An on-line ionization mass spectrometry method for rapid detection of nicotine and cotinine in urine

Abstract

An on-line ionization mass spectrometry method for rapid detection of nicotine and cotinine in urine uses membrane extraction surface pyrolysis desorption electrospray ionization mass spectrometry to perform on-line analysis of nicotine and cotinine in urine. The device involved includes a membrane extraction system, a surface pyrolysis desorption electrospray ionization source, and a mass spectrometer to achieve membrane extraction enrichment of analytes in urine samples, pyrolysis desorption and ionization of analytes, and mass spectrometry detection of analytes. In this method, urine samples are injected into the membrane extraction system, and the components in the urine are extracted and enriched on the membrane. The pre-heated nebulizing gas and electrospray reagent ions perform pyrolysis desorption on the membrane surface, and at the same time complete the release and ionization process of the extracted components. Nicotine and cotinine ions enter the mass spectrometer for analysis. This method can complete the extraction and enrichment of the target substances on-line, and also inhibits the introduction of inorganic salts in urine, overcomes the influence of inorganic salts on the mass spectrometer, combines membrane extraction with surface pyrolysis desorption electrospray ionization mass spectrometry, and can achieve rapid analysis of nicotine and cotinine in urine samples.

Description

Technical Field

[0001] The present invention belongs to the field of detection of biomarkers for tobacco smoke exposure, and particularly relates to an on-line ionization mass spectrometry method for rapidly detecting nicotine and cotinine in urine. This method uses membrane extraction surface pyrolysis desorption electrospray ionization mass spectrometry to rapidly analyze nicotine and cotinine in urine. Background Art

[0002] The issue of smoking and health has attracted increasing widespread attention from the general public, and the risk assessment of tobacco products has become a research hotspot in the field of health. Biomarkers are detectable indicators that reflect the interaction between the body and environmental factors (physical, chemical, or biological). The Tobacco Control Research Group of the World Health Organization (WHO) clearly stated in the report "The Scientific Basis for Tobacco Product Regulation" that biomarkers can become a useful tool for regulators to evaluate whether the risk of tobacco has been reduced. Exposure biomarkers can reflect the actual exposure level of harmful substances in smokers and have been widely used in the risk assessment research of tobacco products.

[0003] Nicotine and cotinine are key biomarkers for evaluating tobacco exposure. Detecting nicotine and cotinine in urine samples of smokers can reflect the tobacco exposure situation of smokers. At the same time, because urine samples are convenient to collect and non-invasive, they are relatively ideal biological samples and have been widely used in smoking epidemiology research. Currently, many studies have been conducted on the detection methods of nicotine and cotinine at home and abroad. The most commonly used methods mainly include gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS / MS). Although the above off-line methods can complete the quantitative analysis of urine samples, they often require a cumbersome pretreatment process. At the same time, due to the long chromatographic separation time, the sample analysis efficiency is limited. In addition, the half-life of cotinine is short and it will continue to decompose under the action of active enzymes in urine, and the change rate within 1 day can reach 30%. Therefore, the rapid analysis and detection of urine samples is of great significance.

[0004] In recent years, open direct ionization mass spectrometry technology has been developed. Since the emergence of desorption electrospray ionization (DESI) and direct analysis in real time (DART), various open direct ionization techniques have continuously emerged. Since these ionization mass spectrometry techniques can directly perform ionization analysis on samples without or with only a simple sample treatment process, the operation steps are greatly simplified and the detection efficiency is improved, which makes it possible to directly ionize and analyze urine samples. However, there are often certain amounts of inorganic salts in urine samples. Inorganic salts have a strong competitive ionization effect in the electrospray system, resulting in a strong ion suppression effect, significantly reducing the sensitivity of the analytes. Secondly, the presence of inorganic salts will generate a series of ion adduct peaks, making the interpretation of the spectrum more complex. In addition, too many salts will corrode and contaminate the hardware of the mass spectrometry system, and in severe cases, it will cause hardware damage and require timely cleaning.

[0005] To achieve the direct ionization analysis of nicotine and cotinine in urine samples, the present invention combines a PDMS membrane injection system with surface thermal desorption electrospray ionization mass spectrometry, which can realize the direct on-line analysis of nicotine and cotinine in urine samples. On the one hand, the PDMS membrane is a hydrophobic membrane, which can extract and transfer the organic components to be detected in urine to the other side of the membrane, while about 95% of the water, urea, and inorganic salts in urine cannot pass through the membrane, thus completing the on-line extraction and enrichment of organic components and suppressing the adverse effects of inorganic substances on ionization and mass spectrometry analysis. On the other hand, the desorption electrospray is directly set at a certain angle on the membrane surface, which can directly ionize the organic substances to be detected and then perform mass spectrometry analysis. Compared with other analytical techniques: the combination of membrane extraction and surface thermal desorption electrospray ionization mass spectrometry in the present invention can directly and rapidly analyze nicotine and cotinine in urine samples without pretreatment of urine samples and without chromatographic separation. The whole process is < 30 s, with the advantages of simple operation and high test efficiency. Summary of the Invention

[0006] The object of the present invention is to develop an on-line ionization mass spectrometry method for rapidly detecting nicotine and cotinine in urine. The membrane extraction surface thermal desorption electrospray ionization mass spectrometry is used to perform on-line analysis of nicotine and cotinine in urine. The devices involved include a membrane extraction system, a surface thermal desorption electrospray ionization source, and a mass spectrometer, which respectively realize the membrane extraction and enrichment of analytes in urine samples, the thermal desorption and ionization of analytes, and the mass spectrometry detection of analytes. In this method, the urine sample is injected into the membrane extraction system, and the components in the urine are extracted and enriched on the membrane. The ionization source is rotated from the non-working position to the working position, so that the preheated nebulizing gas and the electrospray reagent ion pair perform thermal desorption on the membrane surface for a specific time, and at the same time complete the release and ionization process of the extracted components. The nicotine and cotinine ions enter the mass spectrometer for analysis.

[0007] The specific steps of this method include:

[0008] (1) Preparation of internal standard working solution: Weigh accurately a certain amount of nicotine-d3 and cotinine-d3 reference standards, dissolve them with methanol, make up the volume to a certain value, and shake well;

[0009] (2) Preparation of standard solutions: Weigh accurately a certain amount of nicotine and cotinine reference standards, dissolve them with methanol, make up the volume to a certain value, and shake well to obtain a stock solution; Gradually dilute the above stock solution and add the above internal standard working solution to obtain standard solutions with different concentrations;

[0010] (3) Membrane extraction surface desorption electrospray ionization method: Use a 5 ml syringe to accurately aspirate 3 ml of the test solution. The test solution is a standard solution or a urine sample. Inject the test solution into the membrane extraction system at a flow rate of 500 μL / s with an injection pump. After the injection is completed, rotate the ionization source from the non-working position to the working position to perform surface desorption electrospray ionization on the membrane. The time for surface desorption electrospray ionization is 10 s - 30 s;

[0011] (4) Mass spectrometry analysis: The product ions of surface desorption electrospray ionization enter the ion trap mass spectrometer through the mass spectrometry inlet to complete the analysis.

[0012] The above membrane injection system includes a membrane, a membrane injection bottom plate, a sealing ring, and a membrane fixing plate; The membrane used is a PDMS membrane or other hydrophobic membranes, with a thickness of 25 - 100 μm; The membrane injection bottom plate is provided with a membrane extraction inlet and a membrane extraction outlet.

[0013] Preferably, the membrane is a rectangular PDMS membrane with a thickness of 50 μm.

[0014] In the above surface desorption electrospray ionization method, the included angle between the axis of the electrospray ionization source and the membrane plane is 20° - 80°, the included angle between the axis of the mass spectrometry inlet and the membrane plane is 0 - 30°, the distance between the electrospray cone orifice and the mass spectrometry inlet is 5 - 10 mm, and the intersection point of the electrospray axis and the axis of the mass spectrometry inlet is the center of the membrane surface; The electrospray reagent is a 50% (v / v) methanol aqueous solution, with a flow rate of 0.4 mL / min; The electrospray voltage is 3 - 5 kV, and the nebulizing gas is nitrogen, with a pressure of 300 - 500 kPa.

[0015] In the above surface desorption electrospray ionization method, the surface desorption ionization source is connected to a rotating fixed disk through a support rod. By rotating, the axis of the ionization source, the axis of the support rod, and the axis of the mass spectrometry inlet can be in the same plane, which is the working position; Based on the working position, rotate the surface desorption ionization source counterclockwise by 90°, which is the position to be worked. Through the rotating fixed disk, the surface desorption ionization source can be switched between the position to be worked and the working position to achieve surface desorption electrospray ionization at a specific time.

[0016] The above-mentioned electrospray atomization gas needs to be preheated before entering the electrospray ionization source, and the heating temperature is 50 - 300 °C.

[0017] The above-mentioned mass spectrometry method for on-line detection of nicotine and cotinine in urine uses an open ion trap mass spectrometry. The ion transfer temperature is 100 °C - 500 °C, in positive ion fragmentation mode. Collision-induced dissociation is carried out with 35% collision energy. The quantitative ion of nicotine is m / z 163 - 132; the quantitative ion of nicotine-d3 is m / z 166 - 132; the quantitative ion of cotinine is m / z 177 - 80; the quantitative ion of cotinine-d3 is m / z 180 - 83.

[0018] The present invention has the following beneficial effects:

[0019] The present invention rapidly enriches and samples the organic components of urine samples through a membrane extraction system, without the need to pre-treat urine samples to eliminate matrix effects and the influence of inorganic salts on ionization and mass spectrometry, and the operation is simple; there is no separation process of gas chromatography or liquid chromatography, and the analysis time is short; this method realizes the direct and rapid analysis of nicotine and cotinine in urine samples, and the whole process is < 30 s, having the advantages of simple operation and high test efficiency. Description of the Drawings

[0020] Figure 1 . Schematic diagram of membrane extraction surface desorption electrospray ionization mass spectrometry.

[0021] Figure 2 . Schematic diagram of the working position and non-working position of the membrane extraction surface desorption electrospray ionization source.

[0022] Figure 3 . First-stage fragmentation mass spectrometry diagram of the mass spectrometry detection of nicotine, nicotine-d3, cotinine, and cotinine-d3.

[0023] Figure 4 . Mass spectrometry response of nicotine in urine samples for five repeated tests.

[0024] In the drawings: 11. PDMS membrane, 12. Membrane injection bottom plate, 121. Membrane extraction inlet, 122. Membrane extraction chamber, 123. Membrane extraction outlet, 13. Membrane fixing plate, 14. Sealing ring, 21. Sample injection port, 22. Atomization gas inlet, 23. Electrospray cone tube, 24. DC high-voltage power supply, 251. Clamping piece, 252. Support rod, 253. Rotating fixing disk, 3. Mass spectrometry inlet. Detailed Embodiments

[0025] The present invention will be further described below in conjunction with the drawings and specific embodiments:

[0026] Example 1

[0027] The device used in the method of the present invention is membrane extraction surface pyrolysis desorption electrospray ionization mass spectrometry, including a membrane extraction system, a surface pyrolysis desorption electrospray ionization source, and a mass spectrometer, to achieve membrane extraction enrichment of analytes in urine samples, pyrolysis desorption and ionization of analytes, and mass spectrometry detection of analytes. The membrane extraction system, as Figure 1 shown, includes a PDMS membrane 11, a membrane inlet bottom plate 12, a membrane fixing plate 13, and a sealing ring 14; the membrane inlet bottom plate 12, the PDMS membrane 11, the sealing ring 14, and the membrane fixing plate 13 are arranged in sequence from bottom to top to achieve the sealing and fixation of the PDMS membrane 11. The PDMS membrane 11 is a circular membrane with a thickness of 50 μm and a radius of 8 mm; the membrane inlet bottom plate 12 is internally provided with a membrane extraction inlet 121, a membrane extraction chamber 122, and a membrane extraction outlet 123. Among them, the membrane extraction inlet 121 is directly connected to the urine sample pipeline, and the membrane extraction outlet 123 is directly connected to the waste liquid bottle. When the urine sample passes through the membrane extraction chamber 122, it contacts the surface of the PDMS membrane 11 and undergoes extraction enrichment.

[0028] The surface pyrolysis desorption electrospray ionization source, as Figure 1 shown, includes a sample injection port 21, an atomizing gas inlet 22, an electrospray cone tube 23, and a DC high-voltage power supply 24. The electrospray ionization source is arranged at a certain angle with the PDMS membrane 11 and the mass spectrometry inlet 3. The angle α between the central axis of the electrospray ionization source and the membrane plane is 60°, and the angle β between the central axis of the mass spectrometry inlet and the membrane plane is 10°. The electrospray cone tube 23 is 5 mm away from the mass spectrometry inlet, and the intersection point of the electrospray central axis and the mass spectrometry inlet central axis is the center of the membrane surface; as Figure 1 and Figure 2 shown, the surface pyrolysis ionization source is connected to a rotating fixed disk 253 through a clamping member 251 and a support rod 252. By rotating, the ionization source central axis, the support rod axis, and the mass spectrometry inlet 3 central axis can be in the same plane, which is the working position; based on the working position, rotate the surface pyrolysis ionization source counterclockwise by 90°, which is the position to be worked.

[0029] The electrospray reagent is a 50% (v / v) methanol aqueous solution with a flow rate of 0.4 mL / min; the electrospray voltage is 4.5 kV, the atomizing gas is nitrogen, and the pressure is 400 kPa. The atomizing gas needs to be preheated before entering the electrospray ionization source, and the heating temperature is 150 °C.

[0030] The specific steps of the method include:

[0031] (1) Preparation of the internal standard working solution: Accurately weigh a certain amount of nicotine-d3 and cotinine-d3 standard products, dissolve them with methanol, make up the volume, and shake well;

[0032] (2) Preparation of standard solutions: Weigh accurately a certain amount of nicotine and cotinine reference standards, dissolve them with methanol, make up the volume and shake well to obtain the stock solution; Gradually dilute the above stock solution and add the above internal standard working solution to obtain standard solutions with different concentrations;

[0033] (3) Use a 5 ml syringe to accurately aspirate 3 ml of the test solution. The test solution is a standard solution or a urine sample. Inject the test solution into the membrane extraction system with a flow rate of 500 μL / s using an injection pump. After the injection is completed, rotate the ionization source from the non-working position to the working position to perform surface pyrolysis desorption electrospray ionization on the membrane; The time for pyrolysis desorption electrospray ionization is 30 s;

[0034] (4) Mass spectrometry analysis: The product ions of pyrolysis desorption electrospray ionization enter the open ion trap mass spectrometry through the mass spectrometry inlet to complete the analysis.

[0035] Perform mass spectrometry analysis on the standard working solutions of nicotine, nicotine-d3, cotinine, and cotinine-d3 at 200 μg / L using the above method. Set the ion transfer temperature to 300 °C, positive ion fragmentation mode, and use 35% collision energy for collision-induced dissociation to obtain the first-order fragmentation mass spectra of the four components, as Figure 3 shown. Finally, determine that the quantitative ion of nicotine is m / z 163—132; the quantitative ion of nicotine-d3 is m / z 166-132; the quantitative ion of cotinine is m / z 177-80; the quantitative ion of cotinine-d3 is m / z 180-83.

[0036] Perform five tests on a urine sample of a smoker. The mass spectrometry response of nicotine is as Figure 4 shown. The RSD of the 5 test results is 6.1%, indicating that the membrane extraction surface pyrolysis desorption electrospray ionization mass spectrometry has good stability when detecting urine samples.

[0037] Example 2

[0038] Accurately weigh 10 mg each of nicotine and cotinine reference standards, dissolve them with methanol and transfer them to a 10 ml brown volumetric flask, and make up the volume with methanol to obtain the stock solution of the reference standards. Accurately weigh 1 mg each of nicotine-d3 and cotinine-d3 reference standards, dissolve them with methanol and transfer them to a 100 ml brown volumetric flask, and make up the volume with methanol to obtain the internal standard working solution. Use the stepwise dilution method and add a certain amount of the internal standard working solution to obtain standard solutions containing internal standards with different concentrations.

[0039] Perform membrane extraction surface pyrolysis desorption electrospray ionization mass spectrometry on each standard solution in sequence. Specifically, use a 5 ml syringe to accurately draw 3 ml of the standard solution, and inject the sample into the membrane extraction system at a flow rate of 500 μL / s with an injection pump. After the injection is completed, turn on the electrospray, and rotate the ionization source from the non-working position to the working position to perform surface pyrolysis desorption electrospray ionization on the membrane. The time for pyrolysis desorption electrospray ionization is 30 s; detect the quantitative ions of nicotine and cotinine according to the mass spectrometry conditions in Example 1, and use the internal standard method to obtain the quantitative linear equations and correlation coefficients of nicotine and cotinine. The results show that the linear quantitative range of this method is slightly smaller than that of the LC-MS / MS method reported in the literature. However, since the nicotine and cotinine in the urine samples of smokers are usually higher than 100 ng / ml, this method can still meet the detection of urine samples of smokers. Importantly, the operation of detecting samples by this method is simple, and the whole process is <30 s, with a high test efficiency.

[0040] Table 1. Linear equations and correlation coefficients of nicotine and cotinine

[0041]

[0042] Example 3

[0043] Use the established method to quickly detect the urine samples of 2 non-smokers and 2 smokers. The results are shown in Table 2. The results show that the contents of nicotine and cotinine in the urine samples of the 2 non-smokers were not detected, indicating that their contents were significantly lower than 50 ng / ml; the contents of nicotine and cotinine in the urine samples of the 2 smokers were both higher than 200 ng / ml. 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 smokers.

[0044] Table 2. Test results of nicotine and cotinine in urine samples of smokers and non-smokers

[0045]

Claims

1. An on-line ionization mass spectrometry method for rapid detection of nicotine and cotinine in urine, characterized in that: This method directly analyzes nicotine and cotinine in urine by membrane extraction surface pyrolysis desorption electrospray ionization mass spectrometry. The devices involved include a membrane extraction system, a surface pyrolysis desorption electrospray ionization source, and a mass spectrometer, which respectively achieve the membrane extraction and enrichment of analytes in urine samples, the pyrolysis desorption and ionization of analytes, and the mass spectrometry detection of analytes. In this method, urine samples are injected into the membrane extraction system, and the components in the urine are extracted and enriched on the membrane. The ionization source is rotated from the non-working position to the working position, so that the preheated nebulizing gas and the electrospray reagent ions thermally desorb the membrane surface for a specific time, while completing the release and ionization process of the extracted components. Nicotine and cotinine ions enter the mass spectrometer for analysis. The specific steps of this method are as follows: (1) Preparation of internal standard working solution: Weigh accurately a certain amount of nicotine-d3 and cotinine-d3 standard products into a volumetric flask, and make up to the mark with methanol; (2) Preparation of standard solutions: Weigh accurately a certain amount of nicotine and cotinine standard products into a volumetric flask, add the internal standard working solution, and make up to the mark with methanol; Gradually dilute and add the internal standard working solution to obtain standard solutions with different concentrations; (3) Membrane extraction surface pyrolysis desorption electrospray ionization method: Use a 5 mL syringe to accurately aspirate 3 mL of the sample, and inject the sample into the membrane extraction system at a flow rate of 500 μL / s with an injection pump. After the injection is completed, rotate the ionization source from the non-working position to the working position to perform surface pyrolysis desorption electrospray ionization on the membrane. The time for surface pyrolysis desorption electrospray ionization is 10 s - 30 s; (4) Mass spectrometry analysis: The product ions enter the ion trap mass spectrometer through the mass spectrometry inlet to complete the analysis; The mass spectrometer used is an open ion trap mass spectrometer; Among them, the membrane extraction system includes a membrane, a membrane injection bottom plate, a sealing ring, and a membrane fixing plate. The membrane injection bottom plate, the membrane, the sealing ring, and the membrane fixing plate are arranged in sequence from bottom to top to achieve the sealing and fixing of the membrane. The membrane used is a PDMS membrane or other hydrophobic membranes, with a thickness of 25 - 100 μm; The membrane injection bottom plate is provided with a membrane extraction inlet and a membrane extraction outlet; Among them, the surface pyrolysis desorption electrospray ionization source is connected to a rotating fixed disk through a support rod. By rotating, the central axis of the ionization source, the axis of the support rod, and the central axis of the mass spectrometry inlet can be in the same plane, which is the working position; Based on the working position, rotate the surface pyrolysis ionization source counterclockwise by 90°, which is the non-working position; Through the rotating fixed disk, the surface pyrolysis desorption electrospray ionization source can be switched between the non-working and working positions to achieve surface pyrolysis desorption electrospray ionization for a specific time. The angle between the central axis of the electrospray ionization source and the membrane plane is 20 - 80°, the angle between the central axis of the mass spectrometry inlet and the membrane plane is 0 - 30°, the distance between the electrospray cone and the mass spectrometry inlet is 5 - 10 mm, and the intersection point of the electrospray central axis and the mass spectrometry inlet central axis is the center of the membrane surface; The electrospray reagent is a 50% v / v methanol aqueous solution, with a flow rate of 0.4 mL / min; The electrospray voltage is 3 - 5 kV, the nebulizing gas is nitrogen, and the pressure is 300 - 500 kPa.

2. The mass spectrometry method according to claim 1, wherein: The nebulizing gas needs to be preheated before entering the electrospray ionization source, and the heating temperature is 50 - 300 °C.

3. The mass spectrometry method according to claim 1, wherein: The mass spectrometry used is an open ion trap mass spectrometry. The ion transfer temperature is 100°C - 500°C. The positive ion fragmentation mode is adopted, and collision-induced dissociation is carried out with 35% collision energy. The nicotine quantification ion is m / z 163-132; the nicotine-d3 quantification ion is m / z 166-132; the cotinine quantification ion is m / z 177-80; the cotinine-d3 quantification ion is m / z 180-83.

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