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

Abstract

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.

Development and validation of a liquid chromatography–mass spectrometry procedure after solid-phase extraction for detection of 19 doping peptides in human urine

“…Finally, novel, previously unknown macromolecules (i.e., TB500 17-23 fragment), with no current approval for human therapeutic use (i.e., agents under preclinical or clinical development, designer drugs, or compounds approved only for veterinary use), but illegally marketed (e.g., via Internet websites) [5] are included in section S0 ''Non-approved substances''. Different analytical approaches have already been developed and published to detect these compounds in nutritional supplements [6][7][8][9][10] and in doping control samples (either blood [11][12][13][14][15][16][17][18] or urine [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]), employing immunological [26,[35][36][37], electrophoretic [16][17][18][19]27], or liquid chromatography-mass spectrometry techniques [6-15, 20-25, 28-34] or a combination of different analytical technologies. In general, the most effective analytical approach (combined sample pretreatment and the instrumental method) has to take into ...…”

Expanded test method for peptides >2 kDa employing immunoaffinity purification and LC‐HRMS/MS

“…[1] Most existing analytical procedures to determine these target peptides in blood or urine employ liquid chromatography coupled to mass spectrometry (LC-MS) due to its distinguished specificity. [2,[5][6][7][8][9][10][11][12][13][14][15][16][17][18] The principle of combining several of these compounds into one screening method was also realized recently and has already been implemented into routine doping controls of important sport events. [8,17,19] Methods based on bioassays (ELISA, RIA, etc.)…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…[70] Detection limits of 1 fmol/mL of urine were accomplished, which proved adequate for the identification of Synacthen in post-administration study urine specimens; however, stability issues with Synacthen outlined the necessity of appropriate pre-analytical conditions of doping control samples to ensure reproducible results, as observed also in earlier studies.…”