Analysis of carnitine biosynthesis metabolites in urine by HPLC-electrospray tandem mass spectrometry.
BACKGROUND
We developed a method to determine the urinary concentrations of metabolites in the synthetic pathway for carnitine from N(6)-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 N(6)-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 micromol/L. The interassay imprecision (CV) for urine samples with added compounds was 6-12%. The intraassay imprecision (CV) was 1-5% (3-10 micromol/L). Recoveries were 94-106% at 10-20 micromol/L and 98-103% at 100-200 micromol/L. The mean (SD) excretions of N(6)-trimethyllysine and 3-hydroxy-N(6)-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 N(6)-trimethyllysine, all urines of newborns contained deuterium-labeled N(6)-trimethyllysine, 3-hydroxy-N(6)-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 N(6)-trimethyllysine, albeit at a low rate.
Vaz
Frédéric M
FM
Academic Medical Center, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Department of Clinical Chemistry, Emma Children's Hospital, PO Box 22700, 1100 DE Amsterdam, The Netherlands. f.m.vaz@amc.uva.nl
Melegh
Bela
B
Bene
Judit
J
Cuebas
Dean
D
Gage
Douglas A
DA
Bootsma
Albert
A
Vreken
Peter
P
van Gennip
Albert H
AH
Bieber
Loran L
LL
Wanders
Ronald J A
RJ
D002331:Carnitine / Q000096:biosynthesis / Q000378:metabolism* / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium; D006801:Humans; D007231:Infant, Newborn; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization
C003712:trimethyllysine; D003903:Deuterium; D008239:Lysine; D002331:Carnitine