Compound libraries were purchased from Enzo Life Sciences (Farmingdale, NY, USA). The standards were supplied in DMSO (96-well plate format) and were diluted to 1 µM in 50:50 acetonitrile/water (v/v) for MRM optimization and 25 nM in 95:5 water/acetonitrile (v/v) for UPLC analysis. MRM optimization was performed using the MassLynx v4.1 application manager QuanOptimize. A UPLC separation was achieved on a CORTECS C18+, 1.6 µm, 2.1 x 100 mm Column at a flow rate of 0.5 mL/min and gradient from 5% B to 95% B over 10 minutes, with a one-minute hold and re-equilibration for 10-column volumes at a total run time of 16 minutes. The mobile phase consisted of (A) water and (B) acetonitrile, each with 0.1% formic acid and injection volume was 5 µL. The retention times were measured using the TargetLynx Application Manager, and were the average of five-replicate injections.
One hundred seven compounds commonly used in CYP inhibition assays were analyzed using the same column, mobile phase, and mass spectrometer by both the ACQUITY UPLC I-Class and the ACQUITY UPLC I-Class PLUS Systems. Figure 1 shows chromatograms for eight representative compounds on each LC system with tabulated results in Table 1. The retention times between the two systems were comparable for all the compounds tested. Retention time reproducibility is illustrated in Figure 2 for representative compounds, and was less than 0.6% and 0.5% RSD for the 107 compounds for the ACQUITY UPLC I-Class and ACQUITY UPLC I-Class PLUS Systems, respectively. These results demonstrate the reproducibility of the ACQUITY I-Class PLUS System and gives confidence in each analysis.
