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”.