Simultaneous quantification of acylcarnitine isomers containing dicarboxylic acylcarnitines in human serum and urine by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract

Tandem mass spectrometry (MS/MS) has become a prominent method for screening newborns for diseases such as organic acidemia and fatty acid oxidation defects, although current methods cannot separate acylcarnitine isomers. Accurate determination of dicarboxylic acylcarnitines such as methylmalonylcarnitine and glutarylcarnitine has not been carried out, because obtaining standards of these acylcarnitines is difficult. We attempted the individual determinations of acylcarnitines with isomers and dicarboxylic acylcarnitines by applying high-performance liquid chromatography (HPLC). Chromatographic separation was performed by gradient elution using a mixture of 0.08% aqueous ion-pairing agent and acetonitrile as the mobile phase. Mass transitions of m/z 161.8-->84.8 for carnitine and m/z 164.8-->84.8 for deuterated carnitine were monitored in positive ion electrospray ionization mode. One carnitine and 16 acylcarnitines were quantified. The limit of quantitation (LOQ) was 0.1 micromol/L for methylmalonylcarnitine and 0.05 micromol/L for the other acylcarnitines. Intra-day and inter-day coefficients of variance (CVs) were <8.3% and <8.8%, respectively, for all acylcarnitines in serum, and both were <9.2% in urine. Mean recoveries were >90% for all acylcarnitines. Human samples were quantified by this method. After addition of deuterated acylcarnitines as internal standards, acylcarnitines in serum or urine were extracted using a solid-phase extraction cartridge. In healthy adult individuals, isobutyryl-, 2-methylbutyryl- and isovalerylcarnitine were detected in serum and urine. Dicarboxylic acylcarnitines were detected in urine. High concentrations of methylmalonylcarnitine and propionylcarnitine were found in both the serum and the urine of a patient with methylmalonic acidemia. The described HPLC/MS/MS method could separate most acylcarnitine isomers and quantify them, potentially allowing detailed diagnoses and follow-up treatment for those diseases.

Quantification of Carnitine and Acylcarnitines in Biological Matrices by HPLC Electrospray Ionization– Mass Spectrometry

“…The described cation-exchange solid-phase extraction process isolates both carnitine and acylcarnitines with high recoveries yet excludes potential interferences [e.g., this procedure removes palmitic acid (23 )]. Other investigators who also have used cation-exchange solid-phase extraction have reported high recoveries of acylcarnitines from urine and plasma (28 ). The use of HPLC separation in acylcarnitine analysis before detection has been shown to distinguish false positives from true positives (29 ), and our sequential ion-exchange/reversed-phase HPLC method adds even more selectivity (23 ).…”

Acylcarnitine Profiles during Carnitine Loading and Fasting Tests in a Japanese Patient with Medium-Chain Acyl-CoA Dehydrogenase Deficiency

“…Acylcarnitines (acetylcarnitine [C2], C6, C8-N, C10) were quantifi ed using our HPLC-ESI/MS-MS method (Maeda et al 2007). These acylcarnitines and labeled acylcarnitines used as internal standard (IS) were prepared by reacting carnitine hydrochloride with octanoylchloride or decanoylchloride in trifl uoroacetic acid.…”

“…The current newborn screening using ESI-MS/MS cannot separate acylcarnitine isomers to obtain accurate diagnosis and detailed metabolic status. To resolve this issue, we developed a new analytical method using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) which can quantify most acylcarnitine isomers (Maeda et al 2007). For example, the C8 signal for the diagnosis of MCADD covers the isomers n-octanoylcarnitine (C8-N) and valproylcarnitine (C8-V).…”

Application of a Second-Tier Newborn Screening Assay for C5 Isoforms

“…The second-tier test described allows rapid separation and quantification of various C5 acylcarnitines which may underlie elevated C5 levels in routine newborn screening in the original blood sample (Janzen et al 2013). Maeda et al (2008Maeda et al ( , 2007 previously described the chromatographic separation of acylcarnitines from serum and urine. However, their method needs a second sample from the patients (urine or serum) and the sample preparation requires the use of solid phase extraction material (SPE).…”