4.5. Blood Sample Collection and Analytical Method Development
BLOOD
Blood sampling was meticulously conducted across a total of 18 distinct time points for each participant, encompassing both pre-dose and post-dose periods. These samples were obtained via a heparin-locked catheter inserted into the participant’s vein, ensuring a controlled and consistent collection process. Throughout the trial, a cumulative volume of 280 mL of blood was meticulously gathered for analysis. The collected blood was subjected to centrifugation at 3000 rpm and 4 °C for a duration of 10 min to isolate the plasma. This plasma was then carefully stored in a refrigerator at −80 °C until transfer to a specialized clinical trial laboratory, DT&CRO in Yongin, Republic of Korea, where further analysis was conducted.To prepare the plasma samples for analysis, a systematic protocol was followed. Initially, 50 mL of plasma was combined with 10 µL of DHA-d5 (100 g/mL), 50 µL of 10% Tween 80, and 500 µL of acetonitrile within a glass tube. The mixture underwent vortexing at 2500 rpm for a duration of 3 min. Subsequently, it was treated with a 500 µL combination of hydrochloric acid and acetonitrile (1:4, The measurement of the total plasma concentration of EPA and DHA involved the use of sophisticated equipment. An ultra-fast liquid chromatography system from AB SCIEX in Los Angeles, USA, was employed for this purpose. This system featured a C18 reversed-phase column (3 µm, 2.1 mm × 50 mm; Waters, Milford, CT, USA) connected to a TQ 5500 tandem mass spectrometry system (AB SCIEX, Los Angeles, CA, USA) equipped with an electrospray ionization source. The mobile phase consisted of a mixture of 2 mM ammonium acetate (0.1% formic acid) and acetonitrile (15:9, 