Quantification of 4-Beta-Hydroxycholesterol in Human Plasma Using Automated Sample Preparation and LC-ESI-MS/MS Analysis

详细信息    查看全文

• 作者：Angela K. Goodenough ; Joelle M. Onorato ; Zheng Ouyang ; Shu Chang ; A. David Rodrigues ; Sreeneeranj Kasichayanula ; Shu-Pang Huang ; Wesley Turley ; Richard Burrell ; Marc Bifano ; Mohammed Jemal ; Frank LaCreta ; Adrienne Tymiak ; David Wang-Iverson
• 刊名：Chemical Research in Toxicology
• 出版年：2011
• 出版时间：September 19, 2011
• 年：2011
• 卷：24
• 期：9
• 页码：1575-1585
• 全文大小：871K
• 年卷期：v.24,no.9(September 19, 2011)
• ISSN：1520-5010

文摘

It has recently been proposed that plasma levels of 4尾-hydroxycholesterol (4尾HC) may be indicative of cytochrome P450 3A4 (P450 3A) activity and therefore could be used to probe for P450 3A-mediated drug鈥揹rug interactions. With this in mind, we describe a highly sensitive and precise liquid chromatography鈥揺lectrospray ionization鈥搕andem mass spectrometry method for the measurement of 4尾HC in human plasma with a lower limit of quantification established at 2 ng/mL using 50 渭L of plasma. The entire sample preparation scheme including saponification and derivatization of 4尾HC to the corresponding dipicolinyl ester (DPE) was completed in less than 8 h using an automated sample preparation scheme enabling higher-throughput capabilities. Chromatographic resolution of 4尾HC from 4伪-hydroxycholesterol and other endogenous isobaric species was achieved in 11-min using an isocratic gradient on a C18 column. Because of endogenous concentrations of 4尾HC in plasma, a stable isotope labeled (SIL) analogue, d7-4尾HC, was used as a surrogate analyte and measured in the standard curve and quality control samples prepared in plasma. A second SIL analogue, d4-4尾HC, was used as the internal standard. The intraday and interday accuracy for the assay was within 6% of nominal concentrations, and the precision for these measurements was less than 5% relative standard deviation. Rigorous stability assessments demonstrated adequate stability of endogenous 4尾HC in plasma and the corresponding DPE derivative for the analysis of clinical study samples. The results from clinical samples following treatment with a potent P450 3A inducer (rifampin) or inhibitor (ketoconazole) are reported and demonstrate the potential future application for this highly precise and robust analytical assay.