Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs

A gas chromatography-mass spectrometry (GC-MS) method to determine eight anabolic steroids (diethylstilbestrol, methyltestosterone, norethindrone, 17α-ethynylestradiol, estradiol, 6α-methyl-17α-hydroxy-progesterone, estradiol benzoate, and chlormadinone acetate) was developed. Muscle samples were extracted with liquid-liquid extraction and clean-up was performed in two steps, the extracts obtained were derivatized with heptafluorbutyric (HBF) anhydride and analyzed by GC-MS. In the above method, the linear scope was 2.5-50 μg kg−1. The range of the recoveries was 78.5–148% for diethylstilbestrol, 70.8–109% for methyltestosterone, 69.8–87.2% for norethindrone, 67.7–120% for 17α-ethynylestradiol, 82.8–103% for estradiol, 70.3–99.2% for 6α-methyl-17α-hydroxy-progesterone, 73.0–104% for estradiol benzoate, and 72.9–91.8% for chlormadinone acetate. The range of the coefficients of variation within batches was 0.4–12%; the range of the coefficients of variation between batches was 6.4–11%. The limit of detections and the detection capability were 0.99 and 3.30 μg kg−1 for diethylstilbestrol, 1.05 and 3.50 μg kg−1 for methyltestosterone, 1.19 and 3.97 μg kg−1 for norethindrone, 0.94 and 3.13 μg kg−1 for 17α-ethynylestradiol, 1.45 and 4.83 μg kg−1 for estradiol, 1.56 and 5.20 μg kg−1 for 6α-methyl-17α-hydroxy-progesterone, 1.92 and 6.40 μg kg−1 for estradiol benzoate, and 2.41 and 8.03 μg kg−1 for chlormadinone acetate, respectively. These results showed that the method was widely available, accurate, and sensitive.