Key Points
- Millions of sepsis cases arise each year; despite this, traditional diagnostic methods can be heavily demanding, typically causing missed bacteremia diagnoses.
- A new process for quantifying procalcitonin was created using on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS).
- The method was proven to be effective, and future research efforts will aim to transition from PCT protein analysis to PCT-derived peptide analysis using targeted MS/MS measurements.
University of Barcelona researchers have developed a new aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) system for analyzing sepsis protein biomarkers. Their findings were published in the Journal of Chromatography A (1).
Sepsis is a condition where the body improperly reacts to an infection, turning infection-fighting processes on itself and causing organ issues (2). According to 2020 data from the World Health Organization (WHO), there were 48.9 million cases of sepsis and 11 million sepsis-related deaths worldwide, representing 20% of all global deaths (3). Moreover, for every 1000 hospitalized patients, approximately 15will develop sepsis as a complication of receiving health care.
Traditional diagnostic methods, such as blood cultures, can face significant limitations, such as large sample requirements, lengthy processing times, and reduced sensitivity with smaller sample volumes. These concerns can typically cause missed bacteremia diagnoses; as such, there is an urgent need for new analytical approaches that can rapidly and accurately detect infections.
Various immune-based techniques, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CLIA) have been used to detect and quantify procalcitonin (PCT) with high sensitivity. However, immunoassays often have constraints that can affect their reliability, such as issues with reproducibility across different platforms and the potential for both false negatives and positives. Mass spectrometry (MS)-based techniques are believed to offer significant potential for enhancing the diagnosis of sepsis and related infections by enabling unequivocal identification of PCT, a key biomarker for sepsis.
In this study, the researchers introduced an integrated analytical method for the purification, enrichment, separation, identification, and quantification of PCT. This process involves on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). The method involves solid-phase extraction (SPE) micro cartridges containing magnetic bead particles functionalized with a PCT-selective aptamer.
Optimizing the analytical parameters can be challenging due to specific properties of PCT, which adopts rigid structure, partly due to disulfide bonds. 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.
After sample clean-up pretreatment was optimized, the method successfully detected PCT in serum, even in the presence of interfering sample matrix components. While the achieved LODs are within the higher range of PCT concentrations in septic shock patients, further sensitivity improvements are needed to detect lower concentrations. Future research will aim to transition from PCT protein analysis to PCT-derived peptide analysis using targeted MS/MS measurements; this is due to peptide-based MS approaches having potential to significantly increase sensitivity compared to intact proteins.
The AA-SPE-CE-MS method presented in this study was shown to be a promising alternative to immunoassays and other biosensors, offering more reliable and accurate quantification of PCT in clinical practice. With further sensitivity enhancements, the method could be greatly expanded upon for enabling early disease diagnosis and monitoring.
References
(1) Pont, L.; Cebrián, B.; Giménez, E.; Benavente, F. On-Line Aptamer Affinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for Procalcitonin Analysis as a Sepsis Protein Biomarker. J. Chromatogr. A 2025, 1758, 466183. DOI: 10.1016/j.chroma.2025.466183
(2) Sepsis. Mayo Clinic 2023. https://www.mayoclinic.org/diseases-conditions/sepsis/symptoms-causes/syc-20351214 (accessed 2025-7-24)
(3) Sepsis. World Health Organization 2024. https://www.who.int/news-room/fact-sheets/detail/sepsis (accessed 2025-7-24)
