Simple methodology coupling microwave-assisted extraction to SPE/GC/MS for the analysis of natural steroids in biological tissues: Application to the monitoring of endogenous steroids in marine mussels Mytilus sp. Original Research Article

A simple analytical procedure for the simultaneous determination of eight endogenous steroids (testosterone, androstenedione, 17β-estradiol, estrone, pregnenolone, progesterone, dihydroandrostenedione, and dihydrotestosterone) in aquatic molluscs by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC–MS) has been developed. After a microwave-assisted extraction, samples were further extracted and purified using two successive SPE (EnviChrom-P and NH2) cartridges. Steroids 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. Recoveries were in the range 85–114%, although slightly lower for dihydrotestosterone, and the repeatability of the procedure, expressed as the coefficient of variation, was lower than 16%. The limits of detection determined in digestive glands of mussels were in the range 0.1–0.4 ng g−1 wet weight for all the steroids. The developed procedure was then applied to the monitoring of steroid profiles in the digestive glands of mussels from the Arcachon Bay (France) during two reproductive cycles. In parallel, two physiological parameters (lipid content and the condition index of mussels) were also monitored, as well as the seawater temperature and salinity. Only progesterone and pregnenolone were detected in the digestive glands of mussels, and the seasonal variations of progesterone levels seemed to be related to the spawning periods of Mytilus sp. in the Bay. The current challenge for the determination of natural steroids in aquatic invertebrates is also briefly discussed.