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After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 60°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 61°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 62°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 63°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 64°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 65°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 66°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 67°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 68°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 69°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 70°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample. , After incubating at 37°C for 20 minutes to separate the analytes in the spot urine, the sample is loaded onto a C18 Sep-Pak column activated with a mixture of methanol and water. Subsequently, purification and extraction are carried out, followed by evaporation and concentration at 71°C using a nitrogen concentrator. Once the evaporation and concentration process is complete, the solvent is used to reconstitute the sample.

LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 5 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 6 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 7 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 8 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 9 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 10 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 11 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 12 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 13 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 14 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 15 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility. , LC separations were performed on a Hypersil GOLD VANQUISH column (100 × 2.1 mm, particle size 1.9 μm, Thermo fisher). The column temperature and flow rate were set at 30°C and 0.3 ml/min, respectively. The mobile phase used contained 0.1% formic acid in ammonium formate buffer (0.1mM) (A) and acetonitrile (100, v/v) (B). The linear gradient was as follows. 1.0% B for 1 min, 1.0-10% B for 1 min, 10-100% B for 8 min, 100% B for 2 min, 100-1.0% B for 8 min. The injection volume of the sample was adjusted according to the positive ionization polar mode and It was 16 μl using partial loop mode for both negative ionization polar modes. Quality control (QC) samples pooling equal aliquots of samples were measured regularly throughout the run to ensure data reproducibility.

To obtain MS spectral data, LC-ESI-MS/MS analysis was performed on a TSQ Altis Plus (Thermo scientific) coupled with a UPLC system (Thermo scientific).