Development of an analytical approach for identification and quantification of 5-α-androst-16-en-3-one in human milk

► We developed a method for the quantification of androstenone in human milk. ► The quantification of androstenone in human milk was performed by stable isotope dilution assay. ► Concentrations of androstenone in human milk samples ranged from 26 to 155ng/kg. A method was developed for the quantification of 5-α-androst-16-en-3-one in human breast milk based on application of a stable isotope dilution assay using 5α-androst-16-en-3-one-6, 6-d2. The procedure includes extraction of the human milk by hexane with subsequent clean-up of the obtained extract by gel permeation and silica gel column chromatography. The extracted samples were analyzed by gas chromatography–mass spectrometry. Using this method 5-α-androst-16-en-3-one could be identified and for the first time quantified in a concentration range of 26–155ng/kg in human milk.