Analysis of hormonal steroids in fish plasma and bile by coupling solid-phase extraction to GC/MS

An analytical procedure for the simultaneous determination of twelve endogenous steroids (testosterone, androstenedione, 17 beta-estradiol, estrone, pregnenolone, progesterone, dihydroandrostenedione, dihydrotestosterone, 11 alpha-ketotestosterone, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one) in plasma and bile samples by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC-MS) has been developed. After enzymatic hydrolysis for bile samples only, samples were concentrated and purified using two successive SPE (C-18 and NH2) cartridges. Analytes were derivatized with a mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/mercaptoethanol/ammonium iodide (NH4I) and determined by GC-MS in selective ion monitoring mode. For most of the steroids monitored, recoveries were in the range 90-120% in plasma and in the range 60-70% in bile, and the reproducibility was below 10% for the complete procedure. Limits of detection obtained ranged from 0.1 to 0.4 ng/g in fish plasma and from 1.6 to 14 ng/g in fish bile. The developed method was successfully applied to the determination of plasma steroids in flounders (Platichthys flesus) collected from two French estuaries.