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Lipid extractions were performed by slightly modifying the Matyash method. 100% ice-cold methanol (300 µL) containing 0.1% butylated hydroxytoluene and H2O(50 uL) was added in the plasma(50 uL). After adding 1 mL of methyl-tert-butyl ether with 0.1% butylated hydroxytoluene, the samples were shaken at 20 to 23°C for 1 h. In total, 250 µL of water was added and vortexed for 10 min. Thereafter, phase separation was performed via centrifugation at 14,000g for 15 min at 4°C. For targeted lipidomics analysis, the upper (220 µL) and lower (110 µL) phases were pooled and dried using N2 purge. , Human plasma samples were analyzed using the AbsoluteIDQ® p180 Kit (Biocrates Life Sciences AG, Innsbruck, Austria) according to the manufacturer's instructions. Briefly, 10 μL of plasma sample was loaded onto the filter spot of the kit plate preloaded with internal standards and dried under a nitrogen stream for 30 min at room temperature. Samples were derivatized with 50 μL of 5% phenyl isothiocyanate (PITC) solution (PITC:pyridine:ethanol:water) for 25 min at room temperature and subsequently dried under nitrogen for 60 min. Metabolites were extracted using 300 μL of 5 mM ammonium acetate in methanol with shaking at 450 rpm for 30 min. Extracts were collected by centrifugation at 500 × g for 2 min. For FIA-MS/MS analysis, 10 μL of extract was diluted with 490 μL of FIA solvent (methanol containing proprietary FIA mobile phase additive). , Human plasma samples were analyzed using the AbsoluteIDQ® p180 Kit (Biocrates Life Sciences AG, Innsbruck, Austria) according to the manufacturer's instructions. Briefly, 10 μL of plasma sample was loaded onto the filter spot of the kit plate preloaded with internal standards and dried under a nitrogen stream for 30 min at room temperature. Samples were derivatized with 50 μL of 5% phenyl isothiocyanate (PITC) solution (PITC:pyridine:ethanol:water) for 25 min at room temperature and subsequently dried under nitrogen for 60 min. Metabolites were extracted using 300 μL of 5 mM ammonium acetate in methanol with shaking at 450 rpm for 30 min. Extracts were collected by centrifugation at 500 × g for 2 min. For LC-MS/MS analysis, 150 μL of extract was diluted with 150 μL of water. , Plasma samples were prepared by mixing 105 μL of plasma with 105 μL of Bruker IVDr plasma buffer (1:1, v/v). The mixture was homogenized by a single pipetting step. No additional extraction, protein precipitation, centrifugation, or vortex mixing was performed. , Ice-thawed plasma samples (50 μL) were transferred to polypropylene tubes, followed by the addition of 200 μL of ice-cold ethanol:methanol (1:1, v/v). The samples were vortex-mixed for 10 min and centrifuged at 16,000 × g for 10 min at 4°C. Subsequently, 200 μL of the supernatant was transferred to a new polypropylene tube containing 100 μL of distilled water and vortex-mixed for 10 s. After a second centrifugation at 16,000 × g for 10 min at 4°C, 120 μL of the resulting supernatant was transferred to autosampler vials for positive- and negative-ionization analyses, respectively.

Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS, LC-MS 8060, Shimadzu, Kyoto, Japan) with a Kinetex C18 column (100 x 2.1mm i.d., 2.6 μm particle size; Phenomenex, Torrance, CA, USA). Mobile phase A and B consisted of 10 mM ammonium acetate in water/methanol (1/9, v/v) and 10 mM ammonium acetate in methanol/isopropanol (1/1, v/v), respectively. The gradient elution condition was as follows: 30% B (0 min), 95% B (5 min), 95% B (15 min), 30% B (15.1 min) and 30% B (20 min). The sample injection volume was 2 μL and the flow rate was maintained at 0.2 mL/min. , Flow injection analysis (FIA)-MS/MS was performed using the AbsoluteIDQ® p180 Kit on a Waters ACQUITY UPLC system coupled to a Waters Xevo TQ-XS mass spectrometer. No chromatographic column was used during FIA analysis. The FIA solvent consisted of methanol containing the proprietary FIA mobile phase additive supplied with the kit. Samples were injected at a volume of 10 μL using an FTN autosampler. The FIA method was operated at flow rates ranging from 0.03 to 0.80 mL/min according to the validated Biocrates gradient program, with a total run time of 2.0 min per sample. , LC-MS/MS analysis was performed using the AbsoluteIDQ® p180 Kit (Biocrates Life Sciences AG, Innsbruck, Austria) on a Waters ACQUITY UPLC system equipped with an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 75 mm). The column oven temperature was maintained at 50 °C and the autosampler temperature at 10 °C. The injection volume was 2 μL. Mobile phase A consisted of water containing 0.2% formic acid, and mobile phase B consisted of acetonitrile/water (95:5, v/v) containing 0.2% formic acid. The flow rate was set at 0.8 mL/min and increased to 0.9 mL/min during the column washing step. Gradient elution was performed as follows: 0–0.45 min, 100% A; 0.45–3.30 min, 85% A/15% B; 3.30–5.90 min, 30% A/70% B; 5.90–6.05 min, 100% B; 6.05–6.42 min, 100% B at 0.9 mL/min; 6.42–6.52 min, 100% B; and 6.52–7.30 min, re-equilibration at 100% A. The total chromatographic run time was 7.3 min per sample. , Chromatographic separation was performed using an UltiMate 3000 UHPLC system (Thermo Scientific, San Jose, CA, USA) equipped with an HPG-3400RS pump, a WPS-3000 autosampler, and a TCC-3000 column oven. An ACQUITY UPLC HSS T3 column (100 × 2.1 mm, 1.7 μm, 130 Å; Waters, Milford, MA, USA) was maintained at 40°C. The autosampler temperature was set at 4°C, and the injection volume was 4 μL. The mixer volume was 150 μL. The mobile phases consisted of water containing 0.1% formic acid as mobile phase A and methanol containing 0.1% formic acid as mobile phase B. The flow rate was maintained at 0.35 mL/min. The gradient program was as follows: 5% B at 0 min, maintained at 5% B until 2 min, increased to 95% B from 2 to 12 min, maintained at 95% B until 16 min, decreased to 5% B from 16 to 18 min, and maintained at 5% B until 20 min for column re-equilibration. The same chromatographic conditions were applied for both positive- and negative-ionization analyses.

The mass operating conditions were as follows: desolvation temperature, 250 ℃; heat block temperature, 400 ℃; spray voltage 4 kV; drying gas (N2) flow rate, 10 L/min; collision gas, argon; nebulizing gas (N2) flow rate, 3 L/min; collision gas pressure, 230 kPa; and detector voltage, 1.66 kV. , Mass spectrometric detection for FIA-MS/MS analysis was performed using a Waters Xevo TQ-XS triple quadrupole mass spectrometer operated in positive electrospray ionization mode (ESI+). Data were acquired in multiple reaction monitoring (MRM) mode using the validated AbsoluteIDQ® p180 Kit acquisition method (KIT3-FIA_8316). Instrument parameters were as follows: capillary voltage, 3.0 kV; cone voltage, 50 V; source offset voltage, 30 V; desolvation temperature, 350 °C; desolvation gas flow, 650 L/h; cone gas flow, 150 L/h; nebulizer pressure, 7.0 bar; and collision gas setting, 2. Compound-specific MRM transitions and acquisition parameters supplied by Biocrates were used for targeted metabolite analysis. , Mass spectrometric detection for the LC-MS/MS analysis was performed using a Waters Xevo TQ-XS triple quadrupole mass spectrometer coupled to a Waters ACQUITY UPLC system. Data acquisition was conducted in multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) operated in positive ion mode (ESI+). Instrument parameters were set according to the validated AbsoluteIDQ® p180 Kit LC method as follows: capillary voltage, 3.0 kV; cone voltage, 50 V; source offset voltage, 30 V; desolvation temperature, 350 °C; desolvation gas flow, 650 L/h; cone gas flow, 150 L/h; nebulizer pressure, 7.0 bar; and collision gas setting, 2. Compound-specific MRM transitions, collision energies, and retention time windows supplied with the AbsoluteIDQ® p180 Kit LC acquisition method (KIT2-LC_8316) were used for targeted metabolite quantification. , Mass spectrometric analysis was performed using a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a heated electrospray ionization (HESI) source. Data were acquired separately in positive- and negative-ionization modes over an m/z range of 66.7–1,000. The spray voltage was set to 4.0 kV in positive mode and 3.5 kV in negative mode. The capillary temperature and auxiliary gas heater temperature were maintained at 320°C and 350°C, respectively. The sheath gas, auxiliary gas, and sweep gas flow rates were set to 45, 15, and 1 arbitrary units, respectively, and the S-lens RF level was set to 45. Data were acquired using full-scan mass spectrometry with data-dependent MS/MS acquisition (Full MS/dd-MS²). For each full-scan event, the five most abundant precursor ions were selected for fragmentation. Previously fragmented precursor ions were dynamically excluded for 0.25 min. Full-scan spectra were acquired in profile mode at a resolution of 70,000, with an automatic gain control target of 3 × 10⁶ and a maximum injection time of 80 ms. Data-dependent MS/MS spectra were acquired in centroid mode at a resolution of 17,500, with an automatic gain control target of 1 × 10⁵ and a maximum injection time of 50 ms. Precursor ions were isolated using an isolation window of 1.0 m/z and fragmented using stepped normalized collision energies of 30, 40, and 50.

NMR spectra were acquired using a Bruker Avance IVDr 600 MHz NMR spectrometer (Bruker BioSpin, Germany). Measurements were performed using a CP2.1 TCI 600S3 H-C/N-D-05 Z XT probe. Plasma samples were analyzed at 310 K according to the Bruker IVDr workflow. NMR experiments included one-dimensional 1H NOESY presaturation (noesygppr1d), two-dimensional J-resolved spectroscopy (jresgpprqf), and one-dimensional 1H spectroscopy with presaturation (zgpr). For the NOESY experiment, spectra were acquired at a proton resonance frequency of 600.302 MHz with a spectral width of 17,857.143 Hz, 98,304 data points, 32 transients (scans), and a relaxation delay of 4 s.