Investigations into the analysis of intact drug conjugates in animal sport doping control – Development and assessment of a rapid and economical approach for screening greyhound urine

• Published in: Drug testing and analysis 2020-06, Vol.12 (6), p.731-742
• Main Authors: Gray, Bob, Tuckley, Lisa, Cutler, Charlotte, Biddle, Simon, Hudson, Simon, Gower, Simon, Vanhaecke, Lynn
• Format: Article
• Language: English
• Subjects: Adrenal Cortex Hormones - urine; Anabolic Agents - urine; anabolic steroids, doping control, glucuronide conjugates, greyhound urine, UPLC‐MS/MS; Animals; Chromatography, High Pressure Liquid; Dog racing; Dogs; Doping in Sports - methods; Drug testing; Glucuronides - urine; Metabolites; Norethindrone - urine; Reference Standards; Reproducibility of Results; Sports; Steroids - urine; Substance Abuse Detection - methods; Tandem Mass Spectrometry; Testosterone Congeners; Urine
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.2779

Animal sport doping control laboratories are constantly reviewing ways in which they can improve their service offering whilst ensuring that they remain economically viable. This paper describes the development and assessment of a rapid and economical method for the detection of intact glucuronide conjugates of three anabolic steroids and their metabolites along with three corticosteroids in canine urine. The analysis of intact drug conjugates for animal sport doping control is generally not performed routinely as it presents a number of analytical challenges, not least of which is the lack of availability of appropriate reference standards. Here, we report the development of a UHPLC–MS/MS method using APCI in the negative ion mode for the detection of intact phase II conjugates, including the importance of in vitro incubations in order to provide appropriate reference materials. Cross‐validation of the developed method demonstrated that the detection capability of the intact phase II conjugates of stanozolol, boldenone, nandrolone, and their metabolites along with the corticosteroids dexamethasone and methylprednisolone was equivalent to that achieved in routine race‐day screens. The new process has been in operation for approximately 2 years and has been used to analyze in excess of 13500 canine urine samples, resulting in a number of positive screening findings. To the best of our knowledge, this is the first reported use of a routine screen for intact drug conjugates within animal sport doping control. The use of intact phase II conjugates for animal sport doping control is discussed and a method presented for the detection of glucuronide conjugates of anabolic steroids and corticosteroids in canine urine.