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 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.
Center for Mitochondrial Diseases, †Department of Pharmacology and ‡Department of Medicine, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
Paul E
PE
Minkler
Center for Mitochondrial Diseases, †Department of Pharmacology and ‡Department of Medicine, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
Maria S K
MS
Stoll
Center for Mitochondrial Diseases, †Department of Pharmacology and ‡Department of Medicine, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
Stephen T
ST
Ingalls
Center for Mitochondrial Diseases, †Department of Pharmacology and ‡Department of Medicine, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
Janos
J
Kerner
Center for Mitochondrial Diseases, †Department of Pharmacology and ‡Department of Medicine, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
Charles L
CL
Hoppel
D000818:Animals; D001622:Betaine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood / Q000652:urine; D002331:Carnitine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D003921:Diabetes Mellitus, Experimental / Q000175:diagnosis; D006801:Humans; D008698:Mesylates / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry*; D051381:Rats; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization; D014297:Trimethylsilyl Compounds / Q000737:chemistry
D008698:Mesylates; D014297:Trimethylsilyl Compounds; C116917:acylcarnitine; C053353:trimethylsilyl trifluoromethanesulfonate; D001622:Betaine; C002889:gamma-butyrobetaine; D002331:Carnitine