Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

• Published in: Analytical and bioanalytical chemistry 2013-11, Vol.405 (27), p.8829-8836
• Main Authors: Morand, Réjane, Donzelli, Massimiliano, Haschke, Manuel, Krähenbühl, Stephan
• Format: Article
• Language: English
• Subjects: Acetylcarnitine - blood; Amino acids; Analytical Chemistry; Biochemistry; Carnitine; Carnitine - analogs & derivatives; Carnitine - blood; Cation exchange; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatography; Chromatography, High Pressure Liquid; Extraction; Food Science; Humans; Laboratory Medicine; Limit of Detection; Liquid chromatography; Mass spectrometry; Measurement; Metabolic disorders; Methanol - chemistry; Methods; Methyl alcohol; Monitoring/Environmental Analysis; On-line systems; Online; Palmitoylcarnitine - blood; Physiological aspects; Plasma; Reproducibility of Results; Research Paper; Scientific imaging; Solid Phase Extraction - methods; Toxicology; Trapping
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-013-7309-z

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine. Figure A method is presented for the analysis of carnitine and acylcarnitines in urine which includes a precipitation step, on-column extraction and LC-MS/MS. The run time is 8 minutes and the method was validated for carnitine, acetylcarnitine, octanoylcarnitine and palmitoylcarnitine. Analysis of a patient sample with medium-chain acyl-CoA dehydrogenase deficiency is shown.