Journal
ANALYTICAL CHEMISTRY
Volume 87, Issue 17, Pages 8994-9001Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.5b02198
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- Center for Mitochondrial Diseases in the Department of Pharmacology at Case Western Reserve University School of Medicine
- Center for Inherited Disorders of Energy Metabolism (CIDEM), University Hospitals Case Medical Center, Cleveland, OH
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A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four. components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoroinethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.
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