Qualitative and quantitative lipidomic profiling of cardiolipin, oxidized cardiolipin, and monolysocardiolipin from human skeletal muscle by UPLC-Orbitrap-MSn

• Published in: Analytica chimica acta Vol. 1350; p. 343825
• Main Authors: Yuan, Zhi-Xin, Egan, Josephine M., Bektas, Arsun, Tian, Qu, Lyashkov, Alexey, Ferrucci, Luigi, Ramsden, Christopher E., Moaddel, Ruin
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 08.05.2025
• Subjects: Cardiolipins; Human skeletal muscle; Lipidomics; Monolysocardiolipins; Oxygenated cardiolipins; RP-UPLC/HR-MSn; Monolysocardiolipins; Lipidomics; Cardiolipins; Oxygenated cardiolipins; Human skeletal muscle; RP-UPLC/HR-MSn
• ISSN: 0003-2670; 1873-4324; 1873-4324
• DOI: 10.1016/j.aca.2025.343825

Cardiolipins (CL) are a mitochondria-specific family of phospholipids that play central roles in mitochondrial function. Imbalance in CL metabolism, especially excessive CL oxidation, leads to mitochondrial dysfunction, apoptosis, and inflammation, contributing to age-related diseases. As of yet no comprehensive methods have been developed to assess CL, oxidized CL (oxCL), and monolyso-CL (MLCL) species. To fill this critical research gap, we combined untargeted and targeted lipidomic approaches to analyze CL species in human skeletal muscle samples. The method enabled in-depth structural characterization using exact mass measurement followed by multistage fragmentation (MSn) to achieve unequivocal structural elucidation at the molecular species level and sn-position level for some species. This novel methodology identified intact mono- and di-oxygenated L4CL species and allowed the differentiation of isomeric 9/13-HODE-L3CL and 9(10)/12(13)-EpOME-L3CL with unprocessed total lipid extracts. Overall, 220 molecular species (125 CL, 30 oxCL, and 65 MLCL) were detected. Our method includes a quantitation strategy that leveraged on establishing three-leveled matrix-matched calibration curves normalized by using internal standard M4CL and its isotopologues M+1 (13C-M4CL) and M+3 (13C3-M4CL) respectively. The analytical performance was also evaluated and found to be highly sensitive with LLOQs at fmol levels and reproducible with precision RSD <20 % for the majority. This is the first reported method to simultaneously provide broad coverage of CL, oxCL, and MLCL species from a single injection of total lipid extract in a complex biological sample. This method is also the first to demonstrate the presence of sn-positional isomers of CL in human skeletal muscle. It has been successfully applied to a pilot study of skeletal muscle biopsies and provided meaningful results. We anticipate the methodology will facilitate investigations of the cardiolipin lipidome leading to a better understanding of the complex and highly interactive biological processes that regulate mitochondria function and expand minor cardiolipin targets including oxCL and MLCL for biomarker discovery. [Display omitted] •Simple total lipid extraction and sufficient chromatographic separation.•Characterization of 220 species (125 CL, 30 oxCL, and 65 MLCL).•Highly sensitive (fmol level) and reproducible (majority precision <20 %).•Novel method for the simultaneous analysis of a broad range of CL, oxCL, and MLCL.•Application of method to clinical samples.