Integrated Targeted Quantitation Method for Insulin and its Therapeutic Analogs
Ravindra Chaudhari1,*, Eric Niederkofler1, Scott Peterman2, Amanda Leber1, Kwasi Antwi1, Tara Schroeder3, Urban Kiernan1, Kemmons Tubbs1, Bryan Krastins2, Amol Prakash2, Mary Lopez2
1Thermo Fisher Scientific, Tempe, 2Thermo Fisher Scientific BRIMS, Cambridge, 3Thermo Fisher Scientific Somerset, New Jersey, United States
Introduction: The need to detect and quantify insulin and its analogs has become paramount for medical and athletic doping. Conventional insulin assays have an inability to differentiate endogenous insulin from exogenous insulin analogs. The use of LC-MS can overcome this, however, methods to-date lack the analytical sensitivity demanded.
Objectives: Development of a Mass Spectrometric Immunoassay (MSIA) workflow for the high-throughput, analytically-sensitive quantification of insulin and its analogs from human plasma
Methods: Samples were prepared neat and in donor plasma using human insulin and 5 insulin analogs. The analogs were prepared independently and mixed to test selectivity and sensitivity of the enrichment method. Enrichment was performed using MSIA tips derivatized with pan-anti-insulin antibody which recognizes an epitope in the beta chain conserved across all variants. Detection/quantitation was performed using LC-MS on a a Thermo Scientific™ Ultimate™ 3000 LC system coupled to aThermo Scientific™ Q Exactive™ mass spectrometer
Results: The insulin pan-antibody allows capture and detection of all variants from the sample. Full scan MS mode in the analysis stage enables simultaneous detection of multiple insulin analogs and can screen for unsuspected analogs post-acquisition. Accurate intact mass and fragmentation of the analogs confirmed the identity of each variant. With the MSIA workflow, the LLOQ was 15 pM (87 pg/mL) and LOD was < 7.5pM (~47 pg/mL). Reproducibility studies demonstrated inter- and intra-day CV’s of < 3%. Spike and recovery resulted in recoveries of 96-100%. Additionally, the MSIA workflow significantly reduces the background matrix affording shorter LC gradients and LC-MS analysis times
Conclusion: Incorporation of a pan-antibody, used to capture human insulin and 5 commercially available insulin analogues, into a LC-MS detection/quantitation assay enabled differentiation and targeted quantification of human insulin and insulin analogues in a single clinical research assay.
Keywords: Biomarker, Blood, Method