Multi-residue analysis of anabolics in calf urine using high-performance liquid chromatography with diode-array detection

Abstract We describe the development of an HPLC method with diode-array detection (DAD) for the analysis and identification of 20 substances with anabolic properties, that are considered as potential growth promoters, to be used for the analysis of extracts of calf urine samples. The substances 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 with a concave curve was used to achieve a good separation of the compounds with an acceptable analysis time. For the identification, a retention parameter and the UV spectrum were used. The retention parameter was the retention time corrected with a reference mixture. The latter reduced the standard deviations to about 25% of their original values. 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 and to 5–10 ng injected amount for the oestrogens. After extraction from urine the limits of detection were increased by the presence of matrix components, but they were between 5 and 10 ng injected amount for most of the substances.