Search

Monitoring biological samples at trace levels of chemicals from anthropogenic actions such as pesticides, pharmaceuticals, and hormones has become a very important subject. This work describes a method for the determination of eight compounds of different chemical classes in human urine samples. Dispersive liquid-liquid microextraction based on magnetic ionic liquids was used as the sample preparation procedure. The main parameters of the method, such as sample dilution, type, and volume of disperser solvent, amount of magnetic ionic liquids, extraction time, and pH were optimized by univariate and multivariate procedures. Validation was performed using a urine sample of a male volunteer in order to obtain a calibration curve and the main analytical parameters of merit such as limits of detection and quantification. Values varied from 3.0 to 7.5 µg/L and from 10 to 25 µg/L, respectively. Satisfactory precisions of 21% for intraday (n = 3) and 16% for interday (n = 9) were achieved. Accuracy was evaluated by relative recovery assays using different urine samples and ranged from 75 to 130%. Robustness was assured by the Lenth method. The validated procedure was applied to five urine samples from different volunteers and the hormone estrone was found in one sample., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work describes the preparation of an electrochemical immunosensor for ethinylestradiol (EE2) based on grafting of diazonium salt of 4-aminobenzoic acid onto a glassy carbon electrode modified with silver nanoparticles/SiO2/graphene oxide hybrid followed by covalent binding of anti-ethinylestradiol (anti-EE2) to activated carboxyl groups. A competitive immunoassay was developed for the determination of the hormone using peroxidase-labeled ethinylestradiol (HRP-EE2) and measurement of the amperometric response at -200mV in the presence of hydroquinone (HQ) as redox mediator. The calibration curve for EE2 exhibited a linear range between 0.1 and 50ng/mL (r(2)=0.996), with a detection limit of 65pg/mL. Interference studies with other hormones related with EE2 revealed the practical specificity of the developed method for the analyte. A good reproducibility, with RSD=4.5% (n=10) was also observed. The operating stability of a single bioelectrode modified with anti-EE2 was maintained at least for 15 days when it was stored at 4°C under humid conditions between measurements. The developed immunosensor was applied to the analysis of spiked urine with good results., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To predict sewage influent and effluent concentrations of the steroid estrogens 17beta-estradiol, estrone, and 17alpha-ethinylestradiol, a review of human excretion was carried out. This included conjugation and metabolism of the natural and synthetic steroid estrogens within the body, together with quantities excreted in the urine and feces by different members of the population. This has been combined with fate and behavior information for conjugated and unconjugated estrogens in the sewage treatment system to enable sewage works influent and effluent concentration predictions to be made. The model has proved to be reasonably accurate when tested against recent measurements of these steroid estrogens in the influent and effluent of sewage treatment works. The model may be used with river dilution ratios to predict which sewage treatment works are most likely to cause the greatest endocrine disruption due to steroid estrogens.

This study investigated whether the illegal application of ethinylestradiol or clenbuterol in cattle as growth promotors may be concealed by co-treatment with drugs that affect urinary excretion. Therefore, six male veal calves were fed with ethinylestradiol and six different male veal calves were fed with clenbuterol for 13 days. Both groups received the growth promotors twice daily (days -2 to 11) with milk replacer. The calves receiving ethinylestradiol were additionally fed with probenecid on days 7-11, and the calves receiving clenbuterol were additionally fed with trimethoprim (days 7-11). During days 1-11 of the experiment, 24-h urine and blood samples (once daily) were collected and analyses for ethinylestradiol and clenbuterol by specific enzyme immunoassay. In four calves the average urinary excretion of ethinylestradiol during days 7-11 (co-treatment with probenecid) was only about 25% of their average urinary excretion of ethinylestradiol on days 1-6. In the other two calves of this group, the excretion of ethinylestradiol was reduced to 4% on days 7-11 compared with days 1-6. In these two calves several urine samples provided concentrations of ethinylestradiol around the limit of detection. As a consequence, there may be a chance of concealing ethinylestradiol application by co-treatment with probenecid. Co-treatment with trimethoprim led only to a slight reduction of urinary excretion of clenbuterol. The detection of clenbuterol in urine samples from calves which were co-treated with trimethoprim can thus not be prevented.

A method for the detection of ethinylestradiol in cattle urine is described, based on enzymic hydrolysis of the sample, clean-up by means of disposable octadecyl and amino solid-phase extraction columns, fractionation by reversed-phase high-performance liquid chromatography, and detection by gas chromatography-mass spectrometry (selected-ion monitoring). Identification is based on both gas chromatographic and mass spectrometric data. The method has been tested on urine samples for a collaborative study and all the results found were correct.

The extent to which norethisterone is converted to ethynyloestradiol is controversial. To investigate the conversion of norethisterone to ethynyloestradiol we have used a double isotope infusion technique to measure the conversion in vivo. The use of acids or bases was precluded to prevent possible artefactual formation of phenolic metabolites of norethisterone. Transfer constants for the conversion of norethisterone to ethynyloestradiol in two perimenopausal women were 2.26 and 2.34% as measured in blood and 2.27 and 0.38% in urine. Results from this study show that a small but significant proportion of norethisterone is converted to ethynyloestradiol in vivo.

17 alpha-Ethynylestradiol-17 beta (beta-D-glucuronide) [EE217 beta (beta G)], a metabolite of 17 alpha-ethynylestradiol (EE2) identified in urine of women taking EE2 in oral contraceptives, and its synthetic anomer, 17 alpha-ethynylestradiol-17 beta (alpha-D-glucuronide), [EE217 beta (alpha G)], were administered intravenously to female rats in order to determine their effects on bile flow. Both agents induced an immediate, profound and dose-dependent decrease in bile flow which returned to control levels within 1-8 hr. The logarithm of the dose vs the cholestatic response curves for the two anomers were not parallel. EE217 beta (alpha G) was significantly more potent than EE217 beta (beta G) such that the doses inhibiting bile flow by 50% were 1.25 and 11 mumol/kg for the alpha-and beta-anomer respectively.

Chromatographic profiles of the conjugates of 17alpha-ethynylestradiol (EE2) were obtained from the urine of castrate and normal women given tritium labeled EE2 orally. Five distinct radioactive peaks were observed, with considerable quantitative variation between individuals. Glucosiduronate fractions comprised the dominant excreted radioactivity. In vitro incubation of normal human liver also produced five conjugate types. Simultaneous intravenous/oral (14C/3H) administration of EE2 demonstrated conversion to identical urinary conjugate and free steroid products, with a greater excretion of the intravenous material. Authentic 3-glucuronide of EE2 was synthesized and its position in the chromatographic system relative to the urinary conjugate peaks demonstrated.

The biliary and urinary metabolites of [6,7-3H]17 alpha-ethynylestradiol (EE2) in women were studied with reference to the possibility of estimating EE2 2-hydroxylation by analysing urinary metabolites alone. Five subjects received 50 micrograms of 3H-EE2 orally. Bile was obtained by either endoscopy or T-tube drainage. The metabolites excreted in bile and urine were largely glucuronides and arylsulphates, but in variable proportions. The glutathione adduct of 2-hydroxyethynylestradiol was not observed in bile. EE2 was the predominant component of the glucuronide fractions of bile and urine. Additionally, the proportion of glucuronylated EE2 in a subject's urine quantitatively paralleled that in bile. HPLC analyses indicated that the proportions of EE2 and 2-methoxy-EE2 in urine are predictive of EE2 2-hydroxylation in most women. With some subjects, however, urinary analysis alone considerably underestimates the extent of 2-hydroxylation.

A method for separation and analysis of conjugates of ethynylestradiol in urine is described. Steroid conjugates are separated on a lipophilic strong anion exchanger (triethylaminohydroxypropyl Sephadex LH-230), and phenolic steroids released by enzyme hydrolysis or solvolysis ar isolated by chromatography on the same ion exchanger. Steroids carrying an ethynyl group are isolated by chromatography on SP-Sephadex (Ag+). Ethynylestradiol is analyzed by gas chromatography-mass spectrometry of the trimethylsilyl ether, using [9,11,11,12,12-(2)H5] ethynylestradiol and internal standard.

17 alpha-Ethynylestradiol (EE2)[20,21-14C] and [9,11-3H] were administered by intragastric intubation to three adult female rhesus monkeys. Quantification of the tritium isotope indicated that the peak plasma radioactivity (3.4-6.8% of dose) occurred between 30 and 60 min, whereas 36-39% of the administered tritium appeared in the urine in the first 96 hr. Fecal excretion was 9.17.0% during the same period. Fractionation and isolation of the urinary conjugates were accomplished by chromatography on Sephadex LH-20 followed by HPLC. Cochromatography of radioactive fractions with authentic steroid glucuronide standards gave presumptive evidence for the presence of ethynylestradiol and estrone glucuronides. Following hydrolysis, the tritium-labeled natural estrogens, estrone and estradiol-17 beta, were identified with reverse-isotope recrystallization, demonstrating that the de-ethynylation of EE2 had occurred.

Radioactive mestranol (ME) and/or ethynylestradiol (EE) were administered to women in Nigeria, Sri Lanka, and the USA, and the types and patterns of radioactive urinary conjugates examined by Sephadex LH-20 chromatography. There are no differences in the total excretion of urinary radioactivity over 3 days. Consistent geographic differences appear to be present in the proportion of 3-, 17-, and 3,17-glucuronides. If confirmed on larger population samples, these observations may indicate significant geographic differences in the hepatic metabolism of ethynyl estrogens. High performance liquid chromatographic patterns of the urinary aglycone metabolites of ME and EE were examined in a number of women. The separation was accomplished on a Chromegaprep Diol column with a gradient of isopropanol in heptane. Ethynyl estrogen metabolism shows considerable individual variation. EE is usually the principal compound escreted following ME or EE administration. Unmetabolized ME is present in the ME profiles. The profiles of EE and ME are similar, with EE demonstrating a more complex pattern. Oxidative metabolism occurs chiefly at positions 2, 6 and 16 and is fairly extensive in the USA subjects. The Sri Lankan women generally show less of the oxidative products and the Nigerian group display a notable lack of oxidative metabolism. There is no difference in the metabolic patterns of long-term oral contraceptive users vs. non-users. Using silver sulfoethylcellulose column chromatography, from 14.1 to 34.7% of the excreted radiolabeled aglycones are non-ethynyl (i.e., either D-homo or de-ethynylated estrogens).

Metabolites of 17alpha-ethynylestradiol (EE2) were obtained from human urine following ingestion of tritium-labeled EE2. Over 95% of the recovered activity was found as conjugated steroids and these were separated into four groups by chromatography of the urine extract on Sephadex LH-20 with chloroform-methanol (1/1) + 0.01M NaCl. The two major conjugate fractions appeared to be almost exclusively glucosiduronates. Enzymatic hydrolysis liberated at least ten different EE2 metabolites as shown by chromatography on Sephadex LH-20 with benzene-methanol (85/15). After additional separation and purification of these metabolites, positive identification was obtained for nine radioactive compounds by either gas liquid chromatography-mass spectrometry or reverse-isotope recrystallization. Five were ethynyl compounds: EE2, 2-MeO EE2, 16beta-OH EE2, 2-OH EE2 and 6alpha-OH EE2. The other four were de-ethynylated estrogens: estrone, estradiol-17beta, estriol, and 2-Me-O-estradiol-17beta.

Ethinyloestradiol-3H was given intravenously and orally to four and three women, respectively, in a dose of 60 micrograms and 3 mg. To another three female volunteers, 100 micrograms of ethinyloestradiol was administered by both routes in succession. Drug concentration in plasma and total radioactivity in plasma, urine and faeces were measured for different periods of time. Intraindividual comparison of the area under the drug level vs. time curve after intravenous and oral administration of 100 micrograms showed that ethinyloestradiol is subject to an about 60% first-pass effect in women. The time course of ethinyloestradiol concentration in plasma can be described by a 3-compartment model after intravenous injection and by a 2-compartment model after oral administration, because an early disposition phase with a half-life of about 15 minutes only becomes visible after i.v. injection. On an average, the terminal half-life of unchanged ethinyloestradiol level and total radioactivity was calculated to be about 1 day. However, a high variability was found with this parameter as well as with the rate and degree of elimination in urine.

A radiochemical GLC analysis was developed for 3H-labeled ethinyl estradiol in human urine. The technique was applied to the unconjugated and aglycone fractions of urine collected from women who were dosed orally with: (a) single capsules containing 2.0 mg of 3H-quinestrol (900 muCi) and 2.5 mg of unlabeled quingestanol acetate dissolved in sesame oil and (b) single tablets containing 100 microgram of 3H-quinestrol (86 muCi). Unconjugated ethinyl estradiol in Day 1 urine collections represented means of 0.02% of the high quinestrol dose and 0.12% of the low dose. Ethinyl estradiol glucuronide in the same collections represented means of 0.55% of the high drug dose and 1.35% of the low dose. The method could detect 1-ng quantities of 3H-ethinyl estradiol and 3H-quinestrol.

The metabolism of 17 alpha-ethynyl[6,7-3H]estradiol (3H-EE2) (50 micrograms) given orally was studied in two groups of women: (a) six subjects from whom duodenal bile samples were obtained after 4 h by endoscopic aspiration; (b) two subjects with bile-duct (T-tube) drainage. The first group eliminated 16.6 +/- 7.8% (mean +/- S.D.) of the dose in urine over 72 h, the second group 28.6% and 27.5%. Biliary excretion by the latter was 41.9% and 28.3% of the dose, respectively, during the first 24 h after dosing. The metabolites excreted in bile and urine were largely polar conjugates: 1-12% of the 3H was ether extractable. Approx. 70-90% of urinary and biliary 3H was extractable following beta-glucuronidase-arylsulphohydrolase hydrolysis. Both beta-glucuronides and arylsulphates were excreted. Unchanged 3H-EE2 was the principal 3H-labelled component of the glucuronide and arylsulphate fractions of bile, and it was a major component of urinary fractions. 2-Hydroxy-EE2 and 2-methoxy-EE2 were identified as conjugated biliary metabolites.

The pharmacokinetics and metabolism of moxestrol have been compared in the rat, dog and monkey (rhesus and baboon) and, in some instances, confronted with data simultaneously obtained for ethynl estradiol and previously obtained in humans. The apparent initial volume of distribution (AIVD) of total radioactivity after i.v. administration was of the order of body volume in all species under study; the AIVD of intact moxestrol was even higher. This is in agreement with moxestrol's low binding to specific and non-specific plasma proteins. The half-life of total radioactivity elimination was 14-18 h in the rat and rhesus monkey, but longer (43 and 78 h, i.v. and oral respectively) in the baboon. In the dog, the elimination phase could not be distinguished from the distribution phase and had a half-life of 2 h. The half-life of unchanged moxestrol elimination was shorter and very similar in the rhesus, baboon and human (6.6, 7.5 and 8.2 h respectively) and only 1.4 h in the dog. Regardless of the route of administration or the species under study, the clearance and elimination rate of unchanged moxestrol were higher than of total radioactivity implying that metabolites and/or conjugation products were eliminated more slowly than intact product from plasma. Orally administered moxestrol was rapidly absorbed in all species. Since clearance of total radioactivity and of moxestrol was faster after i.v. than oral administration, but the radioactivity levels excreted in the urine were identical for the two routes, a significant first-pass-effect probably occurred in the liver. Radioactivity distribution in tissues was examined in the rat. Total radioactivity was higher 24 h after administration of labelled moxestrol than of labelled ethynyl estradiol in endocrine tissues; it was equivalent or less in the other tissues. For all tissues, the elimination rate of moxestrol was greater than, or equal to, that of ethynyl estradiol. In dog urine, the only product identified was moxestrol; in rhesus or baboon monkey urine, the principal metabolites were catechol estrogens, which were also present in appreciable amount in rat bile (as methyl ethers) but were minor metabolites in human urine. Hydroxylation in position 16 occurred in rats and humans only, in position 15 alpha in humans and, to a much lesser extent, in rats and monkeys. Thus, the metabolic profile of moxestrol in rats most closely resembles that in humans.