Bioformation of boldenone and related precursors/metabolites in equine feces and urine, with relevance to doping control

Despite the impressive innate physical abilities of horses, camels, greyhounds, or pigeons, doping agents might be administered to these animals to improve their performance. To control these illegal practices, anti‐doping analytical methodologies have been developed. This review compiles the analytical methods that have been published for the detection of prohibited substances administered to animals involved in sports over 30 years. Relevant papers meeting the search criteria that discussed analytical methods aiming to detect and/or quantify doping substances in animal biological matrices published from 1990 to 2019 were considered. A total of 317 studies were included, of which 298 were related to horses, demonstrating significant advances toward the development of doping detection methods for equine sports. However, analytical methods for the detection of doping agents in sports involving other species are lacking. Due to enhanced accuracy and specificity, chromatographic analysis coupled to mass spectrometry detection is preferred over immunoassays. Regarding biological matrices, blood and urine remain the first choice, although alternative biological matrices, such as hair and feces, have been considered. With the increasing number and type of drugs used as doping agents, the analytes addressed in the published papers are diverse. It is very important to continue to detect and quantify these drugs, recognizing those that are most frequently used, in order to punish the abusers, protect animals' health, and ensure a healthier and genuine competition.

Section: Resultsmentioning

confidence: 99%

Section: Most Relevant Analytes For Doping Control In Horsesmentioning

confidence: 99%

Doping detection in animals: A review of analytical methodologies published from 1990 to 2019

Moreira

Carmo

Pinho

et al. 2021

“…7,8 Furthermore, storage at room temperature resulted in the formation of isotopelabelled analogues of boldenone and Δ1-progesterone (pregn-1,-4-diene-3,20-dione) from isotope-labelled analogues of testosterone and progesterone (pregn-4-ene-3,20-dione), respectively, in spiked equine urine samples. 8 Inadvertent exposure of a urine sample to faeces may also introduce microbes in the sample and lead to the detection of boldenone, as demonstrated in human urine 9 and bovine faeces. 10 Boldenone has not previously been detected in fresh faecal samples collected from horses, unless mares/fillies were in oestrous 11 or the samples were incubated at room temperature.…”

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confidence: 99%

“…10 Boldenone has not previously been detected in fresh faecal samples collected from horses, unless mares/fillies were in oestrous 11 or the samples were incubated at room temperature. 8 Microbial transformation of Δ1-steroids could theoretically occur in animal feed that contains phytosterols or alternative steroid precursors. Microbes, such as Mycobacterium sp.…”

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confidence: 99%

Differentiation of boldenone administration from ex vivo transformation in the urine of castrated male horses

Viljanto¹,

Kaabia²,

Taylor³

et al. 2022

Self Cite

Boldenone is an anabolic‐androgenic steroid that is prohibited in equine sports. However, in certain situations, it is endogenous or is believed to be formed by microbes in urine, and therefore, an approach for the differentiation is required. Following the identification of Δ1‐progesterone and 20(S)‐hydroxy‐Δ1‐progesterone as potential biomarkers of microbial activity, the presence of six steroids was investigated in the postrace urine of castrated male horses (geldings, n = 158). In line with endogenous findings from several other species when ultrasensitive methods are employed, boldenone was detected at low concentrations in all urine samples (27.0–1330 pg/ml). Furthermore, testosterone and androstenedione were detected in 157 samples (≤12,400 and 944 pg/ml, respectively), boldienone in two samples (≤22.0 pg/ml) and 20(S)‐hydroxy‐Δ1‐progesterone in 20 samples (≤66.0 pg/ml). Δ1‐Progesterone was not detected in any population samples analysed on arrival at the laboratory. The ex vivo transformation of boldienone, boldenone, androstenedione, Δ1‐progesterone and 20(S)‐hydroxy‐Δ1‐progesterone was induced following the storage of urine samples at room temperature for 7 days but not after refrigeration. Because the administration of inappropriately stored feed sources also resulted in an increase in 20(S)‐hydroxy‐Δ1‐progesterone concentrations, a biomarker approach to distinguish steroid administrations was proposed. In situations where the presence of boldenone would exceed a proposed action limit, the presence of Δ1‐progesterone and 20(S)‐hydroxy‐Δ1‐progesterone would be investigated. If either Δ1‐progesterone or 20(S)‐hydroxy‐Δ1‐progesterone would exceed 50 and 100 pg/ml, respectively, for instance, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration.

“…All pre‐ and post‐administration samples were extracted alongside an extracted matrix blank and spiked samples ranging from 0.05–50 ng/ml for oxandrolone, its epimer and the 17,17‐dimethyl‐18‐norandrost‐13‐ene derivative, prepared with pooled equine urine, to enable the estimation of their concentrations. Samples were extracted using Bond Elut Nexus (3 ml, 60 mg) cartridges based on a previously published extraction method for urine samples 29 with modifications made to the elution steps. To individually retain the free and sulfate fractions, samples were eluted into separate tubes using 2 ml of diethyl ether and then 2 ml of solvolysis solvent (EtOAc:MeOH:sulfuric acid [50:10:0.1, v:v:v]).…”

Section: Methodsmentioning

confidence: 99%

In vitro and in vivo metabolism of the anabolic‐androgenic steroid oxandrolone in the horse

Harding¹,

Viljanto²,

Cutler³

et al. 2021

Self Cite

Oxandrolone is an anabolic‐androgenic steroid with favourable anabolic to androgenic ratio, making it an effective anabolic agent with less androgenic side effects. Although its metabolism has been studied in humans, its phase I and II metabolism has not been previously reported in the horse. The purpose of this study was to investigate the in vitro metabolism of oxandrolone (using both equine liver microsomes and S9) and in vivo metabolism following oral administration (three daily doses of 50 mg of oxandrolone to a single Thoroughbred horse), using both gas and liquid chromatography‐mass spectrometry techniques. The in vitro phase I transformations observed included 16‐hydroxylated (two epimers), 17‐methyl‐hydroxylated and 16‐keto metabolites. In addition to parent oxandrolone and these hydroxylated metabolites, the 17‐epimer and a 17,17‐dimethyl‐18‐norandrost‐13‐ene analogue were detected in biological samples following the administration. 16‐keto‐oxandrolone was only observed in urine. The 16‐ and 17‐methyl‐hydroxylated oxandrolone metabolites were predominantly excreted as sulfate conjugates in urine, whereas parent oxandrolone, its epimer and 17,17‐dimethyl‐18‐norandrost‐13‐ene derivative were found predominantly in the unconjugated urine fraction. The most abundant analyte detected in both plasma and urine was parent oxandrolone. However, the longest detection period using the developed analytical method was provided by 17‐hydroxymethyl‐oxandrolone in both matrices. The results of this study provided knowledge of how best to detect the use of oxandrolone in regulatory samples.