Method development for the determination of 24S‐hydroxycholesterol in human plasma without derivatization by high‐performance liquid chromatography with tandem mass spectrometry in atmospheric pressure chemical ionization mode |
| |
| Authors: | Hiroshi Sugimoto Masaaki Kakehi Yoshinori Satomi Hidenori Kamiguchi Fumihiro Jinno |
| |
| Affiliation: | 1. Drug Metabolism and Pharmacokinetics Research LaboratoriesPharmaceutical Research Division, Takeda Pharmaceutical Company Limited;2. Integrated Technology Research Laboratories, Pharmaceutical Research DivisionTakeda Pharmaceutical Company Limited |
| |
| Abstract: | We developed a highly sensitive and specific high‐performance liquid chromatography with tandem mass spectrometry method with an atmospheric pressure chemical ionization interface to determine 24S‐hydroxycholesterol, a major metabolite of cholesterol formed by cytochrome P450 family 46A1, in human plasma without any derivatization step. Phosphate buffered saline including 1% Tween 80 was used as the surrogate matrix for preparation of calibration curves and quality control samples. The saponification process to convert esterified 24S‐hydroxycholesterol to free sterols was optimized, followed by liquid–liquid extraction using hexane. Chromatographic separation of 24S‐hydroxycholesterol from other isobaric endogenous oxysterols was successfully achieved with gradient mobile phase comprised of 0.1% propionic acid and acetonitrile using L‐column2 ODS (2 μm, 2.1 mm id × 150 mm). This assay was capable of determining 24S‐hydroxycholesterol in human plasma (200 μL) ranging from 1 to 100 ng/mL with acceptable intra‐ and inter‐day precision and accuracy. The potential risk of in vitro formation of 24S‐hydroxycholesterol by oxidation from endogenous cholesterol in human plasma was found to be negligible. The stability of 24S‐hydroxycholesterol in relevant solvents and human plasma was confirmed. This method was successfully applied to quantify the plasma concentrations of 24S‐hydroxycholesterol in male and female volunteers. |
| |
| Keywords: | Atmospheric pressure chemical ionization Chromatographic separation 24S‐Hydroxycholesterol Saponification Surrogate matrices |
|
