Quattro ultimaptesi tandem quadrupole mass spectrometer

For steroids quantification by LC-MS/MS analysis, 200 µL of DPS containing IS was added into each 2.0 mL microcentrifuge tube (Eppendorf^®, Hamburg, Germany). Subsequently, 100 µL of serum sample from each patient, calibrators, and QCs was added into a tube with DPS, mixed (20 °C, 15 min) in a Thermomixer (Eppendorf^®), and then centrifuged (4210 rcf, 20 °C, 30 min). The organic layer (175 µL) was recovered, dried in a SpeedVac, and reconstituted with 125 µL of H₂O:MeOH 60:40. The obtained solution was gently mixed in a Thermomixer (20 °C, 15 min) and briefly centrifuged, and the supernatant was transferred into a polypropylene vial (Waters Corporation, Milford, MA, USA). The vials were placed in the autosampler for LC-MS/MS analysis. The LC-MS/MS system consisted of a HPLC Alliance HT 2795 Separations Module coupled to a Quattro UltimaPtESI tandem quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA), operating in positive electrospray ionization mode with MassLynx v4.1 software (Waters). An amount of 50 µL was injected, with a total run time of 18.00 min. A detailed description of the LC-MS/MS analysis for the determination of steroids and the instruments parameters have already been reported [21 (link)]. QuanLynx 4.1 software (Waters Corporation, Milford, MA, USA) was used for data processing and quantification.