AbstractThe high performance liquid chromatographic (HPLC) separation and identification of 12 isomeric and/or highly chemically related steroids with an unsaturated ester moiety at position 17β has been achieved. The main stereochemical features of the steroid skelton cover 3α/β or Δ, and 20E/Z, bearing the alcohol or hemisuccinate group at the 3 position. The isocratic reversed phase C18HPLC separation employed ethanol, methanol and its mixtures with water or 0.01 M phosphoric acid as the mobile phase. The best separaton of the respective alcohols from their hemiscuccinates has been achieved with 20% of aqueous phase content. The best separation among isomeric or related steroids has been achieved with methanol: water 8:2 and 85:15 and similar systems containing phosphoric a