A method and kit for detecting multiple tobacco exposure markers in urine

Abstract

The present invention provides a detection method and a detection kit for multiple tobacco exposure markers in urine, and belongs to the technical field of chemical detection. The detection method provided by the present invention performs enzymatic hydrolysis on urine and then extracts it using a solid medium liquid-liquid extraction column to obtain a test liquid, and then performs liquid chromatography-high-resolution mass spectrometry detection on the test liquid, which can reduce costs, and at the same time has high detection sensitivity, precision and accuracy, a wide linear range, and can accurately detect 14 tobacco exposure markers at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and specifically relates to a detection method and a detection kit for multiple tobacco exposure markers in urine. Background Art

[0002] Nicotine is the main alkaloid in tobacco and is also the substance that causes tobacco dependence. The metabolites of nicotine in the human body are mainly cotinine (COT), trans-3'-hydroxycotinine (OHCOT), nornicotine (NNIC), norcotinine (NCOT), nicotine nitrogen oxide (NNO), cotinine nitrogen oxide (CNO) and three glucuronide conjugates: nicotine glucuronide (NIC-G), cotinine glucuronide (COT-G) and trans-3'-hydroxycotinine glucuronide (OHCOT-G). After the three glucose conjugates are hydrolyzed into free states by β-glucuronidase, the sum of the concentrations of the above 7 nicotine and their metabolites is called total nicotine equivalent, which can more accurately describe and evaluate the nicotine intake level than the traditional method of only using the sum of NIC, COT and OHCOT. Neonicotinoids (ANAT) and anabasine (ANAB) are minor alkaloids in tobacco. E-cigarettes and nicotine replacement therapy usually directly add nicotine and do not contain minor alkaloids. Therefore, they can be used as specific detection indicators to distinguish between people who use traditional cigarettes and e-cigarettes, and to reflect the smoking cessation compliance of people who use nicotine replacement therapy. Tobacco-specific nitrosamines (TSNAs) are substances unique to tobacco, including 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol (NNAL), N'-nitrosonornicotine (NNN), N'-nitrosoneonicotinoids (NAT) and N'-nitrosoanaabasine (NAB). NNK and NNN are listed as Class I carcinogens by IARC (International Agency for Cancer Research). NAB has been found to have carcinogenic properties in animal experiments. NNAL can be used as a biomarker to distinguish occasional smokers and people exposed to secondhand smoke. Therefore, measuring these 14 tobacco exposure markers helps to understand the real smoking situation of the population, helps to assess the nicotine load level of the population, and provides a scientific and technical basis for studying the mechanism of smoking harm and formulating relevant tobacco control policies.

[0003] Commonly used methods for detecting tobacco exposure markers in urine at home and abroad include gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Most tobacco exposure markers in urine are non-volatile organic substances. When measured by gas chromatography-mass spectrometry, they need to undergo complex derivatization before they can be measured. Therefore, liquid chromatography-mass spectrometry is more suitable for the detection of tobacco exposure markers in human urine. Pretreatment methods for the detection of tobacco exposure markers include direct dilution, liquid-liquid extraction (LLE) and solid phase extraction (SPE). The direct dilution method is usually diluted at a large multiple due to the serious matrix effect of urine, which is not conducive to the detection of samples from non-smoking populations. LLE is usually cumbersome, time-consuming and labor-intensive, easily interfered by the urine matrix, and prone to emulsification. The pretreatment process of SPE requires activation, equilibration and elution of the solid phase extraction column. The steps are relatively cumbersome and the amount of organic solvent used is relatively large. Tobacco-specific nitrosamines have very low concentrations in urine (approximately pg / mL), so molecular imprinting extraction columns and solid phase extraction are often used in combination during pretreatment, which has a high detection cost. In addition, the existing technology does not have a method for simultaneously detecting 14 tobacco exposure markers. Summary of the invention

[0004] The purpose of the present invention is to provide a method and a detection kit for detecting multiple tobacco exposure markers in urine. The detection method provided by the present invention has high sensitivity and can accurately detect 14 tobacco exposure markers at the same time.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for detecting multiple tobacco exposure markers in urine, comprising the following steps:

[0007] (1) Mixing urine with a buffer solution and an enzyme for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0008] (2) mixing the enzymatic hydrolyzate obtained in step (1) with an alkali solution and a mixed internal standard solution to obtain a mixed solution;

[0009] (3) extracting the mixed solution obtained in step (2) using a solid medium liquid-liquid extraction column, eluting with an eluent to obtain an eluent, blowing the eluent with nitrogen, and then adding water to redissolve it to obtain a test solution;

[0010] (4) The test solution obtained in step (3) is subjected to liquid chromatography-high resolution mass spectrometry to obtain the content of tobacco exposure markers in urine.

[0011] Preferably, the buffer solution in step (1) comprises an acetic acid-ammonium acetate buffer solution or a formic acid-ammonium formate buffer solution; and the pH value of the buffer solution is 5-6.

[0012] Preferably, in step (1), the volume ratio of urine to buffer solution is (2-2.5):1.

[0013] Preferably, the enzyme in step (1) comprises β-glucuronidase / arylsulfatase, H-1 type β-glucuronidase or H-2 type β-glucuronidase; and the concentration of the enzyme after mixing the urine, buffer solution and enzyme is 500~3000U / 0.45mL.

[0014] Preferably, the enzymatic hydrolysis temperature in step (1) is 35-42° C., and the enzymatic hydrolysis time is ≥4 h.

[0015] Preferably, the concentration of the alkali solution in step (2) is 10-20 mol / L; and the volume ratio of the enzymatic hydrolysate to the alkali solution is (5-7):1.

[0016] Preferably, the volume ratio of the mixed solution in step (3) to the mass ratio of the solid medium liquid-liquid extraction column is 400 μL: (350-1000) mg.

[0017] Preferably, the eluent in step (3) is dichloromethane or an isopropanol-dichloromethane mixed solution; the volume ratio of isopropanol to dichloromethane in the isopropanol-dichloromethane mixed solution is: (1-10): (90-99).

[0018] Preferably, the liquid chromatography conditions for liquid chromatography-high resolution mass spectrometry detection in step (4) are as follows: mobile phase A is aqueous ammonia with a volume concentration of 0.05-0.2%, mobile phase B is methanol, the flow rate is 200-400 μL / min, gradient elution is adopted, and the gradient elution conditions are as follows: 0-5.17 min 10-20% B, 5.17-12 min 30-50% B, 12-13 min 40-100% B, 13-14 min 100% B, 14-15 min 10-20% B, the chromatographic column is a C18 chromatographic column, the length of the chromatographic column is 120-160 mm, the inner diameter of the chromatographic column is 1-3 mm, and the filler particle size is 1.5-2.0 μm;

[0019] The mass spectrometry conditions for liquid chromatography-high-resolution mass spectrometry detection in step (4) are: spray voltage of 3-4 kV, capillary temperature of 300-400°C, resolution of 10000-75000, sheath gas of 40-50 arb, auxiliary gas of 10-20 arb, auxiliary gas temperature of 300-400°C, and segmented scanning.

[0020] The present invention also provides a detection kit, comprising: alkaline solution, buffer solution, 96-well deep well plate, 96-well solid medium liquid-liquid extraction plate, eluent, mixed standard solution of 14 tobacco exposure markers, mixed deuterated internal standard solution of 14 tobacco exposure markers and enzyme.

[0021] The present invention provides a method for detecting multiple tobacco exposure markers in urine, comprising the following steps: (1) mixing urine with a buffer solution and an enzyme, performing enzymolysis, and obtaining an enzymolysis solution; (2) mixing the enzymolysis solution obtained in step (1) with an alkali solution and a mixed internal standard solution to obtain a mixed solution; (3) extracting the mixed solution obtained in step (2) with a solid medium liquid-liquid extraction column, eluting with an eluent to obtain an eluate, blowing the eluate with nitrogen, and then adding water to redissolve to obtain a test solution; (4) performing liquid chromatography-high-resolution mass spectrometry on the test solution obtained in step (3) to obtain the content of tobacco exposure markers in urine. The present invention performs enzymolysis on urine and then extracts it with a solid medium liquid-liquid extraction column to obtain a test solution, and then performs liquid chromatography-high-resolution mass spectrometry on the test solution to determine nicotine metabolites, tobacco minor alkaloids, and tobacco-specific nitrosamines together, while having good detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the chromatogram of 14 tobacco exposure markers in actual urine of Test Example 1;

[0023] Figure 2 The chromatograms of the 14 tobacco exposure marker standard solutions in comparative test example 1 are shown;

[0024] Figure 3 The chromatograms of the 14 tobacco exposure marker standard solutions in Comparative Test Example 2 are shown;

[0025] Figure 4 The chromatograms of the 14 tobacco exposure marker standard solutions in Comparative Test Example 3 are shown;

[0026] Figure 5 is the chromatogram of the 14 tobacco exposure marker standard solutions in Test Example 2;

[0027] Figure 6 The graph is a ratio of the measured values ​​of 14 tobacco exposure markers and the free state values ​​of different enzymes in Examples 3, 5, and 6 at 1000U enzyme addition (37°C);

[0028] Figure 7 It is a ratio diagram of the measured values ​​and free state values ​​of 14 tobacco exposure markers of β-glucuronidase / arylsulfatase at different enzyme addition amounts in Examples 2 to 4;

[0029] Figure 8 The graph is a ratio of the measured values ​​and free state values ​​of 14 tobacco exposure markers at different enzymatic hydrolysis times at 1000U of β-glucuronidase / arylsulfatase in Examples 3 and 7 to 9. DETAILED DESCRIPTION

[0030] The present invention provides a method for detecting multiple tobacco exposure markers in urine, comprising the following steps:

[0031] (1) Mixing urine with a buffer solution and an enzyme for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0032] (2) mixing the enzymatic hydrolyzate obtained in step (1) with an alkali solution and a mixed internal standard solution to obtain a mixed solution;

[0033] (3) extracting the mixed solution obtained in step (2) using a solid medium liquid-liquid extraction column, eluting with an eluent to obtain an eluent, blowing the eluent with nitrogen, and then adding water to redissolve it to obtain a test solution;

[0034] (4) The test solution obtained in step (3) is subjected to liquid chromatography-high resolution mass spectrometry to obtain the content of tobacco exposure markers in urine.

[0035] Unless otherwise specified, the present invention has no particular limitation on the sources of the raw materials, and commercially available products known to those skilled in the art may be used.

[0036] The detection method of the present invention is used to detect 14 tobacco exposure markers in urine, including nicotine (NIC), cotinine (COT), trans-3-hydroxycotinine (OHCOT), nornicotine (NNIC), norcotinine (NCOT), nicotine nitrogen oxide (NNO), cotinine nitrogen oxide (CNO), anabasine (ANAB), anabasine (ANAT), 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol (NNAL), 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N'-nitronornicotine (NNN), N-nitrosoneonicotinoid (NAT) and N-nitrosoanabasine (NAB).

[0037] The invention mixes urine with a buffer solution and an enzyme, performs enzymolysis, and obtains an enzymolysis solution.

[0038] In the present invention, the buffer solution preferably includes an acetic acid-ammonium acetate buffer solution or a formic acid-ammonium formate buffer solution, more preferably an acetic acid-ammonium acetate buffer solution.

[0039] In the present invention, the pH value of the buffer solution is preferably 5 to 6, more preferably 5.2 to 5.8, and further preferably 5.6 to 5.7.

[0040] In the present invention, the volume ratio of the urine to the buffer solution is preferably (2-2.5):1, more preferably (2-2.3):1.

[0041] In the present invention, the enzyme preferably includes β-glucuronidase / arylsulfatase, H-1 type β-glucuronidase or H-2 type β-glucuronidase, more preferably β-glucuronidase / arylsulfatase. In the present invention, the ratio of the enzyme activity of β-glucuronidase to arylsulfatase in the β-glucuronidase / arylsulfatase is preferably 5:1.

[0042] In the present invention, the concentration of the enzyme after the urine, buffer solution and enzyme are mixed is preferably 500-3000U / 0.45mL, more preferably 1000-2500U / 0.45mL, and further preferably 1000-2000U / 0.45mL.

[0043] In the present invention, the temperature of the enzymolysis is preferably 35-42°C, more preferably 36-40°C; the time of the enzymolysis is preferably ≥4h, more preferably 4-24h, and further preferably 12-24h; the enzymolysis is preferably carried out under light-proof conditions. The present invention controls the amount of each raw material, the enzymolysis temperature and time within the above range, so that the enzymolysis can be fully carried out and the detection accuracy can be further improved.

[0044] In the present invention, the enzymatic hydrolysis is preferably performed in a 96-well plate. The present invention uses a 96-well plate for enzymatic hydrolysis, which has high throughput and can process 96 samples at a time.

[0045] After obtaining the enzymatic hydrolyzate, the present invention mixes the enzymatic hydrolyzate with an alkali solution and a mixed internal standard solution to obtain a mixed solution.

[0046] In the present invention, the alkali solution preferably includes potassium hydroxide solution or sodium hydroxide solution.

[0047] In the present invention, the concentration of the alkali solution is preferably 10-20 mol / L, more preferably 12-18 mol / L, and further preferably 15-16 mol / L.

[0048] In the present invention, the volume ratio of the enzymatic hydrolysate to the alkaline solution is preferably (5-7):1, more preferably 6:1.

[0049] In the present invention, the internal standard substances in the mixed internal standard solution preferably include nicotine-D4 (NIC-D4), cotinine-D3 (COT-D3), trans-3'-hydroxycotinine-D3 (OHCOT-D3), nicotine nitrogen oxide-D3 (NNO-D3), cotinine nitrogen oxide-D3 (CNO-D3), neonicotinoid-D4 (ANAT-D4), anabasine-D4 (ANAB-D4), nornicotine-D4 (NNI C-D4), norcotinine-D4 (NCOT-D4), 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol-D3 (NNAL-D3), 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone-D4 (NNK-D4), N'-nitronornicotine-D4 (NNN-D4), N-nitrosoneabacin-D4 (NAT-D4), and N-nitrosoanabasine-D4 (NAB-D4).

[0050] In the present invention, the concentration of each internal standard substance in the mixed internal standard solution is preferably 10-250 ng / mL, more preferably 20-200 ng / mL.

[0051] In the present invention, the volume ratio of the enzymatic hydrolyzate to the mixed internal standard solution is preferably (5-7):1, more preferably 6:1.

[0052] The present invention preferably uses a 12-position liquid displacer to pump 2 to 10 times to uniformly mix the enzymatic hydrolyzate with the alkali solution and the mixed internal standard solution, and more preferably uses a 12-position liquid displacer to pump 3 times to uniformly mix the enzymatic hydrolyzate with the alkali solution and the mixed internal standard solution.

[0053] In the present invention, the mixing is preferably performed in a 96-well plate.

[0054] After obtaining the mixed solution, the present invention extracts the mixed solution using a solid medium liquid-liquid extraction column, elutes with an eluent to obtain an eluate, blows the eluate with nitrogen, and then adds water to redissolve it to obtain a test solution.

[0055] The present invention has no particular limitation on the source of the solid medium liquid-liquid extraction column (SLE column), and commercially available products known to those skilled in the art can be used. In an embodiment of the present invention, the solid medium liquid-liquid extraction column is from Thermo Fisher, model 400 μL, 400 mg, pH=9.

[0056] In the present invention, the volume ratio of the mixed solution to the mass ratio of the solid medium liquid-liquid extraction column is preferably 400 μL: (350-1000) mg, more preferably 400 μL: (380-600) mg, and further preferably 400 μL: 400 mg.

[0057] In the present invention, the eluent is preferably dichloromethane or an isopropanol-dichloromethane mixed solution, more preferably an isopropanol-dichloromethane mixed solution.

[0058] In the present invention, the volume ratio of isopropanol to dichloromethane in the isopropanol-dichloromethane mixed solution is preferably: (1-10): (90-99), more preferably 5:95.

[0059] In the present invention, the volume ratio of the mixed solution to the eluent is preferably 1:(2-2.5), more preferably 1:(2.1-2.3), and further preferably 1:2.25.

[0060] The present invention preferably places a 96-well extraction plate with a solid medium liquid-liquid extraction column on a 96-well collection plate, then adds a mixed solution, lets stand for 4 to 6 minutes, then adds an eluent, lets stand for 4 to 6 minutes, and then uses a 96-well positive pressure solid phase extraction device with 3.5 to 5.5 psi nitrogen pressure for 40 to 70 seconds to obtain a primary eluent.

[0061] After obtaining the primary eluate, the present invention preferably adds an eluent to perform secondary elution on the solid medium liquid-liquid extraction column after extraction to obtain a secondary eluate.

[0062] In the present invention, the parameters during the secondary elution are preferably the same as the parameters during the elution, and will not be described in detail here.

[0063] In the present invention, the primary eluent and the secondary eluent are preferably combined to obtain an eluent, the eluent is nitrogen blown, and water is added to redissolve it to obtain a test solution.

[0064] In the present invention, the nitrogen flow rate during nitrogen blowing is preferably ≤12 L / min.

[0065] In the present invention, the nitrogen blowing is preferably performed until the mixture is dried.

[0066] In the present invention, the water is preferably pure water.

[0067] In the present invention, the volume ratio of the enzymatic hydrolysate to water is preferably (2-3):1.

[0068] In the present invention, the re-dissolution is preferably performed by vortex oscillation. The present invention has no particular limitation on the operation of the vortex oscillation, as long as the components can be mixed uniformly.

[0069] After obtaining the test liquid, the present invention performs liquid chromatography-high-resolution mass spectrometry detection on the test liquid to obtain the content of tobacco exposure markers in urine.

[0070] In the present invention, when the urine is urine of non-smokers, the injection volume is preferably 5 to 20 μL, more preferably 10 μL.

[0071] In the present invention, when the urine is urine of smokers, the preferred injection volume of the present invention is 1 μL to detect nicotine (NIC), cotinine (COT), trans-3-hydroxycotinine (OHCOT), nornicotine (NNIC), norcotinine (NCOT), nicotine nitrogen oxide (NNO), cotinine nitrogen oxide (CNO), anabasine (ANAB) and anabasine (ANAT).

[0072] In the present invention, when the urine is urine from smokers, the preferred injection volume of the present invention is 60 μL to detect the content of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol (NNAL), 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N'-nitronornicotine (NNN), N-nitrosoneanatabine (NAT) and N-nitrosoanabasine (NAB) in urine. The mass concentrations of 14 tobacco metabolites in urine samples from smokers vary greatly. For 5 TSNAs, increasing the injection volume increases the response and improves the detection sensitivity; for nicotine and its metabolites, reducing the injection volume ensures the stability of the instrument state during high-throughput sample detection.

[0073] In the present invention, the liquid chromatography conditions during the liquid chromatography-high resolution mass spectrometry detection are preferably: mobile phase A is ammonia water with a volume concentration of 0.05-0.2%, mobile phase B is methanol, the flow rate is 200-400 μL / min, gradient elution is adopted, and the gradient elution conditions are: 0-5.17 min 10-20% B (mobile phase A volume percentage of 80-90%, mobile phase B volume percentage of 10-20%), 5.17-12 min 30-50% B (mobile phase A volume percentage of 50-70%, mobile phase B volume percentage of 30-50%), 12-13 min 40-100% B (mobile phase A volume percentage of 0-60%, mobile phase B volume percentage of 40-100%), 13-14 min 100% B, 14-15 min 10~20%B (mobile phase A volume percentage of 80~90%, mobile phase B volume percentage of 10~20%), the chromatographic column is a C18 chromatographic column, the length of the chromatographic column is 120~160mm, the inner diameter of the chromatographic column is 1~3mm, and the particle size of the filler is 1.5~2.0μm; more preferably: mobile phase A is ammonia water with a volume concentration of 0.1%, mobile phase B is methanol, the flow rate is 300μL / min, gradient elution is adopted, and the gradient elution conditions are: 0~5.17min 15%B, 5.17~12min 40%B, 12~13min 40~100%B, 13~14min 100%B, 14~15min 15%B, the chromatographic column is a C18 chromatographic column, the length of the chromatographic column is 150mm, the inner diameter of the chromatographic column is 2mm, and the particle size of the filler is 1.9μm. In an embodiment of the present invention, the brand and model of the chromatographic column may specifically be Gemini® 3µm NX-C18 110A.

[0074] In the present invention, the mass spectrometry conditions during the liquid chromatography-high-resolution mass spectrometry detection are preferably: using a heatable electrospray ion source (HESI source), parallel reaction monitoring (PRM), positive ion scanning mode, a spray voltage of 3~4kV, a capillary temperature of 300~400°C, a resolution of 10000~75000, a sheath gas of 40~50arb, an auxiliary gas of 10~20arb, an auxiliary gas temperature of 300~400°C, and segmented scanning; more preferably: using a heatable electrospray ion source (HESI source), parallel reaction monitoring (PRM), a positive ion scanning mode, a spray voltage of 3.5kV, a capillary temperature of 350°C, a resolution of 70000, a sheath gas of 45arb, an auxiliary gas of 15arb, an auxiliary gas temperature of 350°C, and segmented scanning.

[0075] In the present invention, the parent ion (m / z) of nicotine (NIC) among the 14 tobacco exposure markers during mass spectrometry detection is preferably 163.12298, the quantitative daughter ion (m / z) is preferably 132.08027, the qualitative daughter ion (m / z) is preferably 130.06509, the relative collision energy (NCE) is preferably 5-15, more preferably 10; the retention time is preferably 11.82 min, and the segmented scanning time is preferably 9-14 min; the parent ion (m / z) of cotinine (COT) is preferably 177.10205, the quantitative daughter ion (m / z) is preferably 80.05003, the qualitative daughter ion (m / z) is preferably 98.06039, the relative collision energy ( The NCE is preferably 40-50, more preferably 45; the retention time is preferably 7.41 min, and the segmented scanning time is preferably 6-9 min; the parent ion (m / z) of trans-3-hydroxycotinine (OHCOT) is preferably 193.09703, the quantitative daughter ion (m / z) is preferably 80.05006, the qualitative daughter ion (m / z) is preferably 134.05997, and the relative collision energy (NCE) is preferably 45-55, more preferably 50; the retention time is preferably 4.13 min, and the segmented scanning time is preferably 3-6 min; the parent ion (m / z) of nornicotine (NNIC) is preferably 149.10730, the quantitative daughter ion (m / z) is preferably 80 .05006, the qualitative daughter ion (m / z) is preferably 130.06511, the relative collision energy (NCE) is preferably 50-60, more preferably 55; the retention time is preferably 8.99 min, and the segmented scanning time is preferably 6.5-12 min; the parent ion (m / z) of norcotinine (NCOT) is preferably 163.08659, the quantitative daughter ion (m / z) is preferably 80.04994, the qualitative daughter ion (m / z) is preferably 118.06512, the relative collision energy (NCE) is preferably 65-75, more preferably 70; the retention time is preferably 6.09 min, and the segmented scanning time is preferably 5-9 min; the parent ion (m / z) of nicotine nitrogen oxide (NNO) is preferably 163.08659, the quantitative daughter ion (m / z) is preferably 80.04994, the qualitative daughter ion (m / z) is preferably 118.06512, the relative collision energy (NCE) is preferably 65-75, more preferably 70; the retention time is preferably 6.09 min, and the segmented scanning time is preferably 5-9 min; ion (m / z) is preferably 179.11777, the quantitative daughter ion (m / z) is preferably 132.08060, the qualitative daughter ion (m / z) is preferably 117.05737, the relative collision energy (NCE) is preferably 45-55, more preferably 50; the retention time is preferably 2.43min, and the segmented scanning time is preferably 1.5-4min; the parent ion (m / z) of cotinine nitrogen oxide (CNO) is preferably 193.09715, the quantitative daughter ion (m / z) is preferably 96.04465, the qualitative daughter ion (m / z) is preferably 79.04213, the relative collision energy (NCE) is preferably 55-65, more preferably 60; the retention time is preferably 1.92min, the segmented scanning time is preferably 1-3min; the parent ion (m / z) of anabasine (ANAB) is preferably 163.12298, the quantitative daughter ion (m / z) is preferably 80.04944, the qualitative daughter ion (m / z) is preferably 107.06065, the relative collision energy (NCE) is preferably 5-15, more preferably 10; the retention time is preferably 11.11min, and the segmented scanning time is preferably 9-14min; the parent ion (m / z) of anabasine (ANAT) is preferably 161.10721, the quantitative daughter ion (m / z) is preferably 163.12298, the relative collision energy (NCE) is preferably 35-45, more preferably 40; the retention time is preferably 9.40 The parent ion (m / z) of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol (NNAL) is preferably 210.12354, the quantitative daughter ion (m / z) is preferably 93.05762, the qualitative daughter ion (m / z) is preferably 149.08322, the relative collision energy (NCE) is preferably 25-35, more preferably 30; the retention time is preferably 7.66 min, and the segmented scanning time is preferably 6.5-10 min; the parent ion (m / z) of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) is preferably 208.10797, and the quantitative daughter ion (m / z) is preferably 122.060 22, the qualitative daughter ion (m / z) is preferably 148.07533, the relative collision energy (NCE) is preferably 15-25, more preferably 20; the retention time is preferably 8.22 min, and the segmented scanning time is preferably 7-10 min; the parent ion (m / z) of N'-nitronornicotine (NNN) is preferably 178.09726, the quantitative daughter ion (m / z) is preferably 148.09909, the qualitative daughter ion (m / z) is preferably 120.06814, the relative collision energy (NCE) is preferably 15-25, more preferably 20; the retention time is preferably 7.70 min, and the segmented scanning time is preferably 6.5-10 min; the parent ion (m / z) of N-nitrosoneonicotinoid (NAT) is preferably The preferred m / z of N-nitrosoanabasine (NAB) is 192.11337, the preferred m / z of N-nitrosoanabasine (NAB) is 162.11501, the preferred m / z of N-nitrosoanabasine (NAB) is 133.08836, the preferred m / z of N-nitrosoanabasine (NAB) is 190.09741, the preferred m / z of N-nitrosoanabasine (NAB) is 160.09901, the preferred m / z of N-nitrosoanabasine (NAB) is 106.05286, the preferred m / z of N-nitrosoanabasine (NAB) is 15-25, more preferably 20; the preferred m / z of N-nitrosoanabasine (NAB) is 9.34 min, the preferred m / z of N-nitrosoanabasine (NAB) is 8.5~12min。.

[0076] In the present invention, the parent ion (m / z) of nicotine-D4 (NIC-D4) among the 14 internal standard substances during mass spectrometry detection is preferably 167.14807, the quantitative daughter ion (m / z) is preferably 136.10526, the qualitative daughter ion (m / z) is preferably 134.08969, the relative collision energy (NCE) is preferably 5-15, more preferably 10; the retention time is preferably 11.82 min, and the segmented scanning time is preferably 9-14 min; the parent ion (m / z) of cotinine-D3 (COT-D3) is preferably 180.12088, the quantitative daughter ion (m / z) is preferably 80.05006, the qualitative daughter ion (m / z) is preferably 101.07922, the relative collision energy ( The NCE is preferably 70-80, more preferably 75; the retention time is preferably 7.41 min, and the segmented scanning time is preferably 6-9 min; the parent ion (m / z) of trans-3'-hydroxycotinine-D3 (OHCOT-D3) is preferably 196.11588, the quantitative daughter ion (m / z) is preferably 80.05007, the qualitative daughter ion (m / z) is preferably 134.06000, and the relative collision energy (NCE) is preferably 65-75, more preferably 70; the retention time is preferably 4.13 min, and the segmented scanning time is preferably 3-6 min; the parent ion (m / z) of nornicotine-D4 (NNIC-D4) is preferably 153.13228, the quantitative daughter ion (m / z) is preferably 84.07513, the qualitative daughter ion (m / z) is preferably 134.09009, the relative collision energy (NCE) is preferably 60-70, more preferably 65; the retention time is preferably 8.99 min, and the segmented scanning time is preferably 6.5-12 min; the parent ion (m / z) of norcotinine-D4 (NCOT-D4) is preferably 167.11170, the quantitative daughter ion (m / z) is preferably 84.07502, the qualitative daughter ion (m / z) is preferably 121.08398, the relative collision energy (NCE) is preferably 65-75, more preferably 70; the retention time is preferably 6.09 min, and the segmented scanning time is preferably 5-9 min; nicotine nitrogen oxide-D3 (NNO-D 3) preferably has a parent ion (m / z) of 182.13661, a quantitative daughter ion (m / z) of 132.08066, a qualitative daughter ion (m / z) of 117.05742, a relative collision energy (NCE) of 45-55, more preferably 50; a retention time of 2.43 min, and a segmented scanning time of 1.5-4 min; a parent ion (m / z) of cotinine nitrogen oxide-D3 (CNO-D3) preferably has a parent ion (m / z) of 196.11598, a quantitative daughter ion (m / z) of 96.04468, a qualitative daughter ion (m / z) of 79.04212, a relative collision energy (NCE) of 55-65, more preferably 60; a retention time of 1.The parent ion (m / z) of anabasine-D4 (ANAB-D4) is preferably 167.14808, the quantitative daughter ion (m / z) is preferably 84.08075, the qualitative daughter ion (m / z) is preferably 111.08568, and the relative collision energy (NCE) is preferably 5-15, more preferably 10; the retention time is preferably 11.11min, and the segmented scanning time is preferably 9-14min; the parent ion (m / z) of anabasine-D4 (ANAT-D4) is preferably 165.13239, the quantitative daughter ion (m / z) is preferably 167.14808, and the relative collision energy (NCE) is preferably 55-65, more preferably 10. The preferred retention time is 9.40 min, and the preferred segmented scanning time is 8 to 12 min. The preferred parent ion (m / z) of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol-D3 (NNAL-D3) is 213.14204, the preferred quantitative daughter ion (m / z) is 93.05761, the preferred qualitative daughter ion (m / z) is 149.08319, and the preferred relative collision energy (NCE) is 25 to 35, more preferably 30. The preferred retention time is 7.66 min, and the preferred segmented scanning time is 6.5 to 10 min. The preferred parent ion (m / z) of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone-D4 (NNK-D4) is The molecular weight of the molecular weight of N'-nitronornicotine-D4 (NNN-D4) is preferably 212.13289, the quantitative daughter ion (m / z) is preferably 126.08501, the qualitative daughter ion (m / z) is preferably 152.10045, the relative collision energy (NCE) is preferably 15-35, and more preferably 20; the retention time is preferably 8.22 min, and the segmented scanning time is preferably 7-10 min; the parent ion (m / z) of N'-nitronornicotine-D4 (NNN-D4) is preferably 182.12248, the quantitative daughter ion (m / z) is preferably 152.12369, the qualitative daughter ion (m / z) is preferably 124.09319, the relative collision energy (NCE) is preferably 15-25, and more preferably 20; the retention time is preferably 7.70 min, The segmented scanning time is preferably 6.5-10 min; the parent ion (m / z) of N-nitrosoneonicotinoid-D4 (NAT-D4) is preferably 194.12151, the quantitative daughter ion (m / z) is preferably 164.12463, the qualitative daughter ion (m / z) is preferably 110.07777, and the relative collision energy (NCE) is preferably 15-25, more preferably 20; the retention time is preferably 9.34 min, and the segmented scanning time is preferably 8-11 min; the parent ion (m / z) of N-nitrosoanabasicine-D4 (NAB-D4) is preferably 196.13812, the quantitative daughter ion (m / z) is preferably 166.14050, and the qualitative daughter ion (m / z) is preferably 136.10716, the relative collision energy (NCE) is preferably 5-15, more preferably 10; the retention time is preferably 9.72 min, and the segmented scanning time is preferably 8.5-12 min. .

[0077] The present invention preferably uses water as a solvent to prepare a series of tobacco exposure marker standard solutions containing internal standard substances, and then performs liquid chromatography-high resolution mass spectrometry detection to compare the standard tobacco exposure marker ion peak area A with the corresponding internal standard quantitative ion peak area A S The ratio (A / A S ) as the y-axis, and the concentration C of the tobacco exposure marker standard solution as the x-axis to draw a standard curve, specifically as formula (1).

[0078] A / A S =a+b×C formula (1);

[0079] Where: A is the peak area of ​​the standard tobacco exposure marker ion; A S is the peak area of ​​the corresponding internal standard quantitative ion; a is the intercept of the standard curve; b is the slope of the standard curve; C is the concentration of the tobacco exposure marker standard solution, ng / mL.

[0080] In the present invention, the determination result of the tobacco exposure marker in urine is preferably automatically calculated by a data processing system according to the internal standard method, specifically as formula (2).

[0081] ρ = C' × V1 / V (2);

[0082] Where: ρ is the actual concentration of tobacco exposure markers in urine, ng / mL; C' is the concentration of sample solution calculated by the standard curve, ng / mL; V1 is the fixed volume, μL; V is the sample volume, μL.

[0083] The present invention has no special limitation on the operation of using water as solvent to prepare a series of tobacco exposure marker standard solutions containing internal standard substances, and the technical solutions well known to those skilled in the art can be adopted.

[0084] The invention performs enzymatic hydrolysis on urine and then extracts the urine using a solid medium liquid-liquid extraction column to obtain a test liquid, and then performs liquid chromatography-high-resolution mass spectrometry detection on the test liquid. In the pretreatment process, no pretreatment of the small column is required, the operation is simple, and the treatment is performed using a 96-well plate. The invention has high throughput and can process 96 samples at a time. The invention has a wide linear range of pg / mL-30000ng / mL, and the method of controlling the injection volume, using segmented scanning and reducing the collision energy solves the problem of high-concentration samples and mass spectrometry signal saturation. The standard curve has good linearity, and the urine of non-smokers and smokers can be accurately quantified at the same time. At the same time, the detection sensitivity, precision and accuracy are high, and 14 nicotine metabolites, tobacco minor alkaloids and tobacco-specific nitrosamine substances can be accurately detected at the same time. The smoking status in the past 3-5 days can be evaluated, and the smoking status in the past 3-5 months can be reflected. It can also distinguish whether the smoked cigarette is an electronic cigarette or an ordinary cigarette.

[0085] The present invention also provides a detection kit, comprising: alkaline solution, buffer solution, 96-well deep well plate, 96-well solid medium liquid-liquid extraction plate, eluent, mixed standard solution of 14 tobacco exposure markers, mixed deuterated internal standard solution of 14 tobacco exposure markers and enzyme.

[0086] In the present invention, the detection kit is used to detect 14 tobacco exposure markers in urine.

[0087] In the present invention, the alkali solution preferably includes potassium hydroxide solution or sodium hydroxide solution; the concentration of the alkali solution is preferably 10-20 mol / L, more preferably 12-18 mol / L, and further preferably 15-16 mol / L.

[0088] In the present invention, the buffer solution preferably includes an acetic acid-ammonium acetate buffer solution or a formic acid-ammonium formate buffer solution, more preferably an acetic acid-ammonium acetate buffer solution; the pH value of the buffer solution is preferably 5-6, more preferably 5.2-5.8, and further preferably 5.6-5.7.

[0089] In the present invention, the eluent is preferably dichloromethane or an isopropanol-dichloromethane mixed solution, more preferably an isopropanol-dichloromethane mixed solution.

[0090] In the present invention, the volume ratio of isopropanol to dichloromethane in the isopropanol-dichloromethane mixed solution is preferably: (1-10): (90-99), more preferably 5:95.

[0091] In the present invention, the enzyme preferably includes β-glucuronidase / arylsulfatase, H-1 type β-glucuronidase or H-2 type β-glucuronidase, more preferably β-glucuronidase / arylsulfatase.

[0092] In the present invention, the ratio of the enzyme activities of β-glucuronidase and arylsulfatase in the β-glucuronidase / arylsulfatase is preferably 5:1.

[0093] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0094] The instruments and equipment used in each embodiment and comparative example are: QE-PLUS liquid chromatography-high resolution mass spectrometer (ThermoFisher, USA), 96-well positive pressure solid phase extraction device (Waters, USA), YND300 96-well nitrogen blowdown instrument (Shanghai Xiyang Company), vortex oscillator (Scientific Industries, USA), SLE+96-well 400 mg solid medium liquid-liquid extraction extraction disk pH=9 (ThermoFisher, USA), pH / conductivity / ion comprehensive tester-S40K (SenenMulti, Switzerland), Gemini® 3µm NX-C18 110A chromatographic column (150×2.0mm, 1.9µm, Phenomenex, USA), Hypersil GOLD chromatographic column (100×2.1mm, 1.9µm, ThermoFisher, USA).

[0095] The raw materials or reagents are as follows: Standards: Nicotine standard (1 mg / mL, dissolved in methanol, purchased from TMRM), Cotinine standard (1 mg / mL, dissolved in methanol, purchased from Supelco), Trans-3'-hydroxycotinine standard (1 mg / mL, dissolved in methanol, purchased from Supelco), Nornicotine standard (1 mg / mL, dissolved in methanol, purchased from Supelco), Norcotinine standard (5 mg, solid, purchased from TRC), Nicotine nitrogen oxide standard (1 mg, solid, purchased from TRC), Cotinine nitrogen oxide standard Standard product (10 mg, solid, purchased from TRC), neonicotinoid standard (100 μg / mL, dissolved in methanol, purchased from Neochema), anabasine standard (1 mg / mL, dissolved in hydrochloric acid, purchased from Supelco), NNAL standard (5 mg, solid, purchased from TRC), NNK standard (10 mg, solid, purchased from TRC), NNN standard (10 mg, solid, purchased from TRC), NAT standard (5 mg, solid, purchased from TRC), NAB standard (10 mg, solid, purchased from TRC).

[0096] Internal standards: Nicotine-D4 internal standard (100 μg / mL, dissolved in methanol, purchased from Supelco), cotinine-D3 internal standard (1 mg / mL, dissolved in methanol, purchased from Supelco), trans-3'-hydroxycotinine-D3 internal standard (100 μg / mL, methanol, purchased from Supelco), nornicotine-D4 internal standard (100 μg / mL, dissolved in methanol, purchased from Supelco), norcotinine-D4 internal standard (1 mg, solid, purchased from TRC), nicotine-NOx-D3 internal standard Internal standard (1 mg, solid, purchased from BePure), cotinine N-oxide-D3 internal standard (5 mg / mL, solid, purchased from TRC), neonicotinoid-D4 internal standard (1 mg, solid, purchased from TRC), anabasine-D4 internal standard (100 μg / mL, dissolved in hydrochloric acid, purchased from Sigma), NNAL-D3 internal standard (1 mg, solid, purchased from TRC), NNK-D4 internal standard (1 mg, solid, purchased from TRC), NNN-D4 internal standard (1 mg, solid, purchased from TRC), NAT -D4 internal standard (0.5 mg, solid, purchased from TRC), NAB-D4 internal standard (1 mg, solid, purchased from TRC).

[0097] Enzymes: β-glucuronidase / arylsulfatase (100,000 units / mL, arylsulfatase not less than 20,000 units / mL derived from abalone, purchased from CNW), β-glucuronidase (85,000 units / g, H1 type, derived from Roman snail, purchased from Sigma), β-glucuronidase (575,000 units / g, H2 type, derived from Roman snail, purchased from Sigma).

[0098] Other reagents: isopropanol, acetonitrile, dichloromethane (chromatographic grade, Fisher, USA), potassium hydroxide, ammonia (superior grade, Sinopharm Group), glacial acetic acid (chromatographic grade, Fisher, USA), ammonium acetate (analytical grade, SCR), and ultrapure water 0.055 μS / cm (prepared by Cascada II ultrapure water machine, PALL, USA) were used in the experiment.

[0099] Urine samples were collected from volunteers. Based on self-reported smoking frequency and the WHO definition of smoking, urine samples were divided into three groups: urine from non-smokers who never smoked, urine from occasional smokers who smoked more than four times a week but less than one cigarette a day on average, and urine from regular smokers who smoked more than one cigarette a day on average for six consecutive or cumulative months.

[0100] Solution preparation:

[0101] Ammonium acetate-acetic acid buffer solution (pH=5.6): Weigh 148g of ammonium acetate, transfer to a 100mL volumetric flask, dilute to 100mL with pure water to prepare a saturated ammonium acetate solution; add glacial acetic acid to the prepared saturated ammonium acetate solution until the pH value of the solution is adjusted to 5.6 to prepare a saturated ammonium acetate-acetic acid buffer solution with a pH of 5.6, which is stored in a sealed glass bottle.

[0102] Potassium hydroxide solution (15 mol / L): Weigh 84 g and transfer to a 500 mL volumetric flask. Add pure water to make up to 500 mL to obtain a 15.0 mol / L potassium hydroxide solution, which is then stored in a polyethylene bottle.

[0103] Mobile phase A (0.1% ammonia aqueous solution): Pipette 1.0 mL of ammonia water (mass concentration is 25%), transfer to a 1 L volumetric flask, dilute to 1 L with pure water to prepare 0.1% ammonia water, and store in a glass bottle.

[0104] Isopropanol-dichloromethane mixed solution (5:95, v:v): Measure 200 mL of isopropanol and 3800 mL of dichloromethane, mix well to prepare a 5% isopropanol-dichloromethane mixed solution, and store it in a brown glass bottle.

[0105] 14 kinds of tobacco metabolite standard mixed solution: take out 1 mg of NCOT, NNO, CNO, NNAL, NNK, NNN, NAT, and NAB, respectively, dissolve them in 1 mL of methanol to prepare a single standard of 1 mg / mL; take out 200 μL of NNO, CNO, ANAT and 20 μL of ANAB, NNIC, and NCOT in turn, dilute to 2 mL with methanol to prepare 6 mixed standard solutions; take out 200 μL of NNAL, NNK, NNN, NAT, and NAB in turn, dilute to 2 mL with methanol to prepare TSNAs mixed standard solution-1; take out 200 μL of TSNAs mixed standard solution-1, dilute to 2 mL with pure water to prepare TSNAs mixed standard solution-2. Take out 50 μL of NIC, 200 μL of COT, 400 μL of OHCOT, 200 μL of 6 mixed standards, and 20 μL of TSNAs mixed standard solution-2 in turn, and dilute to 2 mL with pure water to prepare 14 tobacco metabolite standard mixed solution-1; take out 750 μL of standard mixed solution-1, and dilute to 5 mL with pure water to prepare 14 tobacco metabolite standard mixed solution-2; take out 100 μL of standard mixed solution-2, and dilute to 5 mL with pure water to prepare 14 tobacco metabolite standard mixed solution-3.

[0106] Mixed internal standard solution of 14 tobacco metabolites: Take out 10 μL of NNAL-D3, NNK-D4, NNN-D4, NAT-D4, and NAB-D4 in turn, dilute to 1 mL with pure water to prepare a TSNAs mixed internal standard solution. Take out 100 μL of NIC-D4, 10 μL of COT-D3, 100 μL of OHCOT-D3, 100 μL of NNIC-D4, 10 μL of NCOT-D4, 10 μL of NNO-D3, 10 uL of CNO-D3, 10 μL of ANAT-D4, 100 μL of ANAB-D4, and 100 uL of TSNAs mixed internal standard solution in turn, and dilute to 10 mL with pure water to prepare 14 kinds of tobacco metabolites mixed internal standard solution-1; take out 2 mL of mixed internal standard solution-1, dilute to 10 mL with pure water to prepare 14 kinds of tobacco metabolites mixed internal standard solution-2. The details are shown in Table 1.

[0107] Table 1 Mixed internal standard solution for 14 tobacco exposure markers-1

[0108]

[0109] Example 1

[0110] The detection method of multiple tobacco exposure markers in urine is as follows: (1) Place the urine on an oscillator and mix at 2000 rpm for 30 seconds, draw 300 μL of the urine to be tested into a 96-mixing plate-1, add 130 μL of ammonium acetate-acetic acid buffer solution (pH=5.6) and 20 μL of β-glucuronidase / arylsulfatase in sequence, add 2000 U of enzyme, vortex and mix, hydrolyze at 37°C in the dark for 12 hours, take out 300 μL of enzymatically hydrolyzed urine and put it into a 96-mixing plate-2, add 50 μL of KOH solution (concentration 15 mol / L) and 50 μL of 14 tobacco exposure markers mixed internal standard solution-1 (the concentration of each internal standard substance is shown in Table 1), use a 12-pipettor to aspirate three times to mix, and add SLE+400 mg The 96-well extraction plate was placed on the 96 collection plate, and then all the mixed solutions were transferred to the extraction plate, allowed to stand for 5 minutes, 0.9 mL of isopropanol-dichloromethane mixed solution (isopropanol volume concentration 5%) was added, allowed to stand for 5 minutes, and a 96-well positive pressure solid phase extraction device was used to press 4.0 psi nitrogen for 55 seconds, and the above elution process was repeated once, and the 96 collection plate for collecting the eluate was placed on a 96-well nitrogen blower, and the nitrogen flow rate was set to 10 L / min, blown dry, and 100 μL of pure water was added to dissolve, the cover was covered, and vortexed to mix to obtain the test solution. For urine samples of non-smokers, 10 μL of the test solution was directly injected for determination; for urine samples of smokers, due to the high concentration, signal saturation is easily generated, and the injection volume is 1 μL for the determination of 7 nicotine and its metabolites and 2 minor alkaloids, and the injection volume is 60 μL for the determination of 5 TSNAs;

[0111] (2) The test solution obtained in step (1) is subjected to liquid chromatography-high-resolution mass spectrometry to obtain the content of tobacco exposure markers in urine; the liquid chromatography conditions for liquid chromatography-high-resolution mass spectrometry are as follows: mobile phase A is ammonia water with a volume concentration of 0.1%, mobile phase B is methanol, the flow rate is 300 μL / min, gradient elution is adopted, and the gradient elution conditions are: 0~5.17min 15%B, 5.17~12min 40%B, 12~13min 100%B, 13~14min 100%B, 14~15min 15%B, the length of the chromatographic column is 150mm, the inner diameter of the chromatographic column is 2mm, the filler particle size is 1.9μm, model: Gemini® 3µm NX-C18110A; the mass spectrometry conditions for liquid chromatography-high-resolution mass spectrometry detection were: a heatable electrospray ion source (HESI source), parallel reaction monitoring (PRM), positive ion scanning mode, spray voltage of 3.5 kV, capillary temperature of 350 °C, resolution of 70000, sheath gas of 45 arb, auxiliary gas of 15 arb, auxiliary gas temperature of 350 °C, and segmented scanning; the detection conditions of 14 tobacco exposure markers and their internal standards during mass spectrometry detection are shown in Table 2; the peak area A of the standard tobacco exposure marker ion is compared with the peak area A of the corresponding internal standard quantification ion S The ratio (A / A S ) as the y-axis, and the standard curve was drawn with the concentration C of the tobacco exposure marker standard solution as the x-axis, and then the content of tobacco exposure markers in urine was calculated. The concentration of the standard solution is shown in Table 3. The internal standard concentration of TSNAs is 10 ng / mL, and the other internal standards are 100 ng / mL.

[0112] Table 2 Detection conditions of 14 tobacco exposure markers and their internal standards by mass spectrometry

[0113]

[0114]

[0115] *: Quantitative ion.

[0116] Table 3 Tobacco exposure marker standard solutions

[0117]

[0118]

[0119] Test Example 1

[0120] According to the detection method of Example 1, actual urine is processed and detected, and the urine is urine of smokers.

[0121] Comparative test example 1

[0122] The 14 tobacco exposure marker standard solutions of S9 in Table 3 were used for liquid chromatography-high-resolution mass spectrometry detection (injection volume was 10 μL), wherein the chromatographic column was a Hypersil GOLD chromatographic column, the mobile phase A was 0.1% ammonia water, the mobile phase B was acetonitrile, the gradient was 9 min, and the gradient elution conditions were: 0-3 min 80% A, 20% B, 3-6 min 100% B, 6-7 min 98% A, 2% B, 7-9 min 80% A, 20% B, the flow rate was 0.3 mL / min, and the other conditions were the same as in Example 1.

[0123] Comparative test example 2

[0124] The 14 tobacco exposure marker standard solutions of S9 in Table 3 were used for liquid chromatography-high-resolution mass spectrometry detection (injection volume of 10 μL), wherein the chromatographic column was a Hypersil GOLD chromatographic column, the mobile phase A was 0.1% ammonia water, the mobile phase B was methanol, the gradient was 9 min, the gradient elution conditions were the same as those in comparative test example 1, and the other conditions were the same as those in example 1.

[0125] Comparative test example 3

[0126] The 14 tobacco exposure marker standard solutions of S9 in Table 3 were used for liquid chromatography-high-resolution mass spectrometry detection (injection volume of 10 μL), wherein the chromatographic column was a Hypersil GOLD chromatographic column, mobile phase A was 0.1% ammonia water, mobile phase B was methanol, the gradient was 15 min, and the gradient elution process and other conditions were the same as in Example 1.

[0127] Test Example 2

[0128] The 14 tobacco exposure marker standard solutions of S9 in Table 3 were used for liquid chromatography-high-resolution mass spectrometry detection (injection volume of 10 μL), wherein the chromatographic column was a Gemini® 3μm NX-C18 chromatographic column, the mobile phase A was 0.1% ammonia water, the mobile phase B was methanol, the gradient was 15 min, and the gradient elution process and other conditions were the same as in Example 1.

[0129] Test Example 1 The chromatograms of 14 tobacco exposure markers in actual urine are as follows Figure 1 As shown, 1 is CNO, 2 is NNO, 3 is OHCOT, 4 is NCOT, 5 is COT, 6 is NNIC, 7 is ANAT, 8 is ANAB, 9 is NIC, 10 is NNAL, 11 is NNK, 12 is NNN, 13 is NAT, and 14 is NAB.

[0130] The chromatograms of the 14 tobacco exposure marker standard solutions in Comparative Test Example 1 are as follows: Figure 2As shown, 1 is CNO, 2 is NNO, 3 is OHCOT, 4 is NCOT, 5 is COT, 6 is NNIC, 7 is ANAT, 8 is ANAB, 9 is NIC, 10 is NNAL, 11 is NNK, 12 is NNN, 13 is NAT, and 14 is NAB.

[0131] The chromatograms of the 14 tobacco exposure marker standard solutions in Comparative Test Example 2 are as follows: Figure 3 As shown, 1 is CNO, 2 is NNO, 3 is OHCOT, 4 is NCOT, 5 is COT, 6 is NNIC, 7 is ANAT, 8 is ANAB, 9 is NIC, 10 is NNAL, 11 is NNK, 12 is NNN, 13 is NAT, and 14 is NAB.

[0132] The chromatograms of the 14 tobacco exposure marker standard solutions in Comparative Test Example 3 are as follows: Figure 4 As shown, 1 is CNO, 2 is NNO, 3 is OHCOT, 4 is NCOT, 5 is COT, 6 is NNIC, 7 is ANAT, 8 is ANAB, 9 is NIC, 10 is NNAL, 11 is NNK, 12 is NNN, 13 is NAT, and 14 is NAB.

[0133] The chromatograms of the 14 tobacco exposure marker standard solutions in Test Example 2 are as follows: Figure 5 As shown, 1 is CNO, 2 is NNO, 3 is OHCOT, 4 is NCOT, 5 is COT, 6 is NNIC, 7 is ANAT, 8 is ANAB, 9 is NIC, 10 is NNAL, 11 is NNK, 12 is NNN, 13 is NAT, and 14 is NAB.

[0134] from Figures 1 to 5 It can be seen that when methanol is used as the organic phase, the overall peak shape of nicotine and its metabolites is better than that of acetonitrile. Therefore, 0.1% ammonia water and methanol are selected as the mobile phase; Hypersil GOLD column cannot separate NIC and ANAB, and the retention time of these two substances is basically the same; Gemini® 3µm NX-C18 column can effectively separate 14 tobacco metabolites, and the chromatographic peak is narrower and the separation effect is better. Therefore, Gemini® 3µm NX-C18 column is selected.

[0135] Example 2

[0136] The amount of enzyme added in step (1) of Example 1 was replaced with 500 U, and the other parameters were the same as those of Example 1.

[0137] Example 3

[0138] The amount of enzyme added in step (1) of Example 1 was replaced with 1000 U, and the other parameters were the same as those of Example 1.

[0139] Example 4

[0140] The amount of enzyme added in step (1) of Example 1 was replaced with 1500 U, and the other parameters were the same as those of Example 1.

[0141] Example 5

[0142] The β-glucuronidase / arylsulfatase in step (1) of Example 3 was replaced by H-1 type β-glucuronidase, and the other parameters were the same as those in Example 1.

[0143] Example 6

[0144] The β-glucuronidase / arylsulfatase in step (1) of Example 3 was replaced by H-2 type β-glucuronidase, and the other parameters were the same as those in Example 1.

[0145] Example 7

[0146] The enzymatic hydrolysis time in step (1) of Example 3 was replaced with 8 h, and the other parameters were the same as those in Example 1.

[0147] Example 8

[0148] The enzymatic hydrolysis time in step (1) of Example 3 was replaced with 24 h, and the other parameters were the same as those in Example 1.

[0149] Example 9

[0150] The enzymatic hydrolysis time in step (1) of Example 3 was replaced with 4 h, and the other parameters were the same as those in Example 1.

[0151] The ratios of the measured values ​​of 14 tobacco exposure markers and the free state values ​​of different enzymes at 1000U in Examples 3, 5 and 6 (37°C) are as follows: Figure 6 shown.

[0152] The ratios of the measured values ​​of β-glucuronidase / arylsulfatase and the free state values ​​of 14 tobacco exposure markers at different enzyme addition amounts in Examples 2 to 4 (37°C) are as follows: Figure 7 shown.

[0153] The ratios of the measured values ​​and free state values ​​of 14 tobacco exposure markers at different enzymatic hydrolysis times in Examples 3, 7, and 9 at 1000 U of β-glucuronidase / arylsulfatase (37°C) are as follows: Figure 8 As shown in the figure, only NNAL was detected in the urine of smokers, and the concentration of NNAL was very low, so the standard deviation of the parallel samples in the figure is larger than that of other substances.

[0154] from Figures 6-8It can be seen that 1000 units of β-glucuronidase / arylsulfatase enzymatic hydrolysis for 12 hours can ensure complete hydrolysis of most samples. Considering that the concentration of urine used in the optimization method is not an extreme value, 2000 units of β-glucuronidase / arylsulfatase enzymatic hydrolysis for 12 hours is most preferred.

[0155] Matrix effects of the method

[0156] Urine samples from non-smokers, light smokers (people who smoke weekly but not daily) and heavy smokers (people who smoke daily) were collected, and 4 samples were collected for each sample and measured by two methods. Condition A: 50 μL of pure water and 50 μL of mixed internal standard solution-2 were mixed and directly injected for measurement; Condition B: except for not adding internal standard, the sample was extracted according to the treatment method of step (1) of Example 1, and then 50 μL of pure water and 50 μL of mixed internal standard solution-2 were added during re-dissolution, and directly injected for measurement. Calculation formula: Matrix effect (ME) = B / A × 100%, usually ME is between 80~120%, and the matrix effect is considered to be insignificant. When measuring 9 substances except TSNAs, the injection volume of urine samples from smokers after treatment was 1 μL, and the injection volume of urine samples from other groups after treatment was 10 μL; when measuring 5 TSNAs, the injection volume of urine samples after treatment was 60 μL. The results are shown in Table 4.

[0157] Table 4 Matrix effects of 14 tobacco exposure markers

[0158]

[0159] As can be seen from Table 4, the matrix effects of the 14 tobacco exposure markers ranged from 89.77% to 110.95%, and the matrix effect of this method was not significant.

[0160] Linear range and detection limit of the method

[0161] In the standard curve, the estimated detected concentration is C concentration. Blank urine samples were spiked with 0.5C concentration, 0.75C concentration, and C concentration, respectively. Each spiked concentration is X, and the corresponding inter-group SD (n=12) is Y. Linear regression is performed, and the intercept with the Y axis is b, the detection limit is 3b, and the quantification limit is 10b. The linear range, detection limit, and quantification limit of 14 tobacco exposure markers are shown in Table 5.

[0162] Table 5 Linear range, detection limit and quantification limit of 14 tobacco exposure markers

[0163]

[0164] As can be seen from Table 5, the 14 tobacco exposure markers had good linearity within their respective concentration ranges, and the coefficient of determination (R2 ) were all greater than 0.995. The detection limits of 14 tobacco exposure markers ranged from 3pg / mL to 1.4ng / mL, and the quantification limits ranged from 10pg / mL to 4.6ng / mL.

[0165] The spike recovery and precision (RSD) of the method

[0166] Non-smoking urine samples were used as background, and different volumes of mixed standard solution were added to prepare low, medium and high concentration spiked urines. Six parallel samples were prepared for each spiked urine. The spiked recoveries and precisions of the 14 tobacco exposure markers are shown in Table 6.

[0167] Table 6 Recovery and precision of 14 tobacco exposure markers

[0168]

[0169] As can be seen from Table 6, the spiked recoveries of 14 tobacco exposure markers were between 82.81% and 120.84%, and the precision (RSD) was between 1.2% and 13.3%. The spiked recovery and precision of this method were good and met the requirements of methodological evaluation.

[0170] Freeze-thaw stability of samples

[0171] After freezing the urine sample to -80℃, take it out and put it to room temperature, then put it back to -80℃ refrigerator and freeze it to -80℃, which is considered one freeze-thaw cycle. This method uses urine samples from non-smokers, occasional smokers and regular smokers, and each urine sample is made into 3 parallel samples, for a total of 5 freeze-thaw cycles. The results are shown in Figure 7.

[0172] Table 7 Freeze-thaw stability of samples of 14 tobacco exposure markers

[0173]

[0174] As can be seen from Table 7, the freeze-thaw stability of the samples was good, the intra-group RSDs of the 14 tobacco exposure markers were between 0.2 and 11.2%, and the inter-group RSDs were between 0.7 and 9.9%.

[0175] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting multiple tobacco exposure markers in urine, characterized in that: The following steps are involved: (1) Mixing urine with a buffer solution and an enzyme for enzymatic hydrolysis to obtain an enzymatic hydrolyzate; (2) mixing the enzymatic hydrolyzate obtained in step (1) with an alkali solution and a mixed internal standard solution to obtain a mixed solution; (3) extracting the mixed solution obtained in step (2) using a solid medium liquid-liquid extraction column, eluting with an eluent to obtain an eluent, blowing the eluent with nitrogen, and then adding water to redissolve it to obtain a test solution; (4) subjecting the test solution obtained in step (3) to liquid chromatography-high resolution mass spectrometry to obtain the content of tobacco exposure markers in urine; The enzyme in step (1) is β-glucuronidase / arylsulfatase; the concentration of the enzyme after mixing the urine, buffer solution and enzyme is 500-3000U / 0.45mL; The buffer solution in step (1) includes an acetic acid-ammonium acetate buffer solution or a formic acid-ammonium formate buffer solution; the pH value of the buffer solution is 5-6; the volume ratio of urine to the buffer solution in step (1) is (2-2.5):1; The concentration of the alkali solution in step (2) is 10-20 mol / L; the volume ratio of the enzymatic hydrolysate to the alkali solution is (5-7):1; The volume ratio of the mixed solution in step (3) to the mass ratio of the solid medium liquid-liquid extraction column is 400 μL: (350-1000) mg; the eluent in step (3) is dichloromethane or an isopropanol-dichloromethane mixed solution; the volume ratio of isopropanol to dichloromethane in the isopropanol-dichloromethane mixed solution is: (1-10): (90-99); The liquid chromatography conditions for liquid chromatography-high resolution mass spectrometry detection in step (4) are as follows: mobile phase A is ammonia water with a volume concentration of 0.05-0.2%, mobile phase B is methanol, the flow rate is 200-400 μL / min, gradient elution is adopted, and the gradient elution conditions are as follows: 0-5.17 min 10-20% B, 5.17-12 min 30-50% B, 12-13 min 40-100% B, 13-14 min 100% B, 14-15 min 10-20% B, the chromatographic column is Gemini ® 3µm NX-C18 110A, the length of the chromatographic column is 120-160 mm, the inner diameter of the chromatographic column is 1-3 mm, and the filler particle size is 1.5-2.0 μm; The mass spectrometry conditions for liquid chromatography-high-resolution mass spectrometry detection in step (4) are: spray voltage of 3-4 kV, capillary temperature of 300-400°C, resolution of 10000-75000, sheath gas of 40-50 arb, auxiliary gas of 10-20 arb, auxiliary gas temperature of 300-400°C, and segmented scanning; The multiple tobacco exposure markers include nicotine, cotinine, trans-3-hydroxycotinine, nornicotine, norcotinine, nicotine nitrogen oxide, cotinine nitrogen oxide, anabasine, neonicotinoids, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone, N'-nitronornicotine, N-nitrosoneonicotinoids and N-nitrosoanabasine.

2. The detection method according to claim 1, characterized in that: The enzymatic hydrolysis temperature in step (1) is 35-42° C., and the enzymatic hydrolysis time is ≥4 h.

3. A detection kit comprising: Alkali solution, buffer solution, 96-well deep well plate, 96-well solid medium liquid-liquid extraction plate, eluent, mixed standard solution of 14 tobacco exposure markers, mixed deuterated internal standard solution of 14 tobacco exposure markers and enzyme.

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