Determination method for determining contents of multiple carbonyl compounds in cigarette smoke

Abstract

The invention discloses a method for determining the content of multiple carbonyl compounds in cigarette smoke, which comprises the following steps: S1, smoking a cigarette, and capturing the carbonyl compounds in the cigarette smoke by using a filter disc on which DNPH-phosphoric acid is applied, wherein carbonyl compounds in the cigarette smoke react with DNPH on the filter disc under the acidic condition, so that the filter disc with the carbonyl compounds is obtained; s2, putting the filter disc with the carbonyl compound into a container, adding an extracting agent to obtain an extracting solution, then filtering the extracting solution by adopting a microfiltration membrane to obtain a filtrate, and adding pyridine and an internal standard solution into the filtrate to obtain a sample solution; and S3, determining the sample solution by using an ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometer to obtain the concentration of each carbonyl compound in the cigarette smoke to be detected. The method provided by the invention can realize simultaneous analysis of 16 carbonyl compounds.

Description

Technical Field

[0001] The present invention relates to the technical field of tobacco chemical analysis, in particular to a method for determining the contents of multiple carbonyl compounds in cigarette smoke. Background Art

[0002] Carbonyl compounds are a significant class of harmful components in cigarette smoke. They are irritants, carcinogenic, or genotoxic, and long-term exposure increases health risks for smokers and those exposed to secondhand smoke. The main carbonyl compounds in traditional cigarette smoke include formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, and butyraldehyde. Compared to traditional cigarettes, new tobacco products, known for their reduced harm, are attracting greater attention to the harmful components. Compared to other harmful components, the levels of carbonyl compounds in the smoke of new tobacco products have not decreased significantly. Furthermore, compounds not found in traditional cigarette smoke, such as glyoxal, methylglyoxal, 2-hydroxypropionaldehyde, and valeraldehyde, have been detected in their smoke. Furthermore, dihydroxyacetone, glycolaldehyde, and glyceraldehyde have also been found in the cracking products of 1,2-propylene glycol and glycerol, the primary atomizers in heated tobacco products. Furthermore, carbonyl compounds are diverse, including monocarbonyl compounds, dicarbonyl compounds, and hydroxycarbonyl compounds, and they exist as isomers, such as butyraldehyde and 2-butanone, glyceraldehyde and dihydroxyacetone, making it difficult to accurately quantify multiple carbonyl compounds simultaneously. Therefore, it is of great significance to establish a method for the simultaneous determination of multiple carbonyl compounds in cigarette smoke. Summary of the Invention

[0003] The present invention aims to solve the technical problem that the existing technology cannot simultaneously measure the contents of 16 carbonyl compounds in cigarette smoke, namely, formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, glyceraldehyde, dihydroxyacetone, 2-hydroxypropionaldehyde, glyoxal, valeraldehyde, methylglyoxal, 2,3-butanedione, and 2,3-pentanedione. The present invention provides a method for measuring the contents of multiple carbonyl compounds in cigarette smoke, which can simultaneously measure the contents of 16 carbonyl compounds in cigarette smoke, namely, formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, glyceraldehyde, dihydroxyacetone, 2-hydroxypropionaldehyde, glyoxal, valeraldehyde, methylglyoxal, 2,3-butanedione, and 2,3-pentanedione.

[0004] To solve the above technical problems, the present invention discloses a method for determining the content of multiple carbonyl compounds in cigarette smoke, wherein the carbonyl compounds include formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, glyceraldehyde, dihydroxyacetone, 2-hydroxypropionaldehyde, glyoxal, valeraldehyde, methylglyoxal, 2,3-butanedione and 2,3-pentanedione. The determination method comprises the following steps:

[0005] S1: smoking a cigarette and capturing carbonyl compounds in the cigarette smoke using a filter disc coated with DNPH-phosphoric acid, wherein the carbonyl compounds in the cigarette smoke react with the DNPH on the filter disc under acidic conditions, thereby obtaining a filter disc with the carbonyl compounds thereon;

[0006] S2: placing the filter disc with the carbonyl compound into a container and adding an extractant to obtain an extract, then filtering the extract through a microporous filter membrane to obtain a filtrate, and adding pyridine and an internal standard solution to the filtrate to obtain a sample solution;

[0007] S3: The sample solution is measured using ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry to obtain the concentration of each carbonyl compound in the cigarette smoke to be tested.

[0008] Preferably, step S3 includes:

[0009] Establish a calibration curve;

[0010] Ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry was used to obtain the ratio of the peak area of ​​each carbonyl compound DNPH derivative in the sample solution to the peak area of ​​the internal standard;

[0011] The measured concentration of each carbonyl compound in the sample solution is obtained based on the calibration curve and each ratio;

[0012] The release amount of each carbonyl compound in cigarette smoke was obtained based on the measured concentration of the DNPH derivative of each carbonyl compound and the following calculation formula:

[0013]

[0014] in:

[0015] Y is the release of each carbonyl compound in cigarette smoke, expressed in micrograms per cigarette (μg / cig);

[0016] X is the measured concentration of the DNPH derivative of each carbonyl compound in the sample solution, expressed in micrograms per milliliter (μg / mL);

[0017] V is the volume of the sample solution in milliliters (mL);

[0018] 1000 is the dilution factor;

[0019] n is the number of cigarettes smoked, in cig;

[0020] M1 is the molar mass of each carbonyl compound in grams per mole (g / mol);

[0021] M2 is the molar mass of each carbonyl compound in grams per mole (g / mol).

[0022] Preferably, establishing the calibration curve in step S3 includes:

[0023] preparing standard working solutions: preparing mixed standard stock solutions of DNPH derivatives of 15 carbonyl compounds and standard stock solutions of DNPH derivatives of glyceraldehyde, respectively, placing the mixed standard stock solution and the standard stock solution of DNPH derivative of glyceraldehyde into two or more containers at different volumes, adding internal standard primary solution to each, and then adjusting the volume with acetonitrile to prepare standard working solutions of different concentrations, wherein the 15 carbonyl compounds include formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, dihydroxyacetone, 2-hydroxypropionaldehyde, glyoxal, valeraldehyde, methylglyoxal, 2,3-butanedione, and 2,3-pentanedione;

[0024] Drawing of calibration curve: Standard working solutions of different concentrations were analyzed using ultra-performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry, and the contents of 16 carbonyl compounds were quantified using the internal standard method. Regression analysis was performed with the ratio of the peak area of ​​each carbonyl compound to the peak area of ​​the internal standard as the ordinate and the mass concentration of each carbonyl compound as the abscissa to establish a calibration curve for 16 carbonyl compounds.

[0025] Preferably, preparing the standard working solution comprises:

[0026] preparing a first mixed standard stock solution of DNPH derivatives of 10 carbonyl compounds: mixing the DNPH derivatives of each of the 10 carbonyl compounds, namely, formaldehyde, acetaldehyde, acetone, propionaldehyde, acrolein, crotonaldehyde, 2-butanone, butyraldehyde, valeraldehyde, and glyoxal, in a mass ratio of 20:60:50:30:10:5:8:6:5:5, and diluting the mixture to a predetermined volume with acetonitrile to prepare a first mixed standard stock solution of the DNPH derivatives of the 10 carbonyl compounds;

[0027] Preparing a mixed standard stock solution of DNPH derivatives of 15 carbonyl compounds: mixing the first mixed standard stock solution with standard stock solutions of the DNPH derivatives of each of the five carbonyl compounds, namely, dihydroxyacetone, 2,3-butanedione, 2,3-pentanedione, 2-hydroxypropionaldehyde, and methylglyoxal, in a volume ratio of 0.4:1:5:25:25:10, and diluting the solution to a predetermined volume with acetonitrile to prepare a mixed standard stock solution of the DNPH derivatives of the 15 carbonyl compounds; and / or

[0028] The standard stock solution of the DNPH derivative of glyceraldehyde is an acetonitrile solution of the DNPH derivative of glyceraldehyde.

[0029] Preferably, the internal standard primary solution is a mixed solution of formaldehyde-2,4-dinitrophenylhydrazone-d3 and acetaldehyde-2,4-dinitrophenylhydrazone-d3, and the concentration of the internal standard primary solution is preferably 35 μg / mL to 45 μg / mL, more preferably 40 μg / mL.

[0030] Preferably, the internal standard solution is a mixed solution of formaldehyde-2,4-dinitrophenylhydrazone-d3 and acetaldehyde-2,4-dinitrophenylhydrazone-d3, and the concentration of the internal standard solution is preferably 375 ng / mL to 425 ng / mL, more preferably 400 ng / mL.

[0031] Preferably, the filter disc is a glass fiber filter disc or a Cambridge filter disc, and / or the extractant is acetonitrile; and / or the filter disc has a rough surface, and the DNPH-phosphoric acid is applied on the rough surface of the filter disc.

[0032] Preferably, the ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry adopts one or a combination of the following detection conditions:

[0033] Mobile phase A was 0.2 mM ammonium acetate aqueous solution;

[0034] The chromatographic column used was a Waters ACQUITY UPLC BEH C18 column with specifications of 50 mm × 2.1 mm, a particle size of 1.7 μm, and a column temperature of 30 °C;

[0035] The injection volume was 3 μL, the flow rate was 0.25 mL / min, the scanning mode was parallel reaction monitoring mode (PRM), the ion source was heated electrospray ionization source (HESI), and the negative ion mode was used;

[0036] Mobile phase B is acetonitrile; and / or

[0037] The elution gradient was as follows: mobile phase B increased from 20% to 40% in 0-2 minutes, increased from 40% to 50% in 2-12 minutes, increased from 50% to 95% in 12-20 minutes and maintained for 3 minutes, decreased to 20% in 0.1 minutes and maintained for 7 minutes.

[0038] Preferably, a blank control group is further provided in step S1, and the blank control group is used to eliminate the interference of carbonyl compounds in the air; and / or step S1 includes: applying a reagent containing DNPH-phosphoric acid to a filter disc twice and drying it, and applying the reagent containing DNPH-phosphoric acid to the filter disc again before smoking the cigarette; preferably, the filter disc applied with DNPH-phosphoric acid is placed on the smoke inlet side with the rough surface of the filter disc facing the smoke inlet side, and another untreated filter disc is placed closely behind it, so that the cigarette smoke passes through the filter disc applied with DNPH-phosphoric acid and the other filter disc in sequence, and preferably the diameter of each filter disc is 44 mm; preferably, the cigarette is smoked using an HCl smoking mode; preferably, after smoking is completed, the filter disc is left to stand for 40 minutes to 60 minutes, more preferably for 40 minutes, to allow the carbonyl compounds in the smoke to fully react with the DNPH on the filter disc.

[0039] Preferably, the reagent containing DNPH-phosphoric acid is a DNPH-2% phosphoric acid / acetonitrile solution with a concentration of 20 mg / mL; preferably, the total amount of DNPH-2% phosphoric acid / acetonitrile solution applied to the filter disc is 4 mL to 5 mL, and the volume ratio of the reagent containing DNPH-phosphoric acid applied three times is 1:1:1. More preferably, 1.5 mL of the reagent containing DNPH-phosphoric acid is applied to the filter disc successively, and each is dried in the dark, and 1.5 mL of the reagent containing DNPH-phosphoric acid is applied to the filter disc again before smoking the cigarette.

[0040] The method for determining the content of multiple carbonyl compounds in cigarette smoke provided by the present invention can simultaneously determine 16 carbonyl compounds in cigarette smoke, including formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, glyceraldehyde, dihydroxyacetone, 2-hydroxypropionaldehyde, glyoxal, valeraldehyde, methylglyoxal, 2,3-butanedione and 2,3-pentanedione, with a more comprehensive range of detection types, higher detection efficiency and more accurate detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A flow chart showing a method for determining the content of multiple carbonyl compounds in cigarette smoke provided by one embodiment of the present invention;

[0042] Figure 2 A schematic diagram showing the connection between a filter disc and a suction unit provided in one embodiment of the present invention;

[0043] Figure 3 HPLC graphs of DNPH derivatives of eight carbonyl compounds in standard working solutions and sample solutions provided in one embodiment of the present invention are shown;

[0044] Figure 4 Shown is a PRM diagram of DNPH derivatives of 16 carbonyl compounds in a standard working solution provided by one embodiment of the present invention;

[0045] Figure 5 Shown is a PRM diagram of DNPH derivatives of 16 carbonyl compounds in a sample solution provided by one embodiment of the present invention;

[0046] Figure 6 FIG2 shows a PRM diagram when a Waters ACQUITY UPLC BEH Shield RP18 chromatographic column is selected according to one embodiment of the present invention;

[0047] Figure 7 A PRM diagram is shown when a Hypersil GOLD C18 chromatographic column is selected according to one embodiment of the present invention;

[0048] Figure 8 A PRM diagram is shown when an InfinityLab Poroshell 120-C18 chromatographic column is selected, provided in one embodiment of the present invention. DETAILED DESCRIPTION

[0049] The following is an explanation of the embodiments of the present invention by specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Although the description of the present invention will be introduced in conjunction with the preferred embodiment, this does not mean that the features of this invention are limited to this embodiment. On the contrary, the purpose of introducing the invention in conjunction with the embodiment is to cover other options or modifications that may be extended based on the claims of the present invention. In order to provide a deep understanding of the present invention, the following description will contain many specific details. The present invention can also be implemented without using these details. In addition, in order to avoid confusion or blurring the focus of the present invention, some specific details will be omitted in the description. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0050] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0051] The terms “first”, “second”, etc. are only used for distinguishing descriptions and should not be understood as indicating or implying relative importance.

[0052] In the description of this embodiment, it should be noted that, unless otherwise specified or limited, the terms "disposed," "connected," and "connected" should be understood broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal connections between two components. Those skilled in the art will understand the specific meanings of the above terms in this embodiment based on specific circumstances.

[0053] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0054] like Figure 1 As shown, Figure 1 A flow chart of a method for determining the content of multiple carbonyl compounds in cigarette smoke provided in one embodiment of the present invention. In this embodiment, the multiple carbonyl compounds include formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, glyceraldehyde, dihydroxyacetone, 2-hydroxypropionaldehyde, glyoxal, valeraldehyde, methylglyoxal, 2,3-butanedione and 2,3-pentanedione. Figure 1 As shown, the determination method includes the following steps:

[0055] S1 (smoke capture step): smoking a cigarette and using a filter disc coated with DNPH-phosphoric acid to capture carbonyl compounds in the cigarette smoke, wherein the carbonyl compounds in the cigarette smoke react with the DNPH on the filter disc under acidic conditions, thereby obtaining a filter disc with the carbonyl compounds thereon;

[0056] S2 (filter disc treatment step): placing the filter disc with the carbonyl compound in a container and adding an extractant to obtain an extract, then filtering the extract through a microporous filter membrane to obtain a filtrate, and adding pyridine and an internal standard solution to the filtrate to obtain a sample solution;

[0057] S3 (quantitative analysis step): the sample solution is measured using ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry to obtain the concentration of each carbonyl compound in the cigarette smoke to be tested.

[0058] Using the above technical solution, a filter treated with an acidic solution of 2,4-dinitrophenylhydrazine (DNPH) is used to capture carbonyl compounds in cigarette smoke. DNPH reacts with carbonyl compounds to form stable 2,4-dinitrophenylhydrazone derivatives, facilitating subsequent analysis and determination. A microporous filter membrane filters the extract to remove impurities. Pyridine, a weak base, is added to the filtrate to neutralize the phosphoric acid in the solution, maintaining a stable pH for the reaction, thereby improving derivatization efficiency and product stability. The detection method provided by the present invention can simultaneously determine 16 carbonyl compounds in cigarette smoke, providing a more comprehensive range of detection types, higher detection efficiency, and more accurate test results.

[0059] Specifically, a blank control group is further provided in step S1, and the blank control group is used to eliminate the interference of carbonyl compounds in the air; and / or step S1 includes: applying a reagent containing DNPH-phosphoric acid to a filter disc twice and drying it, and applying the reagent containing DNPH-phosphoric acid to the filter disc again before smoking the cigarette; preferably, the filter disc applied with DNPH-phosphoric acid is placed on the smoke inlet side with the rough surface of the filter disc (on which DNPH-phosphoric acid is applied) facing the smoke inlet side, and another untreated filter disc is placed closely behind it, so that the cigarette smoke passes through the filter disc applied with DNPH-phosphoric acid and the other filter disc in sequence.

[0060] Preferably, the filter disc is a Cambridge filter disc or a glass fiber filter disc, having a roughened surface to which the DNPH-phosphoric acid is applied; more preferably, the filter disc is a Cambridge filter disc. Preferably, the filter disc has a diameter of 44 mm. Using a Cambridge filter disc with a diameter of 44 mm requires fewer cigarettes to be smoked, uses less solvent, and has higher capture efficiency.

[0061] Preferably, the cigarette is smoked using the HCl smoking mode.

[0062] Preferably, the filter disc is left to stand for 40 to 60 minutes after the puffing is completed; more preferably, the filter disc is left to stand for 40 minutes, which allows the carbonyl compounds in the flue gas to fully react with the DNPH on the filter disc.

[0063] By adopting the above technical solution, the cigarette smoke to be tested passes through the filter disc coated with DNPH-phosphoric acid and the filter disc not coated with DNPH-phosphoric acid in sequence, which not only allows DNPH to fully react with the carbonyl compounds in the cigarette smoke, but also improves the capture efficiency and makes the capture more complete; at the same time, the untreated filter disc can be used to absorb the excess DNPH-phosphoric acid solution, thus preventing the excess solution from penetrating the filter disc and entering the smoking machine to contaminate the smoking machine channel.

[0064] Specifically, the reagent containing DNPH-phosphoric acid is a DNPH-2% phosphoric acid / acetonitrile solution with a concentration of 20 mg / mL.

[0065] Preferably, the total volume of DNPH-2% phosphoric acid / acetonitrile solution applied to the filter is 4 mL to 5 mL, and the volume ratio of the DNPH-phosphoric acid reagent applied three times is 1:1:1. More preferably, the total volume of DNPH-2% phosphoric acid / acetonitrile solution applied to the filter is 4.5 mL, and 1.5 mL of the DNPH-phosphoric acid reagent is applied to the filter in succession, dried in the dark, and then a further 1.5 mL of the DNPH-phosphoric acid reagent is applied to the filter before smoking the cigarette. Controlling the total volume of DNPH-2% phosphoric acid / acetonitrile solution applied to the filter to 4.5 mL ensures complete reaction between the carbonyl compounds and DNPH while preventing excess solvent from contaminating the smoking machine channel during cigarette smoking.

[0066] Specifically, in step S2, acetonitrile is used as the extraction agent, which has high extraction efficiency. The internal standard solution is a mixed solution of formaldehyde-2,4-dinitrophenylhydrazone-d3 and acetaldehyde-2,4-dinitrophenylhydrazone-d3, and the concentration of the internal standard solution is preferably 375 ng / mL to 425 ng / mL, more preferably 400 ng / mL.

[0067] Specifically, step S3 includes:

[0068] Establish a calibration curve;

[0069] Ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry was used to obtain the ratio of the peak area of ​​each carbonyl compound DNPH derivative in the sample solution to the peak area of ​​the internal standard;

[0070] The measured concentration of each carbonyl compound DNPH derivative in the sample solution was obtained based on the calibration curve and each ratio;

[0071] The release amount of each carbonyl compound in cigarette smoke was obtained based on the measured concentration of the DNPH derivative of each carbonyl compound and the following calculation formula:

[0072]

[0073] in:

[0074] Y is the release of each carbonyl compound in cigarette smoke, expressed in micrograms per cigarette (μg / cig);

[0075] X is the measured concentration of the DNPH derivative of each carbonyl compound in the sample solution, expressed in micrograms per milliliter (μg / mL);

[0076] V is the volume of the sample solution in milliliters (mL);

[0077] 1000 is the dilution factor;

[0078] n is the number of cigarettes smoked, in cig;

[0079] M1 is the molar mass of each carbonyl compound in grams per mole (g / mol);

[0080] M2 is the molar mass of each carbonyl compound in grams per mole (g / mol).

[0081] Furthermore, establishing a calibration curve includes:

[0082] preparing standard working solutions: preparing mixed standard stock solutions of DNPH derivatives of 15 carbonyl compounds and standard stock solutions of DNPH derivatives of glyceraldehyde, respectively, placing the mixed standard stock solutions and the standard stock solutions of DNPH derivatives of glyceraldehyde into two or more containers at different volumes, adding internal standard primary solutions to each of the solutions, and then quantifying the volumes with acetonitrile to prepare standard working solutions of different concentrations, wherein the 15 carbonyl compounds include formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, dihydroxyacetone, 2-hydroxypropionaldehyde, glyoxal, valeraldehyde, methylglyoxal, 2,3-butanedione, and 2,3-pentanedione;

[0083] Drawing a calibration curve: Standard working solutions of different concentrations were analyzed using ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry, and the contents of 16 carbonyl compounds were quantified using the internal standard method. Regression analysis was performed with the ratio of the peak area of ​​the DNPH derivative of each carbonyl compound to the peak area of ​​the internal standard as the ordinate and the mass concentration of the DNPH derivative of each carbonyl compound as the abscissa to establish a calibration curve for 16 carbonyl compounds.

[0084] Further, preparing the standard working solution comprises:

[0085] preparing a first mixed standard stock solution of DNPH derivatives of 10 carbonyl compounds: mixing the DNPH derivatives of each of the 10 carbonyl compounds, namely, formaldehyde, acetaldehyde, acetone, propionaldehyde, acrolein, crotonaldehyde, 2-butanone, butyraldehyde, valeraldehyde, and glyoxal, in a mass ratio of 20:60:50:30:10:5:8:6:5:5, and diluting the mixture to a predetermined volume with acetonitrile to prepare a first mixed standard stock solution of the DNPH derivatives of the 10 carbonyl compounds;

[0086] Preparing a mixed standard stock solution of DNPH derivatives of 15 carbonyl compounds: mixing the first mixed standard stock solution with standard stock solutions of the DNPH derivatives of each of the five carbonyl compounds, namely, dihydroxyacetone, 2,3-butanedione, 2,3-pentanedione, 2-hydroxypropionaldehyde, and methylglyoxal, in a volume ratio of 0.4:1:5:25:25:10, and diluting the solution to a predetermined volume with acetonitrile to prepare a mixed standard stock solution of the DNPH derivatives of the 15 carbonyl compounds; and / or

[0087] The standard stock solution of the DNPH derivative of glyceraldehyde is an acetonitrile solution of the DNPH derivative of glyceraldehyde.

[0088] Furthermore, the internal standard primary solution is a mixed solution of formaldehyde-2,4-dinitrophenylhydrazone-d3 and acetaldehyde-2,4-dinitrophenylhydrazone-d3, and the concentration of the internal standard primary solution is preferably 35 μg / mL to 45 μg / mL, more preferably 40 μg / mL.

[0089] Furthermore, the ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry instrument adopts one or a combination of the following detection conditions:

[0090] Mobile phase A was 0.2 mM ammonium acetate aqueous solution;

[0091] The chromatographic column used was a Waters ACQUITY UPLC BEH C18 column with specifications of 50 mm × 2.1 mm, a particle size of 1.7 μm, and a column temperature of 30 °C;

[0092] The injection volume was 3 μL, the flow rate was 0.25 mL / min, the scanning mode was parallel reaction monitoring mode (PRM), the ion source was heated electrospray ionization source (HESI), and the negative ion mode was used;

[0093] Mobile phase B is acetonitrile; and / or

[0094] The elution gradient was as follows: mobile phase B increased from 20% to 40% in 0-2 minutes, increased from 40% to 50% in 2-12 minutes, increased from 50% to 95% in 12-20 minutes and maintained for 3 minutes, decreased to 20% in 0.1 minutes and maintained for 7 minutes.

[0095] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0096] Example 1

[0097] (1) Instruments and reagents

[0098] Instruments: SM450 linear smoking machine (Cerulean, UK); Cambridge filter discs (44 mm diameter, Borgwaldt KC, Germany); CX-572 adsorption tubes (Hefei Zhongwo Instrument Technology Co., Ltd.); Dionex Ultimate 3000 ultra-high performance liquid chromatograph and Q-Exactive Focus quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, USA); ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm), ACQUITY UPLC BEH Shield RP18 column (50 mm × 2.1 mm, 1.7 μm) (Waters, USA); Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 μm, Thermo Fisher Scientific, USA); InfinityLab Poroshell 120-C18 (100 mm × 2.1 mm, 2.7 μm, Agilent, USA); AG104 electronic balance (sensitivity 0.0001 g, Mettler Toledo, Switzerland); HY-8 oscillator (Jiangsu Ronghua Instrument Manufacturing Co., Ltd.); organic phase microporous filter membrane (pore size 0.22 μm, Shanghai Xuntong Machinery Co., Ltd.).

[0099] Reagents and consumables: phosphoric acid (analytical grade, Guangdong Guanghua Chemical Factory Co., Ltd.); sulfuric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); 2,4-dinitrophenylhydrazine (≥99%, Sinopharm Chemical Reagent Co., Ltd.); acetonitrile (HPLC, Merck); ammonium acetate (≥99%, Sigma-Aldrich); pyridine (99%+, ACROS ORGANICS); ethanol, tetrahydrofuran, and chloroform (HPLC, Tianjin Fuyu Fine Chemical Co., Ltd.); water (Watson's).

[0100] Nine carbonyl compound-DNPH derivative standards: formaldehyde-2,4-dinitrophenylhydrazone (>98%), acetaldehyde-2,4-dinitrophenylhydrazone (>98%), acetone-2,4-dinitrophenylhydrazone (>98%), propionaldehyde-2,4-dinitrophenylhydrazone (>97%), crotonaldehyde-2,4-dinitrophenylhydrazone (>98%), 2-butanone-2,4-dinitrophenylhydrazone (>98%), butyraldehyde-2,4-dinitrophenylhydrazone (>96%) and valeraldehyde-2,4-dinitrophenylhydrazone (>98%) were purchased from Tokyo Chemical Industry Co., Ltd., Japan; acrolein-2,4-dinitrophenylhydrazone was purchased from Tanmo Quality Inspection-Standard Material Center (99.6%, standard).

[0101] Seven carbonyl compound standard monomers: glyoxal (39.9% aqueous solution, Chem Service); glyceraldehyde (95%, standard, TRC); dihydroxyacetone (98.5%, standard sample, Tanmo Quality Inspection-Standard Material Center); 2,3-butanedione (≥99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); 2,3-pentanedione (97%, Beijing Bailingwei Technology Co., Ltd.); 2-hydroxypropanal (1 M aqueous solution, Sigma-Aldrich); methylglyoxal (1 mg / mL acetonitrile solution, CHIRON).

[0102] Two carbonyl compounds-DNPH derivatives were used as internal standards: formaldehyde-2,4-dinitrophenylhydrazone-d3 (98% atom) and acetaldehyde-2,4-dinitrophenylhydrazone-d3 (98% atom) (standard products, IsoReag).

[0103] (2) Instrument working conditions

[0104] The chromatographic column was a Waters ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm); the column temperature was 30°C; the injection volume was 3 μL; the flow rate was 0.25 mL / min; the mobile phase A was a 0.2 mM ammonium acetate aqueous solution; the mobile phase B was acetonitrile; the elution gradient program was as follows: mobile phase B increased from 20% to 40% within 0-2 min, from 40% to 50% within 2-12 min, from 50% to 95% within 12-20 min and maintained for 3 min, and then decreased to 20% within 0.1 min and maintained for 7 min; the ion source was the heated electrically heated spray ionization (HESI) source of the QExactive mass spectrometer system in negative ion mode; the sheath gas flow rate was 20 L / min; the auxiliary gas flow rate was 5 L / min; the sweep gas flow rate was 100 mL / min. The gas flow rate was 0 L / min; the spray voltage was -3.0 kV; the capillary heating temperature was 220°C; the auxiliary gas heating temperature was 320°C; the S-lens parameters were 50 (0-11 min) and 80 (11-30 min), respectively; the scanning mode was parallel reaction monitoring (PRM) mode, and the monitored ion pair information is shown in Table 1; the resolution was 17500; and the automatic gain control (AGC target) was 1×10 5 ;The maximum ion injection time (Maximuminjecttime) is 50ms.

[0105] Table 1: Internal standards, retention times, precursor ions, product ions, and collision energies of each derivative

[0106]

[0107] (3) Preparation of DNPH-phosphate reagent

[0108] Accurately weigh 1.0 g of 2,4-dinitrophenylhydrazine into a container, add 1 mL of phosphoric acid and 50 mL of acetonitrile, mix well and set aside to prepare a 20 mg / mL DNPH-phosphoric acid solution.

[0109] (4) Preparation of internal standard solution and internal standard primary solution

[0110] ① Internal standard stock solution: Accurately weigh 10.0 mg of formaldehyde-2,4-dinitrophenylhydrazone-d3 and acetaldehyde-2,4-dinitrophenylhydrazone-d3 respectively into a 10 mL volumetric flask, dilute to the mark with acetonitrile to prepare a 1 mg / mL internal standard stock solution.

[0111] ②Internal standard primary solution: Accurately transfer 1 mL of internal standard stock solution into a 25 mL volumetric flask and dilute to the mark with acetonitrile to prepare a 40 μg / mL internal standard primary solution.

[0112] ③ Internal standard solution: Accurately transfer 1 mL of the first-level internal standard solution into a 100 mL volumetric flask and dilute to the mark with acetonitrile to prepare a 400 ng / mL internal standard solution.

[0113] (5) Preparation of standard working solution

[0114] ① Preparation of the glyoxal-DNPH compound: First, accurately weigh 0.4 g of DNPH into a container. Then, add 2 mL of concentrated H₂SO₄ to the DNPH. While stirring, add the mixture to 3 mL of water, followed by 10 mL of 95% ethanol to prepare a DNPH solution. Add 2 g of a 39.9% aqueous acetaldehyde solution to 15 mL of the freshly prepared DNPH solution. Filter the glyoxal-2,4-dinitrophenylhydrazone through Whatman 41 paper and recrystallize it from 100 mL of a 20:1 (volume ratio) tetrahydrofuran-chloroform solution. After recrystallization, filter the product through a glass fiber filter, wash with cold acetonitrile, and dry to obtain the glyoxal-DNPH compound.

[0115] ② Preparation of the first mixed standard stock solution: Accurately weigh 20 mg, 60 mg, 50 mg, 30 mg, 10 mg, 5 mg, 8 mg, 6 mg, 5 mg, and 5 mg of the DNPH derivatives of 10 carbonyl compounds, namely formaldehyde, acetaldehyde, acetone, propionaldehyde, acrolein, crotonaldehyde, 2-butanone, butyraldehyde, valeraldehyde, and glyoxal, into a 10 mL volumetric flask, and dilute to the mark with acetonitrile to prepare the first mixed standard stock solution.

[0116] ③ Preparation of single-labeled solutions: Accurately weigh 10 mg, 50 mg, 50 mg, and 50 mg of glyceraldehyde, dihydroxyacetone, 2,3-butanedione, and 2,3-pentanedione into four 10 mL volumetric flasks, and dilute to the mark with acetonitrile to prepare single-labeled solutions of glyceraldehyde, dihydroxyacetone, 2,3-butanedione, and 2,3-pentanedione.

[0117] ④ Preparation of standard stock solutions of DNPH derivatives of glyceraldehyde, dihydroxyacetone, 2,3-butanedione, 2,3-pentanedione, 2-hydroxypropionaldehyde and methylglyoxal: Accurately pipette 1.0 mL, 0.2 mL, 2.0 mL, 1.0 mL, 0.1 mL and 1.0 mL of single-standard solution of glyceraldehyde, single-standard solution of dihydroxyacetone, single-standard solution of 2,3-butanedione, single-standard solution of 2,3-pentanedione, 2-hydroxypropionaldehyde solution (1 mol / L aqueous solution) and methylglyoxal solution (1 mg / mL methanol solution) into 6 25 mL volumetric flasks, and add 2.0 mL of The DNPH-phosphoric acid solutions were mixed evenly and reacted at room temperature for 40 minutes. After the reaction, 2.0 mL of pyridine was added to each of the six volumetric flasks, and the volumes were adjusted to the mark with acetonitrile to prepare standard stock solutions of glyceraldehyde DNPH derivatives, dihydroxyacetone DNPH derivatives, 2,3-butanedione DNPH derivatives, 2,3-pentanedione DNPH derivatives, 2-hydroxypropionaldehyde DNPH derivatives, and methylglyoxal DNPH derivatives.

[0118] ⑤ Preparation of mixed standard stock solution: Accurately pipette 0.4 mL, 1 mL, 5 mL, 25 mL, 25 mL, and 10 mL of the first mixed standard stock solution, the standard stock solution of dihydroxyacetone DNPH derivatives, the standard stock solution of 2,3-butanedione DNPH derivatives, the standard stock solution of 2,3-pentanedione DNPH derivatives, the standard stock solution of 2-hydroxypropionaldehyde DNPH derivatives, and the standard stock solution of methylglyoxal DNPH derivatives into a 100 mL volumetric flask, and dilute to the mark with acetonitrile to prepare the mixed standard stock solution.

[0119] ⑥ Preparation of standard working solutions of 15 carbonyl compound DNPH derivatives: Accurately pipette 5 μL, 10 μL, 20 μL, 50 μL, 100 μL, 200 μL, 500 μL, and 1000 μL of the mixed standard stock solution into 8 25 mL volumetric flasks, add 250 μL of the internal standard primary solution to each flask, and dilute to the scale with acetonitrile to prepare 8 standard working solutions of different concentrations.

[0120] ⑦ Preparation of standard working solutions of glyceraldehyde DNPH derivatives: Accurately pipette 0.25 mL of the standard stock solution of glyceraldehyde DNPH derivatives into a 25 mL volumetric flask and dilute to the mark with acetonitrile to prepare the standard primary stock solution of glyceraldehyde DNPH derivatives. Accurately pipette 5 μL, 10 μL, 20 μL, 50 μL, 100 μL, 200 μL, 500 μL, and 1000 μL of the standard primary stock solution of glyceraldehyde DNPH derivatives into eight 25 mL volumetric flasks, respectively, add 250 μL of the internal standard primary solution, and dilute to the mark with acetonitrile to prepare eight standard working solutions of glyceraldehyde at different concentrations.

[0121] (6) Sample information

[0122] Four types of heated cigarette samples with different flavors and heating methods were collected from the market. Detailed information is shown in Table 2.

[0123] Table 2: HTPs sample information

[0124]

[0125] (7) Sample pretreatment

[0126] Apply 1.5 mL of a 20 mg / mL DNPH-2% phosphoric acid / acetonitrile solution evenly to the roughened surface of a 44 mm diameter Cambridge filter disc, place on a grid tray, and allow to air dry in a fume hood. Once dry, apply another 1.5 mL of a 20 mg / mL DNPH-2% phosphoric acid / acetonitrile solution and allow to air dry in a fume hood. Before smoking, apply another 1.5 mL of a 20 mg / mL DNPH-2% phosphoric acid / acetonitrile solution to the dried filter disc to create a pretreated filter disc. Place the pretreated filter disc at the smoke inlet with the roughened surface facing the smoke inlet. Place another untreated 44 mm Cambridge filter disc directly behind it, allowing cigarette smoke to pass through the pretreated filter disc and then the other filter disc.

[0127] like Figure 2 As shown, two cigarettes were inserted into a smoking machine and smoked using the HCl puffing mode (puff volume / puff interval / duration: 55 mL / 30 seconds / 2 seconds, filter vents left open). After the puffs were completed, the trap was removed and allowed to stand for 40 minutes to allow the carbonyl compounds in the smoke to fully react with the DNPH. The Cambridge filter then captured the carbonyl compounds in the smoke of the cigarettes being tested. A blank sample (no cigarette smoke) was included in each puff cycle to eliminate interference from carbonyl compounds in the air.

[0128] Transfer the Cambridge filter disc to a 100 mL Erlenmeyer flask, accurately add 50 mL of acetonitrile, and mechanically shake for 10 minutes to obtain the extract. Filter an appropriate amount of the extract through a 0.22 μm organic phase filter to obtain a filtrate. Add 30 μL of pyridine and 1 mL of internal standard solution to 50 μL of the filtrate and mix thoroughly to obtain the sample solution.

[0129] (8) Sample measurement

[0130] The standard working solutions of the 15 carbonyl compound DNPH derivatives and the standard working solution of the glyceraldehyde DNPH derivatives prepared in step (5) were analyzed by ultra-performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry, and the ratio of the peak area of ​​each carbonyl compound DNPH derivative in the standard working solutions of the 15 carbonyl compound DNPH derivatives and the standard working solutions of the glyceraldehyde DNPH derivatives to the peak area of ​​the internal standard was used as the ordinate, and the mass concentration of each carbonyl compound DNPH derivative was used as the abscissa to perform regression analysis to establish a calibration curve for the 16 carbonyl compound DNPH derivatives.

[0131] The sample solution prepared in step (7) is analyzed by ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry to obtain the ratio of the peak area of ​​each carbonyl compound DNPH derivative to the peak area of ​​the internal standard, and each obtained ratio is substituted into the calibration curve to obtain the measured concentration of each carbonyl compound DNPH derivative in the sample solution.

[0132] The release amount of each carbonyl compound in cigarette smoke was obtained by substituting the measured concentration of the DNPH derivative of each carbonyl compound in the sample solution into the following formula:

[0133]

[0134] in:

[0135] Y is the release of each carbonyl compound in cigarette smoke, expressed in micrograms per cigarette (μg / cig);

[0136] X is the measured concentration of the DNPH derivative of each carbonyl compound in the sample solution, expressed in micrograms per milliliter (μg / mL);

[0137] V is the volume of the sample solution in milliliters (mL);

[0138] 1000 is the dilution factor;

[0139] n is the number of cigarettes smoked, in cig;

[0140] M1 is the molar mass of each carbonyl compound in grams per mole (g / mol);

[0141] M2 is the molar mass of each carbonyl compound in grams per mole (g / mol).

[0142] Since glyceraldehyde will generate dihydroxyacetone, the actual release of dihydroxyacetone needs to be calculated by subtracting the calculated release of glyceraldehyde from the calculated release of dihydroxyacetone.

[0143] (9) Method validation

[0144] ①Linear range, detection limit, quantification limit, intra-day precision and inter-day precision

[0145] Eight different concentrations of 15 standard working solutions of DNPH derivatives of carbonyl compounds and a standard working solution of DNPH derivatives of glyceraldehyde were analyzed using ultra-performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry. Quantification was performed using the internal standard method. Regression analysis was performed with the peak area ratio of each DNPH derivative to the internal standard as the ordinate (Y) and the mass concentration of each DNPH derivative as the abscissa (X, μg / mL). Regression equations and correlation coefficients were obtained for the 16 DNPH derivatives of carbonyl compounds. Six intra-day and inter-day replicates were performed on heated cigarette smoke samples, and intra-day and inter-day precisions were calculated. The standard solution with the lowest concentration within the linear range was injected 10 times, and the standard deviation (SD) of the corresponding concentration was calculated. Three times (3SD) and ten times (10SD) of the obtained standard deviation were used as the limit of detection (LOD) and limit of quantification (LOQ), respectively.

[0146] Table 3: Working curve and correlation coefficient, detection limit, quantification limit, intra-day precision and inter-day precision of the method Note: Y—peak area ratio; X—concentration of DNPH derivatives of carbonyl compounds, μg / mL.

[0147] The results showed that the standard curves of 16 carbonyl compounds had a good linear relationship, r 2 The LOD and LOQ for the 16 carbonyl compounds ranged from 0.16 to 2.93 μg / cg and 0.51 to 9.76 μg / cg, respectively. The intra-day and inter-day precisions for the 16 carbonyl compounds ranged from 0.87% to 5.81% and 1.08% to 13.81%, respectively. Therefore, this method exhibits high selectivity, sensitivity, and precision, making it suitable for quantitative analysis.

[0148] ②Recovery rate and precision

[0149] A typical cigarette sample was selected and processed according to the method in (7) to obtain a sample solution. The standard working solution was added to the sample solution at three addition levels: low, medium, and high. Each sample was measured six times, and the recovery and precision of each addition level were calculated. The recovery calculation formula is as follows:

[0150]

[0151] in:

[0152] R is the recovery rate, in %;

[0153] C_found is the concentration of the sample solution after spiked, in μg / cig;

[0154] C_blank is the concentration of the sample solution before spiking, in μg / cig;

[0155] C_added is the added amount, in μg / cig.

[0156] Table 4: Recovery and precision of 16 carbonyl compounds in cigarette smoke

[0157]

[0158] The results showed that the recoveries of 16 carbonyl compounds ranged from 82.28% to 112.98%, with relative standard deviations (RSDs) ranging from 0.60% to 13.00%, indicating that the method had good accuracy and precision.

[0159] (10) Actual sample analysis

[0160] In this example, the four selected cigarette samples were used to measure the release of 16 carbonyl compounds in cigarette smoke under the intensive inhalation (HCI) mode. The test results are shown in Table 5.

[0161] Table 5: Release of 16 carbonyl compounds in smoke from four heated cigarettes under HCl smoking mode (μg / cig)

[0162]

[0163]

[0164] Note: “-” indicates that the amount in cigarette smoke is below the limit of quantification and not detected.

[0165] The results showed that the release of 2-hydroxypropionaldehyde, 2,3-butanedione, acetaldehyde and methylglyoxal in heated cigarette smoke was high, and the release of 2-hydroxypropionaldehyde in some samples exceeded 1000 μg / cig. #The release levels of the other 14 carbonyl compounds in the samples were all below 1 # , 3 # and 4 # Sample, which may be with 2 # This is related to the lower heating temperature of the sample.

[0166] Example 2: Optimization of the assay method

[0167] In order to investigate the influence of the determination method on the determination results, the HPLC method was used to determine the content of formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and butyraldehyde in cigarette smoke. Except for the determination method, the rest of the methods were the same as in Example 1.

[0168] See also Figure 3 The chromatographic peaks from left to right are formaldehyde (FA), acetaldehyde (AA), acetone (AC), acrolein (ACR), PA (propionaldehyde), crotonaldehyde (CA), 2-butanone (MEK), and butyraldehyde (BA). When measured using the HPLC method, the formaldehyde and propionaldehyde peaks are preceded by interfering peaks, making accurate quantification impossible.

[0169] The standard working solutions of 16 carbonyl compounds ( Figure 4 ) and sample solution ( Figure 5 ) were measured, among which, Figure 4 a From top to bottom, they are formaldehyde (FA), acetaldehyde (AA), acetone (AC), acrolein (ACR), PA (propionaldehyde), crotonaldehyde (CA), 2-butanone (MEK) and butyraldehyde (BA). Figure 4 b From top to bottom: glyceraldehyde (GLA), dihydroxyacetone (DHA), 2-hydroxypropionaldehyde (HPA), glyoxal (GO), valeraldehyde (VA), methylglyoxal (MG), 2,3-butanedione (DA) and 2,3-pentanedione (PD); Figure 5 a and Figure 4 a. Figure 5 b and Figure 4 The order of the identifiers of b is the same.

[0170] The results showed that the peak shapes of the standard working solutions and sample solutions of the 16 carbonyl compounds were good, and the peak positions were relatively matched, which enabled the separation of the 16 carbonyl compounds with high selectivity and good sensitivity.

[0171] HPLC and ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap mass spectrometry were used to analyze 1 # and 2 # The contents of formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and butyraldehyde in the samples were determined.

[0172] Table 6: Measured values ​​of 8 carbonyl compounds in cigarette smoke using different determination methods (μg / cig)

[0173]

[0174]

[0175] The results showed that, in addition to formaldehyde, the HPLC method also measured significantly higher levels of crotonaldehyde and butyraldehyde than the ultra-high performance liquid chromatography-quadrupole orbitrap mass spectrometry method. This was confirmed to be due to interference peaks between the chromatographic peaks of crotonaldehyde and butyraldehyde in the HPLC method. Therefore, ultra-high performance liquid chromatography-quadrupole orbitrap mass spectrometry was selected as the determination method.

[0176] Example 3: Optimization of derivatization conditions

[0177] (1) Phosphoric acid concentration (V / V)

[0178] In order to investigate the effect of phosphoric acid concentration on the derivatization reaction, the volume percentage concentration of phosphoric acid was set to 0.5%, 1%, 1.5%, 2%, 3% and 4%. Except for the phosphoric acid concentration, the rest of the method was the same as in Example 1.

[0179] Table 7: Measured values ​​of 16 carbonyl compounds in cigarette smoke at different phosphoric acid concentrations (μg / cig)

[0180]

[0181] The results show that as the phosphoric acid concentration increases, the measured values ​​of various carbonyl compounds show a trend of first increasing and then decreasing, and the measured value is the highest when the phosphoric acid concentration is 2%. Therefore, the phosphoric acid concentration is selected to be 1.5-2%, preferably 2%.

[0182] (2) Dosage and application method of DNPH-phosphoric acid solution

[0183] In order to investigate the effect of the amount and application method of DNPH-phosphoric acid solution on the determination results of each carbonyl compound, the amount of DNPH-phosphoric acid solution applied was set to 3mL, 3.5mL, 4mL, 4.5mL, 5mL, 5.5mL, and 6mL, respectively. During the application process, since the Cambridge filter disc cannot absorb too much DNPH-phosphoric acid solution at one time, the volume of DNPH-phosphoric acid solution applied to the Cambridge filter disc each time is not more than 2mL. Except for the amount and application method of DNPH-phosphoric acid solution, the other methods are all referred to Example 1. The amount and application method are shown in Table 8, and the experimental results are shown in Table 9.

[0184] Table 8: Dosage and application method of DNPH-phosphoric acid solution

[0185]

[0186]

[0187] Table 9: Measured values ​​of 16 carbonyl compounds in cigarette smoke at different DNPH-phosphoric acid solution dosages and application methods (μg / cig)

[0188]

[0189] Results show that varying the amount and application method of DNPH-phosphoric acid solution has little effect on the detection results of monocarbonyl compounds. However, for dicarbonyl compounds, particularly 2,3-pentanedione, the measured values ​​increase with increasing DNPH-phosphoric acid solution dosage. However, considering the impact of solvent usage during the puffing process, a total amount of 4-5 mL of DNPH-phosphoric acid solution is preferred. Furthermore, the DNPH-phosphoric acid solution should be applied to the filter and dried, and then applied again before smoking the cigarette, with the volume ratio of the three applications being 1:1:1. More preferably, 1.5 mL of DNPH-phosphoric acid solution should be applied separately, dried, and then a further 1.5 mL of DNPH-phosphoric acid solution should be applied before smoking the cigarette. This ensures complete reaction of the carbonyl compounds while preventing excess solvent from contaminating the smoking machine passageway during the cigarette puffing process.

[0190] (3) Reaction time

[0191] In order to investigate the effect of reaction time on the derivatization reaction, the reaction time was set to 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, respectively. Except for the reaction time, the rest of the method was the same as in Example 1.

[0192] Table 10: Measured values ​​of 16 carbonyl compounds in cigarette smoke at different reaction times (μg / cig)

[0193]

[0194]

[0195] The results show that when the reaction time is 40 minutes, the measured values ​​of each carbonyl compound tend to be stable, indicating that the carbonyl compound has reacted completely. Therefore, after the aspiration is completed, it is selected to stand for 40-60 minutes, preferably 40 minutes.

[0196] Example 4: Optimization of liquid phase conditions

[0197] (1) Selection of chromatographic column

[0198] To investigate the effect of the chromatographic column on separation performance, a Waters ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm), a Waters ACQUITY UPLC BEH Shield RP18 column (50 mm × 2.1 mm, 1.7 μm), a Thermo Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 μm), and an Agilent InfinityLab Poroshell 120-C18 column (100 mm × 2.1 mm, 2.7 μm) were selected as research objects. Except for the chromatographic column, the remaining methods were the same as in Example 1.

[0199] See also Figure 6 , the chromatographic peak of acetaldehyde on the Waters ACQUITY UPLC BEH Shield RP18 column has a front tailing phenomenon. Figure 7 and Figure 8 , Hypersil GOLD C18 column ( Figure 7 ) and InfinityLabPoroshell120-C18( Figure 8 ) have analysis times of 75 min and 50 min respectively, which are longer.

[0200] Therefore, the analytical column finally selected was a Waters ACQUITY UPLC BEH C18 column.

[0201] (2) The amount of ammonium acetate added to the mobile phase

[0202] In order to investigate the effect of the amount of ammonium acetate added on the response of each carbonyl compound, the amount of ammonium acetate added was set to 0 mM, 0.1 mM, 0.2 mM, 0.3 mM, and 0.5 mM, respectively. Except for the amount of ammonium acetate added, the rest of the method was the same as in Example 1.

[0203] Table 11: Peak height response values ​​of four carbonyl compounds at different ammonium acetate concentrations

[0204]

[0205] The results showed that as the amount of ammonium acetate added increased, the peak height responses of the carbonyl compounds first increased and then decreased, and the response was highest when the concentration of ammonium acetate was 0.2 mM. Therefore, the preferred concentration of ammonium acetate was 0.2 mM.

[0206] Example 5: Optimization of the capture method of carbonyl compounds in cigarette smoke

[0207] In order to investigate the effect of the capture method on the determination results, the DNPH-filter capture method, the DNPH-solution capture method and the CX-572 capture method were selected. Except for the capture method, the other methods were the same as those in Example 1.

[0208] Table 12: Measured values ​​of 16 carbonyl compounds in cigarette smoke under different capture methods (μg / cig)

[0209]

[0210] Note: “-” indicates that the amount in cigarette smoke is below the limit of quantification and not detected.

[0211] The results showed that the DNPH solution method produced significantly lower levels of glyoxal, methylglyoxal, 2,3-butanedione, and 2,3-pentanedione than the DNPH filter-disc trap method and the CX-572 trap method. This suggests that the DNPH solution trap method is incomplete in capturing dicarbonyl compounds. The CX-572 trap method, on the other hand, requires a specialized trap and elution of carbonyl compounds using a CS2-methanol solution. This is cumbersome and requires high operator skill. In summary, the DNPH filter-disc trap method is the preferred method for capturing carbonyl compounds in cigarette smoke.

[0212] Although the present invention has been illustrated and described with reference to certain preferred embodiments thereof, it should be understood by those skilled in the art that the above description is provided as a further detailed description of the present invention in conjunction with specific embodiments thereof, and that the specific implementation of the present invention is not limited to these descriptions. Those skilled in the art may make various changes in form and details, including simple deductions or substitutions, without departing from the spirit and scope of the present invention.

Claims

1. A method for determining the content of multiple carbonyl compounds in cigarette smoke, characterized in that: The carbonyl compounds include formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, glyceraldehyde, dihydroxyacetone, 2-hydroxypropionaldehyde, glyoxal, valeraldehyde, methylglyoxal, 2,3-butanedione and 2,3-pentanedione, and the determination method comprises the following steps: S1: smoking a cigarette and capturing carbonyl compounds in the cigarette smoke using a filter disc coated with DNPH-phosphoric acid, wherein the carbonyl compounds in the cigarette smoke react with the DNPH on the filter disc under acidic conditions, thereby obtaining a filter disc with the carbonyl compounds thereon; S2: placing the filter disc with the carbonyl compound into a container and adding an extractant to obtain an extract, then filtering the extract through a microporous filter membrane to obtain a filtrate, and adding pyridine and an internal standard solution to the filtrate to obtain a sample solution; S3: Detecting the sample solution using ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry to obtain the concentration of each carbonyl compound in the cigarette smoke to be detected.

2. The assay method according to claim 1, wherein The step S3 comprises: Establish a calibration curve; Obtaining the ratio of the peak area of ​​each carbonyl compound DNPH derivative in the sample solution to the peak area of ​​the internal standard using ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry; Obtaining the measured concentration of each carbonyl compound DNPH derivative in the sample solution based on the calibration curve and each of the ratios; The release amount of each carbonyl compound in cigarette smoke was obtained based on the measured concentration of the DNPH derivative of each carbonyl compound and the following calculation formula: in: Y is the release of each carbonyl compound in cigarette smoke, expressed in micrograms per cigarette (μg / cig); X is the measured concentration of each carbonyl compound DNPH derivative in the sample solution, in micrograms per milliliter (μg / mL); V is the volume of the sample solution, in milliliters (mL); 1000 is the dilution factor; n is the number of cigarettes smoked, in cig; M1 is the molar mass of each carbonyl compound in grams per mole (g / mol); M2 is the molar mass of each carbonyl compound in grams per mole (g / mol).

3. The assay method according to claim 2, wherein Describing the establishment of a calibration curve comprises: preparing standard working solutions: preparing mixed standard stock solutions of DNPH derivatives of 15 carbonyl compounds and standard stock solutions of DNPH derivatives of glyceraldehyde, respectively, filling the mixed standard stock solution and the standard stock solution of the DNPH derivative of glyceraldehyde into two or more containers in different volumes, adding internal standard primary solution to each container, and then adjusting the volume with acetonitrile to prepare standard working solutions with different concentrations, wherein the 15 carbonyl compounds include formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, dihydroxyacetone, 2-hydroxypropionaldehyde, glyoxal, valeraldehyde, methylglyoxal, 2,3-butanedione, and 2,3-pentanedione; Drawing a calibration curve: The standard working solutions of different concentrations were analyzed using ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry, and the contents of 16 carbonyl compounds were quantified using the internal standard method. Regression analysis was performed with the ratio of the peak area of ​​each carbonyl compound DNPH derivative to the peak area of ​​the internal standard as the ordinate and the mass concentration of each carbonyl compound DNPH derivative as the abscissa to establish a calibration curve for the 16 carbonyl compound DNPH derivatives.

4. The assay method according to claim 3, wherein The preparation of the standard working solution comprises: preparing a first mixed standard stock solution of 10 DNPH derivatives of carbonyl compounds: mixing the DNPH derivatives of each of the 10 carbonyl compounds, namely, formaldehyde, acetaldehyde, acetone, propionaldehyde, acrolein, crotonaldehyde, 2-butanone, butyraldehyde, valeraldehyde, and glyoxal, in a mass ratio of 20:60:50:30:10:5:8:6:5:5, and diluting the mixture to a predetermined volume with acetonitrile to prepare a first mixed standard stock solution of the 10 DNPH derivatives of carbonyl compounds; Preparing a mixed standard stock solution of 15 DNPH derivatives of carbonyl compounds: mixing the first mixed standard stock solution with standard stock solutions of the DNPH derivatives of each of the five carbonyl compounds, namely, dihydroxyacetone, 2,3-butanedione, 2,3-pentanedione, 2-hydroxypropionaldehyde, and methylglyoxal, in a volume ratio of 0.4:1:5:25:25:10, and diluting the solution to a predetermined volume with acetonitrile to prepare a mixed standard stock solution of the 15 DNPH derivatives of carbonyl compounds; and / or The standard stock solution of the glyceraldehyde DNPH derivative is an acetonitrile solution of the glyceraldehyde DNPH derivative.

5. The assay method according to claim 4, wherein The internal standard primary solution is a mixed solution of formaldehyde-2,4-dinitrophenylhydrazone-d3 and acetaldehyde-2,4-dinitrophenylhydrazone-d3. Preferably, the concentration of the internal standard primary solution is 35 μg / mL to 45 μg / mL, more preferably 40 μg / mL.

6. The assay method according to claim 1, wherein The internal standard solution is a mixed solution of formaldehyde-2,4-dinitrophenylhydrazone-d3 and acetaldehyde-2,4-dinitrophenylhydrazone-d3. The concentration of the internal standard solution is preferably 375 ng / mL to 425 ng / mL, more preferably 400 ng / mL.

7. The assay method according to claim 1, wherein The filter disc is a Cambridge filter disc or a glass fiber filter disc, and / or the extractant is acetonitrile; and / or the filter disc has a rough surface, and the DNPH-phosphoric acid is applied on the rough surface of the filter disc.

8. The assay method according to claim 1, wherein The ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry instrument adopts one or a combination of the following detection conditions: Mobile phase A was 0.2 mM ammonium acetate aqueous solution; The chromatographic column used was a Waters ACQUITY UPLC BEH C18 column with specifications of 50 mm × 2.1 mm, a particle size of 1.7 μm, and a column temperature of 30 °C; The injection volume was 3 μL, the flow rate was 0.25 mL / min, the scanning mode was parallel reaction monitoring mode (PRM), the ion source was heated electrospray ionization source (HESI), and the negative ion mode was used; Mobile phase B is acetonitrile; and / or The elution gradient was as follows: mobile phase B increased from 20% to 40% in 0-2 minutes, increased from 40% to 50% in 2-12 minutes, increased from 50% to 95% in 12-20 minutes and maintained for 3 minutes, decreased to 20% in 0.1 minutes and maintained for 7 minutes.

9. The assay method according to claim 7, wherein A blank control group is also provided in step S1, which is used to eliminate interference from carbonyl compounds in the air; and / or step S1 includes: applying a reagent containing DNPH-phosphoric acid to a filter disc twice and drying it, and applying the reagent containing DNPH-phosphoric acid to the filter disc again before smoking the cigarette; preferably, the filter disc applied with DNPH-phosphoric acid is placed on the smoke inlet side with the rough surface of the filter disc facing the smoke inlet side, and another untreated filter disc is placed closely behind it, so that the cigarette smoke passes through the filter disc applied with DNPH-phosphoric acid and the other filter disc in sequence, and preferably the diameter of each filter disc is 44 mm; preferably, the cigarette is smoked using an HCl smoking mode; preferably, after smoking is completed, the filter disc is left to stand for 40 minutes to 60 minutes, more preferably for 40 minutes, to allow the carbonyl compounds in the smoke to fully react with the DNPH on the filter disc.

10. The measuring method according to claim 9, wherein The reagent containing DNPH-phosphoric acid is a DNPH-2% phosphoric acid / acetonitrile solution with a concentration of 20 mg / mL. Preferably, the total amount of DNPH-2% phosphoric acid / acetonitrile solution applied to the filter disc is 4 mL to 5 mL, and the volume ratio of the reagent containing DNPH-phosphoric acid applied three times is 1:1:

1. More preferably, 1.5 mL of the reagent containing DNPH-phosphoric acid is applied to the filter disc successively, and each is dried in the dark, and 1.5 mL of the reagent containing DNPH-phosphoric acid is applied to the filter disc again before smoking the cigarette.

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