Abstract
Omega-3 polyunsaturated fatty acids (PUFAs) are important dietary supplements, and their
concentrations are commonly measured to assess PUFA intake from food and supplements.
Increasing evidence indicates that acylcarnitines are markers of the intracellular fatty acid
content, mitochondrial functionality, and fatty acid metabolism. However, the role of PUFAderived acylcarnitines have not been extensively investigated.
To address the need for comprehensive monitoring of PUFAs and their acylcarnitines in
human plasma, we developed and validated a novel liquid chromatography–tandem mass
spectrometry (LC–MS/MS) method for the simultaneous quantification of eicosapentaenoic
acid (EPA), docosahexaenoic acid (DHA), eicosapentaenoyl-L-carnitine (EPAC), and
docosahexaenoyl-L-carnitine (DHAC). Matrix effects were corrected using a background
subtraction approach, and analytes were extracted from blood plasma via simple protein
precipitation with acetonitrile. Detection was performed in multiple reaction monitoring
(MRM) mode using an electrospray ionization source. Chromatographic separation was
achieved in 5 minutes using a reverse-phase C18 column and a gradient mobile phase consisting
of ammonium acetate and acetonitrile.
The method demonstrated high precision and accuracy, with limits of quantification being 2
μM for EPA/DHA and 2 nM for EPAC/DHAC, and coefficient of variation (CV) and bias
values below 10%. The recovery efficiency ranged from 81% to 100%. Stability tests confirmed
that EPA, DHA, EPAC, and DHAC remained stable under various conditions, including up to
6 hours at room temperature and refrigeration, as well as through three freeze-thaw cycles.
However, EPAC and DHAC were unstable during long-term storage. The developed method
was successfully applied to plasma samples from healthy volunteers. Before fish oil
administration, the plasma EPA concentration in healthy volunteers was 4.0 ± 0.5 μM, the DHA
concentration was 7.1 ± 0.5 μM, the EPAC concentration was 3.2 ± 0.3 nM, and the DHAC
concentration was 8.4 ± 0.8 nM. After two weeks of supplementation, EPA and DHA increased
by 20% and 44%, respectively, but decreased below baseline after an additional two weeks. In
contrast, EPAC and DHAC concentrations increased by 60% and remained elevated throughout
the study.
This method is simple, rapid, and cost-effective, making it suitable for high-throughput
analysis in clinical studies. When applied to plasma samples from healthy volunteers, the
method revealed a more pronounced increase in EPAC and DHAC levels compared to EPA and
DHA. These findings suggest that PUFA-derived acylcarnitines could serve as novel and
sensitive biomarkers for PUFA intake.
concentrations are commonly measured to assess PUFA intake from food and supplements.
Increasing evidence indicates that acylcarnitines are markers of the intracellular fatty acid
content, mitochondrial functionality, and fatty acid metabolism. However, the role of PUFAderived acylcarnitines have not been extensively investigated.
To address the need for comprehensive monitoring of PUFAs and their acylcarnitines in
human plasma, we developed and validated a novel liquid chromatography–tandem mass
spectrometry (LC–MS/MS) method for the simultaneous quantification of eicosapentaenoic
acid (EPA), docosahexaenoic acid (DHA), eicosapentaenoyl-L-carnitine (EPAC), and
docosahexaenoyl-L-carnitine (DHAC). Matrix effects were corrected using a background
subtraction approach, and analytes were extracted from blood plasma via simple protein
precipitation with acetonitrile. Detection was performed in multiple reaction monitoring
(MRM) mode using an electrospray ionization source. Chromatographic separation was
achieved in 5 minutes using a reverse-phase C18 column and a gradient mobile phase consisting
of ammonium acetate and acetonitrile.
The method demonstrated high precision and accuracy, with limits of quantification being 2
μM for EPA/DHA and 2 nM for EPAC/DHAC, and coefficient of variation (CV) and bias
values below 10%. The recovery efficiency ranged from 81% to 100%. Stability tests confirmed
that EPA, DHA, EPAC, and DHAC remained stable under various conditions, including up to
6 hours at room temperature and refrigeration, as well as through three freeze-thaw cycles.
However, EPAC and DHAC were unstable during long-term storage. The developed method
was successfully applied to plasma samples from healthy volunteers. Before fish oil
administration, the plasma EPA concentration in healthy volunteers was 4.0 ± 0.5 μM, the DHA
concentration was 7.1 ± 0.5 μM, the EPAC concentration was 3.2 ± 0.3 nM, and the DHAC
concentration was 8.4 ± 0.8 nM. After two weeks of supplementation, EPA and DHA increased
by 20% and 44%, respectively, but decreased below baseline after an additional two weeks. In
contrast, EPAC and DHAC concentrations increased by 60% and remained elevated throughout
the study.
This method is simple, rapid, and cost-effective, making it suitable for high-throughput
analysis in clinical studies. When applied to plasma samples from healthy volunteers, the
method revealed a more pronounced increase in EPAC and DHAC levels compared to EPA and
DHA. These findings suggest that PUFA-derived acylcarnitines could serve as novel and
sensitive biomarkers for PUFA intake.
| Original language | English |
|---|---|
| Pages | 153 |
| Publication status | Published - 2025 |
| Event | FEBS3+ Baltics: Biochemistry at the Fore Line - Vilnius University's Life Sciences Center, Vilnius, Lithuania Duration: 23 Apr 2025 → 25 Apr 2025 https://www.febs3vilnius.lt/ |
Conference
| Conference | FEBS3+ Baltics: Biochemistry at the Fore Line |
|---|---|
| Country/Territory | Lithuania |
| City | Vilnius |
| Period | 23/04/25 → 25/04/25 |
| Internet address |
Field of Science*
- 3.1 Basic medicine
- 3.2 Clinical medicine
Publication Type*
- 3.4. Other publications in conference proceedings (including local)
