The use of isotope dilution for all analytes ensures the accurate quantification,
accounting for analytical variabilities that may be introduced during sample processing and
instrumental analysis. Direct isotopically labelled analogues were used for 8 of the 12 hormones and satisfactory
isotope standards were identified for the remaining 4 hormones. Method detection levels (MDLs)
were determined to describe analyte concentrations sufficient to provide a signal with 99% certainty
of detection. The established MDLs for most analytes were 1–5 ng L−1 in a variety of aqueous matrices.
However, slightly higher MDLs were observed for etiocholanolone, androstenedione, testosterone, levonorgestrel
and dihydrotestosterone in some aqueous matrices. Sample matrices were observed to have
only a minor impact on MDLs and the method validation confirmed satisfactory method stability over
intra-day and inter-day analyses of surface water and tertiary treated effluent samples.