|Novel method for determination of acylcarnitines
|Analysis of Complex Energy Products
|Dr Benjami Jenkins
|Dr Barbora Cervinkova
|University of cambridge
Abstract Information :
Aim: The development of a rapid liquid chromatography separation method hyphenated with accurate mass Orbitrap detection for the semi-quantitative determination of acylcarnitines.
Introduction: Acylcarnitines; as fatty acyl esters of L-carnitine, facilitate the movement of fatty acids into the mitochondria. Their analysis provide biochemical screening for disorders of fatty acid oxidation, also allowing the investigation into the connection between fatty acid metabolism and type 2 diabetes involving possible lipotoxicity affects.
Method: Using a multifaceted sample-preparation procedure to comprehensively extract the diverse range of acylcarnitine species from ex-vivo tissue samples, followed by a targeted sample reconstitution to reduce ion-suppressing lipids whilst maintaining optimal acylcarnitine solubility. Analysis of the acylcarnitines was performed using a Shimadzu Prominence HPLC modular system (Shimadzu UK Limited, Wolverton, United Kingdom) coupled to a Thermo ExactiveTM Orbitrap mass spectrometer (Thermo Fisher Scientific, Hemel Hempstead, United Kingdom). Optimal separation was achieved using an ACE Excel C18-PFP (150mm x 2.1mm, 2 µm) liquid chromatography column employing a binary mobile phase system of water (0.1% formic acid) and acetonitrile (0.1% formic acid) across a hyperbolic aqueous to organic gradient, over a total 6 minute run time. The mobile phase gradient separated acylcarnitine species from free carnitine (C0) to arachidyl carnitine (C20), including mono-, poly-unsaturated and hydroxy-carnitine (OH) species. Accurate mass detection of the protonated acylcarnitines determined the identification and quantification. Monitoring of the ubiquitous daughter fragment ion (85.028 Da, C4H5O2+) quality controlled the identification of the peak during the method development.
Conclusion: This LC-MS method was sensitive enough to comprehensively detect the full profile of acylcarnitine species within ex-vivo muscle and heart tissue samples without applying a complicated derivatisation step into the sample preparation. Chromatographic separation of the acylcarnitine species ensured low abundance analytes were not suppressed by highly abundant signals, this was especially necessary when utilising an accurate mass Orbitrap mass spectrometer for the detector.