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High performance liquid chromatography-mass spectrum-mass spectrum (HPLC-MS-MS) combined detection method for (S)-NNAL and (R)-NNAL in urine

A high-performance liquid chromatography and detection method technology, applied in the field of high-performance liquid chromatography-mass spectrometry-mass spectrometry combined detection, can solve the problems of difficult detection, complex urine sample matrix and the like

Active Publication Date: 2013-06-12
HONGYUN HONGHE TOBACCO (GRP) CO LTD
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Problems solved by technology

[0005] Since the NNK released in the smoke of each Chinese-style cigarette is only a few nanograms, and NNK is metabolized into a variety of 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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  • High performance liquid chromatography-mass spectrum-mass spectrum (HPLC-MS-MS) combined detection method for (S)-NNAL and (R)-NNAL in urine
  • High performance liquid chromatography-mass spectrum-mass spectrum (HPLC-MS-MS) combined detection method for (S)-NNAL and (R)-NNAL in urine
  • High performance liquid chromatography-mass spectrum-mass spectrum (HPLC-MS-MS) combined detection method for (S)-NNAL and (R)-NNAL in urine

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Experimental program
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Effect test

Embodiment 1

[0017] Unless otherwise stated, the percentages used in the present invention are all percentages by weight.

[0018] A high-performance liquid chromatography-mass spectrometry-mass spectrometry detection method for (S)-NNAL and (R)-NNAL in urine, the method comprising the following steps:

[0019] 1. Chemical reagents

[0020] (S)-(+)-α-methoxy-α-trifluoromethylphenylacetic anhydride (CAS No. 20445-33-4), (S)-(+)-α-methoxy-(three 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).

[0021] 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 ...

Embodiment 2

[0039] 1. Derivatization of racemic NNAL: use anhydrous acetonitrile as a solvent to prepare a single standard solution of rac NNAL standard with a concentration of 1 μg / mL, and keep four significant figures; prepare in the same way using anhydrous acetonitrile as a solvent A single standard solution of rac-NNAL-methyl-d3 with a concentration of 1 μg / mL; using anhydrous acetonitrile as a solvent, prepare a mixed standard solution containing rac-NNAL and deuterated rac-NNAL-methyl-d3 according to Table 1; Prepare anhydrous acetonitrile solution of (S)-(+)-α-methoxy-α-trifluoromethylphenylacetic anhydride with a concentration of 10ng / mL; prepare anhydrous solution of triethylamine with a concentration of 10ng / mL Acetonitrile solution; respectively take 0.5mL mixed standard solution in HPLC-MS-MS chromatographic bottles, and then add (S)-(+)-α-methoxy-α-trifluoromethylphenylacetic anhydride according to Table 2 Anhydrous acetonitrile solution and triethylamine acetonitrile soluti...

Embodiment 3

[0043] 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 Mosher reaction respectively, and then After purification and 1H NMR spectrum test, Mosher method was used to determine (S)-NNAL-(S)-(+)-α-methoxy-α-trifluoromethylphenylacetate and (R)-NNAL- Absolute configuration of (S)-(+)-α-methoxy-α-trifluoromethylphenylacetate.

[0044] 2. Derivatization of racemic NNAL and establishment of a standard curve: use anhydrous acetonitrile as a solvent to 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 acet...

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Abstract

The invention relates to a high performance liquid chromatography-mass spectrum-mass spectrum (HPLC-MS-MS) combined detection method for (S)-NNAL and (R)-NNAL in urine. Derivatization and an HPLC-MS-MS method are adopted for determining (S)-NNAL and (R)-NNAL in urine. The HPLC-MS-MS combined detection method comprises the following steps of: determining configuration of (S)-NNAL and (R)-NNAL by adopting a rotation determination and comparison method and taking the configuration as a reference, or determining configuration of (S)-NNAL-(S)-(+)-allpha-methoxy-alpha-trifluoromethyl acetic acid ester and (R)-NNAL-methyl-d3-(S)-(+)-alpha-methoxy-alpha-trifluoromethyl acetic acid ester and taking the configuration as the reference; preparing a mixed standard solution containing rac-NNAL and deuterated rac-NNAL-methyl-d3, then carrying out Mosher derivatization, and then carrying out HPLC-MS-MS analysis on the derivatized mixed standard solution; and finally detecting (S)-NNAL and (R)-NNAL in urine. The HPLC-MS-MS combined detection method provided by the invention has the advantages of low detection limit, good stability, simple pre-treatment, high speed and high accuracy.

Description

technical field [0001] The invention belongs to the technical field of biomarker physical and chemical detection of tobacco-specific nitrosamines in urine, and specifically relates to a high-performance liquid chromatography-mass spectrometry-mass spectrometry detection method for (S)-NNAL and (R)-NNAL in urine . Background technique [0002] Tobacco-specific nitrosamines (TSNAs) are produced through nitrosation of tobacco endogenous alkaloids with nitrate and nitrite, and only exist in tobacco, tobacco products and cigarette smoke. In recent years, as smoking and health issues have attracted widespread attention, the research on various harmful chemical components in smoke has become increasingly in-depth at home and abroad. Since 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) in tobacco-specific nitrosamines may be carcinogenic to animals, the tobacco industry and health agencies are now There is an urgent need to establish a method to accurately evaluate the expos...

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刘亚张敏
Owner HONGYUN HONGHE TOBACCO (GRP) CO LTD
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