Determination of 16β-hydroxystanozolol in urine and faeces by liquid chromatography–multiple mass spectrometry

This paper describes the optimisation of the detection of stanozolol and its major metabolite 16β-hydroxystanozolol in faeces and urine from cattle. Faeces are extracted directly with diisopropyl ether. Urine is first submitted to an enzymatic hydrolysis and then extracted over a modified diatomaceous earth column (Chem-Elut) with a mixture of diisopropyl ether–isooctane. In a final step an acidic back extraction is performed. For the LC–MS–MS detection two approaches are discussed. In a first approach the final extract is detected without derivatization, while the second approach makes use of a derivatization step for 16β-hydroxystanozolol. While the MS–MS spectrum without derivatization exhibits extensive fragmentation, the spectrum of the derivative shows two abundant diagnostic ions with much more reproducible ion ratios. The derivatization method and the method without derivatization enable the detection of 16β-hydroxystanozolol up to 0.03 μg l−1 in urine and 0.07 μg kg−1 in faeces. Until now there is no literature available for the detection of 16β-hydroxystanozolol in faeces and urine at the ppt level.