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

Abstract

AbstractBackground: 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.