Assay recovery %.

Neuroestrogens are synthesized in the brain and regulate social behavior and cognition. In the song sparrow (Melospiza melodia), 17β-estradiol (17β-E2) promotes aggression, even during the non-breeding season, when circulating 17β-E2 levels are non-detectable. Measuring estrogens is challenging due to their low concentrations and the limited sensitivity of many existing assays. Moreover, estrogens other than 17β-E2 are often overlooked. Here, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of eleven estrogens using derivatization with 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS) to enhance sensitivity. We included four 13C-labeled internal standards, which corrected for matrix effects when measuring catecholestrogens and methoxyestrogens. This method is highly specific, sensitive, accurate, and precise, and improves upon our prior protocol, which could measure four estrogens with a single deuterated internal standard. Then, we applied the new method to samples from free-living adult male song sparrows to assess the effects of season and an aggressive encounter on blood and brain estrogens. Subjects were randomly assigned to either a 10-min simulated territorial intrusion (STI; song playback and live decoy) or a control condition (silent speaker and empty cage) during the breeding or non-breeding season, followed by rapid capture and immediate collection of blood and brain tissue. Only estrone and 17β-E2 were present in blood and brain, while the other nine estrogens in the panel were below detection limits. As expected, there was large regional variation in neuroestrogen levels and very low estrogen levels in blood. There was also large seasonal variation, and estrogen levels were lower in the non-breeding season. Despite robust aggression to the STI, estrogen levels did not differ between STI and control subjects in either season. In sum, our novel method enables ultrasensitive measurement of eleven estrogens and will be useful for studies of songbirds and other animals.