이전 사이트 이동

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.

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.

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). LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) system equipped with two binary pumps, an autosampler, a column oven, and a vacuum degasser (Thermo Scientific, TSQ Altis Plus, USA). The column temperature was kept at 30°C throughout the analysis. Mass spectrometry analysis was performed in positive ion mode using electrospray ionization (H-ESI). The ion spray voltage was maintained at 4000V, while the sheath gas (Arb) was set to 45, the aux gas (Arb) to 15, the sweep gas (Arb) to 1, the ion transfer tube temperature to 320°C, and the vaporizer temperature to 350°C. Precursor ions (Q1) and product ions (Q3) were determined for each analyte, and suitable conditions were established. Multiple reaction monitoring (MRM) was then applied to enhance the sensitivity of the analysis. , 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). LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) system equipped with two binary pumps, an autosampler, a column oven, and a vacuum degasser (Thermo Scientific, TSQ Altis Plus, USA). The column temperature was kept at 30°C throughout the analysis. Mass spectrometry analysis was performed in positive ion mode using electrospray ionization (H-ESI). The ion spray voltage was maintained at 4000V, while the sheath gas (Arb) was set to 45, the aux gas (Arb) to 15, the sweep gas (Arb) to 1, the ion transfer tube temperature to 320°C, and the vaporizer temperature to 350°C. Precursor ions (Q1) and product ions (Q3) were determined for each analyte, and suitable conditions were established. Multiple reaction monitoring (MRM) was then applied to enhance the sensitivity of the analysis.