Antibody-Free Inline Dual-Retention Nano-WCX-μSPE-MS/MS for Quantification of Intact Parathyroid Hormone and Antagonistic Fragments in Clinical Serum

Parathyroid hormone (PTH), an 84-amino acid polypeptide critical for calcium/phosphorus homeostasis, exhibits significant analytical challenges in clinical quantification, particularly in chronic kidney disease (CKD) patients due to its low circulating concentrations, biological matrix complexity, and interference from circulating PTH fragments. Traditional immunoassays may either overestimate the PTH level due to cross-reactivity with the PTH 7–84 or fail to quantify the antagonistic fragment. Highly sensitive and selective mass spectrometry (MS)-based methods could be used as the reference measurement procedure (RMP) for PTH assays, once the limitations of quantification thresholds and antibody-dependent workflows are addressed. Here, we introduce the first inline weak cation-exchange microsolid-phase extraction-tandem mass spectrometry (WCX-μSPE-MS/MS) method for simultaneous quantification of PTH 1–84 and its antagonist PTH 7–84 in clinical serum samples. A carboxyl-functionalized polystyrene nanosphere-modified capillary μSPE column with dual weak cation exchange (WCX) and nonpolar retention mechanisms is integrated with electrospray ionization-MS/MS, enabling online purification, enrichment, and detection in a single analysis. The nanoliter-scale capillary extraction can minimize matrix effects and improve sensitivity while greatly eliminating offline sample transfer steps. A limit of detection (LOD) as low as 6.0 pg/mL (PTH 1–84) or 9.0 pg/mL (PTH 7–84) is achieved, with relative standard deviations less than 10%. Our antibody-free strategy greatly reduces solvent/sample consumption, avoids manual errors, and has been successfully applied for accurate quantification of both PTH 1–84 and PTH 7–84 in clinical serum, demonstrating great potential to serve as an RMP for PTH assays, enhancing diagnostic accuracy for hyperparathyroidism, CKD, and renal bone diseases.