4OH-tamoxifen measurement
CAD
SEPARATION, CAD
4-hydroxytamoxifen was separated using revrse phase LC separation with Acquity UPLC column (50 mm×2.1 mm) T3 (C18) bonding and endcapping, packed with high strength silica (HSS) particle substrate. Sample volume of 5 µL was injected using Simadzu Nexera ultra-high performance liquid chromatography (UHPLC) (Shimadzu, Japan) coupled with Q-trap SCIEX 6500 (QQQ) mass spectrometer (SCIEX). The column oven temperature was maintained at 40 °C, with a flow rate of 0.4 mL/min. A linear gradient was applied ranging for 5.0 min ramping from 30% to 95% solvent (0.1% formic acid in acetonitrile). MS parameters for data acquisition using SCIEX 6500 triple quadropole system included: positive Electrospray ionization (ESI); Ion source voltage at 5500 V; Source temperature at 550 °C; collision activate dissociation (CAD) at 8; Nebulizer gas (GS1) at 50; and Auxillary gas (GS2) at 60. 4-hydroxytamoxifen parent ion was monitored (major precursor [M-H]+ ions at m/z 388.0) and daughter ion (major fragmented product ion at m/z 72.2).