title: 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.
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.
authors.affiliation: Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sugimoto
authors.affiliation: Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kakehi
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Satomi
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Kamiguchi
authors.affiliation: Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Jinno
mesh_terms: D001274:Atmospheric Pressure; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D006888:Hydroxycholesterols / Q000097:blood*; D059625:Liquid-Liquid Extraction; D008297:Male; D011786:Quality Control; D053719:Tandem Mass Spectrometry
chemical_list: D006888:Hydroxycholesterols; C044563:24-hydroxycholesterol
keywords: 24S-Hydroxycholesterol; Atmospheric pressure chemical ionization; Chromatographic separation; Saponification; Surrogate matrices