On-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry for procalcitonin analysis as a sepsis protein biomarker.

Sepsis, a life-threatening condition characterized by organ and tissue damage, primarily caused by bacterial infection, remains a leading cause of global mortality. This study introduces an integrated analytical method for the purification, enrichment, separation, identification, and quantification of procalcitonin (PCT), a key biomarker for sepsis, using on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). The method employed SPE microcartridges packed with magnetic bead particles functionalized with a PCT-selective aptamer. Optimizing the analytical parameters was challenging due to the specific properties of PCT, which adopts a rigid structure, partly attributed to the presence of a disulfide bond. The final optimized conditions utilized 1 M acetic acid (pH 2.3) as the background electrolyte (BGE) for the separation, and an aqueous-organic solvent mixture of methanol:water (60:40, v/v) with 0.5 M ammonia (apparent pH 11.3) for the elution. The developed method demonstrated excellent repeatability and reproducibility, with linearity within the 2.5 and 10 µg·mL⁻¹ range and a limit of detection (LOD) of 1 µg·mL⁻¹, yielding a 100-fold enhancement in sensitivity over CE-MS. Additionally, it was successfully applied to analyze PCT in serum samples after a precipitation-based clean-up pretreatment, showcasing its potential as a reliable MS-based analytical methodology for the analysis of intact PCT as a sepsis biomarker.