Method for simultaneously detecting cotinine, phenyl hydroxyacetic acid and phenylglyoxalic acid in human urine based on derivation method

A technology for detecting human body and phenylglycolic acid, applied in the field of bioengineering, can solve the problems of high detection cost, difficult contaminant contact degree, different types, etc., and achieve the effects of high detection sensitivity, convenience and easy availability, and low detection cost.

Inactive Publication Date: 2009-07-08
陈枢青 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] By consulting the literature, we found that there are many methods used by domestic scholars to study the environmental pollutants nicotine and styrene exposure metabolites, including GC, HPLC, GC-MS, thin-layer chromatography, enzyme-linked immunosorbent assay, etc. etc.; the types of instruments used in the method are also different. Taking GC-MS as an example, a combination of Shimadzu GC-14A and American Agilent 6890N-5973MSD are used; there is no special institution for environmental pollutants and The human exposure

Method used

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  • Method for simultaneously detecting cotinine, phenyl hydroxyacetic acid and phenylglyoxalic acid in human urine based on derivation method
  • Method for simultaneously detecting cotinine, phenyl hydroxyacetic acid and phenylglyoxalic acid in human urine based on derivation method
  • Method for simultaneously detecting cotinine, phenyl hydroxyacetic acid and phenylglyoxalic acid in human urine based on derivation method

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Effect test

Embodiment 1

[0039]Take 3 urine samples stored at -20°C, marked as No. 1, 2, and 3 respectively, centrifuge after natural thawing, and carry out the following experimental procedures at the same time: Take 1ml of supernatant urine sample, add 5ul of 2mg / ml benzophenone, Add 50ul of 2mg / ml diphenylamine, add 100ul of 1.2M HCL, extract with 1ml of chloroform, vortex at 1500rpm for 5min, then centrifuge at 2500rpm for 4min, then take the organic phase A; Chloroform was used for the second extraction, vortexed at 1500rpm for 5min, centrifuged at 2500rpm for 4min, and the organic phase B was taken. Mix the two organic phases A and B in the same glass tube, blow dry; then add 50ul acetonitrile to redissolve, add 50ul derivatization reagent MSTFA, derivatize at 50°C for 30min. Take 1ul of the reaction solution for gas chromatography-mass spectrometry determination; GC / MS conditions are: DB-5ms column, EI ion source, voltage 70eV, ion source temperature 230°C, inlet temperature 270°C; SIM method ...

Embodiment 2

[0041] After adjusting the diet and living habits of Person No. 3, take the urine sample of Person No. 3 again, and carry out the following experimental steps: Take 1ml of supernatant urine sample, add 5ul 2mg / ml benzophenone, 50ul 2mg / ml diphenone Aniline, add 100ul 1.2M HCL, extract with 1ml chloroform, vortex at 1500rpm for 5min, then centrifuge at 2500rpm for 4min, then take the organic phase A; add 130ul 1M sodium hydroxide to the remaining liquid, and use 1ml chloroform for the second For secondary extraction, vortex at 1500rpm for 5min, then centrifuge at 2500rpm for 4min, and take the organic phase B. Mix the two organic phases A and B in the same glass tube, blow dry; then add 50ul of acetonitrile to redissolve, add 50ul of derivatization reagent MSTFA, derivatize for 30min at 50°C; take 1ul of the reaction solution for gas chromatography-mass spectrometry . The GC / MS conditions are as follows: DB-5ms column, EI ion source, voltage 70eV, ion source temperature 230°C,...

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Abstract

The invention relates to the technical field of biological engineering, in particular to a method for detecting cotinine, mandelic acid and phenylglyoxalic acid in human urine simultaneously on the basis of a derivation method. The method is characterized in that the method comprises: step one, storing collected urine after centrifugal process; step two, taking the urine obtained in step one and adding the urine into internal label benzophenone, diphenylamine, hydrochloric acid and chloroform to carry out extraction through rotating centrifugation, thereby aspirating and recording organic phase as A and taking residual solution for standby; step three, taking the residual solution obtained in step two and adding the residual solution into sodium hydroxide solution and chloroform to carry out secondary extraction through rotating centrifugation, thereby aspirating and recording organic phase as B; step four, mixing the organic phase A extracted in step two and the organic phase B extracted in step three inside a glass tube, and then blowing dry both organic phases; step five, adding acetonitrile into the glass tube adopted in step four for redissolution and adding derivative reagent to carry out derivative reaction; and step six, taking the reaction solution obtained in step five to carry out gas phase chromatography-mass spectrometry combined measurement. The method has the characteristics of simplicity, convenience, quickness, reliable result, high degree of automation and convenient industrialized application.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for simultaneously detecting codnine, mandelic acid and phenylglyoxylic acid in human urine based on a derivatization method. Background technique [0002] Styrene acts on the P450 enzyme system, and its main metabolites, mandelic acid and phenylglyoxylic acid, can be used as biomarkers of the dose of styrene in humans. However, styrene is widely used in industry, which pollutes the atmosphere, water and soil. In daily life, people inhale styrene mainly due to the pollution of automobile exhaust. With the development of the polymer industry, the number of workers exposed to styrene is increasing. The chemical structure of styrene is similar to the mutagen and carcinogen vinyl chloride, and it is also a potential mutagen after metabolic activation, and its intermediate metabolite 7,8-epoxystyrene is a strong direct mutagen. Cotinine is the main metabolite of nico...

Claims

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Application Information

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IPC IPC(8): G01N30/78G01N30/06
Inventor 吴鸳鸯陈枢青
Owner 陈枢青
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