Development of an SI-traceable magnetic solid-phase extraction coupled with isotope dilution LC-MS/MS method for accurate quantification of alpha-fetoprotein in human serum.

Alpha-fetoprotein (AFP) is a critical biomarker widely used for the screening and diagnosis of hepatocellular carcinoma and germ cell tumors. Although immunoassays are commonly employed for AFP detection, significant discrepancies in measurement results across different laboratories persist due to poor harmonization among methods. To improve the accuracy and harmonization of AFP quantification, this study developed a novel magnetic solid-phase extraction coupled with isotope dilution liquid chromatography-tandem mass spectrometry (MSPE-ID-LC-MS/MS) method for the precise measurement of AFP in serum. The method utilizes high-affinity AFP antibodies cocktails conjugated with magnetic nanoparticles to enhance the recovery of low-abundance AFP, combined with optimized elution conditions to ensure analytical reliability. The protocol involves one-step denaturation and alkylation, followed by tryptic digestion. This enables accurate quantification with SI-traceability using three signature peptides. Method validation demonstrated that intra- and inter-day precisions were <10 %, recovery rates of 99.8-101.4 %, and the limit of quantification (LOQ) of 1.5 ng/mL. This work establishes a metrologically robust reference procedure for AFP measurement and paves the way for standardization of clinical protein assays.