Abstract: A method for the determination of methyltestosterone in tilapia muscle using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed. Deuterium-labeled methyltestosterone was used as internal standard and added to the sample before being extracted with acetonitrile. The extract was partitioned into ethyl acetate and cleaned up on a strong anion exchange column. Subsequently, the eluates were further extracted with n-hexane and cleaned up on a magnesium silicate solid phase extraction cartridge before UPLC-MS/MS determination. The mass spectrometer was operated in the positive ion mode using selected ion monitoring for qualitative and quantitative analysis of methyltestosterone. The limit of detection was 0.4 μg/kg and the limit of quantitation was 1μg/kg. The calibration curve was linear with a correlation coefficient of over 0.999 within the concentration range of 1－60μg/kg. The average recoveries for methyltestosterone were 71.06%－83.06% across three spike levels (n＝5) with a relative standard deviation (CV) between 7.4% and 15.3%. These results demonstrate that this method fully meets the requirements for the determination of methyltestosterone residue in imported and exported tilapia muscles for the purpose of ensuring food safety.

Key words: ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), methyltestosterone, tilapia muscle