The mass spectrometer ionization source settings were optimized for maximum precursor ion yields for each steroid. This was achieved by injecting the tuning solution for each individual compound, all the parameters are summarized in Table 4. Two mass transitions were optimized for each analyte, with a single transition being used to monitor the corresponding deuterated internal standards. The first transition was used to quantify the target analyte and the second to qualify the identity of the target compound using a confirmatory ion ratio. The capillary voltage was 3.5 kV, source temperature was 120 °C, desolvation temperature was 400 °C, and the collision cell gas pressure was 3.5 × 10−3 mbar argon. The inter-channel and inter-scan delay times were 0.02 and 0.1 s, respectively. The dwell time was 0.45 s for CORT, 0.30 for CCONE and ADIONE, 0.25 for 11-DECOL, TESTO and 17-OHP, 0.35 for PROG. The same dwell time parameters as the ones of the endogenous compounds were used for the respective internal standards. Functions 1–7 (Table 4) refer to the MRM experiments created for each analyte.