Table 3_Absolute quantification of tumor necrosis factor-alpha by isotope dilution mass spectrometry.docx

Tumor necrosis factor-α (TNF-α) is an important inflammatory mediator and has been widely recognized as a diagnostic biomarker for various autoimmune and infectious diseases in clinical practice, such as rheumatoid arthritis. In this study, we established an SI-traceable reference method to quantify TNF-α based on liquid chromatography–isotope dilution tandem mass spectrometry (LC-IDMS) with amino acid or peptide analysis. The assays exhibited good linearity (R2 > 0.999), repeatability (RSD < 3%) and accuracy, which had been verified using certified reference materials (CRMs). Stable isotope-labeled version of three amino acids (valine, phenylalanine, and leucine) and two peptides were used as an internal standard to minimize assay variability. For amino acid analysis, TNF-α could be fully hydrolyzed into amino acids after 60 h at 110 °C. The result based on the amino acid analysis was (0.770 ± 0.033) mg/g, expressed as a mass fraction (mg TNF-α per g of total solution), with an expanded uncertainty (k = 2). For peptide analysis, ANALLANGVELR (AR-12) and VVNLLSAIK (VK-9) were chosen as specific peptides. After 36 h of tryptic proteolysis, TNF-α could be completely proteolyzed into AR-12 and VK-9. Based on characteristic peptide analysis, the result was 0.769 ± 0.046 mg/g (k = 2). There was no significant difference between these two analyses, and the concentration of TNF-α was 0.770 ± 0.057 mg/g (k = 2), which was traceable to the International System of Units. Both methods developed in this study can accurately determine the concentration of TNF-α and are useful for detection kit development and instrument calibration. In addition, application data for reagents certified by these methods in cell apoptosis assays and kit evaluation are provided: TNF-α-induced cell apoptosis was significantly attenuated by antagonists, while detection kits based on three different principles exhibited good repeatability (RSD < 9%) and linearity (R2 > 0.999). This accurate, SI-traceable method can improve clinical TNF-α assay standardization and biomarker reliability.