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CN-122016988-A - Simultaneous detection method for nicotine, metabolites, neurotransmitters and lipids in brain tissue sample

CN122016988ACN 122016988 ACN122016988 ACN 122016988ACN-122016988-A

Abstract

The invention provides a method for simultaneously detecting nicotine, metabolites, neurotransmitters and lipids in brain tissue samples, which comprises the steps of firstly injecting nicotine into the abdominal cavity of a rat, then obtaining brain tissue of the rat, preparing brain tissue slices through treatment, then placing the brain tissue slices into a mass spectrum imaging instrument for detection, and analyzing the spatial distribution characteristics of the nicotine, the metabolites, the neurotransmitters and the lipids in the brain after the intraperitoneal injection of the nicotine into the rat based on the obtained rat brain mass spectrum imaging image. The spraying voltage of the mass spectrum imaging instrument is 3.5-6.0 kV, and the spraying 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 the spatial distribution and metabolism information of the three substances in brain tissues, has the characteristics of simple operation, good accuracy, high precision and the like, and provides an accurate and effective analysis method for the spatial distribution and metabolism research of nicotine and metabolites, neurotransmitters and lipids in brain tissues.

Inventors

  • LI QIAN
  • CHEN HUAN
  • HOU HONGWEI
  • FU YANING
  • YIN CHANGFENG
  • CUI LILI
  • WANG GAOGE
  • LI XIN
  • YU HAO

Assignees

  • 国家烟草质量监督检验中心

Dates

Publication Date
20260512
Application Date
20260209

Claims (8)

  1. 1. A method for simultaneous detection of nicotine and its metabolites, neurotransmitters and lipids in a brain tissue sample, comprising: Step one, taking brain to prepare brain tissue slices after injecting nicotine into the abdominal cavity of a rat; Performing mass spectrometry imaging detection on brain tissue slices through a mass spectrometry imaging instrument under the action of a spraying voltage of 3.5-6.0 kV and a spraying solvent, and processing scanning data through data processing software to obtain a rat brain tissue mass spectrometry imaging chart, wherein the spraying solvent is formed by mixing acetonitrile, isopropanol and water according to a volume ratio of 5-7:1-3:1-3; And step three, comparing mass spectrum information in the rat brain tissue mass spectrum imaging diagram with mass spectrum information of compounds in a biological database to obtain spatial distribution information of nicotine, metabolites, neurotransmitters and lipids in different brain regions of the rat.
  2. 2. The simultaneous detection method of claim 1, wherein the nicotinic compound comprises nicotine, nornicotine, norcotinine, a nicotine nitroxide, a cotinine nitroxide, and trans-3' -hydroxy cotinine; The neurotransmitters include dopamine, epinephrine, 5-hydroxytryptamine, adenosine, adenine, hypoxanthine, gamma-aminobutyric acid, taurine, histidine, histamine, carnosine, acetylcholine, choline, arginine, valine, lysine, proline, glutamic acid; the lipid compound comprises arachidonic acid, docosahexaenoic acid and alpha-glycerophosphorylcholine 、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))、PC(16:0/16:0).
  3. 3. The simultaneous detection method according to claim 1 or 2, wherein the first step comprises: Injecting nicotine with the concentration of 1-2 mg/kg into the abdominal cavity of a rat, injecting tribromoethanol with the concentration of 1-mL% into the abdominal cavity of the rat for anesthesia after waiting for 5-15 min, and taking the brain, wherein the integrity of the brain morphology is ensured when taking the brain; Rinsing the complete brain tissue, performing water absorption treatment, quick-freezing the brain tissue in a tinfoil sheet above liquid nitrogen for 2-5 min, then placing the biological brain tissue in a 50mL centrifuge tube, and transferring the biological brain tissue to a-79 to-81 ℃ refrigerator for freezing for more than 1 week so as to ensure the maintenance form of the biological brain tissue; 6-8 hours before brain slicing is carried out, the mouse brain is placed at the temperature of-20 ℃ for thawing, and then the mouse brain is cut by a slicing machine; Fixing the brain tissue on a circular tray, slicing by using a slicing machine, wherein the temperature in the slicing machine is-20 to-24 ℃, the thickness of a brain slice is 9-11 mm, fixing the brain tissue slice on a glass slide, and drying in a vacuum drying oven for 20-40 min before analysis.
  4. 4. The simultaneous detection method according to claim 1 or 2, wherein the mass spectrometry imaging detection method comprises performing mass spectrometry imaging detection by using a AFADESI-MSI platform of an aerodynamic auxiliary desorption electrospray ionization ion source, wherein the mass spectrometry imaging instrument continuously scans a tissue surface in an x-axis direction at a constant speed of 150-170 μm/s and vertically scans the tissue surface at a distance of 180-220 μm in a y-axis direction, using a Full MS mode, wherein the scanning range is 100-1000 Da, the automatic gain control target is (1.8-2.2) ×10 5 , the automatic gain control time is 60-80 MS, the capillary temperature is 340-360 ℃, the spray gas pressure is kept at 0.5-0.7 MPa, the pumping flow rate is 40-50L/min, and the fluidity of the spray solvent is 5-7 μm/min.
  5. 5. The simultaneous detection method according to claim 4, wherein the distances from the mass spectrometer imaging instrument to the brain tissue slice surface and the ion transport tube are respectively 0.5-0.7 mm and 2.5-3.5 mm.
  6. 6. The simultaneous detection method according to claim 5, wherein the distance from the ion source port to the mass spectrum acquisition nozzle of the mass spectrum imaging instrument is 9-11 mm.
  7. 7. The simultaneous detection method according to claim 6, wherein in the second step, after identifying the differential metabolites in the brain by using a spatially resolved metabonomics method, the differential metabolite information is imported into the imaging software MASSIMAGER for ion image reconstruction, and the region-specific property spectrum data spectrogram is obtained by matching the high spatial resolution hematoxylin and eosin images and exported as a. Txt file.
  8. 8. The simultaneous detection method according to claim 4, wherein in the third step, the txt file derived in the second step is imported into marker view1.2.1 software, a mass margin of 10 ppm is set, and the relevant data processing steps of peak pickup, peak alignment and isotope peak removal are performed.

Description

Simultaneous detection method for nicotine, metabolites, neurotransmitters and lipids in brain tissue sample Technical Field The invention belongs to the technical field of detection and analysis, and relates to a method for simultaneously detecting nicotine, metabolites thereof, neurotransmitters and lipids in a brain tissue sample. Background Nicotine, the major alkaloid in tobacco, can rapidly permeate the blood brain barrier after entering the human body and trigger the change of neurotransmitter levels in the brain. Studies have shown that nicotine has a potential protective effect on a variety of neurodegenerative diseases, the effect of which is closely related to its level of exposure in the brain and spatial distribution. Neurotransmitters are chemical messengers that transfer information between neurons or between neurons and effector cells, and their level changes and specific distribution in the brain are closely related to various neurological diseases such as parkinson's disease, depression, and alzheimer's disease, and can be used as important clinical diagnostic, screening, and therapeutic targets. With the continued understanding of the pathogenesis of parkinson's disease, lipids have been found to play an important role therein. Lipids are not only key components of cell membranes, but also play an important role in the process of cell signaling. Studies have shown that metabolic abnormalities of lipids are closely related to the pathological course of various neurodegenerative diseases of the nervous system. In summary, the simultaneous exploration of the content changes and spatial distribution characteristics of nicotine and its metabolites, neurotransmitters and lipids in brain tissue is critical for the deep understanding of the mechanisms of the related diseases. Traditional detection methods, such as electrochemical methods, liquid chromatography-mass spectrometry (liquid chromatogram-mass spectrometry, LC-MS) and gas chromatography-mass spectrometry (gas chromatography-mass spectrometry, GC-MS), generally require complex sample pretreatment, although they allow qualitative and quantitative analysis of drugs metabolized in vivo. The process is easy to cause the loss of spatial in-situ information of the object to be detected in the biological tissue sample, has insufficient spatial resolution capability, and is difficult to accurately acquire the spatial distribution information of the object to be detected in the biological tissue, thereby restricting the further research of the nicotine regulation neurotransmitter, the lipid metabolism level change and the spatial distribution mechanism. In contrast, mass spectrometry imaging (Mass Spectrometry Imaging, MSI) as a non-targeted and highly sensitive imaging method based on mass spectrometry technology can directly provide chemical information and spatial location information of an object to be measured. In addition, MSI has the advantages of simple operation, high sensitivity and large flux, so that the MSI has obvious advantages in biological sample characterization. Therefore, developing an efficient and visual detection method for synchronously analyzing spatial distribution information 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 treating neurodegenerative diseases has become a critical problem to be solved in the art. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to provide a simultaneous detection method for nicotine, metabolites, neurotransmitters and lipids in brain tissue samples, which has the characteristics of simple operation, good accuracy, high precision and the like, and provides an accurate and effective analysis method for spatial distribution and metabolism research of the nicotine, the metabolites, the neurotransmitters and the lipids in brain tissues. In order to achieve the aim of the invention, the invention adopts the following technical scheme: a method for simultaneous detection of nicotine and its metabolites, neurotransmitters and lipids in a brain tissue sample, comprising: Step one, taking brain to prepare brain tissue slices after injecting nicotine into the abdominal cavity of a rat; Performing mass spectrometry imaging detection on brain tissue slices through a mass spectrometry imaging instrument under the action of a spraying voltage of 3.5-6.0 kV and a spraying solvent, and processing scanning data through data processing software to obtain a rat brain tissue mass spectrometry imaging chart, wherein the spraying solvent is formed by mixing acetonitrile, isopropanol and water according to a volume ratio of 5-7:1-3:1-3; And step three, comparing mass spectrum information in the rat brain tissue mass spectrum imaging diagram with mass spectrum information of compounds in a biological database to obtain spatial distribution informati