Development of high speed CYP cocktail inhibition assay using UHPLC-MS/MS

Resorufin (2B6), 1’ -Hydroxy Bufuralol (2D6), (+/-)-4’ -Hydorxy Mephenytoin (2C19), and Oxidized Nifedipine (3A4) were detected by ESI-positive mode. Hydroxy Tolbutamide (2C9) was detected by ESI-negative mode. All compounds were measured by single injection with fast polarity switching (20 msec). In order to improve the sensitivity, the optimization of ionization was carried out. As a result, the calibration curves have shown excellent linearity (R2 > 0.995) ranging from 0.6 to 100 nmol / L for all tested compounds. In conventional method using Shim-pack XR-ODS II (flow rate at 0.2 mL/min), all compounds were eluted within 4.5 min (the cycle time was 7 min). In UHPLC method, the cycle time was successfully reduced within 60 seconds without sacrificing the data quality as the next sample was injected by overlapping with the last part of previous analysis. UHPLC-MS/MS improves the throughput 7 times compared with conventional system.