Method for simultaneously detecting nicotine and metabolites, neurotransmitters and lipids thereof in brain tissue sample

Abstract

The invention provides a method for simultaneously detecting nicotine and metabolites, neurotransmitters and lipids thereof in a brain tissue sample, which comprises the following steps: injecting nicotine into the abdominal cavity of a rat to obtain the brain tissue of the rat, and treating to prepare a brain tissue slice; then putting the brain tissue slice into a mass spectrum imaging instrument for detection; and analyzing the spatial distribution characteristics of nicotine and metabolites, neurotransmitters and lipids thereof in the brain after the rat intraperitoneal injection of nicotine on the basis of the obtained rat brain mass spectrum imaging graph. Wherein the spray voltage of the mass spectrum imaging instrument is 3.5-6.0 kV, and the spray solvent is formed by mixing acetonitrile, isopropanol and water according to the volume ratio of (5-7): (1-3): (1-3). The simultaneous detection method can accurately obtain spatial distribution and metabolism information of the three substances in the brain tissue, has the characteristics of simplicity and convenience in operation, good accuracy, high precision and the like, and provides an accurate and effective analysis method for spatial distribution and metabolism research of nicotine and metabolites thereof, neurotransmitters and lipids in the brain tissue.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis technology, and relates to a method for simultaneous detection of nicotine and its metabolites, neurotransmitters and lipids in brain tissue samples. Background Technology

[0002] Nicotine, the main alkaloid in tobacco, can rapidly cross the blood-brain barrier after entering the human body, triggering changes in neurotransmitter levels in the brain. Studies have shown that nicotine has a potential protective effect against various neurodegenerative diseases, and its effect is closely related to its exposure level and spatial distribution in the brain. Neurotransmitters, as chemical messengers that transmit information between neurons or between neurons and effector cells, are closely related to changes in their levels and specific distribution in the brain, and can serve as important targets for clinical diagnosis, screening, and treatment, such as Parkinson's disease, depression, and Alzheimer's disease. With a deeper understanding of the pathological mechanisms of Parkinson's disease, lipids have also been found to play an important role. Lipids are not only key components of cell membranes but also play a crucial role in cell signaling. Studies have shown that abnormal lipid metabolism is closely related to the pathological progression of various neurodegenerative diseases. In conclusion, simultaneously investigating the changes in the content and spatial distribution characteristics of nicotine and its metabolites, neurotransmitters, and lipids in brain tissue is crucial for a deeper understanding of the mechanisms of related diseases.

[0003] Traditional detection methods, such as electrochemical methods, liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS), while capable of qualitative and quantitative analysis of drugs metabolized in vivo, typically require complex sample pretreatment. This process easily leads to the loss of in-situ spatial information of the analyte in biological tissue samples, resulting in insufficient spatial resolution and difficulty in accurately obtaining its spatial distribution information within biological tissues. This, in turn, limits further research into the mechanisms by which nicotinic acid regulates neurotransmitter and lipid metabolism levels and spatial distribution. In contrast, mass spectrometry imaging (MSI), as a non-targeted and highly sensitive imaging method based on mass spectrometry, can directly provide chemical information and spatial location information of the analyte. Furthermore, the advantages of MSI—simple operation, high sensitivity, and high throughput—make it significantly superior in the characterization of biological samples.

[0004] Therefore, developing an efficient and visualized detection method for simultaneously analyzing the spatial distribution of nicotine and its metabolites, neurotransmitters, and lipids in biological tissue samples (especially brain tissue), and revealing the potential mechanism of action of nicotine in the treatment of neurodegenerative diseases, has become a key problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for the simultaneous detection of nicotine and its metabolites, neurotransmitters, and lipids in brain tissue samples. This simultaneous detection method is characterized by its simplicity of operation, high accuracy, and high precision, providing an accurate and effective analytical method for studying the spatial distribution and metabolism of nicotine and its metabolites, neurotransmitters, and lipids in brain tissue.

[0006] To achieve this objective, the present invention adopts the following technical solution: A method for simultaneous detection of nicotine and its metabolites, neurotransmitters, and lipids in brain tissue samples, comprising: Step 1: After injecting nicotine into rats intraperitoneally, the brain is removed and prepared into brain tissue sections; Step 2: The brain tissue slices are subjected to mass spectrometry imaging using a mass spectrometry imaging instrument under the action of a spray voltage of 3.5-6.0 kV and a spray solvent. The scan data is processed by data processing software to obtain a mass spectrometry imaging image of rat brain tissue. The spray solvent is composed of acetonitrile, isopropanol and water in a volume ratio of 5-7: 1-3: 1-3. Step 3: Compare the mass spectrometry information in the mass spectrometry image of the rat brain tissue with the mass spectrometry information of the compounds in the biological database to obtain the spatial distribution information of nicotine and its metabolites, neurotransmitters and lipids in different brain regions of the rat.

[0007] In one specific embodiment, the nicotine compounds include nicotine, nornicotine, norcotinine, nicotine nitrogen oxides, cotinine nitrogen oxides, and trans-3'-hydroxycotinine; The neurotransmitters include dopamine, adrenaline, serotonin, adenosine, adenine, hypoxanthine, γ-tryptophan, taurine, histidine, histamine, carnosine, acetylcholine, choline, arginine, valine, lysine, proline, and glutamate. The lipid compounds include arachidonic acid, docosahexaenoic acid, α-glycerophosphate choline, PC(20:2(11Z,14Z) / 20:4(5Z,8Z,11Z,14Z)), PC(22:4(7Z,10Z,13Z,16Z) / 16:0), PC(22:2(13Z,16Z) / 14:0), PC(20:1(11Z) / 14:0), PE(P-18:1(9Z) / 20:4(5Z,8Z,11Z,14Z)), and PC(16:0 / 16:0).

[0008] To further ensure the accuracy and precision of the detection, step one includes: Rats were injected intraperitoneally with nicotine at a concentration of 1–2 mg / kg. After waiting 5–15 minutes, the rats were anesthetized by injecting 1 mL of 4% tribromoethanol into the intraperitoneal cavity. The brain was then removed, and the morphology of the brain should be preserved during the removal process. After rinsing the complete brain tissue, it was dehydrated and placed in a foil sheet above liquid nitrogen for quick freezing for 2–5 minutes. Then, the biological brain tissue was placed in a 50 mL centrifuge tube and transferred to a freezer at -79 to -81°C for more than 1 week to ensure that it maintains its shape. Six to eight hours before preparing brain slices, the mouse brains were thawed at -20°C and then cut using a microtome. Brain tissue was fixed on a circular tray and sectioned using a microtome at a temperature of -20 to -24°C. The thickness of the brain slices was 9 to 11 mm. The brain tissue slices were then fixed on glass slides and dried in a vacuum drying oven for 20 to 40 min before analysis.

[0009] In one specific embodiment, step two, the mass spectrometry imaging detection method includes: performing mass spectrometry imaging detection using an AFADESI-MSI platform with an aerodynamically assisted desorption / electrospray ionization source, wherein the mass spectrometry imaging instrument continuously scans the tissue surface along the x-axis at a constant rate of 150–170 µm / s, and vertically scans along the y-axis at intervals of 180–220 µm; using Full MS mode; the scan range is 100–1000 Da; the automatic gain control target is (1.8–2.2) × 10⁻⁶. 5 The automatic gain control time is 60–80 ms; the capillary temperature is 340–360 °C; the spray gas pressure is maintained at 0.5–0.7 MPa; the pumping gas velocity is 40–50 L / min; and the flow rate of the spray solvent is 5–7 μL / min.

[0010] Furthermore, in step two, the distances from the mass spectrometry imaging instrument's sample sprayer to the surface of the brain tissue slice and the ion delivery tube are 0.5–0.7 mm and 2.5–3.5 mm, respectively.

[0011] Furthermore, in step two, the distance from the ion source port to the mass spectrometry acquisition tube port of the mass spectrometry imaging instrument is 9–11 mm.

[0012] In one specific embodiment, in step two, after identifying differential metabolites in the brain using spatially resolved metabolomics, the differential metabolite information is imported into the imaging software MassImager for ion image reconstruction. Region-specific mass spectrometry data is obtained by matching high spatial resolution hematoxylin and eosin images and exported as a .txt file.

[0013] In one specific implementation, in step three, the .txt file exported in step two is imported into Markerview 1.2.1 software, a quality tolerance of 10 ppm is set, and data processing steps such as peak picking, peak alignment, and isotope peak removal are performed.

[0014] Therefore, the technical solution provided by the present invention has the following characteristics: (1) The brain tissue mass spectrometry imaging of the present invention can accurately identify and locate the differential distribution of nicotine and its metabolites, neurotransmitters and lipids in different brain regions in brain tissue samples, which broadens the research on the pharmacokinetic, pharmacodynamic properties and spatial metabolomics of nicotine, and provides an accurate and effective analytical method for the spatial distribution and spatial metabolism research of nicotine and its metabolites, neurotransmitters and lipids in biological brain tissue.

[0015] (2) The brain tissue mass spectrometry imaging of the present invention does not require a complicated pretreatment process for brain tissue samples. It can be directly detected by frozen sectioning, which is simple to operate, has high ionization efficiency, accurate detection results, high sensitivity, and good repeatability. It can realize the detection and analysis of the spatial distribution of small molecule chemical substances in brain tissue. Attached Figure Description

[0016] Figure 1 Figure 1 shows the effect of mass spectrometry parameters on mass spectrometry signal intensity in step two of embodiment 1 of the present invention; Figure A shows the effect of different spray solvents on mass spectrometry signal intensity, and "ACN : IPA : H2O-FA" in this figure refers to "ACN : IPA : H2O-0.1%FA"; Figure B shows the effect of different spray voltages on mass spectrometry signal intensity; Figure C shows the mass spectrometry signal intensity using a spray solvent with a volume ratio of 6:2:2 of MEOH: IPA: H2O. m / z609.1450); Figure D is the mass spectrometry signal intensity diagram of the spray solvent using a volume ratio of 6:2:2 of ACN:IPA:H2O (6:2:2). m / z 609.1450); Figure 2 This is a mass spectrometry image of nicotine and its metabolites in Example 1 of the present invention; Figure 3 This is a mass spectrometry image of neurotransmitters in Embodiment 1 of the present invention; Figure 4 This is a mass spectrometry image of lipids in Example 1 of the present invention; Figure 5 This is a map showing the spatial distribution and inter-group differences of different metabolites in the entire brain region of rats; Figure A is a schematic diagram of a sagittal section of the rat brain and annotation diagrams of each anatomical micro-region. Each of the other sub-figures includes a mass spectrometry imaging thermogram on the left (color scales represent metabolite concentrations, with warm / dark colors indicating high concentrations) and a petal diagram on the right (showing the abundance distribution of the CON group, MPTP group, and LN group in the striatum, prefrontal cortex, and hippocampus). Detailed Implementation

[0017] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely illustrative of the invention and should not be considered as specific limitations thereof. The terminology used in this invention generally has the meanings commonly understood by those skilled in the art, unless otherwise stated. To enable those skilled in the art to better understand the technical solution of the present invention, the invention will be further described in detail below with reference to embodiments.

[0018] As described in the background section, while traditional analytical methods can qualitatively and quantitatively analyze the metabolic processes of drugs in vivo, they struggle to accurately obtain spatial distribution information of analytes in biological tissues. Therefore, this invention utilizes AFADESI (Air-flow-assisted Desorption Electrospray Ionization) technology to establish a method for the simultaneous detection of nicotine and its metabolites, small molecule neurochemicals, and lipids in brain tissue. This method accurately obtains the spatial distribution and metabolic information of these three substances in brain tissue, offering advantages such as ease of operation, high accuracy, and high precision. It provides an accurate and effective analytical method for studying the spatial distribution and metabolism of nicotine and its metabolites, neurotransmitters, and lipids in brain tissue. Example 1

[0019] This embodiment provides a method for the simultaneous detection of nicotine and its metabolites, neurotransmitters, and lipids in brain tissue samples, comprising the following three steps: Step 1: Preparation of biological brain tissue sections The method for preparing the biological brain tissue sections is an existing technology, and specifically may include: SD rats are intraperitoneally injected with nicotine at a concentration of 1 mg / kg, and after waiting 10 min, the rats are anesthetized by intraperitoneal injection of 1 mL of 4% tribromoethanol. The brain is then removed, ensuring its morphology remains intact. The intact brain tissue is rinsed and then blotted for water. It is then placed in a foil sheet above liquid nitrogen and flash-frozen for 2–5 min. The biological brain tissue is then placed in a 50 mL centrifuge tube and transferred to a freezer at -79 to -81°C for more than one week to ensure its morphology is maintained. 6–8 h before preparing the brain sections, the rat brain is thawed at -20°C and then cut using a microtome. The brain tissue is fixed on a circular tray and sectioned using a microtome at a temperature of -20 to -24°C. The brain sections are 10 mm thick. The brain tissue sections are then fixed on glass slides and dried in a vacuum drying oven for 30 min before analysis to obtain the brain tissue sections.

[0020] Step 2: Mass spectrometry imaging detection The brain tissue slices were examined using a mass spectrometry imaging instrument. The scan data was processed by data processing software to obtain mass spectrometry images of the rat brain tissue. Specifically, the mass spectrometry imaging experiment of this invention was performed on an AFADESI-MSI platform equipped with a custom-designed air-flow-assisted desorption electrospray ionization (AFADESI) ion source. The mass spectrometry imaging instrument was a Q-OT-qIT hybrid mass spectrometer (OrbitrapFusion Lumos; Thermo Fisher Scientific, San Jose, California, USA). The mass spectrometry imaging instrument continuously scanned the tissue surface along the x-axis at a constant rate of 150–170 µm / s, and vertically along the y-axis at intervals of 180–220 µm. Full MS mode was used: the scan range was 100–1000 Da; the automatic gain control target was (1.8–2.2) × 10⁻⁶. 5 The automatic gain control time is 60–80 ms; the spray voltage is 3.5–6.0 kV, the capillary temperature is 340–360 °C, the spray gas pressure is maintained at 0.5–0.7 MPa, and the pumping speed is 40–50 L / min; the spray solvent is composed of acetonitrile (ACN), isopropanol (IPA), and water (H2O) in a volume ratio of 5–7:1–3:1–3, and the flow rate of the spray solvent is 5–7 μL / min. The distances from the mass spectrometry imaging instrument's injection nebulizer to the surface of the brain tissue slice and the ion delivery tube are 0.5–0.7 mm and 2.5–3.5 mm, respectively. The distance from the mass spectrometry imaging instrument's ion source port to the mass spectrometry acquisition tube port is 9–11 mm.

[0021] In this embodiment, in step two, the spray solvent is a homogeneous mixture of ACN:IPA:H2O in a volume ratio of 6:2:2, with a flow rate of 6 μL / min; the distance from the ion source spray nozzle of the mass spectrometry imaging system to the tissue section surface is 0.6 mm, and the distance to the ion delivery tube is 3 mm; the distance from the orifice to the mass spectrometry acquisition tube is 10 mm; the mass spectrometry imaging instrument continuously scans the tissue surface along the x-axis at a constant rate of 160 µm / s, and vertically scans along the y-axis at intervals of 200 µm; the specific mass spectrometry analysis parameters are: AFADESI ion source, Full MS mode, scanning range of 100–1000 Da, mass resolution of 120000; automatic gain control target of 2 × 10⁻⁶. 5 The automatic gain control time was 70 ms, the spray voltage was ±6.0 kV, and the capillary temperature was 350°C. The spray gas was nitrogen with a pressure maintained at 0.6 MPa, and the pumping speed was set to 45 L / min.

[0022] After identifying differential metabolites in the brain using spatially resolved metabolomics, the information on differential metabolites was imported into the imaging software MassImager (Chemmind Technologies, Beijing, China) for ion image reconstruction. Region-specific mass spectrometry data were obtained by matching high spatial resolution hematoxylin and eosin (H&E) images and exported as a .txt file.

[0023] Step 3: Analysis of Test Data First, import the .txt file exported in step two above into Markerview 1.2.1 (ABSCIEX) software, set a mass tolerance of 10 ppm, and perform data processing such as peak picking, peak alignment, and isotope peak removal; then compare it with the mass spectrometry information of compounds in the biological database to obtain the spatial distribution information of nicotinic acid and its metabolites, neurotransmitters, and lipids in different brain regions of rats, such as... Figure 1 As shown in Table 1, the target compounds in rat brain tissue sections under Full MS mode are listed in Table 1.

[0024] Table 1. Target compounds in rat brain tissue sections under Full MS mode. target Molecular formula molecular weight Mass-to-charge ratio ( ) Nicotine <![CDATA[C 10 H 14 N2]]> 162.2316 163.1235 Cotinine <![CDATA[C 10 H 12 N2O]]> 176.2151 177.1012 Nicotine <![CDATA[C9H 12 N2]]> 148.2050 149.2170 dactylin <![CDATA[C9H 10 N2O]]> 162.1885 163.2000 Nicotine nitrogen oxides <![CDATA[C6H6N2O2]]> 138.1240 139.1340 Cotinine Nitrogen Oxides <![CDATA[C 10 H 12 N2O2]]> 192.2145 193.2260 trans-3'-hydroxycotinine <![CDATA[C9H 10 N2O2]]> 178.1879 179.1990 dopamine <![CDATA[C8H 11 NO2]]> 153.1784 154.0863 adrenaline <![CDATA[C9H 13 NO3]]> 183.2044 184.0945 5-hydroxytryptamine <![CDATA[C 10 H 12 N2O]]> 176.2151 177.1022 adenosine <![CDATA[C 10 H 13 N5O4]]> 267.2413 268.1093 adenine <![CDATA[C5H5N5]]> 135.1267 136.0617 hypoxanthine <![CDATA[C5H4N4O]]> 136.1115 137.0458 γ-3-aminobutyric acid <![CDATA[C4H9NO2]]> 103.1198 104.0706 Taurine <![CDATA[C2H7NO3S]]> 125.1469 126.0200 Histidine <![CDATA[C6H9N3O2]]> 155.1546 156.0768 histamine <![CDATA[C5H9N3]]> 111.1451 112.0848 Carnosine <![CDATA[C9H 14 N4O3]]> 226.2325 227.1139 Acetylcholine <![CDATA[C7H 16 NO2]]> 146.2074 146.1176 choline <![CDATA[C5H 14 NO]]> 104.1708 104.1069 Arginine <![CDATA[C6H 14 N4O2]]> 174.2010 175.1189 Valine <![CDATA[C5H 11 NO2]]> 117.1463 118.0863 Lysine <![CDATA[C6H 14 N2O2]]> 146.1876 147.1128 proline <![CDATA[C5H9NO2]]> 115.1305 116.0706 glutamic acid <![CDATA[C5H9NO4]]> 147.1293 146.0448 Arachidonic acid <![CDATA[C 20 H 32 O2]]> 304.4740 305.2475 docosahexaenoic acid <![CDATA[C 22 H 32 O2]]> 328.4960 329.5040 α-glycerophosphate choline <![CDATA[C8H 20 NO6P]]> 257.2230 258.1101 Lysophosphatidylethanolamine (LPE) <![CDATA[C 23 H 46 NO7P]]> 479.5950 480.3095 N-(2-hydroxyethyl)lactamide <![CDATA[C5H 11 NO3]]> 133.1470 132.0666 γ-Glu-Met <![CDATA[C 10 H 18 N2O5S]]> 278.3300 277.0863 2-Phenylpropylamine <![CDATA[C9H 13 N]]> 135.2100 136.1120 ADP <![CDATA[C 10 H 15 N5O 10 P2]]> 427.2030 426.0221 GSH <![CDATA[C 10 H 17 N3O6S]]> 307.3280 306.0765 PC(20:2(11Z,14Z) / 20:4(5Z,8Z,11Z,14Z)) <![CDATA[C 48 H 84 NO8P]]> 834.1730 834.6007 PC(22:4(7Z,10Z,13Z,16Z) / 16:0) <![CDATA[C 46 H 84 NO8P]]> 810.1510 810.6007 PC(22:2(13Z,16Z) / 14:0) <![CDATA[C 44 H 84 NO8P]]> 786.1290 786.6007 PC(20:1(11Z) / 14:0) <![CDATA[C 42 H 82 NO8P]]> 760.0910 760.5851 PE(P-18:1(9Z) / 20:4(5Z,8Z,11Z,14Z)) <![CDATA[C 43 H 76 NO7P]]> 750.0550 750.5432 PC (16:0 / 16:0) <![CDATA[C 40 H 80 NO8P]]> 734.0530 734.5694 In step two, parameters such as the spray solvent and spray voltage have a significant impact on the mass spectrometry signal intensity of the analyte in the brain tissue slices. The following single-factor analysis experiment will further illustrate this effect.

[0025] 1.1 Effect of spray solvent on the intensity of the mass spectrometry signal of the target material in brain tissue sections The conditions for this single-factor analysis experiment were basically the same as the simultaneous detection method provided in Example 1, with the main difference being the different spray solvents. The spray solvents used were: ACN, ACN:H2O volume ratio 5:5, ACN:H2O volume ratio 8:2, ACN:IPA:H2O volume ratio 4:4:2, MeOH (methanol), MeOH:H2O volume ratio 5:5, MeOH:H2O volume ratio 8:2, MeOH:IPA:H2O volume ratio 4:4:2, MeOH:IPA:H2O volume ratio 6:2:2, and ACN:IPA:H2O-0.1%FA (0.1% formic acid volume concentration) volume ratio 6:2:2. The experimental results are as follows: Figure 1 A, Figure 1 C and Figure 1 As shown in D.

[0026] Figure 1 A, Figure 1 C and Figure 1 D indicates that the mass spectrometry signal intensity varies significantly depending on the solvent system. When using the ACN:IPA:H2O volume ratio of 6:2:2 as the spray solvent in Example 1, the obtained mass spectrometry signal intensity is significantly better than other solvent combinations. In contrast, when the spray solvent is MeOH:IPA:H2O (6:2:2, v / v / v), the mass spectrometry signal intensity is lower.

[0027] 1.2 Effect of spray voltage on the intensity of the spectral signal of the target substance in brain tissue sections The conditions for this single-factor analysis experiment were basically the same as those for the simultaneous detection method provided in Example 1. The main difference was that the spray voltage was between 2000 and 6000 V. Specifically, tests were conducted at voltage gradients of 2000 V, 3000 V, 4500 V, and 6000 V to evaluate its influence on the signal intensity of the analyte. The results are as follows: Figure 1 As shown in B.

[0028] Figure 1 Results B show that the mass spectrometry signal intensity reaches its highest value when the spray voltage is set to 6000 V, and the signal intensity is lowest at 2000 V.

[0029] Nicotine possesses significant neurobiological activity, influencing related pathological processes and exerting potential neuroprotective effects by regulating multiple signaling pathways in the central and peripheral nervous systems. As an agonist of nicotinic acetylcholine receptors, nicotinic acid has been experimentally proven to improve Parkinson's disease-related symptoms, including promoting dopamine release and increasing the survival rate of GABAergic neurons. Based on this, the drug development value of nicotinic acid in combating neurodegenerative diseases is receiving widespread attention and research. Investigating the changes in endogenous metabolites in the rat brain after nicotinic acid exposure will provide a more solid foundation for a comprehensive understanding of its multiple physiological functions. Figure 2 As shown, the distribution of nicotine and its metabolites (including nornicotine, norcotinine, nicotine nitrosamine, cotinine nitrosamine, and trans-3'-hydroxynorcotinine) in the brain exhibits a clear region-specificity. After absorption, nicotine is widely distributed throughout the brain, but shows significant regional aggregation in the thalamus (TH) and cerebral cortex (CTX). Multiple nicotine metabolites, including nornicotine, norcotinine, nicotine nitrosamine, cotinine nitrosamine, and trans-3'-hydroxynorcotinine, are widely distributed throughout the brain. Notably, nornicotine also shows significant regional aggregation in the TH and CTX brain regions, with distribution characteristics similar to those of nicotine in other brain regions. This distribution pattern suggests that nornicotine may have important biological functions in these specific brain regions, and its mechanism of action may be related to the neuroregulatory function of nicotine.

[0030] Studies have shown that the specific spatial distribution of neurotransmitters in biological brain tissue is closely related to their functional performance. For example... Figure 3As shown, AFADESI mass spectrometry imaging clearly reveals the distribution characteristics of endogenous neurochemicals in different brain regions. For example, dopamine is mainly distributed in the caudate putamen (CP), carnosine is specifically distributed in the olfactory bulb (OB), and adrenaline shows high expression in the hypothalamus (HY). Notably, although neurotransmitters such as γ-aminobutyric acid (GABA), choline, adenosine, taurine, histidine, and lysine are widely distributed throughout the brain, they also exhibit significant regional aggregation. For example, GABA is concentrated in the cerebral aqueduct (CA), middle brain (MB), hypothalamus (HY), CP, and olfactory bulb (OB); taurine is significantly accumulated in CP, OB, and pineal gland (PG); adenosine is mainly distributed in cerebellar (CB), corpus callosum (CC), CP, and OB; and lysine is concentrated in CB, medulla oblongata (MD), and pons (PN).

[0031] Lipids in the brain play important structural and physiological roles in nerve cells, participating in key processes such as neural development, synaptic transmission, and signal transduction. Therefore, lipids play an indispensable role in maintaining brain physiological function and pathological responses, and their distribution and metabolic characteristics are as follows: Figure 4 As shown, docosahexaenoic acid (DHA) is concentrated in prostaglandins (PGs), while arachidonic acid (RAA), α-glycerophosphate choline (α-glycerophosphate choline), and various lipids such as PC (20:2(11Z,14Z) / 20:4(5Z,8Z,11Z,14Z)) exhibit widespread distribution. In particular, PC (20:1(11Z) / 14:0) and (16:0 / 16:0) have high concentrations in brain tissue. Therefore, accurately analyzing the spatial distribution variations of nicotine and its metabolites, along with neurotransmitters and lipids, will provide important evidence for elucidating the pharmacokinetics and pharmacodynamic mechanisms of nicotine.

[0032] Example 2: Feasibility of the Simultaneous Detection Method To further verify the scientific validity, rationality, and feasibility of the simultaneous detection method provided in Example 1, this example uses the simultaneous detection method provided in Example 1. Normal rats (CON group), rats with induced Parkinson's disease (MPTP group), and rats injected intraperitoneally with low concentrations of nicotine (LN group) were used as subjects. The distribution and concentration changes of eleven target compounds, including nicotine and its metabolites, neurotransmitters, and lipids, in their brain regions were measured. The results are shown in the figure below. Figure 5 As shown.

[0033] The animal model was established and administered the following medications: Rats were randomly divided into three groups: the CON group, the MPTP group, and the LN group (nicotine injection concentration 1 mg / kg). Except for the CON group, the other rats received intraperitoneal injection (ip) of MPTP to induce Parkinson's disease-like pathology. The specific modeling protocol was as follows: MPTP 30 mg / kg was injected daily for 7 consecutive days, followed by weekly injections of MPTP to maintain the disease state. After the modeling was completed, the LN group rats further received intraperitoneal injections of 1 mg / kg nicotine once daily for 14 days. Because nicotine is soluble in physiological saline (pH=7.4), the CON group and the MPTP group received the same volume of physiological saline.

[0034] from Figure 5 A shows a schematic diagram of a sagittal section of the rat brain and annotations for each anatomical micro-region.

[0035] Figure 5 B indicates that, based on the experimental results from the LN group, nicotine can rapidly cross the blood-brain barrier and be widely distributed throughout the rat brain after intraperitoneal injection.

[0036] Figure 5 C indicates that the results of the LN group show that cotinine, as the main metabolite of nicotine, is also widely distributed in the whole brain of rats.

[0037] Figure 5 D indicates that, compared with normal mice, the distribution range and content of LPE (0:0 / 18:0) in the brains of rats in the MPTP group were significantly reduced, and intraperitoneal injection of low-concentration nicotine could significantly improve this phenomenon.

[0038] Figure 5 E and 5F showed that the levels of N-(2-hydroxyethyl)lactamide and γ-Glu-Met in the striatum, prefrontal cortex and hippocampus of rats in the MPTP group were significantly higher than those in the CON group. Intraperitoneal injection of nicotine could effectively reverse this upregulation trend and significantly reduce its accumulation level in the brain regions.

[0039] Figure 5The results from group G indicate that 2-Phenylpropylamine is widely distributed in the whole brain of normal mice, as seen in the CON group. However, the abundance of this compound in brain slices from the MPTP group was significantly lower than that in the CON group, suggesting that the synthesis or accumulation of 2-Phenylpropylamine in the Parkinson's disease (PD) model is inhibited. The abundance of this compound in the LN group was significantly higher than that in the MPTP group, indicating that intraperitoneal injection of nicotine can partially restore its level in the mouse brain, further confirming that nicotine intervention can effectively reverse the expression of 2-Phenylpropylamine. These results demonstrate that nicotine can improve the pathological state of the MPTP-induced PD model by regulating the levels of amine metabolites in the brain.

[0040] Figure 5 The results from the H study indicate that histamine is mainly concentrated in the Hyp region (hypothalamus) of the CON group; it is decreased in the MPTP group and partially increased in the LN group; the regulation of histamine may be involved in the neuroprotective effect of the LN group.

[0041] Figure 5 The results showed that ADP was mainly concentrated in the OB and PFC brain regions of rats. The abundance was higher in the CON group, decreased in the MPTP group, and showed a regression phenomenon in the LN group. The reduction in ADP reflects the energy metabolism disorder in the OB and PFC in the MPTP model.

[0042] Figure 5 J indicates that GSH is widely distributed throughout the mouse brain, but is significantly reduced in the MPTP group, while some brain regions in the LN group show recovery; GSH, as a core antioxidant molecule, demonstrates the enhanced oxidative stress in the MPTP model and the antioxidant protection of LN.

[0043] Figure 5 L indicates that: as can be seen from the CON group, dopamine is highly abundant in the striatum, the abundance drops sharply in the MPTP group (lighter color in the heatmap), and the LN group shows significant recovery; combined with the dopaminergic neuron damage mechanism in the PD model in the MPTP group, the pathological characteristics of the model are directly verified.

[0044] therefore, Figure 5 Spatial metabolomics technology clarified the disordered characteristics of different compounds such as nicotine, neurotransmitters, and lipids in the MPTP group. It also proved that intraperitoneal injection of nicotine can intervene in the concentration levels of some key metabolites, further demonstrating that nicotine plays a certain biological role in the treatment of neurodegenerative diseases, and providing technical support for the neuroprotective effect of nicotine. Thus, it is shown that the method for simultaneous detection of nicotine and its metabolites, neurotransmitters, and lipids in brain tissue samples provided in Example 1 of this invention is scientific, reasonable, and has good feasibility and practicality.

[0045] In summary, the detection method provided by the embodiments of the present invention has the following advantages: 1) Compared with other homogenization and protein pretreatment processes, the detection method provided in this embodiment of the invention avoids complex pretreatment processes and completely preserves the spatial structure of rat tissue, enabling in-situ visualization and distribution analysis of target molecules such as drugs, metabolites, and lipids; 2) Compared with detection methods such as LC-MS, the detection method provided in this embodiment of the invention can be directly detected on the surface of rat tissue sections (such as brain region, liver, tumor tissue) to accurately locate the microscopic distribution of the target compound; 3) Compared with other traditional detection methods (such as ELISA, which can only detect 1 to 2 target compounds at a time, and require multiple slides and repeated experiments to analyze multiple components), the detection method provided in this embodiment of the invention can detect multiple target compounds simultaneously on the same rat tissue slide; it meets the analytical needs of rat micro-samples such as brain tissue subregions, and provides more comprehensive molecular spatial information for rat model-related research such as drug metabolism and neuroscience.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A method for simultaneous detection of nicotine and its metabolites, neurotransmitters, and lipids in brain tissue samples, comprising: Step 1: After injecting nicotine into rats intraperitoneally, the brain is removed and prepared into brain tissue sections; Step 2: The brain tissue slices are subjected to mass spectrometry imaging using a mass spectrometry imaging instrument under the action of a spray voltage of 3.5-6.0 kV and a spray solvent. The scan data is processed by data processing software to obtain a mass spectrometry imaging image of rat brain tissue. The spray solvent is composed of acetonitrile, isopropanol and water in a volume ratio of 5-7: 1-3: 1-3. Step 3: Compare the mass spectrometry information in the mass spectrometry image of the rat brain tissue with the mass spectrometry information of the compounds in the biological database to obtain the spatial distribution information of nicotine and its metabolites, neurotransmitters and lipids in different brain regions of the rat.

2. The simultaneous detection method according to claim 1, characterized in that, The nicotine compounds include nicotine, nornicotine, norcotinine, nicotine nitrogen oxides, cotinine nitrogen oxides, and trans-3'-hydroxycotinine. The neurotransmitters include dopamine, adrenaline, serotonin, adenosine, adenine, hypoxanthine, γ-aminobutyric acid, taurine, histidine, histamine, carnosine, acetylcholine, choline, arginine, valine, lysine, proline, and glutamate. The lipid compounds include arachidonic acid, docosahexaenoic acid, α-glycerophosphate choline, PC(20:2(11Z,14Z) / 20:4(5Z,8Z,11Z,14Z)), PC(22:4(7Z,10Z,13Z,16Z) / 16:0), PC(22:2(13Z,16Z) / 14:0), PC(20:1(11Z) / 14:0), PE(P-18:1(9Z) / 20:4(5Z,8Z,11Z,14Z)), and PC(16:0 / 16:0).

3. The simultaneous detection method according to claim 1 or 2, characterized in that, Step one includes: Rats were injected intraperitoneally with nicotine at a concentration of 1–2 mg / kg. After waiting 5–15 minutes, the rats were anesthetized by injecting 1 mL of 4% tribromoethanol into the intraperitoneal cavity. The brain was then removed, and the morphology of the brain should be preserved during the removal process. After rinsing the complete brain tissue, it was dehydrated and placed in a foil sheet above liquid nitrogen for quick freezing for 2–5 minutes. Then, the biological brain tissue was placed in a 50 mL centrifuge tube and transferred to a freezer at -79 to -81°C for more than 1 week to ensure that it maintains its shape. Six to eight hours before preparing brain slices, the mouse brains were thawed at -20°C and then cut using a microtome. Brain tissue was fixed on a circular tray and sectioned using a microtome at a temperature of -20 to -24°C. The thickness of the brain slices was 9 to 11 mm. The brain tissue slices were then fixed on glass slides and dried in a vacuum drying oven for 20 to 40 minutes before analysis.

4. The simultaneous detection method according to claim 1 or 2, characterized in that, The mass spectrometry imaging detection method includes: mass spectrometry imaging detection using an AFADESI-MSI platform with an aerodynamically assisted desorption / electrospray ionization source, wherein the mass spectrometry imaging instrument continuously scans the tissue surface along the x-axis at a constant rate of 150–170 µm / s, and vertically scans along the y-axis at intervals of 180–220 µm; Full MS mode is used: the scan range is 100–1000 Da; the automatic gain control target is (1.8–2.2) × 10⁻¹⁰. 5 The automatic gain control time is 60–80 ms; the capillary temperature is 340–360°C; the spray gas pressure is maintained at 0.5–0.7 MPa; the pumping gas velocity is 40–50 L / min; and the flow rate of the spray solvent is 5–7 μL / min.

5. The simultaneous detection method according to claim 4, characterized in that, The distances from the mass spectrometry imaging instrument's sample injector to the surface of the brain tissue slice and the ion delivery tube are 0.5–0.7 mm and 2.5–3.5 mm, respectively.

6. The simultaneous detection method according to claim 5, characterized in that, The distance from the ion source port to the mass spectrometry acquisition tube port of the mass spectrometry imaging instrument is 9–11 mm.

7. The simultaneous detection method according to claim 6, characterized in that, In step two, after identifying differential metabolites in the brain using spatially resolved metabolomics, the differential metabolite information is imported into the imaging software MassImager for ion image reconstruction. Region-specific mass spectrometry data are obtained by matching high spatial resolution hematoxylin and eosin images and exported as a .txt file.

8. The simultaneous detection method according to claim 4, characterized in that, In step three, the .txt file exported in step two is imported into the Markerview 1.2.1 software, a quality tolerance of 10 ppm is set, and relevant data processing steps such as peak picking, peak alignment, and removal of isotope peaks are performed.

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