A survey of 17α-ethinylestradiol and mestranol residues in Hawkesbury River, Australia, using a highly specific enzyme-linked immunosorbent assay (ELISA) demonstrates the levels of potential biological significance.

This study reports on the potential status of 17α-ethinylestradiol (EE2) and mestranol (MeEE2) residues in aquatic environments in New South Wales (NSW), Australia, based on the analysis by a specific ELISA we developed. Polyclonal antibodies were raised against the EE2 hapten with a linker attached at the C3-position to direct the antibody binding towards the ring D of EE2/MeEE2. Using this approach, an ELISA highly specific to EE2 and MeEE2 was successfully developed, showing less than 3.1% cross-reactivity (% CR) with other major steroidal sex hormones and their derivatives. The assay performed with the limit of detection (LOD) of 0.04 ± 0.01µg/L for both EE2 and MeEE2, and the limit of quantitation (LOQ) of 0.05 ± 0.01ng/L when it was coupled with the SM2-Biobeads solid phase extraction. Prior to conducting the survey study, it was validated against the gas chromatography-mass spectrophotometry (GC-MS) method, which showed high correlation with R2 of 0.934. Fresh surface water samples collected at different sites along Hawkesbury River in New South Wales (NSW) were analyzed for the EE2/ MeEE2 residues using the developed ELISA. The EE2/MeEE2 levels were found to range between 4.1 and 8.3ng/L in Emigrant Creek, NSW, where the primary activity was macadamia plantation, and higher levels between 15 and 29ng/L in South Creek, NSW, Greater Western Sydney at sites upstream and downstream of the municipal sewage treatment plants.

Effect-directed analysis of a hospital effluent sample using A-YES for the identification of endocrine disrupting compounds.

An effect-directed analysis (EDA) approach was used to identify the compounds responsible for endocrine disruption in a hospital effluent (Basque Country). In order to facilitate the identification of the potentially toxic substances, a sample was collected using an automated onsite large volume solid phase extraction (LV-SPE) system. Then, it was fractionated with a two-step orthogonal chromatographic separation and tested for estrogenic effects with a recombinant yeast (A-YES) in-vitro bioassay. The fractionation method was optimized and validated for 184 compounds, and its application to the hospital effluent sample allowed reducing the number of unknowns from 292 in the raw sample to 35 after suspect analysis of the bioactive fractions. Among those, 7 of them were confirmed with chemical standards. In addition, target analysis of the raw sample confirmed the presence of mestranol, estrone and dodemorph in the fractions showing estrogenic activity. Predictive estrogenic activity modelling using quantitative structure-activity relationships indicated that the hormones mestranol (5840 ng/L) and estrone (128 ng/L), the plasticiser bisphenol A (9219 ng/L) and the preservative butylparaben (1224 ng/L) were the main contributors of the potential toxicity. Derived bioanalytical equivalents (BEQs) pointed mestranol and estrone as the main contributors (56 % and 43 %, respectively) of the 50 % of the sample's explained total estrogenic activity.

Ultrasonic-assisted derivatization of estrogenic compounds in a cup horn booster and determination by GC-MS.

Cup horn boosters are miniaturized ultrasound baths that maximize efficiency and precision. The optimization of an ultrasonic-assisted derivatization step by means of a cup horn booster and the determination of estrone, 17beta-estradiol, estriol, 17alpha-ethynyl estradiol and mestranol was developed by GC-MS. Different derivatization reagents and solvents were studied for maximizing the di-derivatization of 17alpha-ethynyl estradiol under ultrasound energy. Only N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane in pyridine gave satisfactory results and this mixture was further used in the optimization of the ultrasound assisted derivatization. The experiment designs included sonication time (1-10 min), sonication power (20-80%), sonication cycles (1-9), derivatization reagent volume (25-125 microL) and solvent volume (25-125 microL). Once the optimum conditions were fixed, the effect of organic matter and the frequency of the water bath change were studied. Finally, the validation of the analytical method was carried out using spiked natural and synthetic waters. Recoveries (natural (138-70%) and synthetic (112-89%)), the LODs (0.35-1.66 ng/L), and LOQs (1.16-5.52 ng/L) and the precision (0.2-5.3%) of the method were studied. This is the first work in the literature where a cup horn booster is used with the aim of minimizing derivatization time during the determination of estrogenic compounds.

Microwave-accelerated derivatization for the simultaneous gas chromatographic-mass spectrometric analysis of natural and synthetic estrogenic steroids.

A rapid microwave-accelerated derivatization process for the GC-MS analysis of steroid estrogens, estrone (E1), 17beta-estradiol (E2), estriol (E3), 17alpha-ethynylestradiol (EE2) and mestranol (MeEE2), was developed. Under microwave irradiation, the five estrogenic hormones studied were simultaneously derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+trimethylchlorosilane (TMCS) in pyridine solution. Effects of irradiation time (15-120 s) and power level (240-800 W) on the yield of the derivatization were investigated. The derivatization under the irradiation of 800 W microwave for 60s produced comparable results when compared with the conventional heating process in a sand bath for 30 min at 80 degrees C in terms of derivatization yield, linearity and precision for all steroid hormones tested. The calibration curves are linear between 3.00 and 3.00 x 10(2) microg mL(-1). The square of the regression coefficients (R(2)) range from 0.979 to 1.000. The applicability of the method was evaluated on spiked river and distilled water samples at two concentrations, 25.0 and 2.00 x 10(2) ng mL(-1). The recoveries obtained by using microwave heating (60s, 800 W) were similar to those by conventional heating. When combined solid-phase extraction (SPE) with the application of the microwave-accelerated derivatization proposed here, the detection limits of 0.02-0.1 ng L(-1) for the steroid hormones have been achieved. The results demonstrated that microwave-accelerated derivatization is an efficient and suitable sample preparation method for the GC-MS analysis of estrogenic steroids.

Occurrence and photochemical degradation of 17alpha-ethinylestradiol in Acushnet River Estuary.

17alpha-Ethinylestradiol (EE2), a major constituent of common contraceptive pills, and three other estrogenic hormones, estrone (E1), 17beta-estradiol (E2) and mestranol (MeEE2) have been determined in Acushnet River Estuary seawater using a GC-MS technique. Among three estrogenic compounds detected, EE2 has the highest concentration, up to 4.7 ng/l, at which EE2 may affect lobster and other fish abundance in the coastal seawater due to its high biological activity on fish feminization. Two natural estrogenic hormones, E1 and E2 have also been found in the estuary at concentrations up to 1.2 ng/l and 0.83 ng/l, respectively. Although EE2 is persistent to microbial degradation, it can undergo a rapid photodegradation in estuarine seawater under natural sunlight irradiation, with a half-life of less than 1.5 days in spring sunny days.

Sterol ratios as a tool for sewage pollution assessment of river sediments in Serbia.

In this work, source pollution tracing of the sediments of the Danube River and its tributaries in Serbia was performed using sterol ratios. Improved liquid chromatography-tandem mass spectrometry method, which enabled complete chromatographic separation of four analytes with identical fragmentation reactions (epicoprostanol, coprostanol, epicholestanol and cholestanol), was applied for the determination of steroid compounds (hormones, human/animal and plant sterols). A widespread occurrence of sterols was identified in all analyzed samples, whereas the only detected hormones were mestranol and 17α-estradiol. A human-sourced sewage marker coprostanol was detected at the highest concentration (up to 1939 ng g(-1)). The ratios between the key sterol biomarkers, as well as the percentage of coprostanol relative to the total sterol amount, were applied with the aim of selecting the most reliable for distinction between human-sourced pollution and the sterols originated from the natural sources in river sediments. The coprostanol/(cholesterol + cholestanol) and coprostanol/epicoprostanol ratios do not distinguish between human and natural sources of sterols in the river sediments in Serbia. The most reliable sterol ratios for the sewage pollution assessment of river sediments in the studied area were found to be coprostanol/(coprostanol + cholestanol), coprostanol/cholesterol and epicoprostanol/coprostanol. For the majority of sediments, human-derived pollution was determined. Two sediment samples were identified as influenced by a combination of human and natural biogenic sources.

Simple high-performance liquid chromatographic assay for norethindrone--mestranol in combination tablets.

A simple, sensitive, and specific high-performance liquid chromatographic procedure was developed to assay norethindrone--mestranol combination tablets. The method involves a chloroform extraction of a single pulverized tablet. After centrifugation, and aliquot of the supernate was injected into a modular high-performance liquid chromatograph. The effluent from the silica column was monitored serially with a fixed-wavelength UV detector (254 nm) for norethindrone quantitation and a fluorescence detector (230 nm for excitation and 280 nm cutoff filter for emission) for mestranol quantitation. Progesterone was used as an internal standard. The method was employed successfully in content uniformity studies of several brands of commercially available tablets.

Quantitative analysis of ethynodiol diacetate and ethinyl estradiol/mestranol in oral contraceptive tablets by high-performance liquid chromatography.

A procedure is described for the assay of ethynodiol diacetate and ethinyl estradiol/mestranol by HPLC using two UV detectors at 210 and 280 nm. The system was acetonitrile 38% (v/v) in water as mobile phase on a 250 x 3.2-mm i.d. RP-2 column, with butylated hydroxytoluene as the internal standard. There was greater than 99% recovery from synthetic preparations and the coefficient of variation was greater than 2.0% for formulations.

Dissolution testing of norethindrone:ethinyl estradiol, norethindrone:mestranol, and norethindrone acetate:ethinyl estradiol combination tablets.

Dissolution of oral contraceptive combination products from six manufacturing firms was studied utilizing the rotating basket method at 100 rpm in 600 mL of 0.1 M HCl and 0.02% sodium lauryl sulfate (SLS). Most of the combination products of norethindrone (NE):ethinyl estradiol (EE) dissolved satisfactorily in water using the paddle method which was first proposed by U.S.P., whereas three of 18 tested products showed better dissolution in acidic aqueous medium containing SLS. Acidic medium with surfactant was also found to be suitable for combination products of norethindrone (NE):mestranol (ME) and norethindrone acetate (NEAc):ethinyl estradiol (EE). A modified U.S.P. assay procedure, a reversed-phase high-performance liquid chromatographic (HPLC) method with a mobile phase of acetonitrile and phosphate buffer, was used to analyze NE and EE concurrently. The NEAc and ME were analyzed separately in these combination products. The NEAc was found to hydrolyze to some extent (approximately 20% in 8 h) in acidic dissolution medium at room temperature, but less so at 4 degrees C. The NE was identified as the sole degradation product of NEAc hydrolysis and was also measured to account for the total amount of NEAc dissolved. A simple dissoluting testing method which utilizes a single dissolution medium was applicable for all oral contraceptive combination tablets surveyed.

Ketonitrophenols from mestranol and related compounds.

Mestranol (17alpha-ethynylestradiol 3-methyl ether), when placed on a carrier such as powdered silica gel and exposed to the atmosphere, is converted to a yellow product. The compound formed was shown to be 1alpha-ethynyltetrahydro-1beta-hydroxy4 - (2 - hydroxy - 5 - methoxy - 3 - nitrophenethyl) - 7a - methyl-5(4H)-indanone. The 3-methyl ethers of three other steroids having aromatic A rings yielded products of a similar type. Identical compounds were prepared from the respective steroids by treatment with nitrating agents in acetic acid. This reaction in acetic acid is light catalyzed. An independent synthesis of a model compound, 6-(2-hydroxy-5-methoxy-3-nitrophenyl)-3-hexanone, established the position of the constituents on the aromatic ring as well as the location of the carbonyl. The mechanism proposed for the formation of these products is an initial oxidation of the 1-substituted tetralin to form a hydroperoxide, which is ionically decomposed to form a ketophenol. The phenol is then nitrated in the ortho-position.

The changing role of ultraviolet spectroscopy in drug analysis.

New applications in the identification of minor components in drugs by rapid scanning diode-array UV spectrophotometers as high-performance liquid chromatographic (HPLC) detectors are exemplified by (a) identification and quantification of alpha-chloro-4-methoxycinnamic acid methyl ester as the byproduct of the Darzens reaction between 4-methoxybenzaldehyde and chloroacetic acid methyl ester; and (b) identification of 4-androsten-17-one and 3 beta-phenyl-5 alpha-androstan-17-one as impurities in 5 alpha-androst-2-en-17-one. The advantages of the use of derivative spectrophotometry are illustrated by the following examples: (a) determination of flumecinol (3-trifluoromethyl-alpha-ethylbenzhydrol) in an oily emulsion formulation; (b) determination of RGH-6148 (2-benzylthiazolidinone) in a suspension used in toxicological studies; and (c) determination of mestranol as an impurity in norethisterone.

Behaviour and occurrence of estrogens in municipal sewage treatment plants--II. Aerobic batch experiments with activated sludge.

Aerobic batch experiments containing a diluted slurry of activated sludge from a real sewage treatment plant (STP) near Frankfurt/Main were undertaken, in order to investigate the persistence of natural estrogens and contraceptives under aerobic conditions. The batch experiments showed that while in contact with activated sludge the natural estrogen 17 beta-estradiol was oxidized to estrone, which was further eliminated in the batch experiments in an approximate linear time dependence. Further degradation products of estrone were not observed. 16 alpha-hydroxyestrone was rapidly eliminated, again without detection of further degradation products. The contraceptive 17 alpha-ethinylestradiol was principally persistent under the selected aerobic conditions, whereas mestranol was rapidly eliminated and small portions of 17 alpha-ethinylestradiol were formed by demethylation. Additionally, two glucuronides of 17 beta-estradiol (17 beta-estradiol-17-glucuronide and 17 beta-estradiol-3-glucuronide) were cleaved in contact with the diluted activated sludge solution and thus 17 beta-estradiol was released. The glucuronidase activity of the activated sludge was further confirmed by the cleavage of 4-methylumbelliferyl-beta-D-glucuronide (MUF-beta-glucuronide) in a solution of a activated sludge slurry and Milli-Q-water (1:100, v/v). The turnover rate obtained was approximately steady state, with a turnover rate of 0.1 mumol/l for the released MUF. Hence, it is very likely that the glucuronic acid moiety of 17 beta-estradiol glucuronides and other estrogen glucuronides become cleaved in a real municipal STP, so that the concentrations of the free estrogens increase.

Contribution to the standardisation of a gas chromatographic method for the analysis of an oestrogenic and progestational combination.

An experimental procedure is described in order to standardize a gaschromatographic method proposed for a collaborative study organised by the F.I.P. Committee for Laboratories and Official Drug Control Services, on the analysis of some oestro-progestinic formulations. The assayed combination was a standard mixture of lynestrenol, mestranol and d, 1-alpha-tocopherol: testosterone was included as internal standard; two combinations, differing only in the internal standard content as specified in the text, were used. The variability of the instrumental factor was investigated performing some sets of single experiments, each consisting of 5 successive injections of the same standard mixture, on the same day. For each single experiment and for each substance the mean value Rm of the ratios R (R=area of the peak for compound cpi/area of the peak of the internal standard) was estimated with its relative standard deviation (% s.d.). For each set of experiments the % s.d. values were elaborated, following the Bartlett test, in order to obtain the sigma intra % value for each set, as a measure of the instrumental factor. An experimental evidence was observed for the relationship existing between the instrumental factor value (sigma intra %) and the internal standard content, in the operating conditions fixed for the present gas chromatographic analysis, a lowering of the instrumental factor beeing observed with the higher level of testosterone content (internal standard) in the mixture.

[Evaluation of some thin-layer chromatography procedures for the identification and detection of impurities of hormonal steroids]. / Studio collaborativo per la valutazione di aleune metodiche di cromatografia in strato sottile per i'dentificazione ed il saggio di purezza di steroidi ad attività ormonale

PIP: The thin-layer chromatography methods described in Volume 2 of the "European Pharmacopeia" were tested to determine the most suitable methods for the identification of hormonal steroids and for the detection of impurities in these substances. The methods are based on partition chromatography for identification and on adsorption chromatogr aphy for the detection of impurities. Other adsorption methods developed by the authors were also tested. The study was conducted on 44 steroids, belonging to the groups of androgens, estrogens, progestinic and corticoid steroids. 9 chromatographic methods based on adsorption, 2 in bidimensional succession, and 7 methods based on partit ion were used. The work was carried out in 6 laboratories. The findings are summarized in 18 tables and illustrated in 13 figures. They were also processed by statistical methods in order to assess the reproducibility of the tests.^ieng

[Infrared spectrophotometry in quantitative drug analysis. II. Assay of some steroids]. / Zastosowanie spektrofotometrii w podczerwieni w ilosciowej analizie leków. II. Oznaczanie niektórych pochodnych sterydowych.

PIP: Infrared spectrometry was applied to the determination of mestranol, chlormadinone, estradiol benzoate, progesterone, testosterone propionate, and methyltestosterone. Except for mestranol, all of the steroids were determined in the range of 1656-1735 cm (-1) on the ground of the absorption of the carbonyl groups. Mestranol can be determined at 3308 cm (-1) (ethyl group variations) or at 1502 cm (-1) (variations of the aromatic ring system). The asorption band 1495 cm (-1) has been shown to be suitable for assaying estradiol benzoate. A relative error of .58% to 3.26% has been charged for the methods used. Methyltestosterone and progesterone can also be assayed by using the absorption bands reflecting the vibrations of the methyl groups, at 1361 cm (-1) and 1380 cm (-1). However, the assays at the latter wave lengths are found to be less accurate, with relative error at 3.49% and 4.41% and precision error at 1.19% and 1.39%, respectively. All measurements were performed in a medium of highly purified chloroform.^ieng

[Simultaneous determination using gas chromatography of mestranol and norethisterone in estrogen-progestins combination for oral use]. / Determinazione simultanea mediante gas-cromatografia del mestranolo e del noretisterone nelle associazioni estrogeno-progestiniche per uso orale.

PIP: The article describes a new method to determine the quantity of mestranol and of norethisterone in a combined oral contraceptive. Gas chromatography was used simultaneously to detect both agents, after they were extracted with ethyl acetate from the total compound of the tablet.^ieng