This study aimed to develop and optimize a method for the extraction and analysis of moenomycin antibiotics (a.k.a. flavomycin) in corn-based feed premix and in chicken litter. Moenomycin-A was isolated from chicken litter using pressurized liquid extraction followed by a solid-phase extraction (SPE) clean-up step. The highly lipophilic nature of moenomycin necessitated the use of the less hydrophobic sorbent, C(4)-based SPE cartridge, and a higher temperature elution solvent, methanol at 50 degrees C, in order to obtain satisfactory percent recoveries. After clean-up, the sample was analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Various reversed-phase columns were examined, including C(18), CN, perfluorinated C(6), and a porous graphitic carbon. The set of conditions that gave the highest separation efficiency while still maintaining symmetrical peak shape was the C(18) column using the H(2)O+0.3% HCOOH and acetonitrile mobile phase.