Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in entire male horses

Abstract

Nandrolone (17β-hydroxy-4-estren-3-one) is amongst the most misused endogenous steroid hormones in entire male horses. The detection of such a substance is challenging with regard to its endogenous presence. The current international threshold level for nandrolone misuse is based on the urinary concentration ratio of 5α-estrane-3β,17α-diol (EAD) to 5(10)-estrene-3β,17α-diol (EED). This ratio, however, can be influenced by a number of factors due to existing intra- and inter-variability standing, respectively, for the variation occurring in endogenous steroids concentration levels in a single subject and the variation in those same concentration levels observed between different subjects. Targeting an efficient detection of nandrolone misuse in entire male horses, an analytical strategy was set up in order to profile a group of endogenous steroids in nandrolone-treated and non-treated equines. Experiment plasma and urine samples were steadily collected over more than three months from a stallion administered with nandrolone laurate (1 mg/kg). Control plasma and urine samples were collected monthly from seven non-treated stallions over a one-year period. A large panel of steroids of interest (n = 23) were extracted from equine urine and plasma samples using a C18 cartridge. Following a methanolysis step, liquid-liquid and solid-phase extractions purifications were performed before derivatization and analysis on gas chromatography-tandem mass spectrometry (GC-MS/MS) for quantification. Statistical processing of the collected data permitted to establish statistical models capable of discriminating control samples from those collected during the three months following administration. Furthermore, these statistical models succeeded in predicting the compliance status of additional samples collected from racing horses.

Two complementary methods to control gonadotropin‐releasing hormone vaccination (Improvac®) misuse in horseracing: Enzyme‐linked immunosorbent assay test in plasma and steroidomics in urine

“…In this case, the method focused on the expected effects of the treatment on a specific biological pathway or tissue. This approach has been recently used successfully to improve the detection of nandrolone in stallions . In this study anabolic androgenic steroids that have effects on steroid biosynthesis and metabolism are kinetically monitored by a set of known biological molecules.…”

“…The steroidomic approach is a semi‐quantitative determination of a set of relevant steroid molecules involved in the steroid metabolism pathway recognized to establish a good assumption of misuse. This kind of steroid approach has been previously studied in humans, in cattle and more recently by Kaabia et al in horse . Thanks to statistical data processing by specific software, it becomes possible to establish models capable of discriminating treated from untreated animals.…”

“…This kind of steroid approach has been previously studied in humans, [6][7][8][9] in cattle [10] and more recently by Kaabia et al in horse. [11] Thanks to statistical data processing by specific software, it becomes possible to establish models capable of discriminating treated from untreated animals. Moreover, the simple screening of GnRH antibodies in plasma with the follow-up of the steroid profile in urine is supposed to be a reliable complementary analysis to define the status of a male animal with a gelding or female steroid profile.…”

Mass Spectrometry for the Detection of Endogenous Steroids and Steroid Abuse in (Race) Horses and Human Athletes

“…Biomarker monitoring can already be considered a new era in human anti-doping [69] and different biomarker approaches are being developed for equine purposes at the moment. As such, longitudinal monitoring of biomarkers can reveal non-physiological responses independently of the used doping technique or substance and may cause sanctioning of illicit practices [70].…”

Evaluation of nandrolone and ractopamine in the urine of veal calves: liquid chromatography‐tandem mass spectrometry approach

“…Usually, nandrolone is distributed in the ester form as phenylpropionate, [7] laureate, [8][9][10] or decaonate. [11] This type of administration decreases the rate of absorption of the drug, leading to a prolonged anabolic effect.…”