Supercritical fluid extraction of methyltestosterone, nortestosterone and testosterone at low ppb levels from fortified bovine urine

Abstract

A multi-residue supercritical fluid extraction (SFE) method is proposed for the isolation of nortestosterone, testosterone and methyltestosterone from bovine urine. Prior to SFE, bovine urine was hydrolyzed and then fortified with the three steroids at 100 ng/ml and 50 ng/ml each for HPLC analysis and 25 ng/ml and 12.5 ng/ml each for GC–MS analysis. The samples then were mixed with an adsorbent material, placed in an SFE extraction vessel prepacked with a 3-ml SPE column containing neutral alumina and the testosterones were extracted from the urine matrix using unmodified supercritical CO 2 at 27.2 MPa and 40°C. The steroids were retained in-line on the neutral alumina sorbent in the SPE column while co-extracted artifactial material was trapped off-line after CO 2 decompression. After SFE, the SPE column was removed from the extraction vessel, and the trapped steroids were eluted from the neutral alumina sorbent with 3 ml of a methanol–water mixture. Eluates were used directly without post-SFE clean-up either for HPLC analysis (detection limit 50 ng/ml) or for GC–MS analysis (detection limit 5 ng/ml after steroid derivatization). The multi-residue SFE recoveries ( n=6) for nortestosterone, testosterone and methyltestosterone from hydrolyzed bovine urine by GC–MS analysis were 90.8±6%, 93.9±3% and 92.5±5%, respectively for each steroid at the 12.5 ng fortification level.