Acyl picolinyl esters provide excellent data to identify the structures of acyl groups. However, the mechanisms for the formation of fragment ions from picolinyl esters are unsettled. Proposed structures for fragment ions have focused on long-chain groups and may not accommodate results from medium-and short-chain acyl groups. Using deuterium-labeled organic acids, we have investigated the mechanisms for the formation of fragment ions. Based on these studies, we propose a new mechanism that is consistent with the experimental data. We then tested the mechanisms by analyzing selected acylcarnitines. Transesterification of acylcarnitines was performed by reaction with 3-pyridylcarbinol and potassium tert-butoxide in dichloromethane to produce acyl picolinyl esters. The picolinyl esters were separated and detected by gas chromatography/electron ionization-mass spectrometry. Each mass spectrum contained a series of peaks with m/z differences of 12, 13, or 14 u depending on the acyl group's chemical structure. [4] in which the fragment ions of an acyl picolinyl ester result from the interaction of alkyl hydrogens with the ionized nitrogen on the pyridine ring. The mechanism invoking a McLafferty rearrangement, proposed by Harvey for the formation of the fragment ions from long-chain acyl groups, is not supported by data from Christie's laboratory [4, 5]. Furthermore, these mechanistic studies have not been conducted using either short-or medium-chain acyl groups.Acyl group identification is an important component in many analytical methods. Several reports detail procedures to identify the acyl groups of acylcarnitines.Lowes and Rose [6,7] reported that by converting acylcarnitines into acyloxylactones, partial, but not complete, structural information of the acyl group was obtained. Libert et al. [8 -10] used a procedure whereby acylcarnitines were extracted from urine and hydrolyzed, followed by conversion of the organic acids into acyl picolinyl esters. These acyl picolinyl esters were then characterized by GC/MS, which provided better structural data than in the case of acyloxylactones. However, the presence of organic acids in urine makes questionable the interpretation of the source of acyl groups as being exclusively from acylcarnitines. Although acyl picolinyl esters usually are produced from organic acids, transesterification has been reported to produce acyl picolinyl esters from lipids by direct reaction with 3-pyridylcarbinol, thus eliminating the need to hydrolyze the lipids first [11].In this paper, we propose mechanisms for the formation of the fragment ions of acyl picolinyl esters based on experiments using deuterium-labeled organic acids. We applied these results to the characterization of acyl groups of several acylcarnitine species following their transesterification to acyl picolinyl esters. The transesterification was performed by reacting acylcarnitines with 3-pyridylcarbinol and potassium tert-