Multi-residue Analysis of Anabolic Steroids and Related Substances Using High Performance Liquid Chromatography with Diode Array Detection

We describe the development of an HPLC-DAD method for the analysis and identification of 20 anabolic steroids and related substances, that are considered as potential growth promoters, to be used for the analysis of extracts of calf urine samples. First, a suitable column was selected and then a gradient was developed. The compounds are separated on an RP-Select B column using a mobile phase consisting of a mixture of acetonitrile and water. Gradient elution from 43-76% acetonitrile in water using a concave curve was used to achieve a good separation of the compounds with an acceptable analysis time. For the identification of substances, a retention parameter and the UV spectrum were used. The retention parameter was the retention time corrected with a reference mixture. The limits of detection of the HPLC system ranged from 0.5-5 ng injected amount for the androgens, progestagens, stilbenes and resorcylic acid lactones to 5-10 ng injected amount for the oestrogens. Introduction Anabolic steroids and some related substances with comparable activities, all of which are here referred to as anabolic steroids, have been used as growth promoters during fattening of cattle for a long time [1,2]. This treatment may result in residues of