Isolation and purification of in vivo-generated 25-hydroxyvitamin D2 by preparative high-performance liquid chromatography.

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  • OKANO Toshio
    Department of Hygienic Chemistry, Kobe Women's College of Pharmacy
  • MATSUYAMA Naoko
    Department of Hygienic Chemistry, Kobe Women's College of Pharmacy
  • KOBAYASHI Tadashi
    Department of Hygienic Chemistry, Kobe Women's College of Pharmacy
  • KURODA Eizo
    Department of Pediatrics, Kobe University School of Medicine
  • KODAMA Socihi
    Department of Pediatrics, Kobe University School of Medicine
  • MATSUO Tamotsu
    Department of Pediatrics, Kobe University School of Medicine

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  • Isolation and Purification of In Vivo G

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Abstract

In order to obtain a standard compound of 25-hy-droxyvitamin D2 (25-OH-D2), a method for isolating in vivo-generated 25-OH-D2 from the blood of rats or rabbits was established by using several steps of preparative high-performance liquid chromatography (HPLC). When the unsaponifiable matter of the plasma obtained from rats or rabbits receiving a large dose of vitamin D2 was applied to the preparative HPLC using a Zorbax SIL column, a peak denoted as peak X was observed on the chromatogram. Since the peak X was thought to be due to 25-OH-D2 from the experiments of time course and dose-response, it was purified by subjecting it to successive preparative HPLC using several kinds of columns. From the results of ultraviolet (UV) absorption spectrum, gas chromatography-mass spectrometry (GC-MS) and mass chromatography, the purified peak X compound was confirmed to be 25-OH-D2. The proposed method for isolating in vivo-generated 25-OH-D2 is very convenient, because the time to perform each HPLC is very short though several steps of HPLC are used.

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