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Quantitation of P450 3A4 endogenous biomarker – 4 β ‐hydroxycholesterol – in human plasma using LC/ESI‐MS/MS
Author(s) -
Huang MikeQingtao,
Lin Weisheng,
Wang Weimin,
Zhang Wei,
Lin Zhongping John,
Weng Naidong
Publication year - 2014
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.3131
Subject(s) - chemistry , chromatography , electrospray ionization , analyte , calibration curve , electrospray , tandem mass spectrometry , mass spectrometry , human plasma , detection limit
ABSTRACT 4 β ‐Hydroxycholesterol (4 β ‐HC) has been proposed as a new endogenous biomarker for cytochrome P450 3A4/5 activity. Therefore, it is important to have a robust method for its accurate determination in human plasma. Here a liquid chromatography–tandem mass spectrometry with electrospray ionization (LC/ESI‐MS/MS) assay for the quantitation of 4 β ‐HC in human plasma is described. While the calibration standards were prepared in a surrogate matrix for human plasma, the quality control samples were prepared in human plasma to mimic the incurred study samples. In order to achieve accurate determination of 4 β ‐HC, the chromatographic separation of 4 β ‐HC from its isomers, especially 4 α ‐hydroxycholesterol (4 α ‐HC), was crucial. In the absence of an authentic 4 α ‐HC standard at the time of this study, an alternative selectivity test strategy was developed to confirm the separation. After being alkalized with potassium hydroxide, the human plasma sample (50 μL) was extracted with hexane, derivatized into picolinyl esters using picolinic acid, extracted again with hexane, and then analyzed by LC/ESI‐MS/MS. The calibration curve range was 5–500 ng/mL and the chromatographic separation was achieved on a 50 × 2.1 mm Thermal Hypersil Gold column with a gradient elution. The assay accuracy, precision, linearity, selectivity and analyte stability throughout the analysis were established. The validated assay was successfully applied to a Phase I clinical study for the measurement of 4 β ‐HC in human plasma. Copyright © 2014 John Wiley & Sons, Ltd.