A very specific high-performance liquid chromatography–mass spectrometric method for the determination of natural tetracyclines was developed in order to characterise the degradation products of oxytetracycline in sediments. First, extraction used a clean up step with a Bond Elut CertifyÒ LRC cartridge. A 3 mm SpherisorbÒ ODS1 column was then used with a methanol, acetonitrile and oxalic acid mobile phase gradient. Chromatographic resolution in these conditions was 3.31 between oxytetracycline and tetracycline. Two liquid chromatography–mass spectrometry methodologies based on a particle beam and a frit fast atom bombardment interface were developed. In the first approach, ionisation was performed in the negative chemical mode using methane as reacting gas. In the other case, glycerol–thioglycerol mixture was used as matrix to ensure good sensitivity. MS–MS experiment was performed to determinate oxytetracycline fragmentation pattern in the perspective of degradation product study.