Development of Analytical Procedure for the Determination of 17β-Testosterone, 11-Ketotestosterone and 17β-Estradiol in the Sea Trout (Salmo trutta L.) Gonads

Steroid hormones, such as 17β-testosterone, 11-ketotestorenone and 17β-estradiol, play an essential role not only in reproductive function but also are potential biomarkers of numerous additional functions in teleost fish. The presence of endocrine disruptor compounds in aquatic ecosystems has raised concern about their effect on hormone levels in fish target organs. Since hormones are present in very low concentrations in biological material, their determination still remains a challenge. A new analytical procedure has been developed to determine 17β-testosterone, 11-ketotestosterone and 17β-estradiol in the sea trout female and male gonads by liquid chromatography-tandem mass spectrometry (LC-MS/MS) system equipped with an ESI source operating in both positive and negative mode. Chromatographic separation of analytes was accomplished in Poroshell 120 EC-C18 (150 mm × 2.1 mm, 2.7 µm) column under isocratic elution conditions. The mobile phase consisted of acetonitrile, methanol and water (20:50:30/v/v/v) at a flow rate of 0.2 mL/min. Analytes were extracted from the gonad matrix with ethyl acetate, and co-extractives impurities were successfully removed by QuEChERS (quick, easy, cheap, effective, rugged and safe) method. The procedure was validated with good sensitivity, linearity, accuracy, and precision. Limits of quantifications were from 0.15 to 0.75 ng/g, linearity was obtained with correlation coefficient R > 0.99, accuracy was from 94.0 to 109.5%, precision expressed as RSD ranged from 1.7 to 27.2% (repeatability) and from 2.2 to 37.1% (reproducibility). Finally, the method was applied to determining 17β-testosterone, 11-ketotestosterone and 17β-estradiol in real samples of the female and male sea trout gonads, 8 and 22 samples, respectively.