Lc 10ad vp hplc system

Chromatographic separation was performed using an UPLC system (Shimadzu LC-10 AD VP HPLC system, Columbia, MD) equipped with a Phenyl/Hexyl Column (2.6 μm, 150 × 3.0 mm, Accucore Thermo Scientific, Waltham, MA) at 40°C. The eluents were 20% methanol in water with 0.2 mM ammonium fluoride (eluent A) and methanol (eluent B). 50 μL of sample solution was injected and the analytes were eluted using the gradient from 40% to 72.5% of eluent B at the flow rate of 450 μl/min with a total of run time of 10 minutes. Mass spectrometric analysis was performed using a triple quadrupole tandem mass spectrometer (AB Sciex 5500, Foster City, CA) with electrospray ionization switching in negative ion mode for E2 and positive mode for TT. Selected reaction monitoring (SRM) was used with quantitation ion transitions (QI) of m/z 271 → 145 for E2, 274 → 148 for ¹³C₃-E2, 289 → 97 for TT, and 292 → 100 for ¹³C₃-TT. For monitoring confirmation ion transitions (CI), m/z 271 → 183 for E2, 274 → 186 for ¹³C3-E2, 289 → 109 for TT, and 292→112 for ¹³C3-TT were used. The declustering potential (DP), entrance potential (EP) and collision cell exit potential (CXP) were −140 V, −12 V, −17 V for E2 and 240 V, 8 V, 12 V for TT, respectively. The collision energy (CE) was −51 eV for all E2 and ¹³C3-E2 transitions and 27 eV for all TT and ¹³C3-TT transitions.