noise ratio of 3 times using the lowest-grade standard working solution. Spike recoveries were calculated at low, medium, and high spike levels. Five replicates were added to each spike level, and the test precision was calculated based on the replicate test results. The linear range, linear coefficient, detection limit, average spike recovery, and average precision of this method are shown in Table 1.

[0067] Table 1. Linear range, linear coefficient, detection limit, average spike recovery and average precision of each target compound

[0068]

[0069]

[0070] (7) Actual sample analysis

[0071] According to the above determination method, 4 tobacco samples were selected and the target substance contents were measured as shown in Table 2:

[0072] Table 2. Nicotine and organic acid content in typical tobacco samples (mg / g)

[0073]

[0074] Example 2: Chromatographic column selection experiment

[0075] Considering the strong polar chemical properties of organic acids, four chromatographic columns were selected for comparison: Phenomenex's Kinetex HILIC (4.6×150 mm, 2.6 μm) and Luna C18 (2) (4.6×150 mm, 3 μm), Waters' Xbridge BEHAmide (2.1×100 mm, 2.5 μm), and Thermo Scientific Acclaim Trinity P1 (3×100 mm, 3 μm). The specific experimental process is shown in Example 1.

[0076] The study found that:

[0077] ①. Based on the recommended mobile phase (methanol-ammonium formate solution), the Kinetex HILIC column did not perform well for the separation of organic acids. Furthermore, malic acid, tartaric acid, citric acid, and nicotine were not retained on the column. Optimizing the mobile phase conditions did not improve the separation of unretained compounds. The SCIENTIFIC Acclaim Trinity P1 column performed well for the separation of organic acids, but salicylic acid, malic acid, citric acid, and nicotine were not retained on the column.

[0078] ② The Luna C18(2) column retained all organic acids and nicotine, but the retention times of most organic acids were concentrated, resulting in poor separation. Further optimization of the mobile phase elution ratio and time did not significantly improve the results.

[0079] ③. Based on the recommended mobile phase (acetonitrile-ammonium acetate solution) for the chromatographic column, all target compounds were retained on the XbridgeBEH Amide column. By further optimizing the mobile phase and gradient elution conditions, a relatively ideal separation effect was achieved.

[0080] Therefore, the chromatographic column finally determined was the Xbridge BEH Amide (2.1×100mm, 2.5μm) chromatographic column.

[0081] Example 3: Mobile phase selection experiment

[0082] To investigate the effect of the mobile phase on the detection method, the following mobile phases were selected for study: water, acetonitrile, 5 mmol / L ammonium acetate aqueous solution, 5 mmol / L ammonium acetate aqueous solution (pH 9.0), acetonitrile / water (V:V = 95 / 5) solution containing 5 mmol / L ammonium acetate, and acetonitrile / water (V:V = 95 / 5, pH 9.0) solution containing 5 mmol / L ammonium acetate. Except for the mobile phase, the remaining methods were similar to those in Example 1.

[0083] turn out:

[0084] ① When water or acetonitrile is used as the mobile phase, citric acid, tartaric acid, and succinic acid in the target compounds are not retained, affecting the retention effect of the compounds.

[0085] ②. Ammonium acetate (5 mmol / L ammonium acetate aqueous solution and acetonitrile / water (V:V = 95 / 5) solution containing 5 mmol / L ammonium acetate) was added to the aqueous phase and organic phase, respectively. All the target compounds were retained, while the responses of citric acid and tartaric acid were lower.

[0086] ③. Adjust the pH of the mobile phase so that the pH of both the aqueous phase and the organic phase is 9.0 (5 mmol / L ammonium acetate aqueous solution and acetonitrile / water (V:V=95 / 5) solution containing 5 mmol / L ammonium acetate), which achieved relatively ideal results.

[0087] Therefore, the final mobile phase composition was determined as Scheme ③.

[0088] Example 4: Solvent selection experiment

[0089] Considering that tobacco contains target substances such as nicotine, citric acid, and malic acid, and organic acids are easily soluble in water, methanol and water were used as extraction solvents. The content of nicotine, citric acid, and malic acid in tobacco was used as evaluation indicators. 25 mL of pure water, 25 mL of methanol, and different volumes of water and methanol were used as extraction solvents to analyze the effect of solvents on tobacco extraction. Except for the solvent, the rest of the methods were referred to Example 1.

[0090] The results are shown in Table 3:

[0091] Table 3 Effects of pure water and methanol on the extraction of nicotine, citric acid and malic acid in tobacco samples

[0092]

[0093] The study found that when tobacco is extracted using a solvent containing pure water, the contents of malic acid, citric acid and nicotine are all high, so it is appropriate to use three water-containing extraction methods.

[0094] Example 5: Selection test of extraction method

[0095] On the basis of Example 4, the spiked recoveries in tobacco were compared using pure water extraction and mixed extraction with different volumes of pure water and methanol.

[0096] turn out:

[0097] ①. In the 25ml pure water extraction method, the recovery rates of sorbic acid, tartaric acid and nicotine were low, and the citric acid content was high.

[0098] ②. In the 10mL pure water immersion and 15mL methanol extraction method, the recovery rates of tartaric acid and nicotine were low, while the recovery rate of citric acid was high.

[0099] ③. The nicotine content in the 5mL pure water soaking and 20mL methanol extraction method is relatively low.

[0100] After comparing the three extraction methods, the final method was determined to be Scheme ③.

[0101] Example 6: Selection experiment of acidic water

[0102] To investigate the effect of water acidity on nicotine extraction, formic acid and acetic acid were added to pure water at concentrations of 0.2% and 1%, respectively. The method followed Example 3, with the exception of the acid component and concentration, to compare the effects of acidity and concentration on the extraction of nicotine and organic acids.

[0103] The study found that:

[0104] ①. After adding 0.2% and 1% formic acid into pure water, the recovery rates of benzoic acid and tartaric acid were high. When the formic acid concentration was 0.2%, the nicotine recovery rate was low. When the formic acid concentration was 1%, the nicotine recovery rate was high, exceeding the range.

[0105] ② After adding 0.2% and 1% acetic acid to pure water, the nicotine recovery rate was moderate.

[0106] ③. The nicotine and citric acid contents in tobacco extracted at an acetic acid concentration of 0.2% were higher than those at an acetic acid concentration of 1%.

[0107] Therefore, the final acid concentration was determined to be 0.2% acetic acid.

[0108] Example 7: Selection experiment of extraction method

[0109] In order to investigate the effects of different extraction methods on the extraction of nicotine and organic acids, ultrasonic extraction and vortex extraction were used to investigate the extraction efficiency, with both extraction times being 30 min.

[0110] The study found that the organic acid content of the two extraction methods was basically the same, and the malic acid content was slightly higher when using ultrasonic extraction than when using vortex extraction, so the ultrasonic extraction method was selected.

Claims

1. A method for determining the nicotine salt content in tobacco and tobacco products, the method comprising the following steps: (1) Weigh tobacco into a centrifuge tube, add internal standard solution and aqueous solution, and let stand for later use; (2) adding methanol to the tobacco solution obtained in step (1), performing ultrasonic extraction, and setting aside; (3) subjecting the extract obtained in step (2) to liquid chromatography-tandem mass spectrometry analysis; (4) Quantitatively calculate the content of the target substance in the sample using the internal standard curve method; In step (3), the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry analysis include: Ion source: electrospray ion source; Ionization mode: ESI+; Detection mode: MRM mode; cone temperature: 130°C; electrospray voltage: 4000 V+, 3500 V-; cone gas flow rate: 20 psi; heated probe temperature: 350°C; probe gas flow rate: 50 psi; nebulizer gas flow rate: 55 psi; In step (3), the chromatographic column used for the liquid chromatography-tandem mass spectrometry analysis is a Waters Xbridge BEHAmide, 2.1×100 mm, 2.5 μm chromatographic column; In step (3), the mobile phase A used in the liquid chromatography-tandem mass spectrometry analysis was: a 5 mmol / L ammonium acetate solution at pH 9.0; the mobile phase B was: an acetonitrile / water solution at a volume ratio of 95:5 containing 5 mmol / L ammonium acetate at a pH of 9.0; the gradient elution program was as shown in the following table: ； In step (1), the aqueous solution is pure water, formic acid aqueous solution, acetic acid aqueous solution The nicotine salt is a salt formed by benzoic acid, salicylic acid, pyruvic acid, succinic acid, sorbic acid, malic acid, tartaric acid, citric acid and nicotine.

2. The method according to claim 1, characterized in that In step (1), the internal standard solution is a methanol solution of benzoic acid-d5 and nicotine-d3, with concentrations of 500 μg / mL and 400 μg / mL.

3. The method according to claim 1, characterized in that In step (1), the aqueous solution is an aqueous acetic acid solution.

4. The method according to claim 1, wherein In step (1), when the aqueous solution is a formic acid aqueous solution or an acetic acid aqueous solution, the concentration of the solution is 0.2 to 1% by volume.

5. The method according to claim 1, characterized in that In step (1), when the aqueous solution is a formic acid aqueous solution or an acetic acid aqueous solution, the concentration of the solution is 0.2% by volume.

6. The method according to claim 1, wherein In step (1), when the aqueous solution is pure water, the volume is 5 to 15 mL.

7. The method according to claim 1, characterized in that In step (1), when the aqueous solution is pure water, the volume is 5 mL.

8. The method according to claim 1 or 2, characterized in that In step (2), the volume of methanol is 10 to 20 mL.

9. The method according to claim 8, characterized in that In step (2), the volume of methanol is 20 mL.

10. The method according to claim 1 or 2, characterized in that In step (3), the chromatographic conditions of the liquid chromatography-tandem mass spectrometry analysis also include: a column temperature of 40°C; a gradient elution program: an initial flow rate of 0.6 mL / min, a flow rate reduced to 0.4 mL / min at 0.5 min, a flow rate increased to 0.6 mL / min at 7.2 min, and maintained for 1.8 min, with a total running time of 9 min; and an injection volume of 1 μL.

11. The method according to claim 1 or 2, characterized in that In step (3), the MRM mass spectrometry parameters for the target compound and internal standard are as follows:

12. The method according to claim 1 or 2, characterized in that In step (4), the internal standard standard curve method is as follows: a series of standard working solutions containing the target substance are prepared, internal standards benzoic acid-d5 and nicotine-d3 are added, and a standard working curve is prepared with the quantitative ion peak area ratio of the target substance to the internal standard substance in each standard working solution as the vertical coordinate and the content of the target substance in each standard working solution as the horizontal coordinate; the analysis result of step (3) is substituted into the standard curve to obtain the content of the target substance in the solution to be tested, and then the content of each target substance in the sample is further calculated.

13. The method according to claim 12, characterized in that In step (4), the concentration range of each target substance in the series of standard working solutions is: nicotine is 0.10-100 μg / mL, citric acid is 10-100 μg / mL, tartaric acid is 5-100 μg / mL, and other organic acids are 0.1-100 μg / mL.

14. The method according to claim 1 or 2, characterized in that The method comprises the following steps: (1) Weigh tobacco into a centrifuge tube, add an internal standard solution and a 0.2% acetic acid aqueous solution by volume, and let it stand for later use; (2) adding methanol to the tobacco solution obtained in step (1), performing ultrasonic extraction, and setting aside; (3) subjecting the extract obtained in step (2) to liquid chromatography-tandem mass spectrometry analysis; Chromatographic conditions: The chromatographic column was a Waters XBridge BEH Amide column, 2.1 × 100 mm, 2.5 μm, and the column temperature was 40°C. The mobile phase consisted of 5 mmol / L ammonium acetate solution at pH 9.0 and 5 mmol / L ammonium acetate in acetonitrile / water at a volume ratio of 95:5, pH 9.

0. The gradient elution program was as follows: initial flow rate 0.6 mL / min, flow rate decreased to 0.4 mL / min at 0.5 min, flow rate increased to 0.6 mL / min at 7.2 min, and maintained for 1.8 min, for a total run time of 9 min. The injection volume was 1 μL. Mass spectrometry conditions: ion source: electrospray ionization source; ionization mode: ESI+; detection mode: MRM mode; cone temperature: 130°C; spray voltage: 4000 V+, 3500 V-; cone gas flow: 20 psi; heated probe temperature: 350°C; probe gas flow: 50 psi; nebulizer gas flow: 55 psi. The MRM mass spectrometry parameters for the target compound and internal standard are as follows: Note: "*" indicates quantitative ion (4) Quantitative calculation of the target content in the sample using the internal standard curve method The internal standard standard curve method comprises the following steps: preparing a series of standard working solutions containing the target substance, adding internal standards benzoic acid-d5 and nicotine-d3, and preparing a standard working curve using the quantification ion peak area ratio of the target substance to the internal standard in each standard working solution as the ordinate and the content of the target substance in each standard working solution as the abscissa; substituting the analysis result of step (3) into the standard curve to obtain the content of the target substance in the solution to be tested, and then further calculating the content of each target substance in the sample; The concentration ranges of the target substances in the series of standard working solutions are: nicotine is 0.10-100 μg / mL, citric acid is 10-100 μg / mL, tartaric acid is 5-100 μg / mL, pyruvic acid is 1-100 μg / mL, and other organic acids are 0.1-100 μg / mL.

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