Accurate measurement of free carnitine in dried blood spots by isotope-dilution electrospray tandem mass spectrometry without butylation

BACKGROUND: To test the feasibility of free carnitine (FC) determination in dried blood spot specimens (DBS) by stable isotope-dilution electrospray-ionisation tandem mass spectrometry (MS/MS). METHODS: The MS/MS method established for newborn screening, measuring acylcarnitines by positive precursor ion scan of m/z 85 in DBS, was adapted by omitting the butylation and heating step during sample preparation. FC measurement in DBS by this non-butylating MS/MS assay was compared with the butylating MS/MS method and the spectrophotometric Cobas method. RESULTS: FC measurement by the non-butylating MS/MS method meets the demands for a bioanalytical microassay with respect to linearity, detection limit (LOD), accuracy, and precision. Formation of FC was 0-1% and 1-4% in liquid samples and in DBS by the non-butylating MS/MS method, while 3-10% and 8-16% by the butylating method, respectively. Acid-catalysed hydrolysis (butanolysis) in liquid samples was higher for short-chain acylcarnitines (acetyl- and propionylcarnitine). Hydrolysis in DBS was more pronounced for long-chain acylcarnitines. FC concentrations in healthy newborns without butylation were 35% lower than those measured by the established newborn screening assay. CONCLUSIONS: The non-butylating MS/MS assay provides a simple and accurate method for FC determination in DBS and represents a trivial but important adaptation of a method already used in many laboratories.