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Clinical Chemistry 48: 826-834, 2002;
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(Clinical Chemistry. 2002;48:826-834.)
© 2002 American Association for Clinical Chemistry, Inc.

Analysis of Carnitine Biosynthesis Metabolites in Urine by HPLC–Electrospray Tandem Mass Spectrometry

Frédéric M. Vaz1a, Bela Melegh2, Judit Bene2, Dean Cuebas3, Douglas A. Gage4, Albert Bootsma1, Peter Vreken1, Albert H. van Gennip1, Loran L. Bieber5 and Ronald J.A. Wanders1

1 Academic Medical Center, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital, PO Box 22700, 1100 DE Amsterdam, The Netherlands.

2 Clinical Genetics Working Group of Hungarian Academy of Sciences at University of Pécs, Department of Medical Genetics and Child Development, H-7623 Pécs, Hungary.

3 Department of Chemistry, Southwest Missouri State University, Springfield, MO 65804.

4 MSU-NIH Mass Spectral Facility and
5 Department of Biochemistry, Michigan State University, East Lansing, MI 48824.

aAddress correspondence to this author at: University of Amsterdam, Academic Medical Center, Departments of Clinical Chemistry and Pediatrics, Laboratory for Genetic Metabolic Diseases (F0-224), PO Box 22700, 1100 DE Amsterdam, The Netherlands. Fax 31-20-6962596; e-mail f.m.vaz{at}amc.uva.nl.

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. {gamma}-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, {gamma}-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.




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Correction
Frederic M Vaz
Clinical Chemistry Online, 17 Jun 2002 [Full text]

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