• 专利标题:   Solid-phase extraction of modified nucleosides e.g. 2'-O-methyluridine in urine, involves using graphene material as solid-phase extraction column filler, extracting and enriching several modified nucleosides in urine, detecting and analyzing using liquid chromatography tandem mass spectrum.
  • 专利号:   CN115487542-A
  • 发明人:   YUAN Y, JIANG K, HU Y, ZHANG X, GUO C
  • 专利权人:   UNIV ZHEJIANG
  • 国际专利分类:   B01D015/12, B01D015/18
  • 专利详细信息:   CN115487542-A 20 Dec 2022 B01D-015/12 202312 Chinese
  • 申请详细信息:   CN115487542-A CN11073729 02 Sep 2022
  • 优先权号:   CN11073729

▎ 摘  要

NOVELTY - Solid-phase extraction of modified nucleosides in urine involves using graphene material as a solid-phase extraction column filler, extracting and enriching several modified nucleosides in urine, detecting and analyzing using liquid chromatography tandem mass spectrum. The detection sensitivity and accuracy are improved. The detection method involves unfreezing a urine sample at room temperature, centrifuging, taking supernatant, adding an isotope internal standard solution, adding water for dilution, weighing 0.1-1 mg adsorbent filler in a solid phase extraction column, activating and balancing with methanol and water, adding the diluted urine sample into a solid phase extraction column, washing, drying, eluting, collecting eluent, vacuum centrifugal drying, re-dissolving with water, analyzing and detecting using a liquid chromatography tandem mass spectrometry. USE - Solid-phase extraction of modified nucleosides e.g. N6-methyladenosine, N1-methyladenosine, 2'-O-methyladenosine, N6, 2 '-O-dimethyladenosine, 2'-O-methylguanosine, N1-methylguanosine, N2-methylguanosine, N7-methylguanosine, N2, N2-dimethylguanosine, 2'-O-methyluridine, 3-methyluridine, 5-methyluridine, 5, 2'-O-dimethyluridine, 2'-O-methylcytidine, 3-methylcytidine, 5-methylcytidine and N4-acetylcytidine in urine (all claimed), and used for analytical chemistry, metabonomics, clinical medicine and preventive medicine applications. ADVANTAGE - The method ensures extraction of modified nucleosides e.g. N6-methyladenosine (m6A), N1-methylguanosine (m1G), 5-methyluridine (m5U) and 5-methylcytidine (m5C) biological samples such as urine. DETAILED DESCRIPTION - Solid-phase extraction of modified nucleosides in urine involves using graphene material as a solid-phase extraction column filler, extracting and enriching several modified nucleosides in urine, detecting and analyzing using liquid chromatography tandem mass spectrum. The detection sensitivity and accuracy are improved. The detection method involves unfreezing a urine sample at room temperature, centrifuging at 13000 rpm at 4℃ for 15 minutes, taking supernatant into 1.5 mL centrifuge tube, adding an isotope internal standard solution, adding water for dilution, weighing 0.1-1 mg adsorbent filler in a solid phase extraction column, sequentially activating and balancing with methanol and water, adding the diluted urine sample into a solid phase extraction column, washing with water, drying by a vacuum pump, eluting, collecting eluent, vacuum centrifugal drying the eluent, re-dissolving with water, analyzing and detecting mixed standard solutions with different concentrations by using a liquid chromatography tandem mass spectrometry, obtaining a linear regression equation by using a peak area ratio of a standard substance and an isotope internal standard corresponding to the standard substance, measuring a urine sample, and substituting the linear equation to calculate the content of an analyte in the sample.