A validated liquid chromatography–tandem mass spectrometry method for the quantitative determination of 4β-hydroxycholesterol in human plasma

• Published in: Journal of pharmaceutical and biomedical analysis 2011-07, Vol.55 (5), p.1089-1095
• Main Authors: van de Merbel, Nico C., Bronsema, Kees J., van Hout, Mischa W.J., Nilsson, Ralf, Sillén, Henrik
• Format: Article
• Language: English
• Subjects: 4β-Hydroxycholesterol; alkaline hydrolysis; Analysis; Analytical, structural and metabolic biochemistry; Antioxidants - chemistry; APPI; atmospheric pressure; Biological and medical sciences; Biomarker; Biomarkers - chemistry; butylated hydroxytoluene; Calibration; cholesterol; Cholesterol - chemistry; Chromatography, Liquid - methods; CYP3A4/5; Cytochrome P-450 CYP3A - chemistry; Edetic Acid - chemistry; esterification; frozen storage; Fundamental and applied biological sciences. Psychology; General pharmacology; hexane; Humans; Hydrolysis; Hydroxycholesterols - chemistry; Ions; LC–MS/MS; liquid chromatography; Medical sciences; Models, Chemical; Pharmacology. Drug treatments; quantitative analysis; Reproducibility of Results; solid phase extraction; solvents; Spectrometry, Mass, Electrospray Ionization - methods; tandem mass spectrometry; Tandem Mass Spectrometry - methods; toluene
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2011.03.017

A novel liquid chromatography–tandem mass spectrometry method is described for the quantitative determination of the endogenous CYP 3A4/5 marker 4β-hydroxycholesterol in human K 2-EDTA plasma. It is based on alkaline hydrolysis to convert esterified to free 4β-hydroxycholesterol, followed by analyte extraction from plasma by hexane and purification of the hexane extract by normal-phase solid-phase extraction. The analyte is chromatographically separated from endogenous isobaric plasma oxysterols and excess cholesterol by a 16-min reversed-phase gradient on a C18 column; detection is performed by atmospheric pressure photoionization tandem mass spectrometry in the positive ion mode, using toluene as a dopant. Using 400 μl of plasma, 4β-hydroxycholesterol can be quantified in the concentration range 10.0–250 nM. Validation results show that the method is sufficiently selective towards endogenous plasma sterols and capable of quantifying the analyte with good precision and accuracy. The analyte is sufficiently stable in all relevant matrices and solvents; the addition of the anti-oxidant butylated hydroxytoluene to prevent in vitro formation of 4β-hydroxycholesterol from cholesterol during storage or analysis is not necessary, provided that long-term frozen storage of plasma occurs at −70 °C.