Method for simultaneously determining multiple tobacco-specific nitrosamines in human plasma

A nitrosamine and plasma technology, applied in the direction of measuring devices, disease resistance to vector transmission, instruments, etc., can solve the problems of long time-consuming, cumbersome operation process, many steps, etc., to reduce matrix effect, good reproducibility, remove The effect of matrix interference

Active Publication Date: 2018-12-25
SHANGHAI TOBACCO GRP CO LTD +1
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, only a small number of studies have measured NNAL in the plasma of smokers, and there are no reports on the determination methods of NNK, NNN, NAT and NAB in the blood of smokers and non-smokers, and the existing methods need to be fixed offline. Phase extraction, rotary evaporation and other steps of purification and enrichment, many steps, cumbersome operation process, time-consuming

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  • Method for simultaneously determining multiple tobacco-specific nitrosamines in human plasma
  • Method for simultaneously determining multiple tobacco-specific nitrosamines in human plasma
  • Method for simultaneously determining multiple tobacco-specific nitrosamines in human plasma

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Embodiment 1

[0062] This embodiment is a method for simultaneously measuring multiple tobacco-specific nitrosamines in human plasma, such as figure 1 Shown is a schematic diagram of an online two-dimensional SPE processing-high performance liquid chromatography system (elution state);

[0063] The method comprises the steps of:

[0064] 1) Collect 4 mL of fasting cubital venous blood from 15 smokers and put it into a heparin anticoagulant tube. Centrifuge blood samples within 2 hours after collection, centrifuge at 3000 rpm / min at 4°C for 10 minutes, transfer the plasma to a clean EP tube without RNAse, and store in a -70°C refrigerator. After the blood sample is thawed at room temperature, take 0.7mL and add 0.7mL β-glucuronidase (12,000units, prepared with phosphate buffer solution, pH6.8), incubate at 37°C for 24h with shaking in a water bath, and then add 20μL Internal standard, all transferred to In Ultra-4 30K ultrafiltration centrifuge tubes, centrifuge at 14,000 rpm for 10 minu...

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Abstract

The invention relates to a method for simultaneously determining multiple tobacco-specific nitrosamines in human plasma. The method particularly comprises the steps of: 1) collecting a blood sample, obtaining plasma and performing online two-dimensional SPE processing, wherein the two-dimensional SPE processing is performing enrichment and purification on plasma to be detected through combinationof a PRS cation exchange column and a Resin GP reversed phase extraction column; 2) preparing a nitrosamine solution with gradient concentration; 3) performing LC-MS / MS analysis on the standard sample and establishing a linear regression equation on the mass concentration by the ratio of the peak area of the nitrosamine and the peak area of the internal standard; and 4) calculating the content of nitrosamine in the plasma to be detected. According to the method for simultaneously determining multiple tobacco-specific nitrosamines in human plasma, a full-automatic online two-dimensional solidphase extraction sample pretreatment method is adopted, thereby removing the matrix interference to the greatest extent and reducing the matrix effect. The method has the advantages that enrichment and purification is completed in one step, solid phase extraction column can be used for multiple times, the reproducibility is good and the measuring efficiency is high.

Description

technical field [0001] The invention belongs to the field of blood sample biomarker detection, and in particular relates to a method for simultaneously measuring various tobacco-specific nitrosamines in human plasma based on online solid-phase extraction-liquid chromatography-tandem mass spectrometry. Background technique [0002] Tobacco-specific nitrosamines (TSNAs) are a class of strong carcinogens widely present in tobacco and cigarette smoke, mainly including 4-(methylnitrosamine)-1-(3-pyridine)-1-butanone ( NNK), N-nitrosonornicotine (NNN), N-nitrosonornicotine (NAT) and N-nitroso anatabine (NAB). The International Cancer Agency (IARC) classified NNK and NNN as a first-class human carcinogen. In rodents, NNK can lead to lung adenocarcinoma, and the same pathology has been found in non-smokers exposed to environmental smoke. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is the main metabolite of NNK and has similar carcinogenic activity to NNK. NNAL can underg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06Y02A50/30
Inventor 张杰周骏刘兴余白若石杨振东徐同广芦楠张晨
Owner SHANGHAI TOBACCO GRP CO LTD
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