Development of a novel method for the analysis of acyl carnitine profile using liquid chromatography mass spectrometry

Abstract

Carnitine is a low-molecular-weight compound that has an obligate role in the mitochondrial oxidation of long-chain fatty acids (acyl groups) and energy production. Therefore, acylcarnitines are essential diagnostic markers for complex diseases, fatty acid metabolism disorders, and disorders related to amino acids metabolism. Biochemical screening of the acylcarnitine profile is performed in clinical laboratories using tandem mass spectrometry as the analytical platform. Analysis of acylcarnitine species can become challenging because several species occur in an isomeric form. A selective and sensitive UHPLC-MS/MS method has been established to analyze an acylcarnitine profile of acylcarnitines with acyl-chain lengths C4-C18. The method includes four isomeric species, two C4 isomers (butyrylcarnitine and isobutyrylcarnitine) and two C5 isomers (2-methylbutyrylcarnitine and isovalerylcarnitine). The analysis was conducted on a Waters Acquity ultra performance liquid chromatograph (UPLC) coupled with a triple quadrupole mass spectrometer operated in electrospray positive mode. Optimization of separation involved testing various parameters such as mobile phase composition, additives, and effect of ion-pairing reagents. The separation efficiency of the C18 column was compared to amide column to investigate the optimal settings for separation of short-chain polar acylcarnitines. The chromatographic separation was achieved using ACQUITY UPLC HSS C18 (1.8 µm, 2.1 x 100 mm) column. Gradient elution was accomplished with a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. High specificity was obtained with MRM operation mode of MS instrumentation. The process of development MRM method involved a series of testing various parameters such as cone voltage, collision energy and flow rate of the collision gas to achieve the optimal set of conditions. Fragmentation of all acylcarnitines provides a characteristic fragment ion of m/z 85. The limit of detection of the acylcarnitine esters including the isomeric species was 10 pg/ml when measured with the optimized MRM method.