| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1211861 | Journal of Chromatography B | 2016 | 7 Pages |
•Simultaneous quantification of cholesterol and its CYP3A4 dependent metabolite 4ß-OH-cholesterol with one analytical method (LC–MS/MS).•No further derivatization needed; i.e. direct determination (established is so far the formation of piconyl esters).•Similar or even higher sensitivity compared to previously published methods and considerably faster chromatographic run time than available methods (8 min vs. 10–30 min).•Comprehensive method validation according to current bioanalytical guidelines.•Application of the method to monitor induction effects of rifampicin in human and foals.
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®, 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 μl/min. d6-cholesterol and d7-4β-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.The method was validated according to current bioanalytical guidelines considering selectivity, linearity, accuracy, precision, recovery, stability. The analytical range was 5–250 and 50–1000 ng/ml, for 4-hydroxycholesterol and cholesterol, respectively.The method was shown to be selective for both compounds with good linearity over the selected range (r > 0.99) as well as good within- and between day accuracy (error: −1.2–3.7% for 4-hydroxycholesterol and −7.7–9.5% for cholesterol) and within- and between day precision (2.1–14.6% for 4-hydroxycholesterol and 1.1–14.9% for cholesterol). Recovery was found to be over 80% for both analytes while significant stability issues could not be observed. Finally, the validated assay was applied to measure 4-hydroxycholesterol and cholesterol in serum samples of clinical studies in humans and foals that could verify induction of hepatic CYP3A4 (human) and CYP3A89 (foals) after premedication with the known enzyme inducer rifampicin.
