Method for detecting contents of nicotine and cotinine in urine exposed by second-hand smoke of children

Abstract

The invention relates to the technical field of second-hand smoke exposure detection, and particularly discloses a method for detecting the content of nicotine and cotinine in second-hand smoke exposure urine of children. The method comprises the following steps: S1, establishment of a standard working curve: preparing an internal standard solution and a standard solution, diluting with ultrapure water to prepare a mixed internal standard solution and a mixed standard solution, and establishing a working curve by using UPLC-MS / MS; s2, collecting a child second-hand smoke exposed urine sample, and performing pretreatment: taking out the frozen urine sample from a refrigerator at-80 DEG C, putting the frozen urine sample into a refrigerator at 4 DEG C, slowly unfreezing, taking 0.15 ml of the unfrozen urine sample, adding 30 [mu] l of a 5 [mu] g / ml mixed internal standard application liquid, adding water to a constant volume of 1.5 ml, uniformly mixing, centrifuging for 20 minutes at a speed of 12000 rpm, passing through a 0.22 [mu] m nylon 66 porous filter membrane, filling a filtrate into a brown sample injection small bottle, and detecting the urine concentration by UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometer / Mass Spectrometer). According to the method, the sample pretreatment operation procedure is simple, the detection time is short, the detection cost is low, and the requirement for rapid detection of the tobacco metabolism level in urine of school-age children with low second-hand smoke exposure crowds is met.

Description

Technical Field

[0001] The present invention relates to the technical field of second-hand smoke exposure detection, and more specifically, to a method for detecting the contents of nicotine and cotinine in the urine of children exposed to second-hand smoke. Background Art

[0002] The problem of second-hand smoke exposure among children and adolescents has become a serious global public health challenge. Second-hand smoke exposure has an adverse impact on various aspects of children and adolescents, such as the cardiovascular system, circulatory system, and blood pressure. School-age children aged 6 - 12 are in a critical period of growth and development, and their organs and tissues are more sensitive to toxic and harmful substances than the adult population. Tobacco smoke is a mixture of hundreds of chemical substances, including at least 70 carcinogens, such as nicotine, tar, carbon monoxide, aldehydes, etc., which is a Class Ι carcinogen. Nicotine (Nicotine, NIC) is the main alkaloid in tobacco, accounting for more than 90% of the total tobacco alkaloids. It is the main addictive agent in tobacco products and seriously harms human health. The main metabolite of nicotine is cotinine (COT), and COT is further metabolized into trans-3-hydroxycotinine (OHCOT), which is currently recognized as a specific biomarker for evaluating tobacco smoke exposure. Currently, the tobacco exposure levels of smokers and non-smokers can be detected through biological samples such as blood, urine, saliva, and hair. Urine is a relatively ideal biological sample due to its non-invasive nature, convenient collection, and high acceptability.

[0003] Currently, the methods for determining nicotine and cotinine at home and abroad mainly include gas chromatography - mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and liquid chromatography - tandem mass spectrometry (LC-MS / MS). When using traditional methods to determine the second-hand smoke exposure level of children, it is necessary to perform pretreatment such as enrichment and extraction on urine samples, and the operation is relatively complex and cumbersome, with a low degree of automation.

[0004] Ultra-high performance liquid chromatography - tandem mass spectrometry (UPLC-MS / MS) has the characteristics of fast analysis speed, high detection sensitivity, and strong anti-interference ability. In the analysis of complex samples, it can effectively reduce matrix interference and improve the accuracy of detection. The Waters ACQUITY UPLC BEH HILIC liquid chromatography column greatly improves the separation degree, sample throughput, and detection sensitivity compared with conventional chromatography columns. The sample can be directly diluted, vortexed and filtered, and then can be tested on the machine, meeting the rapid detection of the tobacco metabolism level in the urine of school-age children with low second-hand smoke exposure.

[0005] Therefore, the present application aims to provide a method for detecting the contents of nicotine and cotinine in the urine of children exposed to second-hand smoke to solve the above problems. Summary of the Invention

[0006] The object of the present invention is to solve the problems of complex pretreatment operation procedures, long time consumption and high cost in the existing test methods for nicotine, cotinine and trans-3-hydroxycotinine in urine, and to provide a method for detecting the contents of nicotine and cotinine in urine of children exposed to second-hand smoke.

[0007] The above object of the present invention is achieved as follows:

[0008] A method for detecting the contents of nicotine and cotinine in urine of children exposed to second-hand smoke, comprising the following steps:

[0009] S1. Establishment of a standard working curve: Prepare an internal standard standard solution and a standard solution, dilute and configure a mixed internal standard standard solution and a mixed standard solution with ultrapure water respectively, and use UPLC-MS / MS to establish a working curve;

[0010] S2. Collect urine samples of children exposed to second-hand smoke and perform pretreatment:

[0011] Before detection, take out the frozen urine sample from the -80°C refrigerator and place it in the 4°C refrigerator to thaw slowly. Take 0.15 ml of the thawed urine sample, add 30 μl of a 5 μg / ml mixed internal standard application solution, make up the volume to 1.5 ml with water, mix well, centrifuge at 12,000 revolutions for 20 minutes, then pass through a 0.22 μm nylon 66 membrane filter, and load the filtrate into a brown injection vial for UPLC-MS / MS detection of urine concentration.

[0012] Further, step S1 specifically includes the following steps:

[0013] a. Preparation of the standard solution: Use chromatographically pure methanol to dilute the purchased nicotine, cotinine and trans-3-hydroxycotinine standard solutions into stock solutions with a concentration of 1 mg / ml respectively; Take the above three standard stock solutions and dilute them with ultrapure water to a mixed standard solution with a concentration of 5 μg / ml;

[0014] b. Preparation of the internal standard standard solution: Use chromatographically pure methanol to dilute the purchased nicotine-d3 standard solution, cotinine-d3 solid powder and trans-3-hydroxycotinine-d3 standard solution into stock solutions with a concentration of 1 mg / ml respectively; Take the above three internal standard stock solutions and dilute them with ultrapure water to a mixed internal standard standard solution with a concentration of 5 μg / ml;

[0015] c. Plotting of the standard working curve: Dilute the mixed standard application solutions of nicotine, cotinine, and trans-3'-hydroxycotinine with ultrapure water to a series of mixed standard solutions with concentrations of 1.0, 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, and 1000.0 ng / mL; respectively take 0.15 mL of the series of standard solutions, add 30 μL of the 5 μg / mL mixed internal standard application solution, namely the mixed internal standard of nicotine-d3, cotinine-d3, and trans-3'-hydroxycotinine-d3, dilute with water to 1.5 mL, filter through a 0.22 μm nylon 66 membrane filter, and then perform UPLC / MS / MS analysis. Plot the standard curve with the mass concentration of the analyte as the abscissa and the area ratio of the analyte and internal standard peaks as the ordinate.

[0016] Further, the conditions of UPLC-MS / MS in step S2 are as follows:

[0017] 1) Liquid phase conditions: Chromatographic column: ACQUITY UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 μm); Column temperature: 40 °C; Mobile phase: A: 0.1% ammonia water (analytical grade) B: acetonitrile (chromatographic grade); Flow rate: 0.2 mL / min; Injection volume: 2 μL; The mobile phase elution gradient is shown in the following table;

[0018] Table Mobile phase elution gradient

[0019]

[0020]

[0021] 2) Mass spectrometry conditions: Ionization mode: ESI positive ion mode (ESI + ); Ion transfer tube temperature: 325 °C; Nebulizer temperature: 350 °C; Sheath gas flow rate: 35 Arb; Auxiliary gas flow rate: 5 Arb; Purge gas flow rate: 0 Arb; Spray needle voltage: 3000 V; The quantitative ion pairs of nicotine are: 163.225 m / z and 130.125 m / z; The quantitative ion pairs of nicotine-d3 are: 166.212 m / z and 130.083 m / z; The quantitative ion pairs of cotinine are: 177.175 m / z and 80.155 m / z; The quantitative ion pairs of cotinine-d3 are: 180.188 m / z and 80.155 m / z; The quantitative ion pairs of trans-3-hydroxycotinine are: 193.188 m / z and 80.155 m / z; The quantitative ion pairs of trans-3-hydroxycotinine-d3 are: 196.188 m / z and 80.155 m / z.

[0022] Furthermore, the higher the response, the higher the sensitivity and the detection rate of the target substance in the sample. Preferably, the collision energies of nicotine ion pairs 163.225 m / z and 130.125 m / z are 21.39 V; the collision energies of nicotine-d3 ion pairs 166.212 m / z and 130.083 m / z are 21.64 V; the collision energies of cotinine ion pairs 177.175 m / z and 80.155 m / z are 25.05 V; the collision energies of cotinine-d3 ion pairs 180.188 m / z and 80.155 m / z are 25.81 V; the collision energies of trans-3-hydroxycotinine ion pairs 193.188 m / z and 80.155 m / z are 28.67 V; the collision energies of trans-3-hydroxycotinine-d3 ion pairs 196.188 m / z and 80.155 m / z are 29.22 V.

[0023] Furthermore, the retention time of nicotine is 4.12 - 4.14 min, and the retention time of nicotine-d3 is 4.16 - 4.17 min; the retention time of cotinine is 2.09 - 2.11 min, and the retention time of cotinine-d3 is 2.09 - 2.11 min; the retention time of trans-3-hydroxycotinine is 1.86 - 1.88 min, and the retention time of trans-3-hydroxycotinine-d3 is 1.87 - 1.88 min.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The method of the present invention overcomes the problems of the traditional method for determining the second-hand smoke exposure level in children, such as the need for complicated pretreatment operations such as enrichment and extraction of urine samples, with relatively complex and cumbersome operations and low automation. It has a fast analysis speed, high detection sensitivity, strong anti-interference ability, and can effectively reduce matrix interference in the analysis of complex samples.

[0026] 2. In the method of the present invention, the chromatographic column under the liquid phase conditions adopts an ACQUITY UPLC BEH HILIC liquid chromatographic column, which greatly improves the resolution, sample throughput, and detection sensitivity compared with conventional chromatographic columns. The sample can be directly diluted, and after vortex oscillation and filtration, it can be directly tested on the machine, meeting the rapid detection of the tobacco metabolism level in the urine of school-age children with low second-hand smoke exposure.

[0027] In summary, the test method of the present invention has a simple operation procedure for sample pretreatment, short detection time, and low detection cost, and can meet the rapid detection of the tobacco metabolism level in the urine of school-age children with low second-hand smoke exposure. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the peak shape diagram of urine metabolites of primary school students whose caregivers smoke in the embodiment of the present invention;

[0029] Figure 2 It is the urine metabolite peak shape diagram of primary school students whose caregivers do not smoke in the embodiments of the present invention. Specific Embodiments

[0030] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] The implementation of the present invention will be described in detail below in conjunction with specific embodiments.

[0032] As shown in the accompanying drawings of the specification, the following are the preferred embodiments provided by the present invention.

[0033] The instruments and reagents used in the following embodiments of the present invention are as follows:

[0034] Instruments and Reagents: Liquid chromatography-tandem triple quadrupole mass spectrometer (Thermo Company, USA), methanol, acetonitrile (chromatographic grade, Shanghai Anpu Experimental Company), 25% ammonia water (analytical grade, Tianjin Beilian Company).

[0035] Reference Substances: Nicotine, purchased from Shiyan Shuoen Biotechnology Co., Ltd., liquid; Nicotine-d3, purchased from Shiyan Shuoen Biotechnology Co., Ltd., liquid; Cotinine, purchased from Wuhan Angesi Biotechnology Co., Ltd., liquid; Cotinine-d3, purchased from Gladys (Beijing) Biotechnology Co., Ltd., solid powder; trans-3-Hydroxycotinine, purchased from Shiyan Shuoen Biotechnology Co., Ltd., liquid; trans-3-Hydroxycotinine-d3, purchased from Shiyan Shuoen Biotechnology Co., Ltd., liquid.

[0036] Embodiment 1: Investigation on the Method Performance of the Present Application

[0037] The solution of this embodiment provides a method for detecting the contents of nicotine and cotinine in the urine of children exposed to second-hand smoke, and the method includes the following steps:

[0038] Preparation of Standard Solution: Use chromatographic grade methanol to dilute the purchased standard solutions of nicotine, cotinine and trans-3-hydroxycotinine into stock solutions with a concentration of 1 mg / ml respectively. Take the above three standard stock solutions and dilute them with ultrapure water to a mixed standard solution with a concentration of 5 μg / ml.

[0039] Preparation of Internal Standard Standard Solution: Use chromatographic grade methanol to dilute the purchased standard solution of nicotine-d3, solid powder of cotinine-d3 and standard solution of trans-3-hydroxycotinine-d3 into stock solutions with a concentration of 1 mg / ml respectively; take the above three internal standard stock solutions and dilute them with ultrapure water to a mixed internal standard standard solution with a concentration of 5 μg / ml.

[0040] Drawing of the standard working curve: Dilute the mixed standard application solutions of nicotine, cotinine, and trans-3'-hydroxycotinine with ultrapure water to a series of mixed standard solutions with concentrations of 1.0, 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, and 1000.0 ng / mL. Take 0.15 mL of the series of standard solutions respectively, add 30 μL of the 5 μg / mL mixed internal standard application solution (mixed internal standard of nicotine-d3, cotinine-d3, and trans-3'-hydroxycotinine-d3), dilute with water to 1.5 mL, filter through a 0.22 μm nylon 66 pore filter membrane, and then perform UPLC / MS / MS analysis. Draw the standard curve with the mass concentration (μg / L) of the analyte as the abscissa and the area ratio of the analyte and internal standard peaks as the ordinate.

[0041] Urine pretreatment: Take out the frozen urine sample from the -80°C refrigerator before detection, place it in the 4°C refrigerator to thaw slowly. Take 0.15 mL of the thawed urine sample, add 30 μL of the 5 μg / mL mixed internal standard application solution, make up the volume to 1.5 mL with water, mix well, centrifuge at 12000 rpm for 20 minutes, then filter through a 0.22 μm nylon 66 pore filter membrane, and load the filtrate into a brown injection vial for UPLC-MS / MS detection of urine concentration.

[0042] Liquid chromatography conditions: Chromatographic column: ACQUITY UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 μm); column temperature: 40°C; mobile phase: A: 0.1% ammonia water (analytical grade), B: acetonitrile (chromatographic grade); flow rate: 0.2 mL / min; injection volume: 2 μL.

[0043] Mass spectrometry conditions are: Ionization mode: ESI positive ion mode (ESI +) ; Ion transfer tube temperature: 325°C; nebulizer temperature: 350°C; sheath gas flow rate: 35 Arb; auxiliary gas flow rate: 5 Arb; purge gas flow rate: 0 Arb; spray needle voltage: 3000 V. The quantitative ion pairs of nicotine are: 163.225 m / z and 130.125 m / z; the quantitative ion pairs of nicotine-d3 are: 166.212 m / z and 130.083 m / z; the quantitative ion pairs of cotinine are: 177.175 m / z and 80.155 m / z; the quantitative ion pairs of cotinine-d3 are: 180.188 m / z and 80.155 m / z; the quantitative ion pairs of trans-3-hydroxycotinine are: 193.188 m / z and 80.155 m / z; the quantitative ion pairs of trans-3-hydroxycotinine-d3 are: 196.188 m / z and 80.155 m / z.

[0044] The specific implementation is as follows:

[0045] 1. Detect the quantitative ions of nicotine and cotinine according to the established mass spectrometry conditions: Using the internal standard method, obtain the quantitative linear equations and correlation coefficients of nicotine, cotinine, and trans-3-hydroxycotinine. The results show that, as shown in Table 1 below, the correlation coefficients are all greater than 0.99, indicating good linearity and can be used for the accurate quantification of nicotine, cotinine, and trans-3-hydroxycotinine in samples.

[0046] Table 1 Linear equations and correlation coefficients of nicotine, cotinine, and trans-3-hydroxycotinine

[0047]

[0048] 2. Method recovery rate and precision:

[0049] Add a certain amount of nicotine, cotinine, and trans-3-hydroxycotinine standard solutions to the mixed urine for the method recovery rate test. Dilute the nicotine standard solution (5 μg / mL) with chromatographically pure methanol into standard solutions with concentrations of 2.5 ng / mL, 25 ng / mL, and 250 ng / mL respectively; dilute the cotinine standard solution (5 μg / mL) into standard solutions with concentrations of 2.5 ng / mL, 25 ng / mL, and 250 ng / mL respectively, and dilute the trans-3-hydroxycotinine standard solution (5 μg / mL) into standard solutions with concentrations of 2.5 ng / mL, 25 ng / mL, and 250 ng / mL respectively.

[0050] The specific spiking test is as follows: Take 20 urine samples and mix them. After sample treatment according to the method described for urine pretreatment, measure the background value. At the same time, add three different concentrations of nicotine, cotinine, and trans-3-hydroxycotinine to the mixed urine in proportion, and measure the spiked measurement value after treatment according to the urine pretreatment method.

[0051] Calculate according to the formula "(spiked sample measurement value - sample measurement value) ÷ spiked amount × 100%". As shown in Table 2, the average recovery rates of nicotine at different concentrations of spiking are between 106.22% and 133.48%, and the relative standard deviation is less than 8%; the average recovery rates of cotinine at different concentrations of spiking are between 96.92% and 117.76%, and the relative standard deviation is less than 14%; the average recovery rates of trans-3-hydroxycotinine at different concentrations of spiking are between 96.68% and 141.96%, and the relative standard deviation is less than 12%.

[0052] Table 2 Spiking recovery rates and relative standard deviations of nicotine, cotinine, and trans-3-hydroxycotinine (n = 5)

[0053]

[0054] Example 2:

[0055] (1) The standard working curve was plotted in the same way as in Example 1.

[0056] (2) The urine pretreatment was the same as in Example 1.

[0057] (3) The test method of UPLC-MS / MS was the same as in Example 1.

[0058] In this Example 2, the urine of 6 non-smoking children was used as the research object and classified according to the smoking situation of the caregivers: smoking and non-smoking. The contents of nicotine, cotinine and trans-3-hydroxycotinine in children's urine were measured. The results showed that the contents of nicotine, cotinine and trans-3-hydroxycotinine in the urine of children whose caregivers smoked were all higher than those of children whose caregivers did not smoke, and the contents of all 6 samples were lower than 50 ng / ml. The summary is shown in Table 3. It shows that the method established by the present invention can be used for the rapid detection of nicotine and cotinine in urine samples of non-smokers. Randomly selected urine samples of children whose caregivers smoked and did not smoke. The peak area of urine metabolites of primary school students whose caregivers smoked was larger and the peak shape was sharper than that of those whose caregivers did not smoke. As Figure 1 and Figure 2 shown.

[0059] Table 3 Test results of nicotine, cotinine and trans-3-hydroxycotinine in urine samples of 6 non-smoking children

[0060]

[0061] To sum up, through the above embodiments of the present application, compared with the traditional methods in the prior art, the method of the present invention has a fast analysis speed, high detection sensitivity, strong anti-interference ability, can effectively reduce matrix interference in the analysis of complex samples, and improve the detection accuracy; at the same time, in the method of the present invention, an ACQUITY UPLC BEH HILIC liquid chromatography column is used, which greatly improves the resolution, sample throughput and detection sensitivity compared with conventional chromatographic columns. The sample can be directly diluted and vortexed and filtered and then tested on the machine. Therefore, the method of the present invention can meet the rapid detection of tobacco metabolism levels in the urine of school-age children with low second-hand smoke exposure.

[0062] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the contents of nicotine and cotinine in the urine of children exposed to second-hand smoke, characterized in that It includes the following steps: S1. Establishment of standard working curve: Prepare internal standard standard solution and standard solution, dilute and configure mixed internal standard standard solution and mixed standard solution with ultrapure water respectively, and establish the working curve using UPLC-MS / MS; S2. Collect urine samples of children exposed to second-hand smoke and conduct pretreatment: Before detection, take out the frozen urine samples from the -80°C refrigerator, place them in the 4°C refrigerator for slow thawing. Take 0.15 ml of the thawed urine sample, add 30 μl of 5 μg / ml mixed internal standard application solution, make up the volume to 1.5 ml with water, mix well, centrifuge at 12,000 rpm for 20 minutes, then pass through a 0.22 μm nylon 66 membrane filter, and load the filtrate into a brown injection vial for UPLC-MS / MS detection of urine concentration.

2. The method for detecting the contents of nicotine and cotinine in the urine of children exposed to second-hand smoke according to claim 1, wherein Step S1 specifically includes the following steps: a. Preparation of standard solution: Use chromatographically pure methanol to dilute the purchased nicotine, cotinine, and trans-3-hydroxycotinine standard solutions into stock solutions with a concentration of 1 mg / ml respectively; Take the above three standard stock solutions and dilute them with ultrapure water to a mixed standard solution with a concentration of 5 μg / ml; b. Preparation of internal standard standard solution: Use chromatographically pure methanol to dilute the purchased nicotine-d3 standard solution, cotinine-d3 solid powder, and trans-3-hydroxycotinine-d3 standard solution into stock solutions with a concentration of 1 mg / ml respectively; Take the above three internal standard stock solutions and dilute them with ultrapure water to a mixed internal standard standard solution with a concentration of 5 μg / ml; c. Plotting of standard working curve: Dilute the nicotine, cotinine, and trans-3'-hydroxycotinine mixed standard application solution with ultrapure water to a series of mixed standard solutions with concentrations of 1.0, 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, and 1000.0 ng / mL; Take 0.15 ml of each series of standard solutions respectively, add 30 μl of 5 μg / ml mixed internal standard application solution, namely the mixed internal standard of nicotine-d3, cotinine-d3, and trans-3'-hydroxycotinine-d3, dilute to 1.5 ml with water, pass through a 0.22 μm nylon 66 membrane filter, and then perform UPLC / MS / MS analysis. Plot the standard curve with the mass concentration of the analyte as the abscissa and the area ratio of the analyte and internal standard peaks as the ordinate.

3. A method for detecting the contents of nicotine and cotinine in the urine of children exposed to second-hand smoke according to claim 1, characterized in that, The conditions of UPLC-MS / MS in step S2 are as follows: 1) Liquid phase conditions: Chromatographic column: ACQUITY UPLC BEH HILIC; Column temperature: 40°C; Mobile phase: A: 0.1% ammonia water (analytical pure), B: acetonitrile (chromatographically pure); Flow rate: 0.2 ml / min; Injection volume: 2 μl; 2) Mass spectrometry conditions: Ionization mode: ESI positive ion mode; Ion transfer tube temperature: 325 °C; Nebulizer temperature: 350 °C; Sheath gas flow rate: 35 Arb; Auxiliary gas flow rate: 5 Arb; Purge gas flow rate: 0 Arb; Spray needle voltage: 3000 V; The quantitative ion pairs of nicotine are: 163.225 m / z and 130.125 m / z; The quantitative ion pairs of nicotine-d3 are: 166.212 m / z and 130.083 m / z; The quantitative ion pairs of cotinine are: 177.175 m / z and 80.155 m / z; The quantitative ion pairs of cotinine-d3 are: 180.188 m / z and 80.155 m / z; The quantitative ion pairs of trans-3-hydroxycotinine are: 193.188 m / z and 80.155 m / z; The quantitative ion pairs of trans-3-hydroxycotinine-d3 are: 196.188 m / z and 80.155 m / z.

4. A method for detecting the contents of nicotine and cotinine in the urine of children exposed to second-hand smoke according to claim 3, characterized in that The collision energy of the nicotine ion pairs 163.225 m / z and 130.125 m / z is 21.39 V; The collision energy of the nicotine-d3 ion pairs 166.212 m / z and 130.083 m / z is 21.64 V; The collision energy of the cotinine ion pairs 177.175 m / z and 80.155 m / z is 25.05 V; The collision energy of the cotinine-d3 ion pairs 180.188 m / z and 80.155 m / z is 25.81 V; The collision energy of the trans-3-hydroxycotinine ion pairs 193.188 m / z and 80.155 m / z is 28.67 V; The collision energy of the trans-3-hydroxycotinine-d3 ion pairs 196.188 m / z and 80.155 m / z is 29.22 V.

5. A method for detecting the contents of nicotine and cotinine in the urine of children exposed to second-hand smoke according to claim 4, characterized in that, The retention time of nicotine is 4.12 - 4.14 min, and the retention time of nicotine-d3 is 4.16 - 4.17 min; The retention time of cotinine is 2.09 - 2.11 min, and the retention time of cotinine-d3 is 2.09 - 2.11 min; The retention time of trans-3-hydroxycotinine is 1.86 - 1.88 min, and the retention time of trans-3-hydroxycotinine-d3 is 1.87 - 1.88 min.

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