Analysis of carnitine biosynthesis metabolites in urine by HPLC-electrospray tandem mass spectrometry

Frédéric M. Vaz, Bela Melegh, Judit Bene, Dean Cuebas, Douglas A. Gage, Albert Bootsma, Peter Vreken, Albert H. Van Gennip, Loran L. Bieber, Ronald J.A. Wanders

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Background: We developed a method to determine the urinary concentrations of metabolites in the synthetic pathway for carnitine from N6-trimethyllysine and applied this method to determine their excretion in control individuals. In addition, we investigated whether newborns are capable of carnitine synthesis from deuterium-labeled N6-trimethyllysine. Methods: Urine samples were first derivatized with methyl chloroformate. Subsequently, the analytes were separated by ion-pair, reversed-phase HPLC and detected online by electrospray tandem mass spectrometry. Stable-isotope-labeled reference compounds were used as internal standards. Results: The method quantified all carnitine biosynthesis metabolites except 4-N-trimethylaminobutyraldehyde. Detection limits were 0.05-0.1 μmol/L. The interassay imprecision (CV) for urine samples with added compounds was 6-12%. The intraassay imprecision (CV) was 1-5% (3-10 μmol/L). Recoveries were 94-106% at 10-20 μmol/L and 98-103% at 100-200 μmol/L. The mean (SD) excretions of N6-trimethyllysine and 3-hydroxy-N6-trimethyllysine were 2.8 (0.8) and 0.45 (0.15) mmol/mol creatinine, respectively. γ-Butyrobetaine and carnitine excretions were more variable with values of 0.27 (0.21) and 15 (12) mmol/mol creatinine, respectively. After oral administration of deuterium-labeled N6-trimethyllysine, all urines of newborns contained deuterium-labeled N6-trimethyllysine, 3-hydroxy-N6-trimethyllysine, γ-butyrobetaine, and carnitine. Conclusions: HPLC in combination with electrospray ionization tandem mass spectrometry allows rapid determination of urinary carnitine biosynthesis metabolites. Newborns can synthesize carnitine from exogenous N6-trimethyllysine, albeit at a low rate.

Original languageEnglish
Pages (from-to)826-834
Number of pages9
JournalClinical chemistry
Issue number6
Publication statusPublished - Jan 1 2002

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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    Vaz, F. M., Melegh, B., Bene, J., Cuebas, D., Gage, D. A., Bootsma, A., Vreken, P., Van Gennip, A. H., Bieber, L. L., & Wanders, R. J. A. (2002). Analysis of carnitine biosynthesis metabolites in urine by HPLC-electrospray tandem mass spectrometry. Clinical chemistry, 48(6), 826-834.