The use of anabolic steroids as hormonal growth promoters for farm animals is banned in the EU and each member state is responsible for monitoring levels in its own meat. There are a wide range of potential drugs that can be administered but GC/MS and LC/MS techniques have been developed to detect them in body fluids, tissues and excreta. However, these methods are not without their problems. For instance, when natural steroids such as testosterone or estradiol are used, it is difficult to distinguish them from the endogenous steroids in the animal.

The analysis of animal hair has been proposed as an alternative to body fluids with several perceived advantages. As a non-invasive procedure, it causes no stress to the animal and the sampled hair is easy to transport and extract. In addition, it has been shown that the steroids can be detected in their original form, as steroid esters, for example, rather than metabolites.

Although several methods have been reported so far, questions still remain about the uptake and transport mechanism of steroids in the hair. It was originally proposed that the drug became trapped in the growing cells at the base of the follicle then moved along the along the hair shaft during growth. Now, a more complex "multiple-pool" mechanism has been proposed to account for experimental observations. The pathways include transport of the drugs from the sweat and sebaceous glands, absorption from the external environment after hair has emerged from the skin and transfer from adjacent tissues.

There are other factors which may have an effect. The pigment melanin assists the transport of some drugs so that dark hairs accumulate more than light hairs. The route of administration, such as pour-on vs. intramuscular injection, is also believed to affect the levels in hairs.

In order to try and get a clearer picture of the distribution of steroid hormones in cattle hair, a team of scientists has studied the distribution of two steroid esters. Michael O'Keeffe and colleagues from the Ashtown Food Research Centre, Dublin and Queen's University Belfast administered estradiol benzoate (EB) to steers and nortestosterone decanoate (ND) to heifers by intramuscular injection. They collected hair from a series of strips parallel to the spinal column from the shoulder downwards every 2-4 days over 43 days.

The hairs were washed, cut into 4-5-mm lengths and spiked with deuterium-labelled internal standards before digestion with the reducing agent TCEP and sodium dihydrogen orthophosphate. After extraction and clean up by SPE, the extracts were analysed by the authors' own LC/MS/MS method (2009) with detection limits of 6.5 and 5.3 µg/kg for EB and ND, respectively.

The results were disappointing. Both steroids were detected but only sporadically and at relatively low levels. The data were insufficient to determine steroid distributions in the hair with time. Several possible reasons for the low figures were investigated but the washing, drying and grinding steps did not induce any losses and the use of alternative extraction solvents did not increase yields.

In a second study, the team administered a combined dose of EB and ND by intramuscular injection to two steers and performed the extraction and LC/MS/MS analysis as before. In this case, some areas were re-sampled to check levels in new growth.

Similar steroid concentrations of both steroids were found in first and second trim hairs, indicating similar concentrations along the hair length. This suggests that the drug incorporation mechanism is not related to hair growth but is more likely to be similar to the multiple pool method.

The steroid levels decreased with distance of the hair from the injection point and were also related to the time of the original dosage, although this relationship might vary with post-treatment time.

This complex arrangement highlights the difficulties associated with the analysis of steroid esters in cattle hair. While residues can be detected, it is not a foolproof method as some treated animals tested negative. However, it may be of value given good pre-screening procedures to identify potentially treated cattle, so long as the testing site is close to the location of treatment.

Related links:

• Journal of Chromatography A 2009 (Article in Press): "Studies on the persistence of estradiol benzoate and nortestosterone decanoate in hair of cattle following treatment with growth promoters, determined by ultra-high-performance liquid chromatography tandem mass spectrometry"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.