Highly Sensitive and Selective Detection of Cytochrome P450 46A1 Activity by UPLC-MS/MS Method.

Cytochrome P450 46A1 (CYP46A1) is a key enzyme responsible for metabolizing cholesterol to 24-hydroxycholesterol in brain, which might be served as a therapeutic target for several neurodegenerative disorders including Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease (HD). However, an applicable, sensitive and reliable method for the precise measurement of CYP46A1 activities in complex biological samples remains limited. In this study, a novel ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for highly sensitive and selective determination of 24-hydroxycholesterol was developed to characterize CYP46A1 activity. The mass spectrometric detection was performed using multiple reaction monitoring (MRM) of for 24-hydroxcholesterol at m/z 385.2→367.2. The limit of quantification for 24-hydroxycholesterol of this UPLC-MS/MS method was as low as 10 nM, which was quite lower than previous methods. The method also showed favorable accuracy and precision. Meanwhile, the short-term and long-term stability of this method was fully validated. In addition, the method was successfully applied to investigate the kinetic properties of 24-hydroxycholesterol formation by CYP46A1.