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).