Detection of Nandrolone and Boldenone Abuse in the Ovine by GC–MS–MS

Abstract

Under European legislation, the use of anabolic steroids as growth promoters in meat production is prohibited. Currently, there is no internationally accepted method used for the detection of the potentially endogenous steroids nandrolone and boldenone in the ovine. In the current study, a multi-residue GC-MS-MS-based urinary assay has been validated for boldenone as well as the nandrolone metabolites 5a-estrane-3b,17a-diol and epinandrolone. Using a standard addition calibration line approach in pooled bovine urine, the method was linear between the endogenous concentrations and those augmented with 6,000 pg mL -1 . The method was then applied to populations of wether (n = 242) and ewe (n = 237) ovine animals in order to establish urinary thresholds for detecting nandrolone and boldenone abuse. A statistical model (the Chebyshev inequality) was used to produce threshold concentrations for each analyte. Adjustment of the nandrolone metabolite data for specific gravity, a measure of the hydration status of the animal, allowed the effective thresholds to be reduced; potentially leading to a lower number of false positives. Furthermore, the proposed epinandrolone confirmatory thresholds (38,628 and 57,950 pg mL -1 in wethers and ewes, respectively) were found to be effective in detecting abuse of nandrolone for at least 1 month post-dose of this steroid. However, further studies would be required to assess the efficacy of the proposed boldenone confirmatory thresholds (19,857 and 56,080 pg mL -1 in wethers and ewes, respectively) since data on its excretion following administration to the ovine are lacking.

A review of analytical strategies for the detection of ‘endogenous’ steroid abuse in food production

“…-Can be combined with some other screening approaches such as threshold concentrations if the same analytical technique is used. Threshold concentrations (urine, plasma/serum) [42][43][44][46][47][48] -Can be used for both screening and confirmation by using different thresholds.…”

“…However, as analytical limits of detection decrease, it is also likely that further steroids will be discovered as endogenous in food-producing animals. For example, since the time that the review article of Scarth et al [35] was published, the following steroids have been discovered to be endogenous: 19-noretiocholanolone in boars and gilts, [42] a range of different nandrolone metabolites in steers and heifers, [47,80] wethers, rams and ewes [48] as well as prednisolone in cattle. [81] Furthermore, it has recently been demonstrated that some types of wooden crate in which veal calves are housed may contain precursors to Boldenone.…”

“…For example, when applied to data from UK ovine populations, correction of urinary steroid concentration data for SG led to a reduction in the relative standard deviation of the steroid population data and hence a reduction in the calculated thresholds. [48] An alternative to correcting steroid concentrations for SG would be to use the creatinine concentration. However, results from bovine [47] and ovine [48] population studies in the UK failed to demonstrate a reduction in the relative standard deviation of the data following correction for creatinine concentration.…”

“…[48] An alternative to correcting steroid concentrations for SG would be to use the creatinine concentration. However, results from bovine [47] and ovine [48] population studies in the UK failed to demonstrate a reduction in the relative standard deviation of the data following correction for creatinine concentration.…”

Monitoring dehydroepiandrosterone (DHEA) in the urine of Thoroughbred geldings for doping control purposes

“…Since DHEA is an endogenous steroid in all genders in horses, a matrix "standard addition" approach was used to prepare calibration lines and quality control (QC) samples for each precision and accuracy batch. 27,28 Spiking pooled gelding urine at known concentrations and using the slope and intercept of the generated calibration lines enabled the calculation of the endogenous concentration of DHEA in pooled urine. Subsequently, the calibration line and QC samples were adjusted to account for that endogenous concentration to measure concentrations in analysed samples.…”