A semi-automated fluorometric method for total estrogens in pregnancy urine

We report a continuous-flow fluorometric method for total urinary estrogens that involves the Kober reaction, with extraction of the reaction product into dichloroethane containing trichloroacetic acid as described by Hahnel and Jones [Clin. Chim. Acta 16, 185 (1967)]. The dichloroethane extraction gives greater stability to the Kober color and sharper separation of aqueous and organic phases. The analytical system is adjusted to give maximum response to estriol 16alpha,beta-D-glucuronide, the principal estrogen conjugate in urine from late pregnancy, when calibrated with estriol standards. An initial 20-fold dilution of the sample with water increased analytical recovery of estriol conjugates from urine while maintaining adequate fluorescent response. Glucose interference was reduced by dilution and eliminated by treatment with sodium borohydride. Urinary protein up to 20 g/liter did not interfere. Total estriol, as determined by gas-liquid chromatography, comprises about 70% of total urinary estrogens in late pregnancy as measured by our continuous-flow fluorometric method. A reference range is presented based on 209 randomly collected urine specimens in which total urinary estrogens are expressed as a ratio to creatinine.