Simultaneous determination of several natural steroids in blood plasma from perch (Perca fluviatilis) by GC-HRMS

A gas chromatography-high resolution mass spectrometry (GC-HRMS) procedure for the simultaneous determination of 18 endogenous steroid hormones in blood plasma from teleost fish has been developed. Proteins were removed by precipitation in methanol and lipids were removed by a liquid-liquid extraction. The protein and lipid free extract was further purified by using two successive solid phase extraction (SPE) methods (C18 and NH2). The isolated steroid hormones were silylated with a mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA): iodotrimethylsilane (TMSI):dithioerythritol (DTE) prior to determination with GC-HRMS working in selective ion recording mode. A volume of 200 µL blood plasma was sufficient for accurate determination of the steroid hormone levels, which enabled determination in individual fish. The detection limits ranged from 0.0003 to 0.2 ng g-1 blood plasma from perch (Perca fluviatilis), approximately 10 to 100 times lower then previously reported in this field. The recoveries for the entire procedure were in the range 58 to 150% with a variation, expressed as standard deviation (SD), below 10% with some exceptions. Despite the multi-step clean-up procedure, the intra-assay coefficient of variation, i.e. the within-day variation, for most steroid hormones was well below 14%. Finally, the procedure has been successfully applied to the determination of steroid hormones in blood plasma from female perch caught in two Swedish lakes.