Lighter than Water Dispersive Liquid-liquid Microextraction Coupled with High Performance Liquid Chromatography for Determination of Cholecalciferol and Calcifediol from Plasma

In this study, a dispersive liquid–liquid microextraction method using an extraction solvent lighter than water has been developed for the extraction and preconcentration of cholecalciferol and calcifediol from plasma samples followed by high performance liquid chromatography determination. Initially, acetonitrile and sodium chloride (NaCl) are added into the plasma as an extraction solvent and a salting–out agent, respectively. After manual shaking, the mixture is centrifuged. In the presence of sodium chloride, a two–phase system is formed. Then a portion of the upper phase is removed and mixed with n–hexane at µL–level and rapidly injected into distilled water by a syringe. In this process, the analytes are extracted into the fine droplets of n–hexane (as an extraction solvent). Under optimal conditions, enrichment factor was obtained 92 and 94 for calcifediol and cholecalciferol, respectively. The intra– (n=6) and inter–day (n=4) precisions were less than or equal to 8.1% at a concentration of 10 ng mL–1 of each analyte. Finally, this method was applied to the analysis of the analytes in human plasma samples.