Characterization and development of an interleukin-6 reference material by SI-traceable isotope-dilution mass...

Interleukin-6 (IL-6) is a critical mediator of inflammation and is implicated in immune regulation, inflammatory responses, and metabolic processes. In this study, a recombinant IL-6 reference material (RM) was prepared and characterized using proteomic techniques. Quantification of the IL-6 solution was performed with SI-traceable methods: amino acid analysis isotope dilution mass spectrometry (AAA-IDMS) and peptide-based isotope dilution mass spectrometry (peptide-IDMS). The determined mass concentrations were (79.46 ± 1.80) μg/g (k = 2) using AAA-IDMS and (78.26 ± 1.94) μg/g (k = 2) using peptide-IDMS, demonstrating excellent consistency between the two techniques. Homogeneity assessment confirmed uniformity across units. Stability studies indicated that the RM retains stable for at least 12 months at -80 °C. The certified value was assigned based on a nine-laboratory collaborative study using AAA-IDMS. The expanded uncertainty (k = 2) incorporates contributions from characterization, between-unit heterogeneity, and long-term storage stability, resulting a final certified IL-6 mass concentration of (80.09 ± 2.93) μg/g (k = 2). This RM is intended for use in the validation and calibration of IL-6 assays in clinical diagnostics.