16a-Hydroxyestradiol is an estrogen very similar to estriol, but with three C13 in the 2,3,4 positions. Estriol is often used as a marker for fetal health since it is produced by the adrenal cortex of the fetus. 16a-Hydroxyestradiol was analyzed for a study focused on emerging contaminants in water by the Federal Institute of Hydrology of Koblenz, Germany. It was retained on both Primesep 100 and Obelisc R HPLC Columns. Obelisc R is a mixed-mode column which uses a long hydrophobic chain and multiple ionic groups to fine tune retention. Primesep 100 is a reverse phase column with embedded acidic ion pairing groups. Method is LC/MS compatible and will be effective for achieving unique retention for many pesticides.