Quantitative Hormone Analysis Protocol

Hormones were analysed as described previously (Albacete et al., 2008 (link)). Briefly, 0.5g of each sample were homogenized, in triplicate, with liquid nitrogen, and extracted with 2.5ml of methanol:H₂O (80:20). Samples were centrifuged and the precipitates were re-extracted with another 2.5ml of the methanol:H₂O mixture. The two supernatants were mixed and passed through a SepPak Plus C18 cartridge (SepPak Plus, Waters, Millford, MA, USA). Samples were evaporated and the residues were dissolved in a methanol:H₂O (20:80) mixture and filtrated. 8 µl of sample were injected in an Agilent 1100 Series HPLC (AgilentTechnologies, Santa Clara, CA, USA), equipped with a micro-wellplate autosampler and a capillary pump, connected to an Agilent Ion Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA,USA) using an electrospray interface.