Centrifugation-assisted extraction for rapid retinol analysis in human serum using functionalized glass beads

A novel, simplified microextraction technique, the centrifugation-assisted extraction method that uses C18-functionalized glass beads as the sorbent has been developed. This procedure is demonstrated with extracting retinol from a small volume of human serum. First, the beads containing C18-functionalities are added to the liquid serum sample to extract retinol. Upon addition of a high-density perfluorinated liquid, a three-phase system is created. Centrifugation enables density-driven separation, in which the perfluorinated inert liquid forms a physical barrier that cleanly separates the matrix components on the top from the analyte-loaded beads collected at the bottom of the tube. Subsequent desorption of the adsorbed compound of interest using a minimal volume of methanol provides a sample suitable for rapid analysis by high-performance liquid chromatography with a diode-array detector. This method has been validated according to the International Council for Harmonization M10 guideline by the European Medicines Agency. Our procedure demonstrated excellent linearity over the concentration range of 0.26–5 μmol/L (r = 0.9999) achieving a lower limit of quantification of 0.26 μmol/L, which is sufficient for clinical monitoring. The method showed a high accuracy (bias < 12%, calculated against nominal concentrations established by the reference liquid-liquid procedure) and precision (relative standard deviation <7% for within-run and <5% for between-run). The developed method is a rapid, cost-effective, and robust alternative to other techniques for determining retinol in human serum. Combining a functionalized, freely moving sorbent with density-driven separation provides a simple, pump-free microextraction approach that is suitable for routine clinical and analytical laboratories because it significantly reduces solvent usage and simplifies the workflow to a single tube.