Determination of carnitine and acylcarnitines in plasma by high-performance liquid chromatography/electrospray ionization ion trap tandem mass spectrometry

Abstract

A high-performance liquid chromatography/mass spectrometry method was developed for the determination of carnitine, its biosynthetic precursor butyrobetaine, and eight acylcarnitines in plasma. The procedure includes a solid-phase extraction for carnitine and short- and medium-chain acylcamitines, and a liquid-liquid extraction for protein-bound long-chain acylcarnitines, followed by separation on a reversed-phase column in the presence of a volatile ion-pairing reagent. Detection was achieved using an ion-trap mass spectrometer run in the tandem mass spectrometry (MS/MS) mode. The choice of the matrix for calibrators, used for quantification of these endogenous compounds, was also investigated. Validation was performed for standard quality controls diluted with 4% bovine serum albumin solution and for spiked plasma quality control samples at concentrations between 0.5 and 80 μmol/L, depending on the compound. Intra- and inter-day precisions for the determination of carnitine were below 3.4% and accuracies were between 95.2 and 109.0%. Application of the method to the diagnosis of pathological acylcarnitine profiles of metabolic disorders in a patient suffering from methylmalonic aciduria is presented. The method allows quantification of carnitine, butyrobetaine, acetylcarnitine and propionylcarnitine, and semi-quantitative analysis of medium- and long-chain acylcarnitines. In contrast with other methods, no derivatization step is needed. Copyright © 2004 John Wiley & Sons, Ltd.