LC–MS/MS‑based simultaneous quantifcation of acylcarnitines, eicosapentaenoic acid, and docosahexaenoic acid: exploring potential biomarkers in human plasma

Acylcarnitines have emerged as valuable markers of the intracellular fatty acid content, mitochondrial functionality, and fatty acid metabolism. However, acylcarnitines derived from polyunsaturated fatty acids (PUFAs) have not been previously examined. To address the need for monitoring lifestyle intervention and omega-3 supplementation studies, a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), eicosapentaenoyl-L-carnitine (EPAC), and docosahexaenoyl-L-carnitine (DHAC). Matrix effects were normalized using a background subtraction approach, and the analytes were extracted from blood plasma via simple protein precipitation with acetonitrile. Chromatographic separation was achieved within 5 min on a reverse-phase C18 column using a gradient mobile phase composed of ammonium acetate and acetonitrile. The limits of quantification of the method were 2 μM for EPA/DHA and 2 nM for EPAC/DHAC. The method exhibited high precision and accuracy, with coefficient of variation and bias values less than 10%. The stability tests confirmed that the analytes remained stable under various conditions, such as remaining up to 6 h at room temperature and refrigeration and undergoing three freeze–thaw cycles; however, the acylcarnitines were unstable during long-term storage. This method is simple, fast, and cost-effective; thus, it is suitable for high-throughput analysis of samples from clinical studies. A PUFA supplement study in healthy volunteers revealed a more pronounced increase in plasma EPAC and DHAC levels than in EPA and DHA levels; these results indicated that PUFA-derived acylcarnitines were potential novel and sensitive markers of PUFA intake.