Zorbax sshd eclipse plus c18 column

Samples (10 μL) were chromatographed on a ZORBAX SSHD Eclipse Plus C18 column (3×50 mm, 1.8 μm, 959757–302; Agilent, Santa Clara, CA) with a guard column (2.1×5 mm, 1.8 μm, 821725–901; Agilent) using 1290 Infinity II LC (Agilent). Column temperature and the LC flow rate were set at 40°C and 0.4 ml/min. Initial chromatographic condition was maintained at 90% mobile phase A (water with 0.1% formic acid, v/v) and 10% mobile phase B (acetonitrile with 0.1% formic acid, v/v) for one min, then increased to 80% B by 3 min, then to 95% B by 4 min, and then returned to initial condition at 5 min until 8 min for sufficient equilibrium. Unmodified mobile phases A (water) and B (acetonitrile) were used for the analysis of Troglitazone.
MS/MS analyses were performed in positive ion mode (all test drugs except Troglitazone) or negative ion mode (Troglitazone) with an electrospray ionization (ESI) source using 6470 triple-quadruple MS (Agilent). The capillary voltage was set at 3500 V. The nebulizer gas pressure and gas temperature were set at 35 psi and 350°C, respectively. The MS/MS parameters for each test compound are summarized in Supplemental Table 1.

Samples were maintained at 4°C in the auto-sample manager of a Waters Acquity H-class UPLC (Waters, Milford, MA) during analyses. Samples (10 μL) were automatically injected into the UPLC and were chromatographed on a ZORBAX SSHD Eclipse Plus C18 column (3.0×50 mm, 1.8 μm, cat no. 959757-302; Agilent, Santa Clara, CA) with a guard column (2.1×5 mm, 1.8 μm, cat no.821725-901; Agilent, Santa Clara, CA). For each sample analysis, initial chromatographic conditions were 80% solvent A (water with 0.1% formic acid) and 20% solvent B (methanol with 0.1% formic acid). Conditions were maintained from 0–1 min, then increased to 90% of solvent B by 3 mins, then to 98% of solvent B by 4 mins, then to 20% of solvent B by 4.2 mins. Then, 20% of solvent B was maintained until 7 mins to allow sufficient equilibration time prior to the next injection. Flow rate was maintained at 0.4 mL/min. Column temperature was controlled at 25°C.

Media samples (10 μL) were chromatographed on a ZORBAX SSHD Eclipse Plus C18 column (3×50 mm, 1.8 μm, 959757-302; Agilent, Santa Clara, CA) with a guard column (2.1×5 mm, 1.8 μm, 821725-901; Agilent) using 1290 Infinity II liquid chromatograph (LC; Agilent). Column temperature and the LC flow rate were set at 40°C and 0.4 ml/min. Initial chromatographic condition was maintained at 90% mobile phase A (water with 0.1% formic acid, v/v) and 10% mobile phase B (acetonitrile with 0.1% formic acid, v/v) for one min, then increased to 80% B by 3 min, then to 95% B by 4 min, and then returned to initial condition at 5 min until 8 min for sufficient equilibrium. Unmodified mobile phases A (water) and B (acetonitrile) were used for the analysis of Troglitazone.
MS/MS analyses were performed in positive ion mode (all test drugs except Troglitazone) or negative ion mode (for Troglitazone) with an electrospray ionization (ESI) source using 6470 triple-quadruple MS (Agilent). The capillary voltage was set at 3500 V. The nebulizer gas pressure and gas temperature were set at 35 psi and 350°C, respectively. The MS/MS parameters for each test compound are summarized in Supplemental Table 1.