Optimization and application of high-throughput supported liquid extraction for simultaneous determination of carotenoids and fat-soluble vitamins in serum.

The demand for analysis of carotenoids (CAR) and fat-soluble vitamins (FSV) is continuously expanding, but currently used sample preparation methods either require complicated extraction procedure or large sample volume, let alone the reliability of the results. This study aimed to develop a fast, high-efficient, and high-throughput method based on supported liquid extraction (SLE) for the simultaneous extraction of FSV and CAR from human serum before using high-performance liquid chromatography-diode array detector (HPLC-DAD) analysis. The optimization of SLE parameters was achieved through response surface methodology (RSM) based on the Box-Behnken design (BBD) and included serum-water-extraction solvent ratio and eluent volume. Under optimal conditions, the proposed method gives acceptable limits of detection (LOD) (0.005-0.3 μg/mL), good recovery (89.6-110.9%) as well as relative standard deviation (RSD) of less than 10.1% by consuming lower serum sample (100 μL) and less sample preparation time (2 min per sample). Compared with liquid-phase extraction (LLE), the SLE delivers rapid extraction with higher recovery, better reproducibility, and lower matrix effect for CAR and FSV analysis. The method has been successfully applied to quantify CAR and FSV levels in serum of healthy individuals and age-related macular degeneration (AMD) patients, demonstrating the feasibility of the proposed method for epidemiology and routine applications.