Determination of anabolic esters in oily formulations and plasma in husbandry using high-performance liquid chromatography and gas chromatography–mass selective detection

Abstract

Two different analytical methods are described for the analysis of anabolic steroid esters in oily formulations for veterinary use and animal plasma samples, respectively. For the determination of anabolic steroid esters in oily formulations (at mg kg^–1 levels) a reversed-phase high-performance liquid chromatographic method with gradient elution is described. Gradient elution is performed owing to the relatively large variations in polarity of the investigated anabolic steroid esters. For the analysis of anabolic steroid esters in plasma (at ng ml^–1 levels) two different strategies are applied. After solid-phase extraction, the plasma samples are introduced into the high-performance liquid chromatography (HPLC) system where the obtained fractions are then analysed by using gas chromatography–mass selective detection (GC–MSD). An alternative method is direct analysis of plasma samples after solid-phase extraction by using GC–MSD without any further clean-up procedure. Prior to GC–MSD the samples are derivatized to corresponding trifluoroacyl (TFA) derivatives. The calibration graph for HPLC is rectilinear over the range 25–150 ng ml^–1 plasma and the analytical recoveries for medroxyprogesterone acetate (MPA) and testosterone propionate (TP) are more than 95%. The detection limits for both analytes in GC–MS are 2.5 ng ml^–1 plasma for MPA and 0.5 ng ml^–1 plasma for TP with an acceptable signal-to-noise ratio (calculated for the derivatized relative molecular mass). In the analysis of plasma obtained from animal experiments concentrations of 6.5 ng ml^–1 are found for MPA by using GC–MSD and 5.0 ng ml^–1 are found for nortestosterone laurate (NL) by using HPLC.