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The liquid chromatography (LC) with electrochemical detection allows to determine, evaluate and validate the level of estrogens and their metabolites in serum. The method is fast and sensitive, and the hormones can be determined simultaneously, from one serum sample. The proposed method was successfully applied to the determination of following estrogens: estrone (E(1)), 17beta-estradiol (E(2)), estriol (E(3)) and following catecholestrogens: 2-hydroxyestradiol (2-OHE(2)), 4-hydroxyestradiol (4-OHE(2)), 4-hydroxyestrone (4-OHE(1)), 2-methoxyestradiol (2-MOE(2)) and 2-methoxyestrone (2-MOE(1)). The method of LC with electrochemical detection (LC/EC) was applied for the determination of catecholestrogens in serum sample taken from pregnant women. Estrogens and catecholestrogens were extracted from 1 mL of serum by applying diethyl ether under the specified conditions. The parameters of the procedure included using a specific mobile phase, applying the column Symmetry C18 (5 microm, 3.0 mm x 150 mm), equipping the applied electrochemical detector with the working glassy-carbon electrode, as well as applying the reference electrode Ag/AgCl. The calibration studies on this study were performed, and a good analytical performance for E(1), E(2), E(3), 2-OHE(2), 4-OHE(2), 4-OHE(1), 2-MOE(2) and 2-MOE(1) was attained, along with low limits of detection (LOD of 0.18-0.30 ng/mL), satisfactory limit of quantification (LOQ of 0.23-0.92 ng/mL) and excellent linear dynamic range (0.6-8.0 ng/mL). In conclusion, the presented methodology is the sensitive method of the simultaneous measurement of eight estrogens and their metabolites from one sample of blood and it might be clinically applied for testing serum of pregnant women, as well as for further studies on estrogens and their metabolites., ((c) 2009 Elsevier B.V. All rights reserved.)

We have developed a gas chromatography/mass spectrometry (GC/MS) assay to measure 17beta-estradiol (E) and its biologically active metabolites 2-hydroxyestradiol (2OHE) and 4-hydroxyestradiol (4OHE), and 2-methoxyestradiol (2MEOE) and 4-methoxyestradiol (4MEOE) in rat plasma. All analytes are well separated and show a linear relationship between concentration (0.25-5 pg/microl) and signal, and coefficients of variation (CVs) are low. Intra-assay CV for the lowest quality control samples (QCs) (0.375 pg/microl) were on average for 17beta-estradiol 20.5%, for 2-hydroxyestradiol 15.6%, for 4-hydroxyestradiol 16.5%, for 2-methoxyestradiol 16.5%, and for 4-methoxyestradiol 11.5%. The inter-assay CVs for the lowest QCs were for 17beta-estradiol 12.1%, for 2-hydroxyestradiol 7.1%, for 4-hydroxyestradiol 15.5%, for 2-methoxyestradiol 16.7%, and for 4-methoxyestradiol 9.7%. The highest sensitivity for this assay was observed for hydroxyestradiols followed by the methoxyestradiols and 17beta-estradiol. In summary, we describe a convenient, sensitive, and specific assay to measure 17beta-estradiol and its biologically active metabolites.

Enzymatic O-methylation of catechol estrogens in red blood cells has been investigated with respect to species difference. In the presence of S-adenosylmethionine, 2- or 4-hydroxyestradiol (2-OHE2 or 4-OHE2) was incubated with blood lysate obtained from rats (five strains), guinea pigs, mice, rabbits, dogs, monkeys, and humans, respectively. The yielded guaiacols and unchanged substrate were determined by gas chromatography/mass spectrometry in a selected ion monitoring mode employing the corresponding 2H4-labeled compounds as internal standards. The total amounts of guaiacols formed from 2-OHE2 and 4-OHE2 were different, being the highest (79.6% and 38.1%) in monkeys and the lowest (5.1% and 1.9%) in humans. The ratios of isomeric guaiacols formed from 4-OHE2 (4Me/3Me) were 7.6-71, while those from 2-OHE2 (2Me/3Me) were 1.4-3.2. Thus, marked differences in O-methylation of catechol estrogens were observed among animal species, but no significant strain difference was detected in rats.

A simple and sensitive method for the determination of guaiaicol oestrogens enzymatically formed from 2- or 4-hydroxyoestradiol, by means of high-performance liquid chromatography with coulometric detection, has been developed. Catechol and guaiacol oestrogens were efficiently separated on a reversed-phase column, using 0.5% ammonium phosphate buffer (pH 3.0)-acetonitrile (59:41, v/v) as the mobile phase, and detected coulometrically in a screening-oxidation mode at +0.10 V and +0.35 V, respectively. The method was applied to the assay of in vitro enzymic O-methylation of catechol oestrogens. After 2- or 4-hydroxyoestradiol had been incubated with rat red blood cells in the presence of S-adenosylmethionine, the resulting guaiacols and unchanged substrate were percolated through an Extrelut-3 cartridge. The dried eluate was redissolved and directly injected. This simple procedure was as sensitive as the previously reported method using gas chromatography-mass spectrometry in a selected ion monitoring mode.

To investigate the role of catechol estrogens in human parturition, these steroids were analyzed in samples from the maternal venous and umbilical venous and arterial plasma at vaginal (n = 28) and abdominal (n = 28) delivery. To ensure the appropriateness of collection of umbilical artery and venous blood samples, progesterone content was also determined. Although there is no significant difference in maternal vein content of catechol estrogens between the two groups, the umbilical venous (p = 0.03) and arterial (p = 0.002) plasma concentrations are significantly higher at vaginal delivery than those measured at abdominal delivery. In view of the present data and the importance of catechol estrogens in prostaglandin synthesis and in potentiating the activity of catecholamines through competitive inhibition of catechol-O-methyltransferase, it is suggested that catechol estrogens may play a role in triggering the events involved in the onset of labor and delivery in humans.

To provide a definite basis for studies on the biological effects of exogenously administered catecholestrogens, the time courses of the concentrations of these estrogens in serum, pituitary and CNS-tissues were studied in male rats after s.c. injection of either 150 microgram of 4-hydroxyestradiol or 2-hydroxyestradiol (dissolved in 200 microliter sesame oil/ethanol/ascorbic acid; 97.5/2.5/0.1; vol/vol/wt) or equimolar amounts of 4-hydroxyestradiol 3, 4-dibenzoate or 2-hydroxyestradiol 2,3-dibenzoate (dissolved in 200 microliter sesame oil). The injection of free catecholestrogens resulted in bolus-like elevations of the serum and tissue concentrations of the respective compound (max. values up to 9 ng/ml, half-life below 1 h) whereas the injection of catecholestrogen benzoates gave lower (max. values about 1 ng/ml) but prolonged elevations (half-life approx. 24 h and 32 h for 4-OHE2 and 2-OHE2) of the respective free catecholestrogen.