Determination of acetyl gestagenic steroids in kidney fat by automated supercritical fluid extraction and liquid chromatography ion-trap mass spectrometry

Abstract

Acetyl gestagenic steroids are isolated from animal tissues such as bovine kidney fat by automated supercritical fluid extraction (SFE). After the addition of internal standards and sample pretreatment, the analytes are extracted from the matrix by supercritical CO₂ and trapped directly in-line on alumina placed in the extraction vessel. The samples are analysed by liquid chromatography combined with ion-trap mass selective detection (LC-MSⁿ). For quantification, deuterated internal standards are added and single ions of the analytes and internal standards are monitored. For confirmation of the identity of the analytes, two transition ions (one MS² and one MS³) were monitored and the ratios between the ions were calculated and compared with those of standards. The detection capability for the multi-analyte LC-MSⁿ analysis of megestrol acetate (MA), medroxyprogesterone acetate (MPA), chlormadinone acetate (CMA) and melengestrol acetate (MGA) is 0.5 μg kg⁻¹. The mean within-laboratory reproducibility ranges from 16–19% (%RSD) at a concentration level of 0.5 μg kg⁻¹ (n = 9). Running the SFE procedure overnight allows the analysis of 24 samples of fat per day.