Determination of non-cholesterol sterols in serum and HDL fraction by LC/MS-MS: Significance of matrix-related interferences

BACKGROUND: Non-cholesterol sterols (NCS) are promising biomarkers for estimation of cholesterol homeostasis properties. In addition, determination of NCS in high-density lipoprotein (HDL) fraction (HDL-NCS) could provide information on cholesterol efflux. However, matrix effects interfere in liquid chromatography-mass spectrometry (LC-MS) analysis of NCS, thereby impairing the method sensitivity. The aims of this study were development, optimization and validation of LC-MS method for quantification of NCS in serum and HDL-NCS. Additionally, matrix effect interferences and methods application in individual serum samples were examined. METHODS: HDL precipitating reagent was used for HDL isolation. Matrix effect was examined by comparing different surrogates by simple regression analysis. Validation was conducted according to the FDA-ICH guideline. 20 healthy volunteers were recruited for testing of method application. RESULTS: The observed matrix effect was 30%, and matrix comparison showed that cholesterol was the dominant contributor to the matrix effect. Cholesterol concentration was adjusted by construction of the calibration curve for serum and HDL fraction (5 mmol/L and 2.5 mmol/L, respectively). The intraand interrun variabilities for NCSs were 4.7-10.3% for serum NCS and 3.6-13.6% for HDLNCS and 4.6-9.5% for serum NCSs and 2.5-9.8% for HDL-NCS, respectively. Recovery studies showed satisfactory results for NCSs: 89.8-113.1% for serum NCS and 85.3-95.8% for HDL-NCS. CONCLUSIONS: The method was successfully developed and optimized. The matrix interference was solved by customising calibration curves for each method and sample type. The measurement of NCS in HDL fraction was proposed for the first time as potentially useful procedure in biomedical researches.