Deeper insights into the stability of oxylipins in human plasma across multiple freeze-thaw cycles and storage conditions

• Published in: Journal of pharmaceutical and biomedical analysis 2025-03, Vol.255, p.116587, Article 116587
• Main Authors: Moran-Garrido, Maria, Camunas-Alberca, Sandra M., Sáiz, Jorge, Gradillas, Ana, Taha, Ameer Y., Barbas, Coral
• Format: Article
• Language: English
• Subjects: Esterified oxylipins; Free oxylipins; Freeze-thaw; Human plasma; LC-MS/MS; Redox Lipidomics; Stability
• ISSN: 0731-7085; 1873-264X; 1873-264X
• DOI: 10.1016/j.jpba.2024.116587

Oxylipins are signaling lipids derived from the oxidation of polyunsaturated fatty acids (PUFAs). In lipidomic studies, human plasma may be subjected to various storage conditions and freeze-thaw cycles, which may impact the analysis of these compounds. In this study, we used liquid chromatography coupled with mass spectrometry (LC-MS) to examine the influence of up to five freeze-thaw cycles (FTCs) on free and total (mostly esterified) oxylipins in human plasma and the influence of temperature and storage duration (4 °C for up to 120 h and –20 °C and –80 °C for 1–98 days) in the presence or absence of butylated hydroxytoluene (BHT) on extracted oxylipins stored in LC-MS amber vials. In fresh plasma subjected to several FTCs, approximately 48 % of the detected free oxylipins were significantly altered by the third cycle, with increases in cytochrome P450 (CYP450) and lipoxygenase (LOX)-derived compounds and reductions in trihydroxylated oxylipins. In contrast, multiple FTCs did not significantly alter esterified oxylipins. At 4 °C, the extracted oxylipins did not change significantly for up to 120 h (5 days). Oxylipin levels remained stable for 98 days at –80 °C but decreased by 98 days at –20 °C. The antioxidant activity of butylated hydroxytoluene (BHT) did not influence oxylipin stability at 4 °C for 120 h or at –80 °C for 98 days, but it reduced oxylipin degradation at –20 °C at 98 days. Conversely, prostaglandin F2α (PGF2α) exhibited substantial increases at –20 °C and –80 °C, independent of BHT. This study demonstrates that (i) unlike free oxylipins, the esterified oxylipin pool remains stable following repeated FTCs, (ii) extracted oxylipins are stable at 4 °C for up to 120 h and at –80 °C for up to 98 days, but not at –20 °C for 98 days, and (iii) BHT may minimize oxylipin degradation of sample extracts stored at –20 °C. This study provides a framework for measuring oxylipins under various freeze-thaw and storage conditions. •Free oxylipins in human plasma change significantly after 3 freeze-thaw cycles.•Esterified oxylipins in human plasma remain stable after freeze-thaw cycles.•Extracted oxylipins are stable for 5 days at 4 °C, and 98 days at –80 °C.•Extracted oxylipins degrade at –20 °C at 98 days.•BHT reduces oxylipin degradation at –20 °C but has no effect at 4°C or –80 °C.