Evaluation of equine urine reactivity towards phase II metabolites of 17‐hydroxy steroids by liquid chromatography/tandem mass spectrometry

Abstract

Proper storage conditions of biological samples are fundamental to avoid microbiological contamination that can cause chemical modifications of the target analytes. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method through direct injection of diluted samples, without prior extraction, was used to evaluate the stability of phase II metabolites of boldenone and testosterone (glucuronides and sulphates) in intentionally poorly stored equine urine samples. We also considered the stability of some deuterated conjugated steroids generally used as internal standards, such as deuterated testosterone and epitestosterone glucuronides, and deuterated boldenone and testosterone sulphates. The urines were kept for 1 day at room temperature, to mimic poor storage conditions, then spiked with the above steroids and kept at different temperatures (-18 degrees C, 4 degrees C, room temperature). It has been possible to confirm the instability of glucuronide compounds when added to poorly stored equine urine samples. In particular, both 17beta- and 17alpha-glucuronide steroids were exposed to hydrolysis leading to non-conjugated steroids. Only 17beta-hydroxy steroids were exposed to oxidation to their keto derivatives whereas the 17alpha-hydroxy steroids were highly stable. The sulphate compounds were completely stable. The deuterated compounds underwent the same behaviour as the unlabelled compounds. The transformations were observed in urine samples kept at room temperature and at a temperature of 4 degrees C (at a slower rate). No modifications were observed in frozen urine samples. In the light of the latter results, the immediate freezing at -18 degrees C of the collected samples and their instant analysis after thawing is the proposed procedure for preventing the transformations that occur in urine, usually due to microbiological contamination.

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

“…In previous studies, boldienone (androsta-1,4-diene-3,17-dione) and its isotope-labelled analogues were formed in a small number of the inappropriately stored equine urine samples (room temperature) that had been spiked either with testosterone (17β-hydroxyandrost-4-en-3-one) glucuronide or isotope-labelled analogues of testosterone and androstenedione (androsta-4-diene-3,17-dione). 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.…”

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

“…4 In addition, fungal species in corn have been reported to convert androstenedione to boldione and then further to boldenone. For example, Fidani et al 10 showed that boldione was detected in 10% of inappropriately stored equine urine samples spiked with testosterone glucuronide. 6 It has also been reported that black oats, contaminated with fungus and insects, contained boldenone and boldione, and its ingestion resulted in the excretion of boldenone sulfate in equine urine.…”

“…It is theorized that boldenone could be formed in equine urine in particular where postcollection storage conditions are unfavorable (temperature/extended period). For example, Fidani et al 10 showed that boldione was detected in 10% of inappropriately stored equine urine samples spiked with testosterone glucuronide. Additionally, inadvertent exposure of a urine sample to feces may result in the formation of boldenone, as demonstrated in both human urine 11 and in bovine feces.…”

Ex vivo spontaneous generation of 19-norandrostenedione and nandrolone detected in equine plasma and urine

“…This unexpected increase might result from possible conversion of testosterone glucuronide present in urine [27] to androstenedione [28]. Testosterone concentration in all the urine samples was close to LOQ and did not noticeably change with storage time (data not shown).…”