Lipidomics analysis method for toxoplasma gondii based on high performance liquid chromatography tandem high-resolution mass spectrum and application

A technology of high-performance liquid chromatography and high-resolution mass spectrometry, which is applied to the analysis of materials, scientific instruments, and material separation. It can solve problems such as low resolution, interference, and complex sample derivation process, achieving good repeatability and simple pretreatment. , Sample determination and analysis efficiency are high

Inactive Publication Date: 2019-03-08
CHINA AGRI UNIV
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  • Claims
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Problems solved by technology

The classic fatty acid detection method is mainly based on gas chromatography-mass spectrometry, but this technology has many shortcomings such as no tandem ability, low resolution, complicated sample derivatization process, and slow

Method used

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  • Lipidomics analysis method for toxoplasma gondii based on high performance liquid chromatography tandem high-resolution mass spectrum and application
  • Lipidomics analysis method for toxoplasma gondii based on high performance liquid chromatography tandem high-resolution mass spectrum and application
  • Lipidomics analysis method for toxoplasma gondii based on high performance liquid chromatography tandem high-resolution mass spectrum and application

Examples

Experimental program
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Example Embodiment

[0033] Example 1 Establishing a method for detecting Toxoplasma gondii tachyzoite cardiolipin molecule

[0034] Use methanol to prepare 1ppm cardiolipin standard C56:0 solution, using HPLC-Q-TOF-MS (LC-20ADXR high performance liquid chromatography, AB SCIEX Triple TOF TM 5600 type Q-TOF quadrupole tandem time-of-flight mass spectrometry) injection analysis. Chromatographic analysis conditions:

[0035] Injection volume 5μl; column temperature 40℃; flow rate 0.4mL / min; gradient elution program is injection 0-0.5min, adjust mobile phase A:B (80:20, v / v), elution 0.5-1.5min ; Mobile phase A: B (60:40, v / v), elution 1.5-3min; Mobile phase A: B (40:60, v / v), elution 3-4.5min; Mobile phase A: B( 10:90, v / v), eluted for 2min; then stably maintained in mobile phase B to the 17th minute.

[0036] Mobile phase A is acetonitrile: water: methanol (20:60:20, v / v / v), containing 5mM ammonium acetate, mobile phase B is isopropanol: acetonitrile (90:10, v / v), containing 5mM 的ammonium acetate.

[0...

Example Embodiment

[0040] Example 2 Identification of various lipid molecules of Toxoplasma gondii

[0041] 1. Extraction of lipids from Toxoplasma gondii tachyzoites

[0042] Human foreskin fibroblasts HFFs were cultured in DMEM containing 15% FBS. After the cell flasks were filled, the DMEM medium with 1% FBS was replaced with Toxoplasma gondii tachyzoites and cultured for 3 days. After the worms are released, use the cell scraper to break the adherent cells, and then use a syringe with a 26G needle to suck the broken cell suspension and blow the cell bottle wall. After repeated pipetting and pipetting 3 times, filter with an 8μm diameter filter The cell suspension was centrifuged at 2000 rpm for 8 min, the supernatant was discarded, and the precipitate was the purified tachyzoite. Collect at least 1×10 8 A tachyzoite for subsequent sample preparation.

[0043] 2 Preparation of lipid extract samples: Suspend tachyzoites in 1ml PBS buffer, centrifuge at 2000rpm for 8min, wash away the residual mediu...

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Abstract

The invention discloses a lipidomics analysis method for toxoplasma gondii based on high performance liquid chromatography tandem high-resolution mass spectrum and application. The lipidomics analysismethod comprises the following steps: using a chloroform-methyl alcohol-aqueous solution for suspending a to-be-detected toxoplasma gondii sample and then performing ultrasonic disruption; centrifuging extracting solution, performing nitrogen blowing and re-dissolving with methyl alcohol, and then filtering and feeding the sample; using a liquid phase C18 chromatographic column for separating; utilizing mobile phases A and B composed of acetonitrile, isopropanol, ammonium acetate, methyl alcohol and water to elute, and then performing detecting in a full-scan manner respectively under positive and negative ion modes using a QTOF high-resolution mass spectrometer; and screening and identifying the detected data according to the chemical formula of target lipid molecules. The method has theadvantages of sensitivity and accuracy, and being independent of standard compound, and can be efficiently used for lipidomics analysis of the toxoplasma gondii.

Description

technical field [0001] The invention relates to molecular parasitology and analytical chemistry, in particular to a lipidomic analysis method and application of toxoplasma gondii based on high-performance liquid chromatography tandem high-resolution mass spectrometry. Background technique [0002] Toxoplasma gondii (Toxoplasma gondii) is an intracellular parasitic protozoan of the Apicomplexan subphylum, with a wide range of hosts, and can parasitize almost all nucleated cells of warm-blooded animals and humans, forming vacuoles in the cells, and dividing by binary division Toxoplasma gondii tachyzoites transform into bradyzoites, which exist in tissues and organs for a long time in the form of cysts, especially in the brain and nerves. And muscle tissue, so that the host carries Toxoplasma gondii. The pathogenic mechanism of Toxoplasma gondii is complex, and many factors such as host type, gender, age and immune status, as well as the pathogenicity of the strain can affect...

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Application Information

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IPC IPC(8): G01N30/88G01N30/06
CPCG01N30/88G01N30/06G01N2030/062G01N2030/8813
Inventor 刘群傅勇崔霞刘晶
Owner CHINA AGRI UNIV
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