Measuring urinary cortisol and testosterone levels in male Barbary macaques: A comparison of EIA and LC-MS

The development of methods to quantify hormones from non-invasively collected samples such as urine or feces has facilitated endocrinology research on wild-living animals. To ensure that hormone measurements are biologically meaningful, method validations are highly recommended for each new species or sample matrix. Our aim was to validate three commonly used enzyme immunoassays (EIA), one for analysis of cortisol and two for analysis of testosterone, to assess adrenocortical and gonadal activity, respectively, from the urine of male Barbary macaques. We compared EIA and liquid chromatography-mass spectrometry (LC-MS) results to determine if the EIA measurements truly reflect levels of the target hormone and to determine if cross-reactivities with other steroids were potentially confounding results. Furthermore, we conducted a biological validation of testosterone to ensure that both EIA and LC-MS were able to capture physiologically meaningful differences in hormone levels. We found that cortisol measured by EIA correlated strongly with cortisol measured by LC-MS in both adult and immature males, without the need for deconjugation of steroids in the urine. Both testosterone EIAs correlated strongly with LC-MS in adult males, but only if steroids in the urine were deconjugated by enzymatic hydrolysis prior to analysis. However, in immature males, EIA and LC-MS results did not correlate significantly. Further correlation analyses suggest this is likely due to cross-reactivity of the testosterone antibody with other adrenal steroids such as cortisol, DHEA, and likely others, which are present at much higher concentrations relative to testosterone in immature males. Testosterone levels were significantly higher in adult compared to immature males as measured by LC-MS but not as measured by EIA. Taken together, our results suggest that the testosterone EIAs are suitable to assess gonadal activity in adult but not immature males, and only if a hydrolysis of the urine is conducted prior to analysis.