A method for simultaneously determining the contents of nicotine salts and alcohol compounds in an electronic cigarette aerosol

Abstract

The present application relates to a kind of method for simultaneously determining nicotine salt and alcohol compound content in electronic cigarette aerosol, which comprises the following steps: (1) using electronic cigarette smoking machine to smoke electronic cigarette sample, using Cambridge filter to capture target in aerosol;(2) Cambridge filter of target in aerosol captured in step (1) is placed into extraction bottle, internal standard solution and methanol are added, and extraction is shaken, ready for use;(3) the extraction solution obtained in step (2) is subjected to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis and gas chromatography-mass spectrometry (GC-MS) analysis;(4) the content of target in sample is quantitatively calculated by internal standard standard curve method.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis technology, specifically relating to a method for simultaneously determining the content of nicotine salts and alcohol compounds in electronic cigarette aerosols. Background Technology

[0002] Electronic cigarettes, also known as electronic nicotine delivery systems, use a built-in atomizer to atomize the e-liquid in the cartridge and deliver the aerosol to the mouth and lungs. The aerosol generally contains atomizing agents (1,2-propanediol, glycerol), nicotine (nicotine salts), flavoring substances (such as menthol), and impurities such as ethylene glycol and diethylene glycol (Nicotine Tob Res, 2016, 18(5):708-714.). Since its inception, e-cigarettes have primarily used free nicotine (pure nicotine) as the added form, with a content generally not exceeding 2% (Nicotine Tob Res, 2016, 18(5):708-714; J.Sep.Sci.2017, 40(5):1049-1056.). In recent years, e-cigarette brands such as JUUL have widely used nicotine salts, such as nicotine benzoate, in e-cigarettes. This technology uses nicotine-organic acid complex to form protonated nicotine salts, which reduces the irritation of free nicotine and enhances the sensory experience and physiological satisfaction. Its nicotine content is much higher than that of traditional e-cigarette products, generally 3% to 5% (NicotineTob Res, 2019, 22(7):1239-1243.). JUUL has achieved great success in the US market and has now become the e-cigarette brand with the highest market share in the US (Tobacco Control, 2019, 28(2): 146-151.), attracting great attention from the US Food and Drug Administration (FDA).

[0003] Nicotine salts (nicotine and organic acids) and alcohols (1,2-propanediol, glycerol, menthol, ethylene glycol, diethylene glycol) in e-cigarette aerosols have a significant impact on their quality and safety. Establishing a high-throughput detection method for simultaneously determining the content of nicotine salts and alcohols in e-cigarette aerosols is of significant theoretical and practical importance. However, research on the analysis and testing of different types of nicotine salts (nicotine and organic acids) in e-cigarette aerosols is relatively limited, and no analytical method for simultaneously determining the content of nicotine salts and alcohols in e-cigarette aerosols has yet been found. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned technical shortcomings by establishing a method for simultaneously determining the content of 23 nicotine salts and 5 alcohol compounds in e-cigarette aerosols using a combination of liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. This method separates non-volatile organic acids, volatile organic acids, nicotine, and alcohols, and has advantages such as high throughput, good separation, simple operation, high sensitivity, good recovery rate, and good repeatability. It is suitable for the analysis of major nicotine salts and alcohol compounds in e-cigarette aerosols.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for simultaneously determining the content of nicotine salts and alcohol compounds in electronic cigarette aerosol, the method comprising the following steps:

[0007] (1) The electronic cigarette sample was drawn using an electronic cigarette smoking machine, and the target substance in the aerosol was captured using a Cambridge filter;

[0008] (2) Place the Cambridge filter containing the target substance in the aerosol from step (1) into an extraction flask, add internal standard solution and methanol, shake to extract, and set aside.

[0009] (3) Take the extraction solution obtained in step (2) and perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis and gas chromatography-mass spectrometry (GC-MS) analysis;

[0010] (4) The content of the target substance in the sample was quantitatively calculated using the internal standard curve method.

[0011] Preferably, in step (1), the electronic cigarette is inhaled in the standard inhalation mode (ISO), the Canadian deep inhalation mode (HCI), or the CORESTA recommended inhalation mode, etc.

[0012] Preferably, in step (2), non-volatile nicotine salts are analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS), wherein 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, respectively;

[0013] Volatile nicotine salts and alcohols were analyzed using gas chromatography-mass spectrometry (GC-MS), wherein the internal standard solutions were methanol solutions of valeric acid-d9, 1,4-butanediol and 2-methylquinoline, with concentrations of 20 μg / mL, 200 μg / mL and 50 μg / mL, respectively.

[0014] Preferably, in step (2), the volume of methanol is 10 to 50 mL, more preferably 10 mL.

[0015] Preferably, in step (2), the oscillation extraction time is 30 to 60 minutes, preferably 30 minutes.

[0016] Preferably, in step (3), the chromatographic conditions for the liquid chromatography-tandem mass spectrometry analysis include: a Waters XBridge BEH Amide column (2.1×100mm, 2.5μm); and the chromatographic conditions for the gas chromatography-mass spectrometry analysis include: an Agilent DB-624UI column (30m×0.32mm×1.8μm).

[0017] Preferably, in step (3), the chromatographic conditions for the liquid chromatography-tandem mass spectrometry analysis further include: a column temperature of 40°C; a mobile phase of 5 mmol / L ammonium acetate solution (pH 9.0) and an acetonitrile / water solution (V:V = 95 / 5, pH 9.0) containing 5 mmol / L ammonium acetate; a gradient elution program of: 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 run time of 9 min; and an injection volume of 1 μL.

[0018] Preferably, in step (3), the chromatographic conditions for gas chromatography-mass spectrometry analysis further include: injection port temperature: 250℃; transfer line temperature: 230℃; carrier gas: helium (≥99.999%); carrier gas flow rate: 1.5 mL / min; constant flow mode; split ratio: 20:1; injection volume: 1 μL; solvent delay: 1.8 min; the temperature program is: initial temperature 80℃, hold for 1 min, increase to 120℃ at 5℃ / min, hold for 1 min, increase to 230℃ at 8℃ / min, hold for 1 min, total running time 24.75 min, and run for 5 min after reaching 250℃;

[0019] Preferably, in step (3), the mass spectrometry conditions for the liquid chromatography-tandem mass spectrometry analysis are as follows: ion source: electrospray ion source; ionization mode: ESI(+); detection mode: MRM mode; cone temperature: 130℃; electrospray voltage: 4000V(+), 3500V(-); cone gas flow: 20psi; heated probe temperature: 350℃; probe gas flow: 50psi; nebulizer gas flow: 55psi.

[0020] Preferably, in step (3), the mass spectrometry conditions for the gas chromatography-mass spectrometry analysis are as follows: ionization mode: Extractor EI ion source; ionization energy: 70 eV; ion source temperature (TEM): 230℃; quadrupole temperature: 150℃; scanning mode: full scan and selected ion monitoring (SIM).

[0021] Preferably, in step (3), the MRM mass spectrometry detection parameters suitable for liquid chromatography-tandem mass spectrometry analysis of the target compound and internal standard are as follows:

[0022]

[0023]

[0024] Note: "*" indicates quantitative ions.

[0025] Preferably, in step (3), the SIM mass spectrometry detection parameters suitable for gas chromatography-mass spectrometry analysis of the target compound and the internal standard are as follows:

[0026]

[0027]

[0028] Preferably, in step (4), the internal standard curve method of liquid chromatography-tandem mass spectrometry analysis is as follows: prepare a series of standard working solutions containing the target analyte, add internal standards (benzoic acid-d5 and nicotine-d3), and construct a standard working curve with the ratio of the quantitative ion peak area of ​​the target analyte to the internal standard in each standard working solution as the ordinate and the content of the target analyte in each standard working solution as the abscissa; substitute the analysis results of step (3) into the standard curve to obtain the content of the target analyte in the test solution, and then further calculate the content of each target analyte in the sample; wherein, the concentration range of each target analyte in the series of standard working solutions is: nicotine 0.10~100μg / mL, citric acid 10~100μg / mL, tartaric acid 5~100μg / mL, pyruvic acid and lactic acid 1~100ug / mL, and other organic acids 0.1~100μg / mL.

[0029] Preferably, in step (4), the internal standard curve method of gas chromatography-mass spectrometry analysis is as follows: prepare a series of working solutions containing the target analyte, add internal standards (valeric acid-d9, 1,4-butanediol and 2-methylquinoline), and construct a standard working curve with the ratio of the quantitative ion peak area of ​​the target analyte to the internal standard in each standard working solution as the ordinate and the content of the target analyte in each standard working solution as the abscissa; substitute the analysis results of step (3) into the standard curve to obtain the content of the target analyte in the test solution, and then further calculate the content of the target analyte in the sample; wherein, the concentration range of each target analyte in the series of standard working solutions is: benzoic acid and formic acid 5-200 μg / mL, acetic acid 2-200 μg / mL, 1,2-propylene glycol and glycerol 5-2000 μg / mL, the remaining 12 organic acids and 3 alcohols 1-200 μg / mL, and nicotine 0.1 μg / mL-200 μg / mL.

[0030] Preferably, the nicotine salt is a salt formed from formic acid, acetic acid, propionic acid, butyric acid, 2-methylbutyric acid, valeric acid, 3-methylvaleric acid, 4-methylvaleric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, benzoic acid, sorbic acid, citric acid, malic acid, levulinic acid, salicylic acid, tartaric acid, pyruvic acid, lactic acid, and nicotine; the alcohol compound is succinic acid, ethylene glycol, propylene glycol, glycerol, and menthol.

[0031] Preferably, in one specific embodiment, the method includes the following steps:

[0032] (1) The electronic cigarette sample was drawn using an electronic cigarette smoking machine, and the target substance in the aerosol was captured using a Cambridge filter;

[0033] (2) Place the Cambridge filter containing the target substance in the aerosol from step (1) into an extraction bottle, add internal standard solution and 10-50 mL of methanol, shake and extract for 30-60 min, then filter through a 0.22 μm filter membrane for later use.

[0034] (3) Take the extract obtained in step (2) and perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) and gas chromatography-mass spectrometry (GC-MS) analysis;

[0035] The chromatographic conditions for the liquid chromatography-tandem mass spectrometry analysis included: a Waters XBridge BEHAmide column (2.1 × 100 mm, 2.5 μm); a column temperature of 40 °C; a mobile phase of 5 mmol / L ammonium acetate solution (pH 9.0) and an acetonitrile / water solution containing 5 mmol / L ammonium acetate (V:V = 95 / 5, pH 9.0); a gradient elution program of: initial flow rate 0.6 mL / min, decreasing to 0.4 mL / min at 0.5 min, increasing to 0.6 mL / min at 7.2 min, holding for 1.8 min, for a total run time of 9 min; and an injection volume of 1 μL. The mass spectrometry conditions included: ion source: electrospray ionization source; ionization mode: ESI(+); detection mode: MRM mode; and cone temperature (Cone). Temperature: 130℃; Spray Voltage: 4000V (+), 3500V (-); Cone Gas Flow: 20psi; Heated Probe Temperature: 350℃; Probe Gas Flow: 50psi; Nebulizer Gas Flow: 55psi.

[0036] The MRM mass spectrometry detection parameters for the target compound and the internal standard are as follows:

[0037]

[0038]

[0039] Note: "*" indicates quantitative ions.

[0040] The chromatographic conditions for the gas chromatography-mass spectrometry analysis included: an Agilent DB-624UI column (30m × 0.32mm × 1.8μm); injection port temperature: 250℃; transfer line temperature: 230℃; carrier gas: helium (≥99.999%); carrier gas flow rate: 1.5mL / min; constant flow mode; split ratio: 20:1; injection volume: 1μL; solvent delay: 1.8min; temperature program: initial temperature 80℃, hold for 1min, increase to 120℃ at 5℃ / min, hold for 1min, increase to 230℃ at 8℃ / min, hold for 1min, total run time 24.75min, followed by a 5min run after reaching 250℃. The mass spectrometry conditions included: ionization method: Extractor EI ion source; ionization energy: 70eV; ion source temperature (TEM): 230℃; quadrupole temperature: 150℃; scan mode: full scan and selected ion monitoring (SIM). The SIM mass spectrometry detection parameters for the target compound and the internal standard are as follows:

[0041]

[0042]

[0043] (4) The content of the target substance in the sample was quantitatively calculated using the internal standard curve method.

[0044] The internal standard curve method is as follows: prepare a series of standard working solutions containing the target analyte, add internal standards (in LC-MS analysis, the internal standards are benzoic acid-d5 and nicotine-d3; in GC-MS analysis, the internal standards are valeric acid-d9, 1,4-butanediol and 2-methylquinoline), and use the ratio of the quantitative ion peak area of ​​the target analyte to the internal standard in each standard working solution as the ordinate and the content of the target analyte in each standard working solution as the abscissa to construct a standard working curve; substitute the analysis results of step (3) into the standard curve to obtain the content of the target analyte in the test solution, and then further calculate the content of each target analyte in the sample.

[0045] Compared with the prior art, the present invention has at least the following beneficial technical effects:

[0046] This invention, based on LC-MS / MS and GC-MS techniques, investigated chromatographic conditions and established a method for simultaneously determining the content of 23 nicotine salts and 5 alcohol compounds in e-cigarette aerosols. It enables the analysis of non-volatile nicotine salts, volatile nicotine salts, and major alcohols, and has advantages such as high throughput, good separation, simple operation, high sensitivity, good recovery rate, and good repeatability. It is suitable for the analysis of major nicotine salts and alcohols in e-cigarette aerosols. Attached Figure Description

[0047] Figure 1This is a multiple reaction monitoring (MRM) chromatogram of the target analyte and its internal standard in the standard working solution of the LC-MS / MS method in Example 1;

[0048] Figure 2 The chromatograms for four columns used in the GC-MS method are shown below (1. Formic acid; 2. Acetic acid; 3. Propionic acid; 4. Ethylene glycol; 5. 1,2-Propanediol; 6. Butyric acid; 7. 2-Methylbutyric acid; 8. Valeric acid-d9 (internal standard); 9. Valeric acid; 10. 3-Methylvaleric acid; 11. 4-Methylvaleric acid; 12. 1,4-Butanediol (internal standard); 13. Hexanoic acid; 14. Diethylene glycol; 15. Glycerol; 16. Heptanoic acid; 17. Sorbic acid; 18. Menthol; 19. Octanoic acid; 20. Benzoic acid; 21. Nonanoic acid; 22. 2-Methylquinoline (internal standard); 23. Nicotine; 24. Decanoic acid). A: DB-WAX column; B: DB-FFAP column; C: TG-WAXMS A column; D: DB-624UI column. Detailed Implementation

[0049] The technical solution of the present invention will be further described below in conjunction with specific implementation methods.

[0050] Example 1: A method for simultaneously determining the content of 23 nicotine salts and 5 alcohol compounds in electronic cigarette aerosols

[0051] (1) Instruments and reagents

[0052] 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 (equipped with an ESI ion source), with HyStar Software as the data acquisition and processing software; the gas chromatography-mass spectrometry (GC-MS) system was an Agilent 8890-5977B, with Qualitative Analysis 10.0 and Quant-My-Way as the qualitative and quantitative analysis software; an ultrasonic instrument (YM-100S, Yumeng); an electronic balance (AE163, Mettler, Switzerland, sensitivity: 0.0001g); and a Talboys digital display multi-tube vortex mixer.

[0053] Reagents and consumables: Formic acid, acetic acid, propionic acid, butyric acid, 2-methylbutyric acid, valeric acid, 3-methylvaleric acid, 4-methylvaleric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, sorbic acid, benzoic acid, lactic acid, acetylpropionic acid, salicylic acid, succinic acid, citric acid, tartaric acid, malic acid, pyruvic acid, nicotine, benzoic acid-d5, 2-methylquinoline, ethylene glycol, diethylene glycol, 1,2-propanediol, glycerol, menthol, nicotine, valeric acid-d9, and 1,4-butanediol were purchased from TRC and Macklin. Formic acid (chromatographic grade) was purchased from MERCK. Ammonium formate (purity greater than 99%) was purchased from Macklin. Ultrapure water was prepared using the Milli-Q system (Milford, MA, USA).

[0054] (2) Instrument operating conditions

[0055] Chromatographic conditions for liquid chromatography-tandem mass spectrometry (LC-MS / MS): Waters XBridge BEHAmide column (2.1×100mm, 2.5μm), column temperature 40℃; mobile phase A: 5 mmol / L ammonium acetate solution (pH 9.0), mobile phase B: acetonitrile / water solution (V:V=95 / 5, pH 9.0) containing 5 mmol / L ammonium acetate; gradient elution program: initial flow rate 0.6 mL / min, flow rate reduced to 0.4 mL / min at 0.5 min, flow rate increased to 0.6 mL / min at 7.2 min, held for 1.8 min, total run time 9 min; injection volume 1 μL. Mass spectrometry conditions: LC-MSMS: Cone Temperature: 130℃; Spray Voltage: 4000V(+), 3500V(-); Cone Gas Flow: 20psi; Heated Probe Temperature: 350℃; Probe Gas Flow: 50psi; Nebulizer Gas Flow: 55psi; MRM mass spectrometry detection parameters for the target compound and internal standard are as follows:

[0056]

[0057]

[0058] Note: "*" indicates quantitative ions.

[0059] The multiple reaction monitoring (MRM) chromatograms of the target analyte and its internal standard in the standard working solution of the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method are shown below. Figure 1 As shown.

[0060] Chromatographic conditions for gas chromatography-mass spectrometry (GC-MS) analysis: Agilent DB-624UI column (30m × 0.32mm × 1.8μm); injection port temperature: 250℃; transfer line temperature: 230℃; carrier gas: helium (≥99.999%); carrier gas flow rate: 1.5mL / min; constant flow mode; split ratio: 20:1; injection volume: 1μL; solvent delay: 1.8min; temperature program: initial temperature 80℃, hold for 1min, increase to 120℃ at 5℃ / min, hold for 1min, increase to 230℃ at 8℃ / min, hold for 1min, total run time 24.75min, run for 5min after reaching 250℃. Mass spectrometry conditions: ion source temperature (TEM): 230℃; quadrupole temperature: 150℃; ionization energy: 70eV; scan mode: full scan and selected ion monitoring (SIM). The mass spectrometry detection parameters for the target compound and the internal standard are as follows:

[0061]

[0062]

[0063] (3) Sample pretreatment

[0064] Using an electronic cigarette device, the CORESTA recommended aspiration mode (55 mL aspiration volume, 3 s aspiration time, and 30 s aspiration interval) was used to aspirate the electronic cigarette sample. A Cambridge filter (44 mm) was used to capture the target analytes in the aerosol. The Cambridge filter containing the aerosol was placed in an extraction bottle, and 100 μL of internal standard solution and 10 mL of methanol were added. After shaking and extraction for 30 min, the sample was filtered through a 0.22 μm organic filter membrane and transferred to a chromatographic analysis bottle for analysis.

[0065] (4) Preparation of standard working solution

[0066] a. Preparation of standard stock solutions and working solutions for non-volatile organic acids and nicotine

[0067] ① Preparation of internal standard solutions: Accurately weigh 50 mg of benzoic acid-d5 and nicotine-d3 into 10 mL brown volumetric flasks, and dilute to volume with methanol to prepare internal standard stock solutions with a concentration of approximately 5 mg / mL. Take 1 mL of benzoic acid-d5 internal standard stock solution and 0.8 mL of nicotine-d3 internal standard stock solution, place them into the same brown volumetric flask, and dilute to volume with methanol to 10 mL to obtain internal standard solutions (internal standards) with a concentration of 500 μg / mL benzoic acid-d5 and a concentration of 400 μg / mL nicotine-d3. Nicotine-d3 is used to determine nicotine, and benzoic acid-d5 is used to determine organic acids.

[0068] ② Preparation of primary standard stock solution

[0069] Accurately weigh 2.0 g each of benzoic acid, lactic acid, levulinic acid, salicylic acid, succinic acid, sorbic acid, citric acid, tartaric acid, malic acid, pyruvic acid, and nicotine, and place them separately in 25 mL amber volumetric flasks. Dilute to the mark with methanol to prepare a standard stock solution with a concentration of approximately 80 mg / mL. This solution should be stored protected from light at 4℃ to 8℃.

[0070] ③ Preparation of secondary standard stock solution

[0071] Accurately pipette a certain volume of benzoic acid, lactic acid, acetylpropionic acid, salicylic acid, succinic acid, sorbic acid, citric acid, tartaric acid, malic acid, pyruvic acid, and nicotine stock solutions into a 50 mL brown volumetric flask, and dilute to the mark with methanol to prepare a mixed secondary standard stock solution with a concentration of 5 mg / mL.

[0072] ④ Preparation of standard working solutions

[0073] Accurately transfer the mixed secondary standard stock solution and internal standard solution, and prepare standard working solutions with methanol to final concentrations of 0.1, 1, 5, 10, 20, 40, 50, 80, and 100 μg / mL. Among them, the final concentration of benzoic acid-d5 in the internal standard is 5 μg / mL, which is used to determine 10 organic acids, including benzoic acid, lactic acid, levulinic acid, salicylic acid, succinic acid, sorbic acid, malic acid, citric acid, tartaric acid, and pyruvic acid. The final concentration of nicotine-d3 is 4 μg / mL, which is used to determine nicotine.

[0074] b. Preparation of standard stock solutions and working solutions for volatile organic acids, nicotine, and alcohols.

[0075] ① Preparation of internal standard solution

[0076] Accurately weigh 0.2 g of valeric acid-d9, 2.0 g of 1,4-butanediol and 0.5 g of 2-methylquinoline into a 100 mL brown volumetric flask, and dilute to volume with methanol to prepare internal standard stock solutions of different concentrations.

[0077] ② Preparation of standard stock solution

[0078] Accurately weigh 0.1 g each of formic acid, acetic acid, propionic acid, butyric acid, 2-methylbutyric acid, valeric acid, 3-methylvaleric acid, 4-methylvaleric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, sorbic acid, benzoic acid, ethylene glycol, diethylene glycol, menthol, 0.2 g of nicotine, and 1.0 g of 1,2-propanediol and glycerol into 100 mL amber volumetric flasks. Dilute to the mark with methanol to prepare a mixed standard stock solution with a concentration of 1–10 mg / mL. This solution should be stored protected from light at 4℃–8℃.

[0079] ③ Preparation of standard working solutions:

[0080] Accurately transfer 5 μL, 10 μL, 20 μL, 50 μL, 100 μL, 200 μL, 500 μL, 1000 μL, and 2000 μL of the mixed standard stock solution into different 10 mL brown volumetric flasks, and then accurately add 100 μL of internal standard solution to each flask. Dilute to the mark with methanol to obtain a series of standard solutions with 9 different concentrations.

[0081] (5) Sample determination

[0082] The standard working solution obtained in step (4) and the sample solution obtained in step (3) were analyzed by LC-MS / MS and GC-MS, respectively. A standard working curve was constructed with the ratio of the quantitative ion peak area of ​​the target analyte to the internal standard in each standard working solution as the ordinate and the content of the target analyte in each standard working solution as the ordinate. The analytical results of step (3) were substituted into the standard curve to obtain the content of the target analyte in the test solution, and then the content of each target analyte in the sample was further calculated.

[0083] (6) Method Validation

[0084] The limit of detection (LOD) of this method was calculated based on the lowest-level standard working solution and a signal-to-noise ratio of 3. Spike recoveries were calculated at three spiking levels (low, medium, and high), with five parallel samples added at each level. Precision was calculated based on the parallel test results. The linear range, linear coefficient, LOD, average spiked recovery, and average precision of the LC-MS / MS method are shown in Table 1. The linear range, linear coefficient, LOD, average spiked recovery, and average precision of the GC-MS method are shown in Table 2.

[0085] Table 1. Linear range, linearity coefficient, limit of detection, average recovery and average precision of each target analyte (LC-MS / MS)

[0086]

[0087] Table 2. Linear range, linearity coefficient, limit of detection, average recovery and average precision of each target analyte (GC-MS)

[0088]

[0089]

[0090] (7) Actual Sample Analysis

[0091] Based on the above determination method, five electronic cigarette aerosol samples were selected, and the content of the target substance was measured as shown in Table 3 (unit: μg / 100 puffs):

[0092] Table 3. Results of the content of 23 nicotine salts and 5 alcohol compounds in typical electronic cigarette aerosol samples.

[0093]

[0094]

[0095] Example 2: Column Selection Experiment

[0096] a. Column selection for non-volatile organic acids and nicotine

[0097] Considering the highly polar chemical properties of organic acids, four chromatographic columns were selected for comparison: Phenomenex's KinetexHILIC (4.6×150mm, 2.6μm) and Luna C18(2) (4.6×150mm, 3μm), Waters' Xbridge BEHAmide (2.1×100mm, 2.5μm), and Thermo's SCIENTIFIC Acclaim Trinity P1 (3×100mm, 3μm). The specific experimental procedure is shown in Example 1.

[0098] The specific experimental procedure is shown in Example 1.

[0099] Research has found that:

[0100] ① According to the recommended mobile phase (methanol-ammonium formate solution) for the chromatographic column, the Kinetex HILIC column is not ideal for separating organic acids, and malic acid, tartaric acid, citric acid and nicotine are not retained on the column. Optimizing the mobile phase conditions does not improve the separation of the unretained compounds. The SCIENTIFIC Acclaim Trinity P1 column has a significant effect on separating organic acids, but salicylic acid, malic acid, citric acid and nicotine are not retained on the column.

[0101] ② The Luna C18(2) column retained all organic acids and nicotine, but the baseline noise for levulinic acid and lactic acid was relatively high. 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.

[0102] ③ Based on the recommended mobile phase (acetonitrile-ammonium acetate solution) for the chromatographic column, all target analytes were retained on the XbridgeBEH Amide column. Further optimization of the mobile phase and gradient elution conditions resulted in a more ideal separation effect.

[0103] Therefore, the final selected chromatographic column was an Xbridge BEH Amide (2.1×100mm, 2.5μm) column.

[0104] b. Column selection for chromatographic analysis of volatile organic acids, nicotine, and alcohols.

[0105] Considering the chemical properties of organic acids, medium-grade and highly polar chromatographic columns were mainly selected, including Agilent's DB-WAX (30m×0.25mm×0.25μm), DB-FFAP (60m×0.25mm×0.25μm), DB-624UI (30m×0.32mm×1.8μm) and Thermo's TG-WAXMS A (30m×0.25mm×0.25μm). The specific implementation process is shown in Example 1.

[0106] Research has found that:

[0107] ① The DB-WAX column, used for separating 21 compounds including organic acids, alcohols, and nicotine, has two drawbacks: firstly, its response to formic acid is not high enough; secondly, the column has poor repeatability, and peaks are prone to tailing (e.g., Figure 2 (As shown in A).

[0108] ②The DB-FFAP column can achieve baseline separation for organic acids and nicotine, but it has poor response to glycerol, a high detection limit, and peak tailing (e.g., Figure 2 (as shown in B).

[0109] ③ The TG-WAXMS A column has poor response to formic acid and cannot achieve baseline separation of formic acid and propionic acid (e.g., Figure 2 (as shown in C).

[0110] ④ The DB-624UI column exhibits good resolution, peak shape, and stability for 21 compounds, and its response to formic acid is higher than the previous three columns (e.g., Figure 2 (as shown in D).

[0111] Therefore, the final selected chromatographic column is the DB-624UI column.

[0112] Example 3: Mobile Phase Selection Experiment

[0113] To investigate the effect of mobile phase on the detection methods of non-volatile organic acids and nicotine, the following mobile phases were selected for study: water and acetonitrile, 5 mmol / L ammonium acetate solution and acetonitrile / water solution with a concentration of 5 mmol / L ammonium acetate (V:V = 95 / 5), 5 mmol / L ammonium acetate solution (pH 9.0) and acetonitrile / water solution with a concentration of 5 mmol / L ammonium acetate (V:V = 95 / 5, pH 9.0). Except for the mobile phase, the other methods were as described in Example 1.

[0114] turn out:

[0115] ① The use of water and acetonitrile solvents in the mobile phase resulted in the non-retention of citric acid, tartaric acid, lactic acid, and succinic acid in the target compound, affecting the retention effect of the compound.

[0116] ② When ammonium acetate (5 mmol / L ammonium acetate solution and acetonitrile / water solution with a concentration of 5 mmol / L ammonium acetate (V:V = 95 / 5)) was added to the aqueous and organic phases of the mobile phase, the target analytes were retained, while citric acid and tartaric acid showed lower responses.

[0117] ③ Add ammonia to adjust the pH of the mobile phase so that the pH of both the aqueous and organic phases is 9.0 (5 mmol / L ammonium acetate solution (pH 9.0) and acetonitrile / water solution with a concentration of 5 mmol / L ammonium acetate (V:V = 95 / 5, pH 9.0), which achieved a relatively ideal result.

[0118] Therefore, the final determined mobile phase composition is scheme ③.

[0119] Example 4: Experiment on the selection of mobile phase flow rate

[0120] To investigate the effect of mobile phase flow rate on the detection method, the mobile phase flow rate was set to 0.4 mL / min; the initial flow rate was 0.6 mL / min, which was reduced to 0.4 mL / min at 0.5 min, increased to 0.6 mL / min at 7.2 min, and maintained for 1.8 min, with a total running time of 9 min. Except for the mobile phase flow rate, the other methods were the same as in Example 1.

[0121] Research has found that:

[0122] ① Under the experimental conditions of 0.4 mL / min, the separation effect of nicotine and salicylic acid was poor, and the analysis time was too long.

[0123] ② The flow rate was 0.6 mL / min, then decreased to 0.4 mL / min at 0.5 min, and increased to 0.6 mL / min at 7.2 min, maintaining a gradient flow rate mode for 1.8 min, which balanced separation and analysis time.

[0124] Therefore, the final determined mobile phase flow rate conditions were: initial flow rate 0.6 mL / min, flow rate reduced 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.

[0125] Example 5: Experiment on the Selection of Mass Spectrometry Temperature

[0126] To investigate the effect of the cone temperature on the detection method, several cone temperatures of 100℃, 120℃, 130℃, 150℃, 200℃ and 300℃ were selected for the study. Except for the cone temperature, the other methods were the same as in Example 1.

[0127] turn out:

[0128] ① The cone temperature has a significant impact on citric acid, lactic acid, tartaric acid and succinic acid. When the cone temperature is between 100℃ and 130℃, citric acid has the largest response at a cone temperature of 120℃, while tartaric acid, lactic acid and succinic acid have the largest response at a temperature of 130℃.

[0129] ② When the temperature of the cone hole increases from 130℃ to 300℃, the responses of citric acid, lactic acid, tartaric acid and succinic acid decrease.

[0130] Therefore, the final determined cone temperature is 130℃.

Claims

1. A method for simultaneously determining the content of nicotine salts and alcohol compounds in electronic cigarette aerosol, the method comprising the following steps: (1) The electronic cigarette sample was drawn using an electronic cigarette smoking machine, and the target substance in the aerosol was captured using a Cambridge filter; (2) Place the Cambridge filter containing the target substance in the aerosol from step (1) into an extraction flask, add internal standard solution and methanol, shake to extract, and set aside. (3) Take the extract obtained in step (2) and perform high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis and gas chromatography-mass spectrometry (GC-MS) analysis; (4) The content of the target analyte in the sample was quantitatively calculated using the internal standard curve method; in, In step (3), the chromatographic conditions for liquid chromatography-tandem mass spectrometry analysis included: a WatersXBridge BEH Amide column, 2.1×100mm, 2.5μm; a column temperature of 40℃; a mobile phase of 5mmol / L ammonium acetate solution with a pH of 9.0 and an acetonitrile / water solution containing 5mmol / L ammonium acetate at a volume ratio of 95:5 and a pH of 9.0; a gradient elution program of: an initial flow rate of 0.6mL / min, a flow rate reduced to 0.4mL / min at 0.5min, a flow rate increased to 0.6mL / min at 7.2min, maintained for 1.8min, and a total run time of 9min; and an injection volume of 1μL. The chromatographic conditions for gas chromatography-mass spectrometry analysis included: an Agilent DB-624UI column (30 m × 0.32 mm × 1.8 μm); injection port temperature: 250 °C; transfer line temperature: 230 °C; carrier gas: helium; carrier gas flow rate: 1.5 mL / min; constant flow mode; split ratio: 20:1; injection volume: 1 μL; solvent delay: 1.8 min; and a temperature program of: initial temperature 80 °C, hold for 1 min, increase to 120 °C at 5 °C / min, hold for 1 min, increase to 230 °C at 8 °C / min, hold for 1 min, total run time 24.75 min, followed by a 5 min run after reaching 250 °C. The nicotine salt is a salt formed from formic acid, acetic acid, propionic acid, butyric acid, 2-methylbutyric acid, valeric acid, 3-methylvaleric acid, 4-methylvaleric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, benzoic acid, sorbic acid, citric acid, malic acid, levulinic acid, salicylic acid, tartaric acid, pyruvic acid, lactic acid, succinic acid, and nicotine. The alcohols are diethylene glycol, ethylene glycol, propylene glycol, glycerol, and menthol.

2. The method according to claim 1, characterized in that, In step (1), the electronic cigarette is inhaled in the standard vaping mode (ISO), the Canadian deep vaping mode (HCI), or the CORESTA recommended vaping mode.

3. The method according to claim 1 or 2, characterized in that, In step (2), non-volatile nicotine salts are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS), wherein 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, respectively; Volatile nicotine salts and alcohols were analyzed using gas chromatography-mass spectrometry (GC-MS), wherein the internal standard solutions were methanol solutions of valeric acid-d9, 1,4-butanediol and 2-methylquinoline, with concentrations of 20 μg / mL, 200 μg / mL and 50 μg / mL, respectively.

4. The method according to claim 1 or 2, characterized in that, In step (2), the volume of methanol is 10-50 mL; the shaking extraction time is 30-60 minutes.

5. The method according to claim 1 or 2, characterized in that, In step (2), the volume of methanol is 10 mL; the shaking extraction time is 30 minutes.

6. The method according to claim 1 or 2, characterized in that, In step (3), the mass spectrometry conditions for the liquid chromatography-tandem mass spectrometry analysis are as follows: Ion source: electrospray ion source; Ionization mode: ESI(+); Detection mode: MRM mode; Cone temperature: 130℃; Spray voltage: 4000V(+), 3500V(-); Cone gas flow: 20psi; Heated probe temperature: 350℃; Probe gas flow: 50psi; Nebulizer gas flow: 55psi.

7. The method according to claim 1 or 2, characterized in that, In step (3), the mass spectrometry conditions for the gas chromatography-mass spectrometry analysis are as follows: ionization mode: Extractor EI ion source; ionization energy: 70 eV; ion source temperature (TEM): 230℃; quadrupole temperature: 150℃; scanning mode: full scan and selected ion monitoring (SIM).

8. The method according to claim 1 or 2, characterized in that, In step (3), the MRM mass spectrometry detection parameters for the target compound and internal standard suitable for liquid chromatography-tandem mass spectrometry analysis are as follows: Note: "*" indicates quantitative ions.

9. The method according to claim 1 or 2, characterized in that, In step (3), the SIM mass spectrometry detection parameters for the target compound and internal standard suitable for gas chromatography-mass spectrometry analysis are as follows:

10. The method according to claim 1 or 2, characterized in that, In step (4), the internal standard curve method is as follows: prepare a series of standard working solutions containing the target analyte, add internal standards, and in LC-MS analysis, the internal standards are benzoic acid-d5 and nicotine-d3, and in GC-MS analysis, the internal standards are valeric acid-d9, 1,4-butanediol and 2-methylquinoline. Use the ratio of the quantitative ion peak area of ​​the target analyte to the internal standard in each standard working solution as the ordinate and the content of the target analyte in each standard working solution as the abscissa to construct a standard working curve; substitute the analysis results of step (3) into the standard curve to obtain the content of the target analyte in the test solution, and then further calculate the content of each target analyte in the sample; wherein, the concentration range of each target analyte in the series of standard working solutions is: nicotine 0.10~100μg / mL, citric acid 10~100μg / mL, tartaric acid 5~100μg / mL, pyruvic acid and lactic acid 1~100ug / mL, and other organic acids 0.1~100μg / mL.

11. The method according to claim 1 or 2, characterized in that, In step (4), the internal standard curve method of gas chromatography-mass spectrometry analysis is as follows: prepare a series of standard working solutions containing the target analyte, add the internal standard valeric acid-d9, 1,4-butanediol and 2-methylquinoline, and construct a standard working curve with the ratio of the quantitative ion peak area of ​​the target analyte to the internal standard in each standard working solution as the ordinate and the content of the target analyte in each standard working solution as the abscissa; substitute the analysis results of step (3) into the standard curve to obtain the content of the target analyte in the test solution, and then further calculate the content of the target analyte in the sample; wherein, the concentration range of each target analyte in the series of standard working solutions is: benzoic acid and formic acid 5-200 μg / mL, acetic acid 2-200 μg / mL, 1,2-propylene glycol and glycerol 5-2000 μg / mL, the remaining 12 organic acids and 3 alcohols 1-200 μg / mL, and nicotine 0.1 μg / mL-200 μg / mL.

12. The method according to claim 1 or 2, characterized in that, The method includes the following steps: (1) The electronic cigarette sample was drawn using an electronic cigarette smoking machine, and the target substance in the aerosol was captured using a Cambridge filter; (2) Place the Cambridge filter containing the target substance in the aerosol from step (1) into an extraction bottle, add internal standard solution and 10-50 mL of methanol, shake and extract for 30-60 min, then filter through a 0.22 μm filter membrane for later use; (3) Take the extract obtained in step (2) and perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) and gas chromatography-mass spectrometry (GC-MS) analysis; The chromatographic conditions for the liquid chromatography-tandem mass spectrometry analysis were as follows: The column was a Waters XBridge BEH Amide column, 2.1 × 100 mm, 2.5 μm; the column temperature was 40 °C; the mobile phase was a 5 mmol / L ammonium acetate solution at pH 9.0 and an acetonitrile / water solution with a concentration of 5 mmol / L, a volume ratio of 95:5, and a pH of 9.0; the gradient elution program was: initial flow rate 0.6 mL / min, decreasing to 0.4 mL / min at 0.5 min, increasing to 0.6 mL / min at 7.2 min, holding for 1.8 min, for a total run time of 9 min; the injection volume was 1 μL; the mass spectrometry conditions included: ion source: electrospray ionization source; ionization mode: ESI(+); detection mode: MRM mode; cone temperature: 130 °C; electrospray voltage: 4000 V(+), 3500 V(-); cone gas flow rate: [not specified]. Flow: 20psi; Heated Probe Temperature: 350℃; Probe Gas Flow: 50psi; Nebulizer Gas Flow: 55psi; The mass spectrometry detection parameters for the target compound and the internal standard are as follows: Note: "*" indicates quantitative ions; The chromatographic conditions for the gas chromatography-mass spectrometry analysis were as follows: Agilent DB-624UI column (30m × 0.32mm × 1.8μm); injection port temperature: 250℃; transfer line temperature: 230℃; carrier gas: helium; carrier gas flow rate: 1.5mL / min; constant flow mode; split ratio: 20:1; injection volume: 1μL; solvent delay: 1.8min; temperature program: initial temperature 80℃, hold for 1min, increase to 120℃ at 5℃ / min, hold for 1min, increase to 230℃ at 8℃ / min, hold for 1min, total run time 24.75min, followed by a 5min run after reaching 250℃; mass spectrometry conditions included: ionization method: Extractor EI ion source; ionization energy: 70eV; ion source temperature (TEM): 230℃; quadrupole temperature: 150℃; scan mode: full scan and selected ion monitoring (SIM); the mass spectrometry detection parameters for the target compound and internal standard are as follows: (4) The content of the target analyte in the sample was quantitatively calculated using the internal standard curve method. The internal standard curve method is as follows: prepare a series of standard working solutions containing the target analyte, add internal standards, and in LC-MS analysis, the internal standards are benzoic acid-d5 and nicotine-d3, and in GC-MS analysis, the internal standards are valeric acid-d9, 1,4-butanediol and 2-methylquinoline. The ratio of the quantitative ion peak area of ​​the target analyte to the internal standard in each standard working solution is used as the ordinate, and the content of the target analyte in each standard working solution is used as the ordinate to construct a standard working curve. Substitute the analysis results of step (3) into the standard curve to obtain the content of the target analyte in the test solution, and then further calculate the content of each target analyte in the sample.

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