Sample pretreatment method for detecting special nitrosamine in tobacco and tobacco products by LC-MS/MS (liquid chromatogram-mass spectrum/mass spectrum)

A technology for sample pretreatment and tobacco products, which is applied in the field of sample pretreatment for LC-MS/MS detection of specific nitrosamines in tobacco and tobacco products, which can solve the problems of large reagent consumption and long time consumption, and achieve reagent consumption Small size, short extraction time, safe and convenient operation

Active Publication Date: 2014-07-30
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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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  • Sample pretreatment method for detecting special nitrosamine in tobacco and tobacco products by LC-MS/MS (liquid chromatogram-mass spectrum/mass spectrum)
  • Sample pretreatment method for detecting special nitrosamine in tobacco and tobacco products by LC-MS/MS (liquid chromatogram-mass spectrum/mass spectrum)
  • Sample pretreatment method for detecting special nitrosamine in tobacco and tobacco products by LC-MS/MS (liquid chromatogram-mass spectrum/mass spectrum)

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0035] 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 , with a concentration of 5 ng mL -1 NNN-d4, 4 ng mL -1 NNK-d4, 20 ng mL -1 NAT-d4, 0.24 ng mL -1 NAB-d4), capped and sealed, placed in an ultrasonic cleaner, ultrasonically extracted at room temperature for 30 min, ultrasonic power was 100 HZ, and then centrifuged at 8000 rpm for 5 min, 0.8 mL supernatant was taken, and placed in Add 0.8 mL of ethyl acetate to the supernatant, cover and seal, shake at room temperature for 5 min, then centrifuge at 8000 rpm for 5 min, take 0.4 mL of the supernatant and dry it with nitrogen, wash with 0.4 mL of deionized water Reconstitute and transfer to a chromatographic vial for LC-MS / MS analysis.

[0036] By LC-MS / MS analysis, the resulting total ion current...

example 2

[0038] Weigh 20-25 mg flue-cured tobacco shred samples into a glass test tube, add 0.75 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 5 ng mL -1 NNN-d4, 4 ng mL -1 NNK-d4, 20 ng mL -1 NAT-d4, 0.24 ng mL -1 NAB-d4), capped and sealed, placed in an ultrasonic cleaning machine, ultrasonically extracted at room temperature for 30 min, ultrasonic power was 100 HZ, and then centrifuged at 8000 rpm for 5 min, 0.4 mL supernatant was taken, and placed in Add 0.4 mL of ethyl acetate to the supernatant, cover and seal, shake at room temperature for 5 min, then centrifuge at 8000 rpm for 5 min, take 0.2 mL of the supernatant and dry it with nitrogen, wash with 0.2 mL of deionized water Reconstitute and transfer to a chromatographic vial for LC-MS / MS analysis.

[0039] By LC-MS / MS analysis, the resulting total ion current TIC and ...

example 3

[0041] 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 concentrations are respectively 5ngmL -1 NNN-d4, 4 ng mL -1 NNK-d4, 20 ng mL -1 NAT-d4, 0.24 ng mL -1NAB-d4), capped and sealed, placed in an ultrasonic cleaner, ultrasonically extracted at room temperature for 30 min, with an ultrasonic power of 100 HZ, and then centrifuged at 8000 rpm for 5 min, and 1.6 mL of the supernatant was taken in Add 1.6 mL of ethyl acetate to the supernatant, cover and seal, shake at room temperature for 5 min, then centrifuge at 8000 rpm for 5 min, take 0.8 mL of the supernatant and dry it with nitrogen, wash with 0.8 mL of deionized water Reconstitute and transfer to a chromatographic vial for LC-MS / MS analysis.

[0042] By LC-MS / MS analysis, the resulting total ion current TIC...

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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

[0001] technical field [0002] 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 [0003] 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, LC-MS / MS is used for the determination of TSNAs. The sample pretreatment method reported in (CORESTA recommended method N 72 of 61st Tobacco Science Research Conference) 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 Applications(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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