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
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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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