Optimization of an online Immunopurification assay platform of Insulin and Its Mimetics Using ESI-MS/MS for antidoping analysis

Abstract

Insulin is a hormone that regulates blood glucose levels, promotes glycogen storage in muscles, and prevents muscle breakdown, thereby influencing strength and endurance. Due to these effects, World Anti-Doping Agency(WADA) classifies insulin as a growth factor and prohibits its usage. In this study, we developed and optimized an online immunopurification assay platform for insulin and its mimetics (Lispro, Glulisine, Aspart, Degludec, Detemir, Glargine) simultaneously, based on asymmetrical flow field-flow fractionation (AF4) technology. AF4 is a size-based separation technique that operates within a narrow channel and is based on the flow velocity of the solvent. Polyvinylidene fluoride (PVDF) and regenerated cellulose (RC) membranes were used to immobilize antibodies, and protein A was also applied to align the binding site of antibodies. After binding insulin and its mimetics in a sample to antibodies, the pH and organic solvent content of the mobile phase are adjusted to elute target materials from antibodies. By adjusting reaction time conditions, the antigen-antibody interaction efficiency and sensitivity were significantly enhanced for 10 folds, but the effect of temperature was not dramatically changed. Additionally, this platform was enabled to introduce large volume of urine samples (>1 mL), therefore it can analyze insulin within 30 minutes without additional pretreatment, significantly reducing the time from over 4 hours, and can be applied in doping control.