Rapid LC-MS/MS method for the determination of 4-hydroxycholesterol/cholesterol ratio in serum as endogenous biomarker for CYP3A activity in human and foals

摘要

Cytochrome P450 3A (CYP) enzymes are involved in the elimination of many drugs and are known to be regulated by several environmental factors. Thus, it was the aim of this study to develop and validate an analytical method allowing estimation of the hepatic CYP3A enzyme activity using the 4-hydroxycholesterol to cholesterol ratio as an endogenous biomarker in serum. Both compounds were isolated from the biological matrix by liquid-liquid extraction using n-hexane after saponification with ethanolic sodium methoxide solution (2 M) to cleave the steroids from their esterified forms without any kind of further derivatization. Chromatographic separation was achieved on a reversed phase column (SupelcoAcsentis (R), C8) within 7 min using an isocratic elution with ammonium acetate 5 mM (pH = 3.8, 10%) and acetonitrile (90%) at a flow rate of 300 mu l/min. d6-cholesterol and d7-4 beta-hydroxycholesterol were used as internal standards. Detection was done on a triple quadrupole mass spectrometer using the following mass transitions: 369.3/161.5, 369.3/147.1 and 369.3/95.2 for cholesterol; 385.2/367.4, 385.2/109.1 for 4-hydroxycholesterol; 374.4/152.7 and 392.2/108.9 for d6-cholesterol and d7-4-hydroxycholesterol, respectively as the internal standards.