Cardiolipin (CL) is found almost exclusively in the inner mitochondrial membrane. Alteration of CL, namely by oxidative modifications, has been related with various pathological conditions, and with cell apoptosis. Their location, in the mitochondria, makes them even more likely to be oxidized, since that there is a considerable production of ROS in the inner mitochondrial membrane. In spite of the importance of CL oxidation and its biological consequence, there is a limited knowledge of the oxidation products of CL In this study, mass spectrometry coupled with liquid chromatography (LC-MS) was used to identify the specific oxidative modifications of tetra-linoleoyl CL induced by the OH center dot generated under Fenton reaction conditions (H(2)O(2) and Fe(2+)). Short chain products (with fatty acyl shortened) formed during CL oxidation were identified for the first time and were further characterized by LC-ESI-MS/MS. The short chain products identified resulted from beta-cleavage of alkoxyl radicals at C9 and C13. These products were identified as [M-2H](2-) at m/z 691 and 719, with C13 and at m/z 685, 693 and 701, with C9 shortened fatty acyl chains. Detailed analysis of the fragmentation pathways of these precursor ions allowed to identify the MS/MS product ions which allow the unequivocal assignment of the oxidized cardiolipin species, very valuable for their detection in biological samples. Some of these products, namely, the CL short chain product at m/z 685 and 693 were detected in the mitochondria of kidneys obtained from rats treated with gentamicin. Gentamicin is an aminoglycoside antibiotic that induces nephrotoxicity and that has been associated with mitochondrial dysfunction and lipid peroxidation. (C) 2010 Elsevier B.V. All rights reserved.