DETERMINATION OF FREE ESTRIOL IN AMNIOTIC-FLUID BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The estrogen levels of amniotic fluid and maternal plasma may be alter ed in those cases with a fetus which presents Down syndrome. This pape r describes the methodology for determination of free estriol in amnio tic fluid. Fresh amniotic fluid was extracted using solid phase extrac tion techniques. Analtech C18 Spice cartridges were used to extract es triol (E3) prior to chromatography. After elution of the analyte E(3) from the cartridge, the solvent was evaporated under nitrogen in a war m water bath at 60 degrees C. The extract was reconstituted in methano l and E3 separated by high-performance liquid chromatography. A revers ed phase C18 Partisil ODS-3 (4.6 mm X 25 cm) column was developed with acetonitrile - methanol (65:35 v/v) as mobile phase. Subambient tempe rature (-20 degrees C) improved the resolution and aided separation fr om extraneous material. Quantitation followed intrapolation against a standard curve prepared under identical conditions. Measurement of amn iotic fluid E(3) may provide insight into the reported alteration of m aternal plasma E(3) level in Down syndrome. Results so far indicate th at in Down syndrome the amniotic fluid E(3) levels wee below the detec tion limits.