Detection of testosterone, nandrolone and precursors in horse hair

Abstract

Growing interest among several horse-breeder associations has initiated the development of a screening procedure to test for anabolic agents in hair, which has the advantage over blood and urine specimens of allowing long-term detection. An analytical method was established to monitor in tails or manes several anabolic substances available as veterinary medicines or as so-called nutritional supplements (clenbuterol, different esters or prohormones of nandrolone and testosterone). The analytical procedure to detect steroids in hair samples consists of the following steps: decontamination of the hair strand or segment with methanol/water (1:1), milling, extraction of the hair material in an ultrasonic bath using methanol, purification by liquid-liquid extraction (n-pentane/methanol, 25:1) and HPLC cleanup, derivatisation of the relevant LC fractions with MSTFA, and measurement using GC-MS/MS technique. The first objective of our study was the detection of exogenous nandrolone (nortestosterone, NT) in the horse hair; therefore nandrolone-associated compounds [nandrolone dodecanoate administered intramuscularly (i.m.) and a mixture of 4-estrenediol and 4-estrenedione, transdermal] were administered to four geldings. The highest concentrations of NT following i.m. treatment were measured after 10 days in a 2-cm hair segment (up to 18 pg/mg); NT was detectable for up to 120 days and in some cases up to 330 days in tail hair (limit of detection 0.3 pg/mg). Following transdermal application, nandrolone as well as the administered prohormones were identified in tail and mane until the latest sampling at 3 months. Furthermore, untreated stallions (128) were investigated to estimate the range of endogenous levels of NT and testosterone (T) in hair. Maximum values of 3 pg/mg (NT) and 1 pg/mg (T) were quantified originating from endogenous formation in the male horse. Additionally, a possible relationship between steroid concentrations in hair specimens and the age of stallions was appraised. NT and T were not detected in hair samples of control geldings. Following nandrolone treatment of geldings, highest values in hair exceeded the endogenous amount detected in untreated stallions. Therefore comparison of concentrations measured in control samples with the estimated endogenous levels could give a clue to exogenous application in cases of abnormally high amounts of NT or T. The possibility of the evaluation of threshold values is discussed as a means to verify an exogenous administration of NT and T in hair samples. Furthermore, the detection of a synthetic substance in hair, e. g. the parent steroid ester by itself, would be unequivocal proof of an exogenous origin of NT or T and the previous medication of the stallion.

Detection of testosterone propionate administration in horse hair samples

“…This is due to the use of damping gas at the optimized flow rate. As reported before [16,17] testosterone is not detectable in hair samples collected from non-treated geldings. In the present study, testosterone was not detectable in the mane and tail samples obtained before administration.…”

“…Regarding steroid analysis in horse hair samples, boldenone was detected in the mane and tail retrospectively for up to 12 months after boldenone undecylenate administration to two different horses [15]. As recently showed by Anielski et al [16] and Popot et al [17], testosterone has been detected in mane and tail samples from a stallion but was not detected in hair of a gelding before administration of testosterone enanthate. Testosterone was detected in a hair sample taken 1 month after administration.…”

“…The detection limit for testosterone propionate achieved on the two TQ mass spectrometers defined as 3 S/N was about 0.5 pg mg −1 , which is appropriate [5,14,16] for the analysis of anabolic esters in hair.…”

Hair analysis for veterinary drug monitoring in livestock production

“…The presence of anabolic agents such as ␤-agonist and steroidal compounds in hair has been extensively studied in farm animals [15,21,38,39,45,47,48,53,55,61,62].…”

“…The addition of TMIS, 2-mercaptoethanol, DTT, DTE or NH 4 I has also been proposed to improve the efficiency of the derivatization reactions [16,22,33,40,45,54,55,61].…”