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

Departments of¹ Pharmacology, ² Medicine, and ³ Nutrition, Case Western Reserve University School of Medicine, Cleveland, OH.

^aAddress correspondence to this author at: Case Western Reserve University School of Medicine, Department of Pharmacology (4965), 10900 Euclid Ave., Cleveland, OH 44106. Fax (216) 368-5162; e-mail charles.hoppel{at}case.edu.

Background: 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.

Methods: 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 d₃-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d₆-acetyl-, d₃-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine.

Results: 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) µmol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) µmol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) µmol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) µmol/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.

Conclusions: This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.

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