Solid phase extraction for multiresidue analysis of anabolic steroids and related substances from calf urine using C18 and alumina columns

A solid phase extraction method for anabolic steroids and related substances in calf urine is reported, that is suitable as a screening method for illegal growth promoters. Two types of sorbent were used: a reversed phase C18 material and a polar alumina material. After overnight enzymatic deconjugation, the 5-mL sample was first brought on the C18 column. This column was washed with 55% methanol in water and was then eluted with 95% acetone in water. The extract was directly brought on the alumina column. The run-through and an additional elution with 95% acetone in water were collected. The final extracts were analysed with an HPLC-DAD method described previously. The steroids are separated on a Superspher RP-Select B column with a gradient mobile phase consisting of acetonitrile and water. The method was found to be suitable for at least 19 illegally used anabolic steroids, having recoveries ranging from 65-110% at a spiking level of 25 ng/mL. Detection limits ranged from 0.6-80 ng injected amount or 1-160 ng/mL urine.