An analytical method was developed for the simultaneous analysis of 12 natural and synthetic hormones in aqueous matrices. No previous GC–MS method is known that encompasses this full range of estrogenic and androgenic analytes. Furthermore, the use of GC–MS/MS has enabled unambiguous identification and non-interfering quantification of closely eluting chromatographic peaks in a very short analysis time of only 15min. The use of isotope dilution for all analytes ensures the accurate quantification, accounting for analytical variabilities that may be introduced during sampling, extraction, derivatisation, chromatography, ionisation or mass spectrometric detection. Direct isotopically labelled analogues were used for 8 of the 12 hormones. However, satisfactory isotope standards were determined for the remaining 4 hormones, based on structural similarity and observed method recoveries of 80–120% in all sample
matrices.