Determination of Free Estriol in Amniotic Fluid by High Performance Liquid Chromatography

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Abstract
The estrogen levels of amniotic fluid and maternal plasma may be altered in those cases with a fetus which presents Down syndrome. This paper describes the methodology for determination of free estriol in amniotic fluid. Fresh amniotic fluid was extracted using solid phase extraction techniques. Analtech C18 Spice cartridges were used to extract estriol (E3) prior to chromatography. After elution of the analyte E3 from the cartridge, the solvent was evaporated under nitrogen in a warm water bath at 60°C. The extract was reconstituted in methanol and E3 separated by high-performance liquid chromatography. A reversed phase C18 Partisil ODS-3 (4.6 mm × 25 cm) column was developed with acetonitrile—methanol (65:35 v/v) as mobile phase. Subambient temperature (−20°C) improved the resolution and aided separation from extraneous material. Quantitation followed intrapolation against a standard curve prepared under identical conditions. Measurement of amniotic fluid E3 may provide insight into the reported alteration of maternal plasma E3 level in Down syndrome. Results so far indicate that in Down syndrome the amniotic fluid E3 levels wee below the detection limits.