Rapid liquid chromatography-electrospray tandem mass spectrometry method for serum free and total carnitine.

Abstract

The measurement of serum free carnitine (SFC) is useful for the investigation and monitoring of patients suffering from inherited metabolic disorders of fatty acid oxidation. Two tandem mass spectrometry (MS/MS) methods (1)(2) have recently been published to circumvent technical problems in the radioenzymatic reference method and the routine spectrophotometric enzymatic method. The method described by Hardy et al. (1) requires butyl esterification of SFC before MS/MS analysis and suffers from inaccuracy attributable to partial hydrolysis of acylcarnitines during the derivatization procedure. The method of Stevens et al. (2) does not require derivatization, but samples are first applied to cotton fiber filter paper, and SFC is eluted with a methanol–water solution before MS/MS analysis. Although not discussed by Stevens et al. (2), the filter paper serves to reduce serum sample matrix interference, a well-known phenomenon in electrospray ionization MS. The presence of any nonvolatile solutes in the biological sample matrix changes the droplet solution properties during electrospray ionization, causing ion suppression (3). On the other hand, liquid chromatography (LC) conditions have also been used to reduce matrix interference (4). We present an alternative LC MS/MS method for SFC that does not require derivatization and has reduced matrix interference. To 20 μL of serum sample, we added 300 μL of an internal standard solution containing 3.3 μmol/L 2H3-l-carnitine in acetonitrile to precipitate the serum protein in an Eppendorf tube. The mixture was vortex-mixed and sonicated for 10 min to improve the precision of the extraction procedure before centrifugation (7000 g for 5 min). The supernatant was then transferred to a sample vial for LC MS/MS analysis. A series of aqueous calibrators, ranging from 0 to 100 …