Each endogenous steroid (1 mg) was dissolved in ethanol (stock solution) and stored at −20 °C. Stock solutions of each compound were diluted in methanol/water 50:50 with a final concentration of 10 µg/mL (tuning solution) and stored at −20 °C. The internal standard mixture (IS mix) from PerkinElmer® kit was reconstituted with 1.25 mL of ACN and stored at −20 °C. Before the extraction procedure, the Daily Precipitation Solution (DPS) containing Internal Standards was prepared by diluting 1:1000 the IS mix in two consecutive steps with ACN with 0.1% formic acid. After diluting the tuning solution, we obtained the working solutions for each steroid of interest as follow: CORT 50 ng/mL, CCONE, TESTO, 17-OHP and PROG 5 ng/mL, 11-DECOL and ADIONE 10 ng/mL. Calibrators were prepared from each working solution to achieve final concentrations as reported in Table 3. The same procedure was used to obtain QC samples that, considering the small concentration range of interest, resulted to coincide with L2, L4 and L6 calibrator levels (Table 3).