A systematically evaluated SEC-ICP-MS/MS workflow for Mn, Fe, Cu, and Zn speciation in human serum

Species-resolved analysis of essential transition metals in serum may provide biologically and clinically relevant information beyond total elemental concentrations, particularly where altered metal distribution is linked to toxicological or disease-related processes. However, serum metal speciation remains analytically challenging because many metal-protein and metal-ligand interactions are labile, relevant fractions span broad molecular-mass and concentration ranges, and species-specific standards are generally unavailable. This work presents a newly developed size exclusion chromatography-inductively coupled plasma tandem mass spectrometry (SEC-ICP-MS/MS) workflow for combined Mn, Fe, Cu, and Zn speciation in human serum. It integrates bioinert chromatography, mild mobile-phase conditions, and matrix-matched calibration across the chromatographic system. Importantly, its analytical performance was systematically assessed over five measurement days using two quality control materials and two native human serum samples, addressing an aspect that has received limited attention in previous multi-element serum speciation studies. The summed chromatographic response showed excellent linearity, and mean total-element recoveries after chromatographic separation were 96-102 %. Major species exhibited intra-day coefficients of variation (CVs) below 1.5 % and inter-day CVs below 5 %, while remaining fractions stayed below 10 % and 15 %, respectively. Limits of quantification were sufficiently low for all investigated elements, including low-abundance Mn. The method reproducibly resolved four Mn, three Fe, five Cu, and four Zn species, most of which could be tentatively assigned to biologically plausible proteins. Overall, the method provides a systematically evaluated multi-element platform that may help move serum metal speciation beyond predominantly exploratory applications toward more demanding biomonitoring, occupational, and clinical study designs.