[Development and Validation of an Analytical Method for the Determination of Diethylstilbestrol, Dienestrol, Hexestrol, and Their Conjugates in Bovine Muscle].

A reliable and sensitive analytical method was developed for the determination of stilbenes-namely diethylstilbestrol, dienestrol, hexestrol, and their conjugates-in bovine muscle. The method involves the addition of internal standards to samples, extraction with ethanol/water (9 : 1, v/v),enzymatic hydrolysis using β-glucuronidase and aryl sulfatase, cleanup using hydrophilic-modified styrene-divinylbenzene copolymer and aminopropyl silica solid-phase extraction cartridges, and subsequent LC-MS/MS analysis. The method was validated for diethylstilbestrol, dienestrol, and hexestrol in bovine muscle spiked at 0.5 μg/kg for each analyte. The results demonstrated satisfactory analytical performance, with trueness values of 94-100%, repeatability below 5%, and within-laboratory reproducibility below 8%. No interfering peaks were observed, confirming the high selectivity of the method, and matrix effects were negligible. The developed method is applicable not only to diethylstilbestrol and its conjugates regulated in Japan but also to dienestrol and hexestrol, whose use for growth promotion in food-producing animals is prohibited in the EU. Although the conjugates of dienestrol and hexestrol were not directly validated in this study, they were presumed to be quantifiable based on their structural similarity to diethylstilbestrol conjugates.