Rapid measurement of plasma acylcarnitines by liquid chromatography-tandem mass spectrometry without derivatization

Abstract

Background: Tandem mass spectrometry (MS/MS) is being increasingly used to identify and measure acylcarnitines in blood and urine of children suspected of having fatty oxidation disorders and other inborn errors of metabolism. Rapid MS/MS analysis requires simple and efficient sample preparation. We developed a LC-MS/MS method for the online extraction of acylcarnitines in plasma without derivatization that requires only precipitation of proteins by acetonitrile followed by centrifugation, thus increasing efficiency.

Methods: An API-3000 tandem mass spectrometer (SCIEX, Toronto, Canada) equipped with electrospray ionization (ESI), TurboIon Spray source, three Shimadzu LC10AD micropumps and autosampler (Shimadzu Scientific Instruments, Columbia, MD) was used to perform the analysis. Within-day and between-day imprecision was evaluated for 10 analytes in the MRM mode using 3 levels of controls. Accuracy was determined by comparing the method with another MS/MS procedure and by recovery experiments. Sensitivity and specificity were evaluated by identifying patient samples under a wide variety of clinical conditions.

Results: Within-day CVs was <10% for all analytes tested and between-day CVs ranged from 4.4% to 14.2%. The method was linear in the range between 1.0 and 100 micromol/l for C2 and 0.1 and 10 micromol/l for the other acylcarnitines. The results of the comparison study yielded r values ranging between 0.948 and 0.999. Recovery ranged from 84% to 112%. The method correctly identified patients with a variety of fatty acid oxidation disorders and organic acidemias.

Conclusions: Our method is a simple procedure for the analysis of acylcarnitines in plasma with minimal sample preparation. It is thus ideal in a routine clinical setting where efficient processing of clinical samples is necessary to reduce turnaround time under conditions of high-throughput.