LC-MS/MS
To analyze gonadal steroids, we reconstituted samples in HPLC grade
methanol and used the biphenyl LC-MS/MS method (6) (Table 1). We
injected 10 µL of each sample onto the Restek (Bellefonte, PA) Ultra
Biphenyl column (250 mm x 4.6 mm, 5 µm particle size) column and
conducted separation using a gradient of acetonitrile and methanol (both
with 0.1 % volume formic acid) over 36 minutes at a flow rate of 500
µL/min.
We analyzed adrenal steroids using the C18 LC-MS/MS method developed in
this study and described above. Before injecting 10 µL of each sample
onto a C18 column for adrenal steroid analysis, we conducted a solvent
exchange, bringing samples to a 50:50 volume fraction of Milli-Q water
and methanol (described in Galligan et al. (8). Once injected, we
separated steroids using a gradient of methanol and Milli-Q water (both
with 0.1 % volume acetic acid) throughout the 52-minute method at a 250
µL/min flow rate (Table 2).