title: Simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by semi-micro high-performance liquid chromatography with an immobilized cholesterol oxidase as a pre-column reactor: application to bovine adrenal fasciculata cells.
abstract: A method for the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by high-performance liquid chromatography with an immobilized cholesterol oxidation enzyme reactor was developed. Pregnenolone and 17α-hydroxypregnenolone were converted to progesterone and 17α-hydroxyprogesterone, respectively, by the immobilized enzyme packed into the reactor column, and could thus be monitored by UV absorption at 240 nm. The calibration curves for pregnenolone and 17α-hydroxypregnenolone were linear in the range of 0.4-10 and 0.3-10 μg/ml with a correlation coefficient of 0.9993 and 0.9998, respectively. The detection limit at a signal-to-noise ratio of 3 was 0.12 and 0.08 μg/ml for pregnenolone and 17α-hydroxypregnenolone, respectively. The conversion rate of pregnenolone to progesterone and 17α-hydroxypregnenolone to 17α-hydroxyprogesterone was 90.6% and 99.3%, respectively. Intra-day and inter-day precision (in terms of percentage coefficient of variation) were less than 9.3%, with accuracy greater than 94.8%. This method was successfully applied to the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone secreted into the culture medium of bovine adrenal fasciculata cells and of both analytes produced within the cells.
authors.affiliation: Department of Bio-analytical Chemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Akiha-ku, Niigata, Niigata 956-8603, Japan. yamatos@nupals.ac.jp
authors.forename: Susumu
authors.initials: S
authors.lastname: Yamato
authors.forename: Saori
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Natsumi
authors.initials: N
authors.lastname: Yamazaki
authors.forename: Takao
authors.initials: T
authors.lastname: Aketo
authors.forename: Eiichi
authors.initials: E
authors.lastname: Tachikawa
mesh_terms: D006907:17-alpha-Hydroxypregnenolone / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019326:17-alpha-Hydroxyprogesterone / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D016014:Linear Models; D011284:Pregnenolone / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011374:Progesterone / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D015383:Zona Fasciculata / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D006907:17-alpha-Hydroxypregnenolone; D011374:Progesterone; D019326:17-alpha-Hydroxyprogesterone; D011284:Pregnenolone; D002789:Cholesterol Oxidase