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GC-MS-MS (gas chromatography-mass spectrometry-mass spectrometry) detection method for (S)-NNAL ((4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) and (R)-NNAL in urine

A technology of gas chromatography and detection methods, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve problems such as difficult detection and complex urine sample matrix

Active Publication Date: 2013-06-26
HONGYUN HONGHE TOBACCO (GRP) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Since the amount of NNK released in cigarette smoke is only a few nanograms per cigarette, and NNK is metabolized into various substances after entering the human body, and the matrix of the urine sample is complex, the (S)-NNAL and (R The detection of )-NNAL is very difficult, and many related industries at home and abroad cannot carry out the detection of (S)-NNAL and (R)-NNAL in urine samples

Method used

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  • GC-MS-MS (gas chromatography-mass spectrometry-mass spectrometry) detection method for (S)-NNAL ((4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) and (R)-NNAL in urine
  • GC-MS-MS (gas chromatography-mass spectrometry-mass spectrometry) detection method for (S)-NNAL ((4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) and (R)-NNAL in urine
  • GC-MS-MS (gas chromatography-mass spectrometry-mass spectrometry) detection method for (S)-NNAL ((4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) and (R)-NNAL in urine

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Experimental program
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Embodiment 1

[0016] Unless otherwise stated, the percentages used in the present invention are all percentages by weight.

[0017] The gas chromatography-mass spectrometry-mass spectrometry detection method of (S)-NNAL and (R)-NNAL in urine comprises the following steps:

[0018] 1. Chemical reagents

[0019] (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride (CAS No. 20445-33-4), (S)-(+)-α-methoxy-(tri Fluoromethyl)phenylacetic anhydride (CAS No. 85541-57-7), 4-(-Methylnitrosoamino)-1-(3-pyridyl)-butanol (NNAL) (rac NNAL, CAS No. 76014- 81-8), 4-(methyl-d3-nitrosoamino)-1-(3-pyridyl)-1-butanol (rac-NNAL-methyl-d3CAS No. 1020719-61-2), triethyl Amylamine, β-glucosidase (type IX-A, from Escherichia coli)

[0020] Preparation of (S)-NNAL and (R)-NNAL and determination of absolute configuration: preparative OD-H chiral chromatography column, mobile phase isopropanol: n-hexane ratio of 10:90, two effluents were collected Chromatographic peaks were concentrated until their residue wei...

Embodiment 2

[0037] 1. Derivatization of racemic NNAL: use anhydrous acetonitrile as the solvent to prepare a single standard solution of rac NNAL standard with a concentration of 1 μg / mL, and keep four significant figures; prepare separately in the same way using anhydrous acetonitrile as the solvent Single standard solution of rac-NNAL-methyl-d3 with a concentration of 1 μg / mL; using anhydrous acetonitrile as solvent, prepare 8 mixed standards containing rac-NNAL and deuterated rac-NNAL-methyl-d3 according to Table 1 solution; prepare a solution of (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride with a concentration of 10ng / mL in anhydrous acetonitrile; prepare a solution of triethylamine with a concentration of 10ng / mL Anhydrous acetonitrile solution; respectively take 0.5mL of 8 mixed standard solutions in 8 GC-MS-MS chromatographic bottles, and add (S)-(+)-α- Anhydrous acetonitrile solution of methoxy-α-trifluoromethylphenylacetyl chloride and triethylamine acetonitrile solut...

Embodiment 3

[0040] 1. Preparation of (S)-NNAL and (R)-NNAL and determination of absolute configuration: use anhydrous acetonitrile solution of rac NNAL standard, (S)-(+)-α-methoxy-α-tri The anhydrous acetonitrile solution of fluoromethylphenylacetic anhydride and the anhydrous acetonitrile solution of (R)-(+)-α-methoxy-α-trifluoromethylphenylacetic anhydride and triethylamine carry out the Mosher reaction respectively, purified and 1 H NMR spectrum test, using Mosher method to determine (S)-NNAL-(S)-(+)-α-methoxy-α-trifluoromethylphenylacetate and (R)-NNAL-(S) - Absolute configuration of (+)-α-methoxy-α-trifluoromethylphenylacetate.

[0041] 2. Derivatization of racemate NNAL and establishment of standard curve: use anhydrous acetonitrile as solvent, prepare a single standard solution of rac NNAL standard with a concentration of 1 μg / mL, and keep four significant figures; Prepare a single standard solution of rac-NNAL-methyl-d3 with a concentration of 1 μg / mL in acetonitrile; use anhydr...

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Abstract

The invention relates to a GC-MS-MS (gas chromatography-mass spectrometry-mass spectrometry) detection method for (S)-NNAL ((4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) and (R)-NNAL in urine. The GC-MS-MS detection method comprises the following steps of: adding rac-NNAL-methyl-d3 solution, ammonium dihydrogen phosphate buffer solution (pH 6.4, 0.6M) and beta-glucuronidase into a urine sample, mixing uniformly, adding the mixture into a microwave-assisted extraction instrument, carrying out enzymolysis on the urine sample at the constant temperature of 37 DEG C, purifying the urine sample subjected to enzymolysis by using an MIP-NNAL solid-phase extraction columella, concentrating the urine sample under reduced pressure until the urine sample is dried, adding anhydrous acetonitrile solution of triethylamine and anhydrous acetonitrile solution of (S)-(+)-alpha-methoxy-alpha-trifluoromethyl-phenylacetyl chloride, carrying out derivatization, carrying out GC-MS-MS analysis on the sample, and calculating the content of the (S)-NNAL and the (R)-NNAL in the urine sample. The analysis and detection method for the (S)-NNAL and the (R)-NNAL in the urine has the advantages of low limit of detection, good stability and simple pre-treatment, can be used for detecting the (S)-NNAL and the (R)-NNAL in the urine quickly and accurately and fills the gap in the technical field.

Description

technical field [0001] The invention belongs to the technical field of biomarker physical and chemical detection of tobacco-specific nitrosamines in urine, and in particular relates to a gas chromatography-mass spectrometry-mass spectrometry detection method for (S)-NNAL and (R)-NNAL in urine. Background technique [0002] In recent years, people pay more and more attention to smoking and health problems, so the research on various harmful chemical components in smoke is getting more and more in-depth at home and abroad. After years of research, the toxicity evaluation results of the International Agency for Research on Cancer (IARC) concluded that: 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone ( NNK) is carcinogenic to animals, and NNK and its metabolites are potential lung cancer carcinogens. Therefore, there is an urgent need to establish a method to accurately evaluate the exposure of different populations to NNK, and the biomarker method is a recognized and effective ...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
Inventor 蒋举兴段焰青王明锋夏建军王文元史小波
Owner HONGYUN HONGHE TOBACCO (GRP) CO LTD