Validated Method for the Quantification of Free and Total Carnitine, Butyrobetaine, and Acylcarnitines in Biological Samples

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 trifluoromethane...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2015-09, Vol.87 (17), p.8994-9001
Main Authors: Minkler, Paul E, Stoll, Maria S. K, Ingalls, Stephen T, Kerner, Janos, Hoppel, Charles L
Format: Article
Language:English
Subjects:
Accuracy
Analytical chemistry
Animals
Betaine - analogs & derivatives
Betaine - analysis
Betaine - blood
Betaine - urine
Biological
Carnitine
Carnitine - analogs & derivatives
Carnitine - analysis
Carnitine - blood
Carnitine - urine
Cations
Chromatography
Chromatography, High Pressure Liquid
Diabetes Mellitus, Experimental - diagnosis
Extraction
Humans
Ionization
Liquid chromatography
Mass spectrometry
Medical services
Mesylates - chemistry
Muscle, Skeletal - chemistry
Rats
Solid Phase Extraction
Spectrometry, Mass, Electrospray Ionization
Trimethylsilyl Compounds - chemistry
Validation studies
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Summary: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 trifluoromethanesulfonate, (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.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.5b02198