LC/ESI-MS/MS
The UPLC-MS/MS was performed according to the method described previously14 using an Agilent 1290 Infinity II and 6470 triple quadrupole mass spectrometer equipped with an ESI ion source (Agilent Technologies, Inc. Santa Clara, CA). Briefly, the samples were separated with an Agilent Eclipse Plus C18 RRHD 2.1x100 mm, 1.8 μm column by using solvent A (0.1% formic acid in deionized water) and solvent B (0.1% formic acid in MeOH). The elution gradient was from 40-80.0 % B from 0 to 8 min, maintained at 80 % B from 8 to 10 min, 80-40.0 % from 10.0 to 10.10 min and held at 40 % B from 10.1 to 13.1 min. The injection volume was 20 µL. The MRM mode was applied for the detection and quantitation of all steroids with two transitions optimized for each targeted compound (Table S1). The post-column addition of 0.2 mM LiCl in H2O was carried out with an Agilent 1100 binary pump as the auxiliary pump. The column effluent (0.4 mL min-1) and auxiliary solution (0.4 mL min-1) were mixed at the T-connector and passed through two in-line filters (Agilent 1290 inline filter, 0.3μm), which were connected in tandem, prior to reaching to the ion source (Figure S1). The auxiliary pump was off when conducting the “H method”.