We developed and validated a method to measure steroid hormones with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in faecal samples of white-faced capuchins. The method includes the measurement of adrenal and gonadal hormones such as cortisol, testosterone, estrone, progesterone and a number of their faecal metabolites. This method can be used for simultaneous routine measurements of steroids in faecal samples and provides a reference method for the validation of new immunoassays in this matrix. The optimised method consists of an extraction of the dried faecal samples with 80% methanol followed by purification of the extracts by solid phase extraction, solvolytic cleavage of conjugates and liquid-liquid extraction. Extracts were measured by LC-MS/MS with an electrospray interface in positive ionisation mode. Out of 19 steroids spiked into methanol extracts, 14 showed a recovery of 79.8-118.5% with an intra-day precision of 2.5-13.0% and an inter-day precision of 7.2-15.1%. Detection limits for these steroids ranged from 0.3 to 27.0 ng/mL of extract. Five steroids did not fulfil our requirements concerning precision and accuracy and we therefore considered these to not be reliably measurable with this method. While there was no indication of considerable amounts of conjugated forms for most metabolites, 87% of the testosterone was found in the solvolysis fraction, which indicates that the majority of testosterone was conjugated. Therefore, solvolysis turned out to be crucial, especially for the quantification of the total amount of testosterone. The physiological validation of this LC-MS/MS method confirmed known physiologically caused differences in faecal steroid concentrations. This indicates the usefulness of the method in investigating variation in the levels of major steroid hormones in faeces of white-faced capuchins. The possibility to simultaneously measure hormones of the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis makes this method suitable for dealing with questions concerning the cross talk between those axes.
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