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Analysis method for rapidly detecting in-vivo metabolism marker of smoke
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A technology of metabolic markers and analysis methods, which is applied in the field of rapid detection of metabolic markers in smoke gas, to achieve the effects of improving analysis sensitivity, reducing enzyme loss, and improving analysis speed
Active Publication Date: 2018-04-17
ZHENGZHOU TOBACCO RES INST OF CNTC +1
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specific Embodiment 1
[0035] The preparation of specific embodiment 1 β-glucuronidase reactor
[0036] (1) Take 300μL GMA and 100μL TRIM in a 1.5mL centrifuge tube, vortex for 5min;
[0038] (3) Mix 250 μL of GMA-TRIM solution with 750 μL of cyclohexanol-dodecanol solution;
[0039] (4) Add 0.0100g of AIBN to dissolve in the above solution, ultrasonically degas for 20 minutes, vortex for 10 minutes to a clear and transparent solution, pour into the activated capillary column, seal both ends, and polymerize in a constant temperature water bath at 50°C 24h; then wash with methanol;
[0043] Example 2 Collection and pretreatment of enzymatic hydrolyzate
[0044] Take 10 μL concentration is 0.05mg.mL -1 , 0.1mg.mL -1 , 0.2mg.mL -1 , 0.5mg.mL -1 , 0.8mg.mL -1 , 1.0mg.mL -1 The substrate NNAL-O-Glu passed through the organic polymer monolithic column enzyme reactor at a flow rate of 0.6 μL / min, and the enzymatic solution was collected in a 1.5mL centrifuge tube. At the same time, free hydrolyzate with concentration of 0.05mg.mL-1, 0.1mg.mL-1, 0.2mg.mL-1, 0.5mg.mL-1, 0.8mg.mL-1, 1.0mg.mL-1 was prepared in 1.5mL centrifuge tube and place it in a 37°C water bath. After 24 hours, remove the free hydrolyzate in the water bath.
[0045] The enzymatic solution was desalted, concentrated, reconstituted in 1 mL of 0.1% formic acid-water, sonicated for 15 minutes, and centrifuged at 13300 rpm / min 3 times, 10 minutes each time. Vortex at 3000rmp / min for 5 minutes, pipette the supernatant, and filter with a 0.22 μm organic phase needle filter; perform Nano ESI-RPL...
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Abstract
The invention relates to an analysis method for rapidly detecting an in-vivo metabolism marker of smoke. The analysis method comprises a smokesample purification-enrichment pretreatment step and an analysis detection step. A metabolite is cotinine or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). The analysis method is characterized in that the metabolite is firstly hydrolyzed by virtue ofa beta-glucuronidase reactor and then is analyzed and detected by virtue of a nano-flow liquid chromatography-mass spectrometry. The innovation points of the invention are as follows: by preparing online through an enzyme reactor, the hydrolysis effect is relatively good, the experimental steps can be simplified, and the analysis speed and the analysis sensitivity can be increased; a nano-flow liquid chromatographytandem mass spectrometry is applied to the field of smoke metabolites for the first time; and the sample amount required by an experiment is extremely low, only micro-nanogram-scale samples are required under a nano-flow condition, and high-sensitivity detection and high-throughput analysis can be realized.
Description
technical field [0001] The invention relates to a method for detecting metabolic markers in smoke gas, belonging to the technical field of analysis and detection, in particular to an analysis method for rapidly detecting metabolic markers in smoke gas, and the metabolites are cotianine or 4-(methyl sulfide) Nitroamino)-1-(3-pyridyl)-1-butanol (NNAL). Background technique [0002] 4-(Methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific N-nitrosamine (TSNAs) with high carcinogenic activity, Research Institute (IARC) listed as a carcinogen. It is a smoke carcinogen formed by the nitrosylation of nicotine or a small amount of nicotinenitrosylation, and its metabolite 4-(methyl nitroso Amino)-1-(3-pyridyl)-1-butanol (NNAL) and its glycosidic derivative NNAL-Glucs are very valuable biomarkers for studying the mechanism of toxicity and detoxification of NNK in the metabolism of human body thing. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol is the It is the...
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