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

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2002-06, Vol.48 (6), p.826-834
• Main Authors: Vaz, Frederic M, Melegh, Bela, Bene, Judit, Cuebas, Dean, Gage, Douglas A, Bootsma, Albert, Vreken, Peter, van Gennip, Albert H, Bieber, Loran L, Wanders, Ronald J.A
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Carnitine - biosynthesis; Carnitine - metabolism; Carnitine - urine; Chromatography, High Pressure Liquid; Deuterium; Humans; Infant, Newborn; Investigative techniques, diagnostic techniques (general aspects); Lysine - analogs & derivatives; Lysine - metabolism; Male; Medical sciences; Miscellaneous. Technology; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1093/clinchem/48.6.826

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. 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. 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. 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.