Assay for Free and Total Carnitine in Human Plasma Using Tandem Mass Spectrometry

Abstract

Measurements of free and total carnitine in plasma are important in the diagnosis and clinical management of patients with carnitine deficiency syndromes and certain inborn errors of metabolism (1). The most popular analytical methods are based on the enzymatic reaction in which an acetyl group is transferred from acetyl-CoA to carnitine with release of free CoA (2). A spectrophotometric assay based on this method, using a Cobas centrifugal analyzer (Roche Diagnostics), has been in use in this laboratory for the past several years (3). Limitations of the Cobas enzymatic method in our view include the initial filtration step, which requires a minimum sample volume of 300 μL, and the number and cost of the reagents required. Assay results are critically dependent on two labile reagents, the enzyme carnitine acetyltransferase and the color reagent 5,5′-dithiobis(2-nitrobenzoic acid). We have also observed that patients on valproic acid therapy sometimes give total carnitine values that are spuriously low for reasons that are not clear. Another limitation of the Cobas assay is that acylcarnitines with chain lengths >10 are lost in the filtration step (3), and the method will underestimate the total carnitine content of patients with defects in the oxidative degradation of long-chain or multiple acyl-CoAs, especially when metabolically decompensated. We have emphasized previously that the most valuable applications of the routine Cobas assay are the identification of patients with carnitine deficiency and the monitoring of patients on carnitine therapy (3). We have developed a new carnitine assay method based on isotope-dilution tandem mass spectrometry (TMS), which is inherently more straightforward and accurate than previously reported methods. This is true primarily because it has absolute molecular specificity, uses an isotope-labeled internal standard to compensate for any losses or variance resulting from the sample preparation, and has no known chemical interference. The new assay …