Development and validation of a confirmatory method for the determination of stilbene estrogens in ostrich serum

A simple and accurate ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the first time as a confirmatory method for the simultaneous determination of stilbenes - hexestrol and diethylstibestrol in serum. Extraction was based on a simple acid denaturation of protein followed by liquid-liquid extraction using methyl tert butyl ether. Extracts were directly injected into the UHPLC-MS/MS without further purification. Excellent recoveries in the range 82-99% and 91-128% were obtained for hexestrol and diethylstibestrol, respectively. Both within-day repeatability and between-day reproducibility were generally satisfactory with RSD <20%. The linearity of the internal standard based matrix-matched calibration curve measured as the coefficient of regression (r²) was generally >0.99 for both hexestrol and diethylstibestrol. Both matrix effect and uncertainties associated with sample preparation and instrumental analysis were significantly reduced with the use of a deuterated compound (hexestrol-d4) as internal standard. The LOD and LOQ were 0.09 and 0.08 ng/ml, and 0.28 and 0.25 ng/ml, respectively, for hexestrol and diethylstibestrol. The method was found to be suitable for the simultaneous determination of hexestrol and diethylstibestrol in serum.