Aim: Oxysterols are derivatives of cholesterol which are formed by oxidation through numerous chemical reactions and play an important role in many physiological processes and in various degenerative and metabolic diseases, lipid metabolism disorders. In this study, analytical validation of liquid chromatography-tandem mass spectrometric method was evaluated by using various extraction and derivation steps in the measurement of Cholestan-3β,5α,6β-triol and 7-ketocholesterol which are important oxysterols.

Material and Methods: Optimization studies were performed in liquid chromatography-tandem mass spectrometry analyser with positive electrospray ionization in multiple-reaction monitoring mode. The analytical validation of cholestan-3β,5α,6β-triol and 7-ketocholesterol multiplex measurements with dimethylglycine derivatization was evaluated in human plasma samples. For this purpose, linearity, accuracy, repeatability, detection and quantitation limits, recovery and carry-over analysis were studied, and the results were evaluated statistically.

Results: The time of the analysis was 10 minutes for both parameters (cholestan-3β,5α,6β-triol: 3.1 minutes, 7-ketocholesterol: 7.26 minutes). The r2 value of the calibration curves for cholestan-3β,5α,6β-triol was 0.999, for 7-ketocholesterol was 0.994. The intra- and inter-assay variation coefficients assessed at three concentrations were below 15%. The assay accuracies of analytes ranged from 93.76 - 102.25 %. Plasma Cholestan-3β,5α,6β-triol levels were determined in a control group of 25 individuals (min: 15.7, max: 38.61; mean±SD: 25.61±9.2 ng/mL). Although valid results were obtained in calibration and quality control samples for 7-ketocholesterol, reproducible and reliable results were not obtained for plasma samples of healthy controls.

Conclusion: A rapid, sensitive and specific oxysterol analytical method has been tried and made ready for implementation in clinical laboratory by method validation for the diagnosis and/or monitoring of Niemann Pick-C disease and some neurodegenerative disorders including Alzheimer's disease, Multiple Sclerosis and spastic paraplegias in this study.