Measurement of urinary estriol glucuronides during the menstrual cycle by high-performance liquid chromatography

A simple high-performance liquid chromatographic (HPLC) method for measuring estriol-3-glucuronide (E3-3-G) and estriol-16-glucuronide (E3-16-G) in the urine of non-pregnant women is described. Estriol conjugates were extracted from 4 ml of urine with a small cartridge of graphitized carbon black (Carbopack B). After washing, E3-3-G and E3-16-G were desorbed separately by a two-step elution system. After solvent removal, the two glucuronides were quantified by isocratic ion-suppression HPLC with fluorimetric detection. The analytical recovery of the two estriol metabolites was about 95%. The detection limit of the method was 0.6 ng/ml for both analytes in urine, which is well below the concentrations of clinical interest, and the method is not susceptible to substantial interferences. Data relative to urinary levels of E3-3-G and E3-16-G measured by this method on a daily basis in early morning samples from nine women during their menstrual cycles were compared with those reported in the literature and obtained by radioimmunoassay techniques. Moreover, the potential use of defined changes in the concentrations of the two conjugates for predicting the fertile period of women was assessed.