The UHPLC column used for isolating steroids/secosteroids is Waters ACQUITY UPLC HSS T3 Column (100Å, 1.8 µm, 2.1 mm × 100 mm, Part# 186003539).
When setting up an LC-MS/MS run, make sure that TSQ Altis (Figure 2) is correctly informed of the LC flow rate (Figure 1).
Figure 1. LC flow rate in Xcalibur Instrument Setup (pump settings).
Figure 2. LC flow rate fed to TSQ Altis (ion source properties) in Xcalibur Instrument Setup (NOT in FreeStyle).
When an LC-MS/MS sequence finishes running, the vanquish pump should be stopped manually: go to "Direct Control", click "Take Control" at the top, and uncheck the "Flow" box under the "Vanquish Pump" tab.
Figure 3. Stopping the vanquish pump.