The concentrations of twenty kinds of hormones in the follicular fluid were detected by high-performance liquid chromatography–mass spectrometry (HPLC-MS/MS). An Agilent 1200 series high-performance liquid chromatography (HPLC) instrument (Agilent, USA) was utilized. A PAL autosampler (CTC, Swiss) and a Gemini-NX-C18 column (2.0 mm×50 mm, 3 μm, Waters, USA) were used. The ion source was an API-4000 quadrupole electrostatic field orbit trap high-resolution mass spectrometer (Applied Biosystems, USA). The scanning mode was multiple reaction monitoring (MRM) (Agilent-1200 LC system coupled to an API400 mass spectrometer).