Method for detecting 3-hydroxycotinine in urea

A technology of hydroxycotinine and urine, which is applied in the field of physical and chemical testing of tobacco alkaloids, achieves the effects of low detection limit, small impact, and easy popularization and application

Active Publication Date: 2015-04-08
HONGYUN HONGHE TOBACCO (GRP) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

How to realize simultaneous accurate detection of two kinds of 3-hydroxycotinine contents in urine samples and in-depth analysis of the ratio of the two, there is no good solution in the prior art

Method used

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  • Method for detecting 3-hydroxycotinine in urea
  • Method for detecting 3-hydroxycotinine in urea
  • Method for detecting 3-hydroxycotinine in urea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. Preparation of chemical reagents: (3R,5S)-3-hydroxycotinine (CAS No. 34834-67-8), (3S,5S)-3-hydroxycotinine (CAS No. 37096-14-3 ),(3R,5S)-3-hydroxycotinine-d 3 (CAS No. 159956-78-2), beta-glucuronidase (type IX-A, from Escherichia coli).

[0025] 2. Standard curve establishment:

[0026] Table 2 Proportion of mixed standard solution

[0027]

[0028] Using acetonitrile as solvent, prepare (3R,5S)-3-hydroxycotinine, (3S,5S)-3-hydroxycotinine and (3R,5S)-3-hydroxycotinine-d according to Table 2 3 The mixed standard solution, determine the chromatographic peaks of (3R, 5S)-3-hydroxyl cotinine and (3S, 5S)-3-hydroxyl cotinine by retention time; In the HPLC-MS-MS chromatogram (3R, The concentration of 5S)-3-hydroxycotinine and (3R,5S)-3-hydroxycotinine-d 3 The ratio of the concentration is the abscissa, with the quantitative ion-pair peak area of ​​(3R, 5S)-3-hydroxyl cotinine in the chromatogram and (3R, 5S)-3-hydroxyl cotinine-d 3 The ratio of the quantitative i...

Embodiment 2

[0033] Repeat Example 1 with the following differences: Shake the urine sample after thawing at room temperature, accurately pipette 1.0 mL into a 10 mL test tube, and add 20 μL of (3R,5S)-3-hydroxyl at a concentration of 10 mg / mL Cotinine-d 3 solution, then add 0.2mL of sodium phosphate buffer solution (pH6.0, 0.1mol / L) and 100μL of β-glucuronidase (1000U / mL urine), mix thoroughly and place in a microwave-assisted extraction apparatus at 37°C Enzymatic hydrolysis at constant temperature in the dark for 2 minutes. Add 10 mL of ultrapure water to the urine sample after enzymatic hydrolysis, filter it with a 0.2 μm aqueous phase filter membrane, and perform HPLC-MS-MS detection. Use (3R,5S)-3-hydroxycotinine and (3S,5S)-3-hydroxycotinine standard solution working curves to calculate the (3R,5S)-3-hydroxycotinine in the sample to be tested. Tinine and (3S,5S)-3-hydroxycotinine, and then converted to the concentration of (3R,5S)-3-hydroxycotinine in the urine is 3919ng / mL, (3S,5...

Embodiment 3

[0035] Repeat Example 1 with the following differences: Shake the urine sample after thawing at room temperature, accurately pipette 0.5 mL into a 10 mL test tube, add 10 μL of (3R,5S)-3-hydroxyl at a concentration of 10 mg / mL Cotinine-d 3 solution, then add 0.2mL of sodium phosphate buffer solution (pH6.0, 0.1mol / L) and 100μL of β-glucuronidase (1000U / mL urine), mix thoroughly and place in a constant temperature shaker at 37 Enzyme hydrolysis overnight at constant temperature in the dark. Add 10 mL of ultrapure water to the urine sample after enzymatic hydrolysis, filter it with a 0.2 μm aqueous phase filter membrane, and perform HPLC-MS-MS detection. Use (3R,5S)-3-hydroxycotinine and (3S,5S)-3-hydroxycotinine standard solution working curves to calculate the (3R,5S)-3-hydroxycotinine in the sample to be tested. Tinine and (3S,5S)-3-hydroxycotinine, and then converted to the concentration of (3R,5S)-3-hydroxycotinine in the urine is 1793.8ng / mL, (3S,5S)-3 The concentration...

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Abstract

The invention discloses a method for detecting 3-hydroxycotinine in urea. The method comprises the following steps: establishing a (3R,5S)-3-hydroxycotinine and (3S,5S)-3-hydroxycotinine standard solution working curve; carrying out enzymolysis, dilution and filtration on a urea sample and detecting by an HPLC-MS-MS (High Performance Liquid Chromatography-Mass Spectrometry-Mass Spectrometry), so as to convert the concentrations of (3R,5S)-3-hydroxycotinine and (3S,5S)-3-hydroxycotinine in the urea sample; and calculating the ratio of the (3R,5S)-3-hydroxycotinine and the (3S,5S)-3-hydroxycotinine. The method can provide a scientific judgment method for evaluating the metabolism health condition of an individual and evaluating the exposure degree of nicotine in smoke, has the advantages of low detection limit, good stability, rapidness and accuracy and the like, and fills up a blank of the technical field.

Description

technical field [0001] The invention belongs to the technical field of physical and chemical testing of tobacco alkaloids, and in particular relates to a method for detecting (3R, 5S)-3-hydroxycotinine and (3S, 5S)-3-hydroxycotinine in urine. Background technique [0002] Nicotine is the main alkaloid in tobacco, with an average content of 4% in tobacco, accounting for about 95% of the total amount of tobacco alkaloids. Smokers smoke tobacco mainly because nicotine can bring physical and psychological satisfaction. Studies have proved that the physiological effects of nicotine on the human body mainly include the effects on the central nervous system, cardiovascular system and endocrine system. Nicotine can be quickly metabolized into various products in the human body (mainly in the liver, followed by the lungs and kidneys). The main metabolites are 3-hydroxycotinine and its glycosides, cotinine and its glycosides, Nicotine and its glycosides, nornicotine, norcotinine, nic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
Inventor 者为王文元段焰青冯斌夏建军陈兴蒋举兴党立志
Owner HONGYUN HONGHE TOBACCO (GRP) CO LTD
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