Method for screening xanthine oxidase inhibitor by ultra performance liquid chromatography and mass spectrometry

A technology of xanthine oxidase and ultra-high performance liquid phase, which is applied in the field of analytical chemistry, can solve the problems of long time-consuming, easy to be interfered by the background of the spectroscopic method, and a large amount of samples, so as to achieve good specificity and avoid false positives and false positives. Negative results, fast results for sample testing

Active Publication Date: 2011-06-15
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The screening of xanthine oxidase inhibitor generally uses spectral method, and its shortcoming is that spectral method is easily subject to background interference, may obtain false positive or false negative result (Akihiko Nagao, Michiko Sehi, Hidetaka Kobayashi, Biosci.Biotechnol.Biochem63 (1999 ) 1787-1790; Chunmao Lin, Chienshu Chen, Chientsu Chen, Yuchih Liang, and Jenkun Lina, Biochemical and Biophysical Research Communications 294 (2002) 167-172); some people have also used liquid chromatography to overcome the shortcomings of spectroscopic methods Screening of xanthine oxidase inhibitors is performed, but the liquid chromatography method requires a large amount of sample and takes a long time (Akihiko Nagao, Michiko Sehi, Hidetaka Kobayashi, Biosci. Biotechnol. Biochem 63(1999) 1787-1790)

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  • Method for screening xanthine oxidase inhibitor by ultra performance liquid chromatography and mass spectrometry
  • Method for screening xanthine oxidase inhibitor by ultra performance liquid chromatography and mass spectrometry
  • Method for screening xanthine oxidase inhibitor by ultra performance liquid chromatography and mass spectrometry

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Embodiment 1

[0071] The method for screening xanthine oxidase inhibitors by ultra-high performance liquid chromatography and mass spectrometry provided by the invention comprises the following steps:

[0072] (1) Preparation of enzymatic reaction buffer

[0073] The enzymatic reaction buffer includes 50 mmol / L tris, 7 mmol / L hydrochloric acid, 1 mmol / L EDTA disodium salt, pH 8.9;

[0074] (3) Preparation of standard products

[0075] Use 2 mol / L of ammonia solution to prepare the standard products with concentrations of 1 micromol / L, 2 micromol / L, 5 micromol / L, 10 micromol / L, 15 micromol / L or 20 micromol / L liter of standard solution, and each concentration of standard solution contains 1 micromol / liter of sodium tauroursodeoxycholate as an internal standard; the standard is xanthine; xanthine is the xanthine oxidase substrate;

[0076] (3) Enzymatic reactions of reference substances, blank samples and samples

[0077] Preparation of reference substance: total reaction volume 200 microl...

Embodiment 2

[0114] The sample is as follows: the total reaction volume is 200 microliters, xanthine oxidase with a final concentration of 100 nanomoles / liter and isorhamnetin with a final concentration of 20 micromoles / liter are incubated at 37° C. for 30 minutes, and a final concentration of 100 nanomoles is added. Micromoles / L substrate xanthine, react at 37°C for 5 minutes, add 800 microliters of methanol to terminate the reaction, add internal standard sodium tauroursodeoxycholate with a final concentration of 1 micromoles / liter, as a sample; For the determination of ultra-high performance liquid chromatography and mass spectrometry;

[0115] All the other are with embodiment 1;

[0116] According to the detection steps of Example 1, the inhibition rate of xanthine oxidase by the 20 micromol / L isorhamnetin standard substance was detected and calculated to be 73%. Figure 11 and Figure 12 It is the extraction chromatogram of sample in embodiment 2.

Embodiment 3

[0118] The sample is as follows: the total reaction volume is 200 microliters, xanthine oxidase with a final concentration of 100 nanomoles / liter and genistein with a final concentration of 20 micromoles / liter are incubated at 37°C for 30 minutes, and a final concentration of 100 microliters is added. mol / L substrate xanthine, react at 37°C for 5 minutes, add 800 microliters of methanol to terminate the reaction, add internal standard sodium tauroursodeoxycholate with a final concentration of 1 micromol / liter as a sample; Determination by ultra-high performance liquid chromatography and mass spectrometry;

[0119] All the other are with embodiment 1;

[0120] According to the detection procedure of Example 1, the inhibition rate of xanthine oxidase by 20 micromol / liter genistein standard substance was detected and calculated to be 50%. Figure 13 and Figure 14 It is the extraction chromatogram of sample in embodiment 3.

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Abstract

The invention provides a method for screening a xanthine oxidase inhibitor by ultra performance liquid chromatography and mass spectrometry. The method is used for analyzing the in-vitro inhibition rate of the extract or monomer of a natural product on the xanthine oxidase and the catalytic activity of the xanthine oxidase. By using the ultra performance liquid chromatography-mass spectrometry in the xanthine oxidase inhibitor screening, the method is rapid and accurate in sample detection, and the correlation coefficients of the linear equation reaches 0.998. The mass spectrometry has high accuracy and good specificity in detecting the mass electron ratio of a compound, and can be used for screening the xanthine oxidase inhibitor and for the kinetic study of the xanthine oxidase inhibitor without false positive and false negative results which occur in the spectrometry. The results showed that the inhibition rate I of 20 mumol/L allopurinol is 80%; the inhibition rate of 20 mumol/L isorhamnetin is 73%; the inhibition rate of 20 mumol/L genistein is 50%; and the inhibition rate of 0.1 mg/mL aqueous extract of Ginkgo biloba is 27%.

Description

field of invention [0001] The invention belongs to the field of analytical chemistry, and relates to a method for screening xanthine oxidase inhibitors by ultra-high performance liquid chromatography and mass spectrometry, in particular, it relates to the in vitro inhibition rate of xanthine oxidase by natural product extracts or monomers and xanthine An Ultra-Performance Liquid Chromatography-Mass Spectrometry Method for the Catalytic Activity of Oxidases. technical background [0002] Xanthine oxidase is a kind of flavoprotease that exists widely in organisms. It belongs to the molybdenum protease family and is a key enzyme in the nucleic acid metabolism pathway in vivo. Xanthine oxidase catalyzes the oxidation of the substrates xanthine and hypoxanthine to uric acid and produces superoxide anion (O - 2 ) and hydrogen peroxide (H 2 o 2 ). Excessive uric acid concentration can lead to hyperuricemia, and the deposition and crystallization of uric acid in the joints can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/72G01N30/06
Inventor 刘志强刘舒邢俊鹏宋凤瑞郑重刘淑莹
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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