Evaluation of analytical validation of plasma oxysterol measurement by mass spectrometry.

Objective: 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 (LC-MS / MS) method was evaluated by using various extraction and derivation steps in the measurement of cholestan-3β,5α,6β-triol (C-Triol) and 7-ketocholesterol (7-KC) which are important oxysterols. Method: Optimization studies were performed in LC-MS/MS (LC-20 AD UFLC XR, Shimadzu 8040 Corporation, Japan) with positive electrospray ionization (ESI) in multiple-reaction monitoring (MRM) mode. The analytical validation of C-Triol and 7-KC multiplex measurements with dimethylglycine (DMG) derivatization was evaluated in plasma from 25 healthy individuals. For this purpose; linearity, accuracy, repeatability, detection and quantitation limits (LLOD and LLOQ), recovery and carry-over analysis were studied and the results were evaluated statistically. Results: The time of the analysis was 10 minutes (min) for both parameters (C-Triol: 3.2 min, 7-KC: 7.4 min). The values of r-squared (r2) were found for C-triol as 0,999, for 7-KC as 0,994 in generated calibration curves. Concentrations of C-triol and 7-KC were within ± 20% in quality control samples prepared at least two levels for accuracy analysis. Plasma C-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). The standard deviations were found high for 7-KC measurements, it was considered that the plasma matrix interfered or the 7-KC stability in the plasma deteriorated. Conclusion: In this study, oxysterol measurement has been developed and ready for use in our laboratory with a sensitive and specific method. 7-KC was considered to be a useful parameter to reflect the stability of the analyte in the plasma. Key words: Oxysterols, Cholestan-3β,5α,6β-triol, 7-ketocholesterol, LC-MS/MS