Uplc system

Whole-blood and tumour concentrations of RMC-7977 were determined using LC–MS/MS methods performed at WuXi AppTec. Tumour tissue samples were homogenized with a 10× volume of methanol/15 mM PBS (1:2, v:v). Sample preparation and analysis on a Sciex 6500+ triple quadrupole mass spectrometer equipped with an ACQUITY UPLC system were performed as previously described^{12 (link)}. RMC-7977 and internal standard verapamil were detected by positive electrospray ionization using multiple reaction monitoring (RMC-7977: m/z 865.4/706.4; verapamil: m/z 455.2/164.9).

All samples were analyzed by the UPLC-MS/MS system according to our previous work [32 (link)]. Firstly, all chromatographic separations were performed using an ultra performance liquid chromatography (UPLC) system (SCIEX, UK). An ACQUITY UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm, Waters, Milford, MA, USA) was used for the reversed phase separation. The column oven was maintained at 35 °C. The flow rate was 0.4 mL/min and the mobile phase consisted of solvent A (25 mM ammonium acetate and 25 mM NH₄H₂O) and solvent B (IPA:CAN = 9:1, v/v, and 0.1% of formic acid). Gradient elution conditions were set as follows: 0 ~ 0.5 min, 95% of solvent B; 0.5 ~ 9.5 min, 95 to 65% of solvent B; 9.5 ~ 10.5 min, 65% ~ 40% of solvent B; 10.5 ~ 12 min, 40% of solvent B; 12 ~ 12.2 min, 40% ~ 95% of solvent B; 12.2 ~ 15 min, 95% of solvent B. The injection volume for each sample was set at 4 μL.
A high-resolution MS/MS TripleTOF 5600 plus (SCIEX, UK) was used to recognize the metabolites eluted from the column. The TOF was carried out in both positive and negative ion modes. The detail parameters of UPLC-MS/MS analysis were set according to our previous work [32 (link)]. Furthermore, in order to evaluate the stability of the UPLC-MS/MS system during the whole data acquisition process, one quality control sample was detected after every 10 samples.

The levels of E209 were evaluated in whole blood to determine standard PK parameters in the individual animals used in the efficacy study. Peripheral blood samples (5 μg ml⁻¹) were taken at different times (0.25, 0.5, 1, 2, 4, 6, 8 and 23 h) after drug administration, mixed with 25 μl of Milli-Q water and immediately frozen on dry ice. The frozen samples were stored at −80 °C until analysis. Vehicle-treated mice experienced the same blood-sampling regimen. Blood samples were processed by liquid–liquid extraction. Quantitative analysis by Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was performed using a Waters UPLC system and Sciex API4000 mass spectrometer. The lower limit of quantification in this assay was 5 ng ml⁻¹. Blood concentration versus time was analysed by non-compartmental analysis (NCA) using Phoenix ver.6.3 (from Pharsight), from which exposure-related values (C_max and AUC_0–23, AUC_0–t) and t_max were estimated.

The collected intestinal tissue samples for metabonomic analysis. The metabolites were extracted with 120 μL of precooled 50% methanol, vortexed mixed for 1 min, and incubated at room temperature for 10 min. After centrifugation at 4,000 g for 20 min, the supernatants were transferred into new 96 well plates. The samples were stored at -80 °C before the LC-MS analysis. First, all chromatographic separations were performed with an ultra-performance liquid chromatography (UPLC) system (SCIEX, UK). An ACQUITY UPLC T3 column (100 mm * 2.1 mm, 1.8 µm, Waters, UK) was used for the reversed phase separation. A high-resolution TripleTOF 5600plus tandem mass spectrometer (SCIEX, UK) was used to detect metabolites eluted from the column. The Q-TOF was operated in both positive and negative ion modes. LC-MS raw data files were converted into mzXML format and then processed with the XCMS, CAMERA, and metaX toolbox implemented in the R software.

Chromatographic separation was performed using a UPLC system (SCIEX, UK). The analytes were separated on a Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) maintained at 35°C. A TripleTOF5600plus high-resolution tandem mass spectrometer (SCIEX, UK) was used to detect metabolites eluted from the chromatographic column, and Q-TOF was run in positive and negative ion modes. During the entire collection period, the mass accuracy was calibrated every 20 samples. In addition, one QC sample was analyzed for every eight samples to evaluate the stability of the LC-MS. The raw LC–MS data and all metabolite molecules detected in the sample were analyzed by LC-Bio (Hangzhou, China).