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Estriol (EO) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method using HPLC for EO was investigated. After placing 1.0 ml of EO solution at 50 microg/ml and 0.5 g of the lotion into a 10-ml volumetric flask, the methanol was added to make until that volume and this solution was used as the testing solution. Milky lotion was procedured as follows: After placing 1.0 ml of EO solution at 50 microg/ml and 0.5 g of the milky lotion into a 10-ml volumetric flask, the methanol was added to make until that volume. The suspending mixture was moved to a centrifuging tube with a cap. After centrifuging at 3000 rpm for 5 minutes, the supernatant was used as the testing solution. The testing solution of 20 microl was determined by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250 mm), the mixture of water and acetonitrile (31:9) and the detection wavelength of 285 nm. The working curve from 1.0 to 6.0 microg/ml showed a linear line between the concentrations of EO and the peak area. There was no interference of peak of EO from the lotion and milky lotion.

A simultaneous microdetermination of unconjugated estrone, estradiol, estriol and estetrol in serum or amniotic fluid by High Performance Liquid Chromatography with an Amperometric Detector is described. Steroids in serum or amniotic fluid were extracted with 10 volumes of ethyl ether, and then ether extract was evaporated to dryness under N2 gas. After defatting with a mixture of 50% methanol/n-hexane, the methanol phase was evaporated to dryness under N2 gas. The residue was applied to microcolumn packed with 2 ml volume of Sephadex LH-20 in the eluting solvent benzene/methanol (85:15). Fractions contained estrone and estradiol; estriol and estetrol were collected and then evaporated to dryness under N2 gas. The sample solution was applied to HPLC using a reverse phase ODS column and acetonitrile: 0.1M KH2 PO4 47:53 for estrone and estradiol fraction, and 30:70 for estriol and estetrol fraction as a mobile phase, respectively. The fraction of each estrogen was separated completely within a 20 minute period. The limit of detection of estrone, estradiol, estriol and estetrol was 50 pg, respectively.

As estetrol (E4) is believed to be the steroid most likely to wholly dependent on fetal origin, we developed a radiommunoassay for unconjugated and conjugated E4 (E4-U and E4-G) and investigated plasma and urinary levels serially throughout the second half of pregnancy to establish their validity by means of monitoring or screening tests to assess fetal well-being. E4 exhibits a remarkable increase during the latter half of pregnancy. At term, the mean E4-U level in maternal peripheral plasma was 0.67 +/- 0.33 ng/ml, a five fold increase from that at 28 weeks; E4-G was 4.57 +/- 2.84 ng/ml, showing a four fold increase; and E4-G levels in maternal urine were 1.68 +/- 0.96 mg/day, showing a three fold increase from that at 28 weeks. E4-U and E4-G levels showed no diurnal change. The coefficient of the correlation between plasma E4-U and E4-G was 0.699, which is satisfactory, but no correlation was found between urinary and plasma E4 levels. A significant correlation was shown between maternal and umbilical E4-U (r=0.820) and E4-G (r=0.608). No relationships between E4 levels and birth weight were detected. Pre-eclampsia, Rh-isoimmunization and diabetes mellitus are common complications of pregnancy which may cause latent fetal distress. Prenatal fetal assessment was performed by serial daily evaluations of these E4 values. In pre-eclampsia resulting in a small full term baby, E4 levels were mostly below normal mean values or failed to show an increased pattern. In addition, the E4 levels decreased in one case of neonatal death. In Rh-isoimmunization, plasma E4-G levels were lower in the group affected severely by the hemolytic desease. In a patient with diabetes mellitus delivered of a healthy baby, E4 levels were within the range of a normal pregnancy. In order to evaluate fetal and placental reserve capacities as well as feto-placental function, the dehydroepiandrosterone sulfate (DHA-S) loading test was performed by loading selected subjects with 50 mg of DHA-S, then serially measuring the E4 in the maternal plasma and urine. Intravenous infusion of 50 mg of DHA-S was completed in 60 minutes. A rapid and sharp increase of plasma E4 was observed, reaching maximal concentrations at 120 minutes in normal pregnancies. However, urinary levels showed patterns similar to those reported for estriol (E3). In some abnormal pregnancies, no increased or delayed patterns were observed in plasma E4-G levels, while the serial levels remained within the normal range. This possibly suggests that in these pregnancies, fetal functions had been inhibited or had reached their limit. It is concluded that the simultaneous determinations of serial E4 levels accompanied by the DHA-S loading test may be of value in assessing fetal well-being and reserve capacity and may therefore improve fetal and neonatal prognosis in abnormal pregnancies.