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Frozen saliva samples were stored at −80°C until analysis and thawed at 4°C prior to sample preparation. The samples were centrifuged to remove particulate matter and cellular debris. An aliquot of the saliva supernatant was transferred to a clean microcentrifuge tube and mixed with an equal volume of 0.2 M phosphate buffer prepared in D₂O (pH 7.0) containing sodium azide to stabilize the sample and provide a deuterium lock signal for NMR analysis. Trimethylsilyl propionate (TSP) was subsequently added as the internal reference compound. The prepared sample was mixed thoroughly, transferred into a 5 mm NMR tube, and maintained under controlled conditions prior to spectral acquisition to minimize analytical variation. , Frozen liver tissue samples were stored at −80°C until analysis. Approximately 100–200 mg of tissue was transferred into a homogenization tube containing zirconium oxide beads. Methanol-d4 and 0.2 M phosphate buffer prepared in D₂O (pH 7.0) were added, and the samples were homogenized to extract polar metabolites. Following homogenization, additional methanol-d4, phosphate buffer, and chloroform-d3 were added to produce a biphasic extraction system. The mixture was vortex-mixed, incubated under chilled conditions, and centrifuged to separate the aqueous and organic phases. The aqueous phase was collected for NMR analysis, while the organic phase was reserved for lipid analysis. Sample preparation was performed under refrigerated conditions to minimize metabolite degradation and analytical variation. , Urine samples were mixed with Bruker urine buffer at a 9:1 (v/v) ratio, following the manufacturer's recommendation. After buffer addition, the mixture was homogenized by gentle pipetting without vortexing. , 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 tissue samples were analyzed using the AbsoluteIDQ® p180 Kit (Biocrates Life Sciences AG, Innsbruck, Austria) according to the manufacturer's instructions. Briefly, 10 μL of tissue 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. , 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. , To extract urinary metabolites, urine samples were thawed on ice and vortexed thoroughly. Each urine sample aliquot was mixed with cold aqueous methanol or acetonitrile containing internal standard, followed by vortexing to precipitate proteins and extract metabolites. After incubation at 4°C, the samples were centrifuged at 4°C, and the supernatant was collected. The extracts were dried using a centrifugal vacuum concentrator and reconstituted in 75% aqueous acetonitrile. After an additional centrifugation step, the prepared sample solution was transferred into glass LC vials, and 5 µL was injected for LC-QTOF MS analysis in both positive and negative ionization modes. , Plasma metabolites were extracted from 50 µL of plasma using chloroform:methanol (2:1, v/v) with internal standard when required. After addition of water, the samples were vortexed, incubated at 4°C, and centrifuged for phase separation. The upper aqueous phase was collected for polar metabolite analysis, and the lower organic phase was collected for lipid metabolite analysis. The extracts were dried, reconstituted in the appropriate solvent, centrifuged, and transferred into glass LC vials for LC-MS analysis. , To extract metabolites from cancer cell line samples, 8 × 10^6 cells were prepared for each sample. After removal of the culture medium, the cells were washed twice with PBS and detached using trypsin. The harvested cells were transferred to 5-mL tubes, and 2 mL of 80% methanol containing QReSS internal standard was added, followed by vortexing for 1 min. Then, 1 mL of water and 2 mL of chloroform were added to each tube and mixed by vortexing for 1 min. The samples were incubated at 4°C for 1 h and centrifuged at 12,700 rpm for 20 min at 4°C. The supernatant was collected and divided into 300-µL aliquots, followed by drying using a centrifugal vacuum concentrator. The dried extracts were reconstituted in 200 µL of 75% aqueous acetonitrile and centrifuged at 4°C for 10 min. For LC-MS analysis, 100 µL of the prepared sample solution was transferred into a glass LC vial, and 5 µL was injected for both positive and negative ionization modes. QC samples were prepared by pooling 300 µL aliquots from 17 cell samples, and the pooled QC mixture was divided into 300-µL aliquots. , To extract metabolites from cancer cell line samples, 8 × 10^6 cells were prepared for each sample. After removal of the culture medium, the cells were washed twice with PBS and detached using trypsin. The harvested cells were transferred to 5-mL tubes, and 2 mL of 80% methanol containing QReSS internal standard was added, followed by vortexing for 1 min. Then, 1 mL of water and 2 mL of chloroform were added to each tube and mixed by vortexing for 1 min. The samples were incubated at 4°C for 1 h and centrifuged at 12,700 rpm for 20 min at 4°C. The supernatant was collected and divided into 300-µL aliquots, followed by drying using a centrifugal vacuum concentrator. The dried extracts were reconstituted in 200 µL of 75% aqueous acetonitrile and centrifuged at 4°C for 10 min. , To extract metabolites from cancer cell line samples, 8 × 10^6 cells were prepared for each sample. After removal of the culture medium, the cells were washed twice with PBS and detached using trypsin. The harvested cells were transferred to 5-mL tubes, and 2 mL of 80% methanol containing QReSS internal standard was added, followed by vortexing for 1 min. Then, 1 mL of water and 2 mL of chloroform were added to each tube and mixed by vortexing for 1 min. The samples were incubated at 4°C for 1 h and centrifuged at 12,700 rpm for 20 min at 4°C. The supernatant was collected and divided into 300-µL aliquots, followed by drying using a centrifugal vacuum concentrator. The dried extracts were reconstituted in 200 µL of 75% aqueous acetonitrile and centrifuged at 4°C for 10 min. , Saliva samples were thawed on ice, vortexed, and centrifuged to remove debris and viscous materials. The supernatant was mixed with cold methanol or acetonitrile containing internal standard, followed by vortexing, incubation at 4°C, and centrifugation. The final supernatant was dried when necessary, reconstituted in 75% aqueous acetonitrile, centrifuged, and transferred into glass LC vials for LC-MS analysis. , To extract liver metabolites, 50 mg of liver tissue were transferred to each homogenizer tube containing three beads, and the tissue weight was recorded. Then, 600 µL of 50% aqueous methanol containing a 5-fold diluted internal standard was added to each tube. The samples were homogenized, followed by the addition of 600 µL of chloroform, and vigorously vortexed for 1 min. After incubation at 4°C for 15 min, the samples were centrifuged at 12,700 rpm for 20 min at 4°C. The upper supernatant was collected and divided into three aliquots of 160 µL each in new tubes, followed by drying using a Speed Vacuum concentrator. The dried extracts were reconstituted in 200 µL of 75% aqueous acetonitrile and further diluted to an appropriate concentration. For LC-MS analysis, 100 µL of the 2-fold diluted sample solution was transferred into a glass LC vial, and 5 µL was injected for both positive and negative ionization modes. The final concentration of the internal standard in the injected sample corresponded to a 10-fold dilution. , Plasma metabolites were extracted from 50 µL of plasma using chloroform:methanol (2:1, v/v) with internal standard when required. After addition of water, the samples were vortexed, incubated at 4°C, and centrifuged for phase separation. The upper aqueous phase was collected for polar metabolite analysis, and the lower organic phase was collected for lipid metabolite analysis. The extracts were dried, reconstituted in the appropriate solvent, centrifuged, and transferred.

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. , LC separation were performed using a Waters ACQUITY I-Class UPLC system equipped with a SeQuant ZIC-HILIC column (2.1 × 100 mm, 3.5 µm, Merck). The column temperature was maintained at 35°C, and the sample cooler was set to 4°C. The mobile phases consisted of 0.1% formic acid and 10 mM ammonium acetate in 90% acetonitrile (A) and 0.1% formic acid and 10 mM ammonium acetate in 50% acetonitrile (B). The flow rate was 0.4 mL/min, and the injection volume was 5 µL. The gradient elution program was as follows: 1% B from 0 to 2 min, 1–55% B from 2 to 8 min, 55–99% B from 8 to 9 min, 99% B from 9 to 11 min, 99–1% B from 11 to 11.1 min, and 1% B from 11.1 to 15 min for column re-equilibration. The total run time was 15 min. , LC separation were performed using a Waters ACQUITY I-Class UPLC system equipped with a SeQuant ZIC-HILIC column (2.1 × 100 mm, 3.5 µm, Merck). The column temperature was maintained at 35°C, and the sample cooler was set to 4°C. The mobile phases consisted of 0.1% formic acid and 10 mM ammonium acetate in 90% acetonitrile (A) and 0.1% formic acid and 10 mM ammonium acetate in 50% acetonitrile (B). The flow rate was 0.4 mL/min, and the injection volume was 5 µL. The gradient elution program was as follows: 1% B from 0 to 2 min, 1–55% B from 2 to 8 min, 55–99% B from 8 to 9 min, 99% B from 9 to 11 min, 99–1% B from 11 to 11.1 min, and 1% B from 11.1 to 15 min for column re-equilibration. The total run time was 15 min. , CE separation was performed using an Agilent capillary electrophoresis system coupled to a mass spectrometer. The cassette temperature was maintained at 20°C, and the total CE method time was set to 30 min. Prior to sample injection, the capillary was preconditioned by flushing for 300 s. Samples were introduced by pressure injection at 50 mbar for 8 s, followed by an additional pressure step at 50 mbar for 2 s. During analysis, the separation voltage was changed to 30 kV at 0.3 min. The acquired CE-MS data were used for targeted metabolite analysis in cation mode.

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 SCIEX 5600 QTOF MS system equipped with an electrospray ionization (ESI) source. Data were acquired in both positive and negative ionization modes over a mass range of m/z 30–1,000. The ion source parameters were as follows: curtain gas (CUR), 30 psi; nebulizer gas (GS1), 60 psi; heating gas (GS2), 60 psi; and source temperature, 500°C. The IonSpray voltage floating (ISVF) was set to 5,500 V for positive ion mode and 4,500 V for negative ion mode. The collision energy was set to 35 V in positive mode and −35 V in negative mode, with a collision energy spread of 15 V. , Mass spectrometric analysis was performed using an Agilent 6490 triple quadrupole mass spectrometer equipped with an electrospray ionization source. Data were acquired in positive ion mode using a cation MRM method. The source parameters included a gas temperature of 250°C, gas flow of 14 L/min, nebulizer pressure of 10 psi, and capillary voltage of 4,000 V. The sheath gas temperature and flow were set to 300°C and 3.0 L/min, respectively. The MRM segment was acquired from 1.0 min, with a fragmentor voltage of 380 V and a cell accelerator voltage of 5 V. The acquired MRM data were used for targeted metabolite profiling. , Mass spectrometric analysis was performed using a SCIEX 5600 QTOF MS system equipped with an electrospray ionization (ESI) source. Data were acquired in negative ionization modes over a mass range of m/z 30–1,000. The ion source parameters were as follows: curtain gas (CUR), 30 psi; nebulizer gas (GS1), 60 psi; heating gas (GS2), 60 psi; and source temperature, 500°C. The Ionspray voltage floating (ISVF) was set to 4,500 V for negative ion mode. The collision energy was set to −35 V in negative mode, with a collision energy spread of 15 V. , Mass spectrometric analysis was performed using a SCIEX 5600 QTOF MS system equipped with an electrospray ionization (ESI) source. Data were acquired in positive ionization modes over a mass range of m/z 30–1,000. The ion source parameters were as follows: curtain gas (CUR), 30 psi; nebulizer gas (GS1), 60 psi; heating gas (GS2), 60 psi; and source temperature, 500°C. The Ionspray voltage floating (ISVF) was set to 5,500 V. The collision energy was set to 35 V in positive mode, with a collision energy spread of 15 V.

¹H-NMR spectra of saliva samples were acquired using a Bruker AVANCE III HD 800 MHz nuclear magnetic resonance spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a 5 mm CPTCI Z-GRD cryogenic probe. Spectra were recorded using the one-dimensional NOESY-presaturation pulse sequence (noesygppr1d) with water suppression. Instrument parameters were configured according to the UTNM guideline for untargeted metabolomics of saliva samples, including a relaxation delay of 2 s, an acquisition time of 2 s, an FID size of 64K, a sweep width of 20 ppm, and a sample temperature of 25°C. These acquisition parameters were applied consistently to all analytical samples. , 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. Urine samples were analyzed at 300 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. , 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.