Sensitive and selective quantification of elcatonin in equine plasma and urine using LC-FAIMS-MS/MS

The use of drugs for enhancing bone strength is prohibited under international regulations governing horse racing and equestrian sports. Elcatonin, a synthetic peptide with analgesic and bone-strengthening properties, is routinely monitored for compliance. However, its cyclic structure and high molecular weight hinder reliable detection. In this study, we developed and validated a sensitive liquid chromatography-high-field asymmetric waveform ion mobility spectrometry-tandem mass spectrometry method for quantifying elcatonin in equine plasma and urine. Sample preparation was optimised via weak cation exchange solid-phase extraction. A pilot pharmacokinetic and pharmacodynamic study was conducted to confirm the practical applicability of our method by validating linearity, reproducibility, sensitivity, selectivity, recovery, and analyte stability, including enzymatic/proteolytic stability. The method achieved a detection limit of 10 pg/mL for urine and plasma (R² > 0.9995 in both cases), meeting the validation criteria for quantitative analysis. Following administration, elcatonin was detected in plasma (for up to 6 h) but not in urine. Pharmacodynamic analysis revealed that elcatonin administration caused a transient small decrease in the plasma calcium levels without any other observable clinical effects, such as hypocalcaemia. The established method overcomes the challenges of detecting and quantifying cyclic peptides and has the potential to improve the detectability of elcatonin and related molecules relevant to doping control and animal healthcare.