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Human plasma samples from the CKD cohort , were thawed on ice prior to lipid extraction. For extraction, 400 μL of 80% isopropanol (IPA) was added to 50 μL of each sample. The mixtures were vortexed and centrifuged for 5 min at 18,341 × g and 4 °C. Subsequently, 200 μL of the supernatant was transferred into two Eppendorf SafeLock tubes and dried under N2 gas. The dried pellets were reconstituted with 100 μL of 95% acetonitrile (ACN) containing 10 mM ammonium acetate (AmAc) for HILIC-MRM analysis. , Human plasma samples from the CKD cohort , were thawed on ice prior to lipid extraction. For extraction, 400 μL of 80% isopropanol (IPA) was added to 50 μL of each sample. The mixtures were vortexed and centrifuged for 5 min at 18,341 × g and 4 °C. Subsequently, 200 μL of the supernatant was transferred into two Eppendorf SafeLock tubes and dried under N2 gas. The dried pellets were reconstituted with 200 μL of ACN:Water:IPA (40:24:36) containing 10 mM AmAc for RPLC-PRM analysis.

Chromatographic separation was achieved on an ACQUITY Premier BEH Amide column (1.7 μm, 100 × 2.1 mm I.D., Waters, Milford, MA, USA) using mobile phases A (ACN:Water = 95:5) and B (ACN:Water = 50:50), both containing 10 mM AmAc. The injector needle was washed with ACN:Water (95:5) after each injection, and the injection volume was set to 2 μL. The gradient elution program was as follows: the system was initially maintained at 0.1% B for 2 min, followed by an increase to 80% B over 3 min. The gradient was returned to the initial condition (0.1% B) within 0.1 min and equilibrated for 3 min at the starting condition. The flow rate was set to 300 μL/min, the column temperature was maintained at 40 °C, and the sample injection volume was 2 μL. , Chromatographic separation was achieved using an ACQUITY UPLC HSS T3 column (1.8 μm, 100 × 2.1 mm I.D., Waters, Milford, MA, USA) with a mobile phase A comprising ACN:Water of 60:40 and phase B comprising ACN:IPA of 10:90, both containing 10 mM AmAc. A linear gradient elution was applied, beginning at 40% B for 1 min, increasing from 40 to 65% B over 4 min, from 65 to 95% B over 5 min, held at 95% B for 2 min, followed by a rapid return to 40% B over 0.1 min, and re-equilibration at 40% B for 6 min. The flow rate, column temperature, and injection volume were set at 250 μL/min, 50 °C, and 10 μL, respectively.

Optimized ion source parameters included a voltage of 5500 V in positive mode and −4500 V in negative mode, an ion source temperature of 400 °C, and GS1 and GS2 settings of 30 and 35 psi, respectively. Curtain gas and collision-activated dissociation gas pressures were maintained at 20 and 6 psi. A scheduled MRM (sMRM) algorithm with a retention time (tR) window of 1 min was applied. , Ion source parameters were configured with a voltage of −4500 V in negative mode, sheath gas at 60 Arb, auxiliary gas at 15 Arb, sweep gas at 1 Arb, and an ion transfer tube temperature of 380 °C. The vaporizer temperature was maintained at 350 °C. Full MS scan data were acquired over an m/z range of 67–1000 in dual-polarity mode at a resolution of 120,000 FWHM at m/z 200, while product ion scans were obtained over the same m/z range at a resolution of 15,000 FWHM at m/z 200. Both MS scan and product ion scan data were acquired in centroid mode.