Ultrasensitive Quantification of Multiple Estrogens in Songbird Blood and Microdissected Brain by LC-MS/MS

Neuroestrogens are synthesized within the brain and regulate social behavior, learning and memory, and cognition. In song sparrows, Melospiza melodia, 17β-estradiol (17β-E₂) promotes aggressive behavior, including during the nonbreeding season when circulating steroid levels are low. Estrogens are challenging to measure because they are present at very low levels, and current techniques often lack the sensitivity required. Furthermore, current methods often focus on 17β-E₂ and disregard other estrogens. Here, we developed and validated a method to measure four estrogens [estrone (E₁), 17β-E₂, 17α-estradiol (17α-E₂), estriol (E₃)] simultaneously in microdissected songbird brain, with high specificity, sensitivity, accuracy, and precision. We used liquid chromatography tandem mass spectrometry (LC-MS/MS), and to improve sensitivity, we derivatized estrogens using 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). The straightforward protocol improved sensitivity by 10-fold for some analytes. There is substantial regional variation in neuroestrogen levels in brain areas that regulate social behavior in male song sparrows. For example, the auditory area NCM, which has high aromatase levels, has the highest E₁ and 17β-E₂ levels. In contrast, estrogen levels in blood are very low. Estrogen levels in both brain and circulation are lower in the nonbreeding season than in the breeding season. This technique will be useful for estrogen measurement in songbirds and potentially other animal models.