Quantification of Carnitine and Acylcarnitines in Biological Matrices by HPLC Electrospray Ionization- Mass Spectrometry

Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional i...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 2008-09, Vol.54 (9), p.1451-1462
Main Authors: Minkler, Paul E, Stoll, Maria S. K, Ingalls, Stephen T, Yang, Shuming, Kerner, Janos, Hoppel, Charles L
Format: Article
Language:English
Subjects:
Acetylation
Analytical chemistry
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Calibration
Carnitine - analysis
Carnitine - chemistry
Carnitine - metabolism
Chemicals
Chromatography, High Pressure Liquid - methods
Fundamental and applied biological sciences. Psychology
Humans
Investigative techniques, diagnostic techniques (general aspects)
Mass spectrometry
Medical sciences
Metabolic disorders
Molecular Structure
Plasma
Spectrometry, Mass, Electrospray Ionization - methods
Tandem Mass Spectrometry - methods
Tissues
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Summary:Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished. Carnitine and acylcarnitines were isolated by ion-exchange solid-phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, separated by HPLC, and detected with an ion trap mass spectrometer. Carnitine was quantified with d(3)-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d(6)-acetyl-, d(3)-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine. Example recoveries [mean (SD)] were 69.4% (3.9%) for total carnitine, 83.1% (5.9%) for free carnitine, 102.2% (9.8%) for acetylcarnitine, and 107.2% (8.9%) for palmitoylcarnitine. Example imprecision results [mean (SD)] within runs (n = 6) and between runs (n = 18) were, respectively: total carnitine, 58.0 (0.9) and 57.4 (1.7) micromol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) micromol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) micromol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) micromol/L. Standard-addition slopes and linear regression coefficients were 1.00 and 0.9998, respectively, for total carnitine added to plasma, 0.99 and 0.9997 for free carnitine added to plasma, 1.04 and 0.9972 for octanoylcarnitine added to skeletal muscle, and 1.05 and 0.9913 for palmitoylcarnitine added to skeletal muscle. Reference intervals for plasma, urine, and skeletal muscle are provided. This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.
ISSN:0009-9147
1530-8561
DOI:10.1373/clinchem.2007.099226