Synthesis and HILIC/MS Analysis of Acylcarnitines

By: 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.1 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

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.7 In order to develop new analytical methods, the synthetic methodologies for the required metabolites need to be established.8

Table 1. Selected Acyl-L-Carnitines for Genotype-Phenotype-Relationships in Inborn Errors of Metabolism

Chromosome Genotypes Gene/Locus MIM No.* Phenotype MIM No.*
3p21.31 SLC25A20 613698 Carnitine-Acylcarnitine Translocase Deficiency (CACTD) 212138
1p32.3 CPT2 600650 Susceptibility to acute, infection-induced Encephalopathy (IIAE4) 614212
2p23.3 HADHA 600890 Long-chain 3-Hydroxyacyl-CoA dehydrogenase Deficiency 609016
11q25 ACAD8 604773 Isobutyryl-CoA dehydrogenase Deficiency 611283
1p31.1 ACADM 607008 Medium chain Acyl-CoA dehydrogenase Deficiency (ACADMD) 201450
8q24.3 SLC52A2 607882 Brown-Vialetto-Van Laere syndrome 2 (BVVLS2) 614707
4q32.1 ETFDH 231675 Multiple Acyl-CoA dehydrogenase Deficiency IIC (MADD) Glutaric acidemia 231680
19p13.2 CD320 606475 Methylmalonic aciduria due to transcobalamin receptor defect 613646
4q25 HADHSC 601609 Familial Hyperinsulinemic hypoglycemia 4 609975
11q13.3 CPT1A 600528 Carnitine Palmioyltransferase I Deficiency hepatic, type 1A 255120
10q26.13 ACADSB 600301 2-Methylbutyryl-CoA dehydrogenase Deficiency, 2-Methylbutyrylglycinuria 610006

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.

Table 2. LC/MS Instrument and Setting

LC Instrument Dionex® UltiMate® 3000 RSLC
Equilibration Time 10 min
MS Instrument Bruker micrOTOF-Q II™
MS Scan Time 0 to 20, ESI(+)
Gas Flow
Temperatures
Dry Gas 9 L/h, 250 °C
Voltages
Capillary (kV) 4.5


Table 3. LC-MS Analysis of Selected Acyl-L-Carnitines

Analyte Mol. Weight Max m/z
Stearoyl-L-Carnitine 427.66 428.371
Palmitoyl-L-Carnitine 399.61 400.3401
Myristoyl-L-Carnitine 371.4 372.3091
Lauroyl-L-Carnitine 343.5 344.2786
Decanoyl-L-Carnitine 315.45 316.2471
Octanoyl-L-Carnitine 287.4 288.2156
Hexanoyl-L-Carnitine 259.3 260.1846
Isovaleryl-L-Carnitine 245.32 246.17
2-Methylbutyryl-L-Carnitine 245.32 246.17
Isobutyryl-L-Carnitine 231.29 232.1529
Butyryl-L-Carnitine 231.29 232.1529
Propionyl-L-Carnitine 217.26 218.1375
Acetyl-L-Carnitine HCl 203.6 204.1228
L-Carnitine 161.2 162.1107

 

 LC/MS Analysis of Twelve Acyl-L-Carnitines

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

Results and Conclusion

The Ascentis Express OH5 column provided adequate retention and selectivity under HILIC conditions to resolve the majority of the acyl-L-carnitines tested. It may be possible to use MS/MS and particular fragment ions to resolve the two coeluting pairs. As opposed to reversed-phase analysis, in HILIC the analytes elute in the order of decreasing hydrophobicity, or, in this example, decreasing carbon chain length. The acetonitrile and aqueous ammonium acetate mobile phases used are compatible with both MS and CAD detection. For CAD detection the introduction of a reverse gradient post-column is recommended to improve the sensitivity and reduce baseline drift.

We offer an increasing range of acyl-L-carnitines along with the analytical reagents and HPLC columns for their reliable, sensitive analysis by LC/MS.

Materials

     

 References

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