Figure 2.

High-performance liquid chromatography separation of (a) cortisol, (b) corticosterone, (c) 11-deoxycortisol, and (d) 11-deoxycorticosterone. To separate these glucocorticoids, we used reversed-phase HPLC (Gilson 322) with a Waters SymmetryShield C₁₈ column (4.6 mm × 250 mm, 5 μm silica particles). The mobile phase consisted of 50% HPLC-grade methanol in HPLC-grade water with 0.01% glacial acetic acid (solvent A; indicated by the solid gray line at top). At 45 min, 5% isopropanol (solvent B; dashed gray line at the bottom) was added in order to elute the final peaks more rapidly. The solid black line indicates radioinert steroids measured with a UV detector, and the dotted gray line indicates ³H-labeled steroids measured with a radioflow detector (as described in Mensah-Nyagan et al., 2008; Pradhan et al., 2008, 2010a). Detection of radiolabeled steroids occurs 1.6 min later than radioinert steroids because of the extra length of tubing between the UV and radioflow detectors. A fraction collector was then used to collect glucocorticoids for quantification via enzyme immunoassay (EIA) or radioimmunoassay (Taves et al., unpublished results).