Simultaneous determination of methyltestosterone and its metabolite in fish by gas chromatography-mass spectrometry

Methyltestosterone is one of the banned drugs in aquaculture, and it should be monitored in food-producing animals. 17 alpha-Methyl-5 beta-androstane-3 alpha,17 beta-diol, as the main metabolite of methyltestosterone in vertebrates, could be used as another marker for controlling the administration of methyltestosterone, due to its high residual concentration and slow elimination rate. In this study, an analytical method based on gas chromatography-mass spectrometry (GC-MS) was developed and validated for the simultaneous determination of methyltestosterone and its main metabolite in fish. After pretreatment by liquid-liquid extraction with n-hexane and solid phase extraction with C18 and NH2 columns, the target analytes in the muscle tissues were extracted and concentrated, and the influence of the sample matrix was eliminated. Then, the prepared samples were separated and detected with GC-MS in the selected ion monitoring (SIM) mode. Methyltestosterone-D3 was chosen as the internal standard for quantitation. After optimization, the limits of detection for methyltestosterone and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol were 20 mu g kg-1 and 15 mu g kg-1, respectively. The limits of quantitation were both 50 mu g kg-1. The calibration curves showed good linearity in the concentration range from 50.0 ng mL-1 to 500.0 ng mL-1. The correlation coefficients of methyltestosterone and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol were more than 0.9990. The recoveries of the analytes in real samples were in the range of 99.7-116.6% with the relative standard deviation of 5.2-8.3%. The established method could meet the demand for simultaneous detection of methyltestosterone and its major metabolite, and it could be used to provide more information on the abuse of methyltestosterone in food-producing animals. Methyltestosterone is one of the banned drugs in aquaculture, and it should be monitored in food-producing animals.