Determination of carnitine and acylcarnitines in biological samples by capillary electrophoresis–mass spectrometry

Abstract

Free carnitine and acylcarnitines (carnitine esters) play an important role in the metabolism of fatty acids. Metabolic disorders can be detected by abnormal levels of these compounds in biological fluids. Capillary electrophoresis–mass spectrometry has the advantage of combining an efficient separation technique with highly selective detection. Therefore, we have developed a method for the determination of carnitine and several of its esters implementing electrospray capillary electrophoresis–mass spectrometry in the positive ion selected reaction monitoring mode. A sheath-flow interface with a mixture of 2-propanol or methanol, water and acetic acid as sheath liquid and nitrogen as nebulizing gas was used. The zwitterionic analytes migrated as cations in the applied electric field using ammonium acetate–acetic acid or formic acid electrolytes. Separations were performed in aqueous, mixed organic–aqueous and non-aqueous media. The influence of the electrolyte composition on the separation efficiency was investigated. The electrospray conditions have been optimized regarding ion current stability and sensitivity. Ammonium acetate (10 mmol/l)–0.8% formic acid in water or 6.4% formic acid in acetonitrile–water (1:1) were used as running buffers for the determination of carnitine and acylcarnitines in human biological samples. Methanol extracts of dried blood spots were analyzed as well as urine and plasma following sample preparation via solid-phase or liquid–liquid extraction. Recoveries approaching 100% were achieved depending on the analytes and sample preparation procedures employed. Endogenous carnitine and acetylcarnitine were determined at concentrations between 2.7 and 108 nmol/ml in normal human urine and plasma. Other acylcarnitines were detected at levels of below the limit of detection to 12 nmol/ml. Good precision (0.8 to 14%) and accuracy (85 to 111%) were obtained; the achieved limits of quantitation (0.1 to 1 nmol/ml) are sufficient to characterize carnitine and acylcarnitine levels occurring as markers for metabolic disorders.