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HomeSmall Molecule HPLCSynthesis & HILIC/MS Analysis of Acylcarnitines

Synthesis & HILIC/MS Analysis of Acylcarnitines

Rudolf Köhling, Markus Obkirche

Reporter US Volume 34.2

Introduction

L-Carnitine was discovered at the beginning of the last century and plays an important role in fatty acid metabolic pathways. It acts as a carrier of long chain acyl groups from activated fatty acids across the inner mitochondrial membrane into the mitochondrial matrix where they undergo β-oxidation to acetyl CoA to obtain usable energy via the citric acid cycle.The resulting “acyl-L-carnitine” molecule is shown in Figure 1. A number of diseases caused by defects of mitochondrial transport are characterized by specific metabolic dysfunctions and depend on the physiological role of the affected carrier in intermediary metabolism.2 Therefore, the functions and roles of acyl-L-carnitines in various tissues like brain, heart, and muscle continue to attract much interest.3-6

Acyl-L-Carnitine Parent Structure R represents acyl chains of varying length derived from the transported fatty acid.

Figure 1.Acyl-L-Carnitine Parent Structure R represents acyl chains of varying length derived from the transported fatty acid.

Analytical Requirements

Newborn screening programs detect treatable disorders in infants before they become symptomatic. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) has greatly increased the screening possibilities in newborn screening programs by monitoring levels of acylcarnitines in order to detect treatable disorders in infants before symptoms appear. LC/MS/MS can detect several disorders with a single injection, which is important in high throughput laboratories. Measuring different acylcarnitines can be used to detect more than 40 different inborn errors of metabolism, a selection of which is shown in Table 1.

Pure and stable metabolite standards as well as robust and sensitive methods for their analysis are prerequisites for investigating the functions of healthy and diseased biological cells on a molecular pathway level. Human metabolic phenotyping in relation to clinical diagnostics, prognostics and epidemiology is therefore one of the most widely applicable areas for the development of precision medicine.In order to develop new analytical methods, the synthetic methodologies for the required metabolites need to be established.8

Chromosome GenotypesGene/LocusMIM No.*PhenotypeMIM No.*
3p21.31SLC25A20613698Carnitine-Acylcarnitine Translocase Deficiency (CACTD)212138
1p32.3CPT2600650Susceptibility to acute, infection-induced Encephalopathy (IIAE4)614212
2p23.3HADHA600890Long-chain 3-Hydroxyacyl-CoA dehydrogenase Deficiency609016
11q25ACAD8604773Isobutyryl-CoA dehydrogenase Deficiency611283
1p31.1ACADM607008Medium chain Acyl-CoA dehydrogenase Deficiency (ACADMD)201450
8q24.3SLC52A2607882Brown-Vialetto-Van Laere syndrome 2 (BVVLS2)614707
4q32.1ETFDH231675Multiple Acyl-CoA dehydrogenase Deficiency IIC (MADD) Glutaric acidemia231680
19p13.2CD320606475Methylmalonic aciduria due to transcobalamin receptor defect613646
4q25HADHSC601609Familial Hyperinsulinemic hypoglycemia 4609975
11q13.3CPT1A600528Carnitine Palmioyltransferase I Deficiency hepatic, type 1A255120
10q26.13ACADSB6003012-Methylbutyryl-CoA dehydrogenase Deficiency, 2-Methylbutyrylglycinuria610006
Table 1.Selected Acyl-L-Carnitines for Genotype-Phenotype-Relationships in Inborn Errors of Metabolism

Synthesis of Acyl-L-Carnitines

The expansion of the Sigma-Aldrich portfolio of acyl-L-carnitine standards has been successfully achieved by a generalized synthetic approach. L-Carnitine hydrochloride was added to a mixture of the appropriate carboxylic acid and a slight molar excess of acid chloride. The reaction mixture was kept at an elevated temperature until TLC analysis showed an optimum ratio of the corresponding product to its starting material and side products.9 If the suitable acid chloride was not available, the carboxylic acid was reacted in a first step with thionyl chloride and then the carnitine was added consecutively.

To avoid counter ions (e. g. chloride), which are undesirable in some applications, all acyl-L-carnitines were transformed to their inner salts by a subsequent treatment on a weakly basic anion exchanger.10 After precipitation of the raw product, column chromatography was required to remove any traces of carnitine and side products.

LC/MS Analysis of Acyl-L-Carnitines

Since UV detection of acylcarnitines is very insensitive, a method to allow separation, identification, and quantification with complementary detectors, like mass spectrometry (MS) or charged aerosol detection (CAD), is required. Detection by MS provides the required sensitivity, and direct infusion may be sufficient to analyze a range of different acyl-L-carnitines. However, for more detailed analysis and for measuring acylcarnitine isomers that are closely related, separation by liquid chromatography before detection is important.11,12 Liquid chromatography-mass spectrometry has been established as an efficient and robust methodology for analyzing acyl-L-carnitines and hydrophilic interaction liquid chromatography (HILIC) to separate free carnitine and acylcarnitines is fast and does not need derivatization.13-15 In this study, HILIC mode chromatography was explored using an Ascentis® Express OH5 column with a gradient of acetonitrile in 50 mm ammonium acetate. LC and MS instrument and settings appear in Table 2 and Table 3. Final chromatographic conditions appear in Figure 2.

LC InstrumentDionex® UltiMate® 3000 RSLC
Equilibration Time10 min
MS InstrumentBruker micrOTOF-Q II™
MS ScanTime 0 to 20, ESI(+)
Gas Flow
Temperatures
Dry Gas9 L/h, 250 °C
Voltages
Capillary (kV)4.5
Table 2.LC/MS Instrument and Setting
AnalyteMol. WeightMax m/z
Stearoyl-L-Carnitine427.66428.371
Palmitoyl-L-Carnitine399.61400.3401
Myristoyl-L-Carnitine371.4372.3091
Lauroyl-L-Carnitine343.5344.2786
Decanoyl-L-Carnitine315.45316.2471
Octanoyl-L-Carnitine287.4288.2156
Hexanoyl-L-Carnitine259.3260.1846
Isovaleryl-L-Carnitine245.32246.17
2-Methylbutyryl-L-Carnitine245.32246.17
Isobutyryl-L-Carnitine231.29232.1529
Butyryl-L-Carnitine231.29232.1529
Propionyl-L-Carnitine217.26218.1375
Acetyl-L-Carnitine HCl203.6204.1228
L-Carnitine161.2162.1107
Table 3.LC-MS Analysis of Selected Acyl-L-Carnitines
LC/MS Analysis of Twelve Acyl-L-Carnitines on Ascentis Express OH5 (HILIC Mode)

Figure 2.LC/MS Analysis of Twelve Acyl-L-Carnitines on Ascentis Express OH5 (HILIC Mode)

Materials
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