Spironolactone (I), canrenone (II), and potassium canrenoate (III) can be analyzed in the low nanogram range utilizing a fluorescence reaction in sulfuric acid (62% v/v). The fluorescent species, a trienone, was used as an external standard. The methods were highly sensitive (~10 ng./ml. of I-III in plasma) and specific for 17-alkyl-17-hydroxy-4,6-dien-3-one steroids. The 7α-thioacetyl-4-en-3-one (I) was dethioacetylated under mild alkaline or acid conditions to the 4,6-dienone moiety prior to extraction. The ester glucuronide of III was quantitatively hydrolyzed at pH 13 for 10min. at 100°. An estimate of acid-labile conjugates of I-III was achieved by reaction in methanolic 3.6% sulfuric acid for 30min. at 100°. Spiro-γ-lactone congeners (e.g., I and II) were separated from γ-hydroxycarboxylic acids (e.g., III) by extraction into methylene chloride at pH 7-8. The methods were applicable to biological samples such as plasma, bile, urine, and gastric fluids.