Online Pressure Change Focusing-Supercritical Fluid Selective Extraction Chromatography for Analyzing Chiral Drugs in Microliter-Scale Plasma Samples

The online coupling technique of sample preparation with chromatography is a frontier topic in analytical chemistry since it minimizes errors caused by sample loss, shortens analysis time, and reduces solvent consumption. An online pressure change focusing-supercritical fluid selective extraction chromatography (PCF-SFSEC) technique was developed in this study, realizing extraction, purification, separation, and detection in a single run with only microliter-scale samples. The pressure change focusing strategy achieved column-head stacking by decreasing the dissolving capacity of the supercritical fluid, enabling the large volume introduction of extractants into supercritical fluid chromatography without causing peak broadening or distortion. All the extracts could be directly loaded into the chromatography system without split flow. Based on the supercritical fluid selective extraction (SFSE) strategy, the sorbents removed interferences and water from samples, effectively alleviating matrix effects and realizing the direct aqueous sample analysis. The efficiency of online PCF-SFSEC was demonstrated by the enantioselective analysis of 22 chiral drugs in rat plasma, covering eight categories with different pharmacological effects. The entire analysis took 25 min, consuming only 5 μL samples. All analytes in PCF-SFSEC obtained sharp and symmetrical peaks with resolutions higher than 1.0, and 86% had resolutions higher than 1.5. Limits of quantification (LOQs) ranged from 0.0600 to 32.1 μg/L. Recoveries were in the range of 75.8–117.2%. In addition, the developed approach obtained more satisfactory repeatability and significantly reduced matrix effects than conventional methods. The newly established online PCF-SFSEC technique is believed to be a green and powerful tool for the chiral analysis of complex samples.