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Determination of Synacthen in urine for sports drug testing by means of nano-ultra-performance liquid chromatography/tandem mass spectrometry.

Doping control analysis of performance-enhancing peptides in urine represents a challenging requirement in modern sports drug testing. Low dosing, effective metabolism and short half-life lead to target concentrations in the low fmol/mL range in urine. Synthetic adrenocorticotropic hormone (1-24, Syn-ACTH-en) shares all these characteristics and improved analytical performance is required for its sufficient determination by means of liquid chromatography/tandem mass spectrometry (LC/MS/MS). The desired effects for cheating sportsmen are mainly due to enhanced release of corticosteroids as well as androgenic steroids into the circulation after systemic administration of the drug. Immunoaffinity purification with coated magnetic beads and subsequent liquid chromatography with nano-ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (high resolution/high mass accuracy) of Synacthen from urinary specimens is described in the present study. The general proof of principle was obtained by analysis of excretion study urine samples and validation was performed with focus on the limit of detection (3 pg/mL), linearity, precision (<20%), recovery ( approximately 30%), robustness, specificity and stability. For all experiments, the ACTH fragment 1-17 was used as the internal standard.