ESTRIOL AND ITS CONJUGATES IN LATE PREGNANCY DETERMINED BY EXTRACTION WITH CARBOPACK-B AND LIQUID-CHROMATOGRAPHY WITH FLUOROMETRIC DETECTION

Abstract We report a method for measuring estriol and its intact conjugates in urine, serum, and amniotic fluid. A single assay can be done within about 50 min, eight samples assayed in less than 5 h. A 70-microL urine sample is diluted and the estriol conjugates are adsorbed from it onto graphitized carbon black (Carbopack B, Supelco). After two washings, the analytes are desorbed with chloroform/methanol (60/40, by vol) containing tetrapropylammonium bromide. After solvent evaporation, the residue is redissolved in 100 microL of water/acetonitrile and 20 microL is injected into the chromatograph. Or 1 mL of serum or 0.5 mL of amniotic fluid is deproteinized with cold methanol, then passed through the Carbopack column. After three washings, the estriol and its conjugates are desorbed and treated as for urine. Mean analytical recoveries of the analytes in any of these body fluids were within about 92-98%, except for estriol-3-sulfate-16 alpha-glucuronide in serum (mean recovery 88.3%). The limit of sensitivity is well below the concentrations of clinical interest, and the method is not susceptible to substantial interferences.