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A sample pretreatment method for detecting specific nitrosamines in tobacco and tobacco products by LC-MS/MS

A sample pretreatment and tobacco product technology, which is applied in the field of sample pretreatment of specific nitrosamines (TSNAs), can solve the problems of large reagent consumption and long time consumption, and achieve small reagent consumption, short extraction time and safe operation convenient effect

Active Publication Date: 2016-01-20
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The problem to be solved by the present invention is that in the prior art, the sample pretreatment method of using LC-MS / MS to detect tobacco and tobacco product-specific nitrosamines takes a long time and consumes a lot of reagents, so as to provide a method suitable for simple and rapid detection of tobacco and sample pretreatment methods for specific nitrosamines in tobacco products

Method used

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  • A sample pretreatment method for detecting specific nitrosamines in tobacco and tobacco products by LC-MS/MS
  • A sample pretreatment method for detecting specific nitrosamines in tobacco and tobacco products by LC-MS/MS
  • A sample pretreatment method for detecting specific nitrosamines in tobacco and tobacco products by LC-MS/MS

Examples

Experimental program
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Effect test

example 1

[0034] Weigh 45-50 mg of freeze-dried fresh Burley tobacco leaf samples into a glass test tube, add 1.5 mL of 100 mM ammonium acetate buffer solution into the glass tube (4 kinds of deuterated TSNAs internal standards were added to the ammonium acetate buffer solution in advance, the concentrations were respectively 5ngmL -1 NNN-d4, 4ngmL -1 NNK-d4, 20ngmL -1 NAT-d4, 0.24ngmL -1 NAB-d4), capped and sealed, placed in an ultrasonic cleaning machine, ultrasonically extracted at room temperature for 30min, with an ultrasonic power of 100HZ, and then centrifuged at a speed of 8000rpm for 5min, 0.8mL supernatant was taken, and in the supernatant Add 0.8mL ethyl acetate to the solution, cover and seal, shake at room temperature for 5min, then centrifuge at 8000rpm for 5min, take 0.4mL supernatant and dry it with nitrogen, reconstitute with 0.4mL deionized water, transfer to chromatographic analysis LC-MS / MS analysis in vials.

[0035] By LC-MS / MS analysis, the resulting total ion...

example 2

[0037] Weigh 20~25mg flue-cured tobacco shred samples into a glass test tube, add 0.75mL100mM ammonium acetate buffer solution into the glass tube (4 kinds of deuterated TSNAs internal standards are added to the ammonium acetate buffer solution in advance, the concentration is 5ngmL respectively -1 NNN-d4, 4ngmL -1 NNK-d4, 20ngmL -1 NAT-d4, 0.24ngmL -1 NAB-d4), capped and sealed, placed in an ultrasonic cleaning machine, ultrasonically extracted at room temperature for 30min, with an ultrasonic power of 100HZ, and then centrifuged at a speed of 8000rpm for 5min, 0.4mL supernatant was taken, and the supernatant Add 0.4mL ethyl acetate to the solution, cover and seal, shake at room temperature for 5min, then centrifuge at 8000rpm for 5min, take 0.2mL supernatant and dry it with nitrogen, reconstitute with 0.2mL deionized water, transfer to chromatographic analysis LC-MS / MS analysis in vials.

[0038] By LC-MS / MS analysis, the resulting total ion current TIC and extracted ion ...

example 3

[0040] Weigh 90~100 mg of freeze-dried oriental tobacco into a glass test tube, add 3.0 mL of 100 mM ammonium acetate buffer solution into the glass tube (4 kinds of deuterated TSNAs internal standards are added to the ammonium acetate buffer solution in advance, the concentration is 5 ng mL -1 NNN-d4, 4ngmL -1 NNK-d4, 20ngmL -1 NAT-d4, 0.24ngmL -1 NAB-d4), capped and sealed, placed in an ultrasonic cleaning machine, ultrasonically extracted at room temperature for 30min, with an ultrasonic power of 100HZ, and then centrifuged at a speed of 8000rpm for 5min, 1.6mL supernatant was taken, and in the supernatant Add 1.6mL of ethyl acetate to the solution, cover and seal, shake at room temperature for 5min, then centrifuge at 8000rpm for 5min, take 0.8mL of supernatant and dry it with nitrogen, reconstitute with 0.8mL of deionized water, transfer to chromatographic analysis LC-MS / MS analysis in vials.

[0041] By LC-MS / MS analysis, the resulting total ion current TIC and extrac...

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Abstract

The invention discloses a sample pretreatment method for detecting special nitrosamine in tobacco and tobacco products by LC-MS / MS (liquid chromatogram-mass spectrum / mass spectrum). The sample pretreatment method comprises the following steps of: (1) preparing ammonium acetate buffering solution; (2) drying and crushing the tobacco and tobacco products; (3) adding the ammonium acetate buffering solution in the crushed sample, and ultrasonically extracting; (4) carrying out high-speed centrifugation on the extracted sample at room temperature to obtain a first supernatant; (5) adding ethyl acetate in the first supernatant and carrying out extraction and purification, and then centrifuging to obtain a second supernatant; (6) taking the second supernatant, blow-drying by nitrogen and redissolving by deionized water to prepare sample to be detected; and (7) detecting tobacco special nitrosamine in the sample to be detected by virtue of LC-MS / MS. The sample pretreatment method disclosed by the invention has the advantages that the sample pretreatment method is simple and practicable; the tobacco special nitrosamine is dissolved out by the ammonium acetate buffering solution, and purified by the extraction of ethyl acetate solution, and proteins and interference substances are removed to obtain refined sample solution after impurity removal, thus the sample detection is high in accuracy, high in extraction efficiency, simple and convenient to operate, time-saving, and low in reagent consumption.

Description

technical field [0001] The invention relates to a sample pretreatment method for tobacco metabolomics specific nitrosamines (TSNAs) in tobacco and tobacco products, in particular to a sample pretreatment method for LC-MS / MS detection of specific nitrosamines in tobacco and tobacco products. Background technique [0002] Tobacco-specific nitrosamines (TSNAs) include N-nitrosonornicotine (NNN), 4-methylnitrosopyridylbutanone (NNK), N-nitrosoanatabine (NAT) and N- Nitrosobasine (NAB), etc., are a class of harmful substances in tobacco and tobacco products, among which NNN and NNK are defined as Group 1-confirmed human carcinogens by the International Organization for Cancer Research. At present, the determination of TSNAs adopts the analysis method of LC-MS / MS. The sample pretreatment method reported by (CORESTArecommended method N72of61stTobaccoScienceResearchConference) includes the following steps: 1) Sample extraction: Weigh a uniform sample, add ammonium acetate buffer sol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06
Inventor 陈霞周会娜金立锋陈千思刘萍萍翟妞郑庆霞卢鹏王燃魏春阳杨军林福呈
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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