A method for the determination of carnitine and acylcarnitines in urine, plasma, and tissues developed for application to clinical research and diagnosis is presented. Acylcarnitines are extracted with acetonitrile/methanol (3:1), isolated with silica gel cation exchange SPE chromatography, derivatized with pentafluorophenacyl trifluoromethanesulfonate, chromatographed by sequential cation exchange and reversed-phase HPLC and detected by ESI/MS using a quadrupole ion-trap instrument. For quantification, standard curves are generated from MS/MS chromatograms for carnitine and straight-chain, branched-chain, unsaturated and dicarboxylic acylcarnitines. The qualitative MS chromatogram allows for the detection and quantitation of non-standard acylcarnitines. The advantages of this procedure over the popular acylcarnitine tandem MS method are: 1) Chromatographic resolution of constitutional isomers, 2) Accommodation of complex sample matrices without modification, 3) Instantaneous derivatization (extremely mild conditions do not hydrolyze acetylcarnitine). 4) True quantification (authentic standards and multiple point standard curves).

NIH P01 AG15885 and VA Medical Research Service.