Chemical analysis of fish bile extracts for monitoring endocrine disrupting chemical exposure in water: Bisphenol A, alkylphenols, and norethindrone.

The present study determined concentrations of estrogenic bisphenol A (BPA), nonylphenol, octylphenol (4-tert-octylphenol), butylphenol (4-tert-butylphenol), and progestogenic norethindrone by liquid chromatography-tandem mass spectrometry in bile extracts from field fish from the Xin'an River and market fish in Shanghai, China. Compared with the field fish, endocrine disrupting chemical (EDC) concentrations in market fish bile were at relatively high levels with high detectable rates. The average concentrations of BPA, nonylphenol, 4-tert-octylphenol, 4-tert-butylphenol, and norethindrone in field fish bile were 30.1 µg/L, 203 µg/L, 4.69 µg/L, 7.84 µg/L, and 0.514 µg/L, respectively; in market fish bile they were 240 µg/L, 528 µg/L, 76.5 µg/L, 12.8 µg/L, and 5.26 µg/L, respectively; and in the surface water of Xin'an River they were 38.8 ng/L, 7.91 ng/L, 1.98 ng/L, 2.66 ng/L, and 0.116 ng/L, respectively. The average of total estrogenic activity of river water was 3.32 ng/L estradiol equivalents. High bioconcentration factors (BCFs) were discovered for all 5 EDCs (≧998-fold) in field fish bile. Furthermore, the authors analyzed the BCF value of BPA in fish bile after 30-d exposure to environmentally relevant concentrations of BPA in the laboratory, and the analysis revealed that BCF in fish bile (BCF(Fish bile)) changed in an inverse concentration-dependent manner based on the log10-transformed BPA concentration in water. Strikingly, the data from the field study were well fitted within this trend. The data together suggested that analysis of fish bile extracts could be an efficient method for assessing waterborne EDCs exposure for aquatic biota.

Tissue-specific uptake and bioconcentration of the oral contraceptive norethindrone in two freshwater fishes.

The environmental presence of the oral contraceptive norethindrone (NET) has been reported and shown to have reproductive effects in fish at environmentally realistic exposure levels. The current study examined bioconcentration potential of NET in fathead minnow (Pimephales promelas) and channel catfish (Ictalurus punctatus). Fathead minnows were exposed to 50 µg/l NET for 28 days and allowed to depurate in clean water for 14 days. In a minimized 14-day test design, catfish were exposed to 100 µg/l NET for 7 days followed by 7-day depuration. In the fathead test, tissues (muscle, liver, and kidneys) were sampled during the uptake (days 1, 3, 7, 14, and 28) and depuration (days 35 and 42) phases. In the catfish test, muscle, liver, gill, brain, and plasma were collected during the uptake (days 1, 3, and 7) and depuration (day 14) stages. NET tissue levels were determined by gas chromatography-mass spectrometry (GC-MS). Accumulation of NET in tissues was greatest in liver followed by plasma, gill, brain, and muscle. Tissue-specific bioconcentration factors (BCFs) ranged from 2.6 to 40.8. Although NET has been reported to elicit reproductive effects in fish, the present study indicated a low potential to bioconcentrate in aquatic biota.

Electrochemical immunosensor for norethisterone based on signal amplification strategy of graphene sheets and multienzyme functionalized mesoporous silica nanoparticles.

Norethisterone is one kind of widely used anabolic steroid hormones which can help to promote livestock growth and sometime has been illegally used for livestock breeding. The residues of norethisterone in animal food will harm people's health, therefore, it has been banned to use for the growth promotion purposes in livestock. In this study, amino-group functionalized mesoporous silica nanoparticles (MSN) were prepared and used to immobilize Au nanoparticles, which was further utilized for the adsorption of horseradish peroxidase (HRP) and the secondary antibody (Ab2). The resulting nanoparticles, Au-MSN-HRP-Ab2 were used as labels for immunosensors to detect norethisterone antigen. A sandwich-type protocol was used to prepare the immunosensor with the primary antibody (Ab1) immobilized onto thionine (TH) and graphene sheet (GS) modified glassy carbon electrode surface. The sensitivity of the sandwich-type immunosensor using Au-MSN-HRP-Ab2 as labels for norethisterone antigen detection was much higher than that using either Au-MSN-Ab2 or MSN-HRP-Ab2 as labels. Within norethisterone concentration range (0.01-10 ng/mL), a linear calibration plot (Y=0.55671+8.27101X, r=0.9993) was obtained with a detection limit of 3.58 pg/mL under optimal conditions. The proposed immunosensor shows good reproducibility, selectivity, and acceptable stability. This new type of labels for immunosensors may provide many potential applications for the detection of growth hormone in animal derived food.

Doping in sport--2. Quantification of the impurity 19-norandrostenedione in pharmaceutical preparations of norethisterone.

The finding of measurable amounts of 19-norandrostenedione in norethisterone tablets prompted us to develop an assay to quantify this steroid. 19-Norandrostenedione is an anabolic steroid whose use in sport is prohibited by the World Anti-Doping Agency (WADA). The assay was developed using isotope dilution and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 19-norandrostenedione in norethisterone formulations, with [3,4-(13)C(2)]-19-norandrostenedione as the internal standard. The results showed amounts up to 1.01+/-0.01microg (mean+/-S.E.M.) per tablet in those containing 5mg of norethisterone or norethisterone acetate (0.02%, w/w) and up to 0.5+/-0.01microg (mean+/-S.E.M.) per tablet (0.05%, w/w) in oral contraceptive tablets containing 0.35-1.5mg of norethisterone or norethisterone acetate. No tablet tested exceeded the British Pharmacopoeia limit of 0.1% for this impurity.

Voltammetry and quantification of the contraceptive drug norethisterone in bulk form and pharmaceutical formulation.

The electrochemical behavior of norethisterone at the mercury electrode was studied in the universal buffer of various pH values using dc-polarography, cyclic voltammetry and controlled-potential electrolysis. Norethisterone was reduced at the mercury electrode via the consumption of two electrons corresponding to reduction of the 3-keto-delta-4-group in the A-ring of the molecule. The pK(a) value (8.7) of norethisterone was determined from the polarographic and spectrophotometric measurements. A fully validated, simple, sensitive, precise and inexpensive square-wave adsorptive cathodic stripping (SWAdCS) voltammetry procedure was described for trace quantification of bulk norethisterone. The stripping voltammetry peak current of norethisterone in a universal buffer of pH 5 following its accumulation onto the hanging mercury drop electrode (HMDE) at -0.6 V (versus Ag/AgCl/KCl(s)) for 130 s showed a linear response with the concentration over the range 5 x 10(-9) to 3 x 10(-7)M norethisterone. Detection and quantitation limits of 1.5 x 10(-9) and 5 x 10(-9)M bulk norethisterone, respectively, were achieved. The proposed procedure was successfully applied for the assay of norethisterone in Steronate tablets without interference from excipients.

Norethindrone acetate (NA) and ethinyl estradiol (EE) related oxidative transformation products in stability samples of formulated drug product: synthesis of authentic references.

Preparative chemical methods for the synthesis of eight oxidative transformation products of ethinyl estradiol (EE) and norethindrone acetate (NA) are described. The prepared materials are useful as reference materials and standards for pharmaceutical analysis of EE and NA as bulk chemical or in formulated product. All eight products result from oxidation of the A and/or B rings of the parent compounds. Oxidation of the heteroannular 3,5 dienyl acetate derivative of NA resulted in the 6 alpha-hydroxy, 6 beta-hydroxy and 6-keto NA. Oxidation of 6-keto NA led to the preparation of 6 alpha-hydroxy, 6 beta-hydroxy, 6-keto- and Delta(6) EE. Delta(11) EE was prepared from estrone.

Analysis of steroids Part 50. Derivatization of ketosteroids for their separation and determination by capillary electrophoresis.

4-Ene-3-ketosteroids and 17-ketosteroids were quantitatively transformed into the corresponding hydrazones using Girard P and T reagents, respectively. The positively charged derivatives were separated by capillary electrophoresis. The spectrophotometric characteristics of the derivatives permitted their sensitive detection in the 230-280 nm range. The steroids investigated included nortestosterone and its phenylpropionate, norethisterone and its oenanthate, d,l-norgestrel, dehydroepiandrosterone, androstenedione and ethisterone.

Estimation of impurity profiles of drugs and related materials. Part 14: the role of HPLC/diode-array UV spectroscopy in the identification of minor components (impurities, degradation products, metabolites) in various matrices.

The possibility of the rapid identification of drug related minor components by HPLC/diode-array UV spectroscopy is demonstrated by three examples. Hydroxylated impurities (degradation products) of norgestrel (6 alpha and beta, 10 beta-hydroxy derivatives) were identified on the basis of their UV spectra and retention matching with the synthesized impurities. The position of the phenolic hydroxyl groups in the mono- and dihydroxylated metabolites of bisaramil was established by UV spectroscopy and retention matching with the synthesized metabolites. The discrimination between the isomeric 4-ene-3-ketone and 1-ene-3-ketone components in crude 19-nortestosterone, product of the Birch reduction of 3-methoxy-1,3,5(10)-oestratriene-17 beta-ol, was also based on the diode-array UV spectra.

[Estrogens. Contraceptive therapy]. / Estrógenos. Terapia anticonceptiva.

To evaluate the competitive molecular phenomenon of Ethynyl-Estradiol (EE-2) from contraceptive formulations against the endogenous Estradiol (E-2) at the intra and the extracellular compartments, plasma and endometrial samples were simultaneously obtained on different days of the pseudomenstrual cycle from oral contraceptive users under EE-2+ Norgestrel (30 micrograms/+ 500 micrograms) and EE-2+ Norethindrone (50 micrograms + 1.0 mg) in order to quantify EE-2 & E-2. When measuring both molecules it was shown that the chronic administration of steroids regardless of the pharmacological action of the progestin component the lower content of EE-2 (30 micrograms) does not compete substantially at the circulating level permitting the cyclic fashion of the natural estradiol while at the endometrial compartment such phenomenon is not seen thus, a local infertility effect should be reconsidered to anticipate a different approach in the future of contraception.

PIP: 27 women participated in a study of systemic and endometrial estradiol concentrations in oral contraceptive (OC) users. 13 women aged 15-22 used OCs containing ethinyl estradiol 30 mcg and norgestrel 500 mg for 4-24 months, and 14 women aged 18-28 used OCs containing 50 mcg ethinyl estradiol and 1.0 mg norethindrone for 1-28 months. A peripheral blood sample and an endometrial biopsy were obtained at random from each woman on different cycle days to measure the concentration of endogenous estradiol. The ovarian response defined by endogenous plasma and endometrial estrogen levels differed greatly in the two groups. In the 30 mcg group, plasma estrogen levels were similar to those described in an ovulatory menstrual cycle, while this profile was not observed in the 50 mcg group. The endometrial estrogen profile described for women not using hormonal contraception was not observed in either of the two dose groups.

Modification of the U.S.P. dissolution method for the analysis of norethindrone and ethinyl estradiol tablets.

A modification of the U.S.P. dissolution method for the quantitation of norethindrone and ethinyl estradiol tablets is proposed. This modification consists of the use of distilled water as the dissolution medium instead of 0.1 N hydrochloric acid (HCl) and/or neutralization of 0.1 N HCl with sodium carbonate prior to analysis. Statistical analysis of the results indicate that there are no significant differences between the dissolution in 0.1 N HCl and the dissolution in distilled water (p < 0.05) for norethindrone and ethinyl estradiol.

[Analysis of steroids. Part 42. By-products of the ethynylation of 17-ketosteroids (isolation, identification and determination)]. / Adatok a szteránvázas vegyületek analitikájához 42. 17-Ketoszteroidok etinilezésének melléktermékei (elválasztás, azonosítás, meghatározás).

The therapeutically very important 17 alpha-ethynyl steroids are prepared from 17-keto steroids by means of addition of acetylene. Two important side reactions of this procedure are known: the formation of the isomeric beta-ethynyl derivative and the formation of a dimeric product with acetylene bridge. The aim of this paper is to approach this problem from the point of view of impurity profiling of 17 alpha-ethynyl steroids (norethisterone, ethisterone, norgestrel and delta 9(11)-ethisterone) i.e. isolation, identification and quantification of the above mentioned by-products as impurities in the bulk drugs. Capillary gas chromatography is an ideal tool for the separation and quantitative determination of the beta-ethynyl derivatives (20 m long fused silica capillary, I.D 0.2 mm; stationary phase Ultra-2: 5% phenylmethyl silicon gum phase with a film thickness of 0.33 mu; column temperature 240-250 degrees C). The dimeric impurity cannot be determined directly by gas chromatography as it decomposes in the flash heater to the 17 alpha-ethynyl and the 17-keto derivatives. For this reason reversed-phase HPLC was preferred for their separation and quantitation; (column: 250 x 4 mm LiChrosorb RP-18, 10 microns; eluent methanol-water 7:3; UV detector 240 nm). The HPLC method is suitable for the separation and determination of the beta-ethynyl impurities, too. The chemical shifts of the protons and carbon atoms in the vicinity of C-17 in the 1H and 13C NMR spectra of the epimeric 17-ethynyl steroids greatly depend on the configuration of the ethynyl group and for this reason they (especially that of the C-18 are eminently suitable for the characterization of the isomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of norethindrone stability on red delicious apples.

A stability-indicating assay procedure for the determination of norethindrone on the surface of Red Delicious apple slices was developed. The apple slice is homogenized in pH 10 borate buffer solution and norethindrone is then partitioned into chloroform using a diffusion chamber that has a membrane that is permeable to hydrophobic compounds separating the buffer and chloroform. An aliquot of the chloroform extract is then evaporated to dryness and reconstituted in tetrahydrofuran. The reconstituted sample is then chromatographed on a C18 reversed-phase column using a mobile phase consisting of water:tetrahydrofuran:methanol (63:26:11, v/v/v). Detection is in the UV range at 254 nm. The chromatographic system is specific for norethindrone in the presence of its autoxidation products. Stability data indicate that norethindrone is stable for up to 6 h on the surface of Red Delicious apples.

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.

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.

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. I. Optimization for selectivity in reversed-phase chromatography.

The application of solvent optimization to the development of isocratic reversed-phase liquid chromatography has been reported in several publications. Two different approaches to solvent optimization for controlling band spacing for the maximum resolution of samples are "solvent strength" and "solvent type" optimization. To improve the separation selectivity further the combination of these two approaches was examined, as a (global) optimum mobile phase composition requires the optimization of the solvent strength by varying the percentage of organic component and of the solvent selectivity of methanol, acetonitrile, tetrahydrofuran and water. It was found that the combination of "solvent strength" and "solvent type" optimization provides a markedly better separation than either procedure alone.

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. II. Optimization for selectivity in normal-phase systems.

Optimization of selectivity in normal-phase chromatography was studied. Using the same optimization criteria (Rs,min, Dmin, Rsb and Rsa) as in Part I the possible combination of "solvent strength" optimization by variation of the percentage of polar modifier in the less polar mobile phase and "solvent type" optimization by measuring the solvent selectivity of chloroform, acetonitrile, tetrahydrofuran and dioxane was examined. From the results it can be concluded that by replacement of medium-polarity solvents (chloroform, acetonitrile, tetrahydrofuran and dioxane) in the mobile phase, the selectivity of the separation can be significantly improved as the elution order is a function of solvent type belonging to Class P. In the separation of a steroid mixture dioxane provides the best properties for improving band spacing.

Analysis of steroids. XXXVIII. The use of high-performance liquid chromatography with diode-array UV detection for estimating impurity profiles of steroid drugs.

High-performance liquid chromatography (HPLC) diode-array UV-spectrophotometric detection is used for estimating impurity profiles of steroid drugs. It is shown to be a very useful first screening method for the identification of UV-active impurities and degradation products, giving a rapid answer to many questions or at least providing important initial information to complement the results obtained by other spectroscopic techniques. In this paper the estimation of the impurity profiles of ethynyloestradiol, norgestrel, and norethisterone, and of the degradation product of RGH-1113 (3-chloro-, 6,9-difluoro-11 beta,16 alpha,17 alpha,21-tetrahydroxy-1,3,5-pregnatrien-20-one 16,17-acetonide 21-acetate) will be discussed.