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Quantitative detection method of nicotine in cell lysate

A quantitative detection method and cell lysate technology, which is applied in the field of quantitative detection of nicotine in animal cell lysates, can solve problems such as difficult to obtain the nicotine content in whole blood, and the nicotine content has not been known, and achieve improved sensitivity and moderate retention time , strong eluting ability

Active Publication Date: 2020-06-16
CHINA JILIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Therefore, the current research has only quantitatively detected the nicotine content in plasma or serum, urine or hair, and has not yet known the nicotine content in various blood cells (including red blood cells, white blood cells, etc.) or intracellular nicotine content

Method used

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  • Quantitative detection method of nicotine in cell lysate
  • Quantitative detection method of nicotine in cell lysate
  • Quantitative detection method of nicotine in cell lysate

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0059] The preparation method of the cell lysate is: add an equal volume of cell lysis solvent to the cell fluid / whole blood and shake evenly (oscillate at 100±10r / min for 5±0.5 minutes) to obtain the cell lysate; the formula of the cell lysis solvent For: ammonium chloride 8.29g, potassium bicarbonate 1g and EDTA-2Na 37.2mg, dissolved in 1000mL ultrapure water.

[0060] The concentration of cells in the cytosol of hepatocytes is 1×10 6 / ml, the cell concentration in the cell solution of tracheal cells is 1×10 6 / ml; the culture medium used is: DMEM basal medium, containing 1% non-essential amino acid, 1% L-glutamine, 100U / mL penicillin, 100U / mL streptomycin and 10% fetal bovine serum; this belongs to conventional technology.

Embodiment 1

[0061] Embodiment 1, a kind of method for the quantitative detection of nicotine in the cell lysate, with the cell lysate as the object to be tested, carry out the following steps successively:

[0062] 1) Pipette 0.4mL of cell lysate into a container with a cover (centrifuge tube with a cover), add 0.2mL of 1.0M sodium hydroxide solution, mix well, add 3mL of ether, vortex with an oscillator for 2±0.2min, 9000±500r / min high-speed centrifugation for 5±0.5min to obtain the ether layer on the upper layer and the residue on the lower layer respectively;

[0063] Add 2 mL of diethyl ether to the residue and repeat the above-mentioned oscillatory vortex and high-speed centrifugation (oscillator vortex for 2±0.2min, 9000±500r / min high-speed centrifugation for 5±0.5min); that is, the residue is extracted again;

[0064] Combine the ether layers obtained by two centrifugations; dry them in a nitrogen stream at 35±1°C in a water bath, reconstitute the dried product with 0.2mL of 10% (...

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Abstract

The invention discloses a quantitative detection method for nicotine in cell lysate. The quantitative detection method takes the cell lysate as an object to be detected; the cell lysate is prepared by adding a cell lysis solvent into cell sap / whole blood; the quantitative detection method sequentially comprises the following steps: adding a sodium hydroxide solution and ethyl ether into the cell lysate; after drying an obtained ethyl ether layer, re-dissolving and centrifuging to obtain supernatant I; carrying out chromatography treatment on the supernatant I to obtain supernatant II; carrying out high performance liquid chromatography system analysis on the supernatant II; substituting an obtained medicine peak area Y of the nicotine into a formula Y is equal to 74729X-354.61 and converting to obtain the concentration of the nicotine in the cell sap / whole blood to be detected.

Description

technical field [0001] The invention relates to a quantitative detection method for nicotine in animal (including human) cell lysates. Background technique [0002] During the burning of tobacco, more than 5,000 known chemical substances can be produced, among which there are more than a dozen carcinogenic or cancer-promoting substances. The main harmful substances of tobacco are: [0003] Nicotine: Also known as nicotine, it is a highly addictive substance, second only to heroin. Nicotine can act on the brain of the smoker, making the smoker dependent on tobacco, and is the main ingredient leading to tobacco addiction. Nicotine can also cause vasoconstriction, increased blood pressure, and rapid heartbeat, causing coronary artery spasm, damaged vascular intima, and induced angina pectoris and myocardial infarction. [0004] Tobacco tar: commonly known as "tobacco tar", ranging from 5-15mg per cigarette, contains a variety of carcinogens and cancer-promoting substances. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06G01N30/74
CPCG01N30/06G01N30/74
Inventor 葛建年夫照林芳胡华军刘辉军邓同乐张永勇刘军
Owner CHINA JILIANG UNIV
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