A Highly Sensitive Liquid Chromatography-Electrospray Ionization-Time of Flight/Mass Spectrometric Assay for the Quantitation of 4-Beta-Hydroxycholesterol and Its Application to in vivo Cytochrome P450 3a Induction by AGM-130

Abstract

A liquid chromatography-electrospray ionization-time-of-flight/mass spectrometry (LC-ESI-TOF/MS) method was developed for the determination of 4-beta-hydroxycholesterol (4-HC) in rat plasma, to evaluate cytochrome P450 3A (CYP3A) induction liability of a novel cyclin dependent kinases (CDKs) inhibitor, AGM-130. The method consisted of sample preparation without derivatization reagents for high sensitivity such as picolinyl ester, which could be labor-intensive and time consuming. The 4% bovine serum albumin in phosphate-buffered saline as a surrogate matrix was used in the calibration curve and quality control samples for the determination of 4-HC. Stable isotope-labeled (SIL) 4-HC (d7-4-HC) was used as an internal standard. A quadratic regression (weighted 1/concentration), with an equation y=ax(2)+bx+c, was used to fit calibration curves over the concentration range of 3.01-2220ng/mL for 4-HC. For quality control samples at 15.0, 165, and 1820ng/mL from the qualification experiment, the within-run accuracy ranged from 95% to 105% with precision values 10% for 4-HC. This novel LC-ESI-TOF/MS method was successfully applied to evaluate the potential of CYP3A induction by 5-hydroxy-5-nitro-indirubin oxime (AGM-130) in rat.