Acylcarnitine profiling by low-resolution LC-MS

Abstract

Acylcarnitines are fatty acyl esters of L-carnitine and facilitate the entry of long-chain fatty acids into mitochondria via the carnitine shuttle, where they are metabolized via ß-oxidation. Alterations of acylcarnitine species can be diagnostic for fatty acid oxidation disorders and organic aciduria and are thus frequently used to screen newborns. Only a subfraction of all known acylcarnitines is thereby monitored and quantified. Therefore, a method for the simultaneous fast and robust detection of all known acylcarnitines was developed using a single concise liquid chromatography mass spectrometry (LC-MS) approach. Derivatization by 3-nitrophenylhydrazine increased the signal intensity of the acylcarnitines and a linear elution from a reversed phase column was observed that was dependent on the length of the carbon chain. This allowed a precise prediction of the exact elution time for each acylcarnitine class, which depended solely on the chemical nature of the carbon chain. This method can be further used to screen for yet unknown acylcarnitine species and adds a layer of confidence for their correct identification. Altogether 123 acylcarnitines species were used to establish a targeted low-resolution LC-MS method. The method was applied to acylcarnitine profiling in several mouse tissues and fluids, in order to identify large differences in the quantity and composition of acylcarnitines.

Identification and Distribution of Sterols, Bile Acids, and Acylcarnitines by LC–MS/MS in Humans, Mice, and Pigs—A Qualitative Analysis

“…Moreover, using our method, isomeric acylcarnitines were distinguished solely based on the retention times. As an example, three isomeric acylcarnitines (with monoisotopic mass 245.162 Da) eluted in the following order: 2-methylbutyroylcarnitine, followed by iso-valeryl carnitine and finally valeryl carnitine (Figures 6 and 7), in accordance with the rule that branched carbon chains elute first [30]. However, these three acylcarnitines had the same MS/MS fragmentation pattern and could only be distinguished based on the elution order (Figure 7).…”

Analytical approaches for studying oxygenated lipids in the search of potential biomarkers by LC-MS

“…It is able to increase their chromatographic retention time (RT) on reversedphase columns [100] and resolve better structural isomers and nominal isobaric species in their detection [101,102] and quantitation [97]. This is also the case of derivatization based on dimethylglycinetype esters to selectively label the hydroxy group at C3 in oxysterols (entry 45 [67]) or the formation of acylhydrazones using 3nitrophenylhydrazine (3NPH) and its labeled version [13C6]3NPH (entry 12) [39] for quantitation of acyl-L-carnitines.…”

“…Additionally, it has been performed with the aim of searching specific metabolites or for their structural characterization (including fragmentation pattern) to later move into targeted analysis (entries 4, 22, 31, 39) [26,61,33,55]. Other purposes for moving from non-targeted to targeted analysis include the validation of findings using a different analytical technique (entries 12, 21) [39,47].…”

“…Regarding SP (2OH), they have been studied in metabolic syndrome [31], AD [19], neurological outcome in cardiac arrest [32] and cholangiocarcinoma [33]. As for acyl-L-carnitines, considerable effort has been taken to develop new methods for their comprehensive profiling including CAR (3OH) and CAR (DC) species [20,21,35,38,34,39,36,37]. Various CAR (3OH) and CAR (DC) have been found to be implicated in inborn errors of metabolism [20], diabetes mellitus and metabolic syndrome [21,38,34] among others (Supporting Table S1.1).…”

A facile synthesis of isotope labeled acylcarnitines

“…8 The newborn screening panel uses flow injection tandem mass spectrometry to analyze more than 30 different acylcarnitines and amino acids extracted from dried blood spots on filter papers. 7,9,10 In order to quantitatively determine the concentrations of these metabolites, internal standards with a distinct mass pattern from the native metabolites are required. 11 Ideally, for each target molecule, the corresponding internal standard that shares the same structure and only differs in the isotope labeling should be provided to minimize potential errors caused by matrix effect, ionization efficiency, and other factors.…”