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To extract adducts from urine, 1 mL of each sample was transferred into a 1.5 mL microcentrifuge tube. Prior to sample loading, the Sep-Pak C18 column(waters seppak vac 1cc(100mg)C18 cartridges) was conditioned by passing 1 mL each of methanol and water. The sample was then loaded onto the column, followed by purification and extraction using 1 mL each of water and a methanol/ethyl acetate mixture. The eluted solution was evaporated and concentrated at 60°C for approximately 20 minutes using a nitrogen concentrator. After evaporation and concentration, the sample was reconstituted with the solvent used in the pretreatment process, filtered through a syringe filter, and transferred to an autosampler vial for analysis.

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).