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Determination of estradiol and its degradation products by liquid chromatography
Abstract
A novel HPLC method for simultaneous determination of estradiol and its seven degradation products in topical gel was developed. Zorbax SB-CN (150 mm x 4.6 mm, 5 microm) analytical column and mobile phase composed of acetonitrile, phosphoric acid 0.085%, and tetrahydrofurane (27:63:10, v/v/v) at flow-rate 1.0 ml min(-1) were used for the chromatographic separation using UV detection at 225 nm. The active substance estradiol was separated from all its known degradation products successfully. Two degradation products estrone and Delta(9(11))-estrone were not separated sufficiently, their peaks were evaluated as a sum of two components. The method was validated according to ICH guideline recommendations and thereafter it was successfully applied for stability tests of topical cream Estrogel HBF in the quality control laboratory. Limits of detection for degradation products ranged from 1.03 x 10(-5) to 1.14 x 10(-4) mg ml(-1), limits of quantitation for degradation products were in the range 3.43 x 10(-5) to 3.81 x 10(-4) mg ml(-1). The developed method is selective, precise, accurate and sensitive enough for determination of estradiol and its known degradation products.
... It may also be used as replacement therapy for female hypogonadism or primary ovarian failure. The decrease of 17 b-estradiol at menopause is often accompanied by vascular instability and rise in incidence of heart disease and an increasing risk of osteoporosis (Havlikova et al., 2006). ...
... HPLC method for 17 b-estradiol and 17 b-estradiol-3-acetate using C 18 column and mobile phase of acetonitrile-water (50:50, v/v) has been reported (Russell et al., 2000). The other one is the reverse phase HPLC method (Havlikova et al., 2006) which utilized acetonitrile-0.085% phosphoric acid-tetrahydrofurane (27:63:10, v/v/v) as mobile phase system for 17 b-estradiol from its solid dosage form. ...
... In comparison with earlier reported and official method for the estimation of 17 b-estradiol in pharmaceutical formulation the proposed HPLC method gave a lower LOD and LOQ values (Havlikova et al., 2006;The United States, 2000). Also, the HPLC method is found to be superior to earlier reported methods, as the mobile phase is simple to prepare and economical. ...
In this study, new, rapid UV spectrophotometry (UV) and reversed phase high performance liquid chromatography (HPLC) methods were developed for the determination of 17 β-estradiol in pure and in pharmaceutical dosage form. The solvent system, wavelength of detection and chromatographic conditions were optimized in order to maximize the sensitivity of both the proposed methods. The linear regression equations obtained by least square regression method were y = 0.0184x + 0.0059 for the UV method and y = 56742x − 3403.6 for the HPLC method. The developed methods were successfully employed with a high degree of precision and accuracy for the estimation of total drug content in a commercial tablet of 17 β-estradiol. The results obtained from the UV method were compared with those obtained by using HPLC. The proposed methods are highly sensitive, precise and accurate and can be used for the reliable quantitation of 17 β-estradiol in pharmaceutical dosage form.
... [1] Many analytical methods including chromatography have been developed for the determination of estradiol hemihydrates and its derivatives from a variety matrices e.g., body fluids, pharmaceutical formulations and environmental water samples (aquatic environment). [2][3][4][5][6]Among different modes of liquid chromatography, such as high performance liquid chromatography (HPLC) coupled to mass spectrometry (HPLC-MS and HPLC-MS/MS) or to other detectors, instrumental thin-layer chromatography (TLC) is still widely applied in clinical laboratory field as well as in pharmaceutical and also environmental analysis of synthetic and natural steroids like for example estradiol and its derivative metabolites. [2][3][4][5][6]As was described in excellent review paper by Bhawani et al., [7] which has been dedicated to the TLC determination of different steroids, the modern instrumental thin-layer chromatography coupled to densitometry (TLC-densitometry) is a very important tool in steroid analysis from different materials. ...
... [2][3][4][5][6]Among different modes of liquid chromatography, such as high performance liquid chromatography (HPLC) coupled to mass spectrometry (HPLC-MS and HPLC-MS/MS) or to other detectors, instrumental thin-layer chromatography (TLC) is still widely applied in clinical laboratory field as well as in pharmaceutical and also environmental analysis of synthetic and natural steroids like for example estradiol and its derivative metabolites. [2][3][4][5][6]As was described in excellent review paper by Bhawani et al., [7] which has been dedicated to the TLC determination of different steroids, the modern instrumental thin-layer chromatography coupled to densitometry (TLC-densitometry) is a very important tool in steroid analysis from different materials. [7] The main advantage of TLC method in comparison with other chromatographic techniques is its low cost per analysis, speed, flexibility and allowing the detection of many simultaneously samples in one step (onto one TLC plates). ...
Thin layer chromatography (TLC) combined with densitometry was employed to determine the limit of detection (LOD) and quantification (LOQ) of estradiol hemihydrate. Average values of both LOD and also LOQ were determined on the basis of the standard deviation of the response and slope of special calibration curve accordance with the procedure required by validation guidelines. Influence of chromatographic conditions, such as different sorbents and various mobile phase compositions on determining of LODs and LOQs of examined steroid by NP-TLC and RP-TLC techniques was estimated. Of all chromatographic plates and mobile phases used, those which gave the best results (the lowest LOD and LOQ value) using NP-TLC system are silica gel 60 F254 plates developed with mobile phase consisted of dichloromethane - ethyl acetate (8:2, v/v). In the case of RP-TLC method, a mixture of methanol - water (9:1, v/v) as mobile phase enabled detection of estradiol hemihydrate at the lowest concentration level on RP8 F254s precoated plates. The results of LODs and LOQs obtained in this study confirm that the proposed TLC-densitometric method is suitable for the quantification of estradiol hemihydrate in the samples containing this steroid in the range of 0.093 to 3.930 µg per spot. Described method could be an important tool in the pharmaceutical as well as in clinical or environmental analysis of examined steroid hormone.
... An Agilent 1200-4 Series High-Performance Liquid Chromatography (HPLC) system equipped with binary solvent pump, autosampler, photodiode array detector, thermostated column compartment and Chemstation chromatographic software was used for estimating concentration of estradiol. The methods described elsewhere in the literature were used with a few modifications [23,24]. Waters X-Select™ C18 column (3.5 μm, 4.6 × 150 mm) maintained at 35°C was used to elute estradiol. ...
... The authors acknowledge the funding support from the CDER's Regulatory Science and Review Enhancement Grant (Grant # RSR [13][14][15][16][17][18][19][20][21][22][23][24][25]. Surgically discarded human skin samples were provided by the Cooperative Human Tissue Network which is funded by the National Cancer Institute. ...
The objective of this study was to develop a level A in vitro-in vivo correlation (IVIVC) for drug-in-adhesive (DIA) type estradiol transdermal drug delivery systems (TDDS). In vitro drug permeation studies across human skin were carried out to obtain the percent of estradiol permeation from marketed products. The in vivo time versus plasma concentration data of three estradiol TDDS at drug loadings of 2.0, 3.8 and 7.6mg (delivery rates of 25, 50 and 100μg/day, respectively) was deconvoluted using Wagner-Nelson method to obtain percent of in vivo drug absorption in postmenopausal women. The IVIVC between the in vitro percent of drug permeation (X) and in vivo percent of drug absorption (Y) for these three estradiol TDDS was constructed using GastroPlus® software. There was a high correlation (R(2)=1.0) with a polynomial regression of Y=-0.227X(2)+0.331X - 0.001. These three estradiol TDDS were used for internal validation whereas another two products of the same formulation design (with delivery rates of 60 and 100μg/day) were used for external validation. The predicted estradiol serum concentrations (convoluted from in vitro skin permeation data) were compared with the observed serum concentrations for the respective products. The developed IVIVC model passed both the internal and external validations as the prediction errors (%PE) for Cmax and AUC were less than 15%. When another marketed estradiol TDDS with a delivery rate of 100μg/day but with a slight variation in formulation design was chosen, it did not pass external validation indicating the product-specific nature of IVIVC model. Results suggest that the IVIVC model developed in this study can be used to successfully predict the in vivo performance of the same estradiol TDDS with in vivo delivery rates ranging from 25 to 100μg/day.
Copyright © 2015. Published by Elsevier B.V.
... 17 b-estradiol and its semi-synthetic esters are primarily used as menopausal hormones. [2] 17 b-estradiol may also be used as replacement therapy for female hypogonadism or primary ovarian failure. The decrease of 17 b-estradiol at menopause is often accompanied by vascular instability and rise in incidence of heart disease and an increasing risk of osteoporosis. ...
... The decrease of 17 b-estradiol at menopause is often accompanied by vascular instability and rise in incidence of heart disease and an increasing risk of osteoporosis. [2] Several methods have been reported for the determination of 17 b-estradiol including voltammetry, [3] high performance liquid chromatography (HPLC), [1,[4][5][6][7][8] and liquid chromatography-tandem mass spectrometry (LC-MS-MS). [9] Many other gas chromatography-mass spectrophotometry (GC-MS) methods have been published quantifying 17 b-estradiol and its metabolites. ...
A new method was developed for the determination of 17 β-estradiol in rabbit plasma by reversed-phase high performance liquid chromatography (RP-HPLC) with fluorescence (FL) detection. The method employed one-step extraction of 17 β-estradiol from plasma matrix with a mixture of water and ethylacetate (1.9:5, v/v) using estriol as an internal standard. The mobile phase consisted of a mixture of metanol and water (70:30, v/v) flowing at a flow rate of 1.0 mL min−1. Calibration curve was linear between concentration range of 125–6000 ng mL−1. Average recovery of 17 β-estradiol and the internal standard from the biological matrix was more than 94.9 and 92.5%, respectively. The intra-day and inter-day precision and accuracy were between 3.74–8.12 and 3.72–8.80%, respectively. Also, the method was successfully applied to determine of 17 β-estradiol in New Zealand white rabbits.
... Furthermore, the determination of estra-diol is also of interest in environmental monitoring because of its high endocrine disrupting potency, and the extensive use of therapies with synthetic hormone in humans and other animals [2]. Gas chromatography [3,4] and high performance liquid chromatography [5,6] have been used for the determination of estradiol and related compounds in biological, pharmaceutical, and water samples. Also, electrochemical methods involving voltammetry [7,8] and chronoamperometry [9,10] were reported. ...
... All the steps involved in the immunosensor preparation were monitored by electrochemical impedance spectroscopy using 5 mM Fe(CN) 6 4− /Fe(CN) 6 3− as redox probe in 0.1 M KCl (Fig. 2). As expected, modification of the SPCE with ABA gave rise to a large increase in the electron transfer resistance, R CT , which varied from 279 to 907 as a consequence of the electrostatic repulsion between the redox probe and the negatively charged carboxylate groups. ...
This work describes the preparation of an electrochemical immunosensor for estradiol based on the surface modification of a screen printed carbon electrode with grafted p-aminobenzoic acid followed by covalent binding of streptavidin (Strept) and immobilization of biotinylated anti-estradiol (anti-estradiol-Biotin). The hormone determination was performed by applying a competitive immunoassay with peroxidase-labelled estradiol (HRP-estradiol) and measurement of the amperometric response at -200 mV using hydroquinone (HQ) as redox mediator. The calibration curve for estradiol exhibited a linear range between 1 and 250 pg mL(-1) (r=0.990) and a detection limit of 0.77 pg mL(-1) was achieved. Cross-reactivity studies with other hormones related with estradiol at physiological concentration levels revealed the practical specificity of the developed method for estradiol. A good reproducibility, with RSD=5.9% (n=8) was also observed. The operating stability of a single bioelectrode modified with anti-estradiol-Biotin-Strept was nine days when it was stored at 8°C under humid conditions between measurements. The developed immunosensor was applied to the analysis of certified serum and spiked urine samples with good results.
... Note that the difference between E2 and E1 is merely one hydroxyl substitution that is oxidized into a ketone in the estrone (E1). Both estrogenic species should be monitored in the effluents, according to pharmacopoeia regulations (Havlíková et al., 2006). Furthermore, both the E2 and E1 ...
The continuous removal of TOC and the degradation efficiency of carbamazepine and 17β-estradiol were investigated using actual secondary municipal-effluent RO-retentate solutions. A specific set of operating parameters were applied within the supercritical water oxidizing conditions: temperature range 420-480 °C, 25.1 MPa, hydraulic retention time (HRT) of 1-2 min, excess oxidant molar-ratio of 3-10 and presence of a homogenous catalyst (IPA) at 50-100 mg/L. > 99% organic carbon mineralization, along with complete degradation of model pollutants, was observed at 450 °C/1 min/OC= 5-10 and 100 mgIPA/L. The outlet estrone concentration, 1.03 ± 1.14 ng/L, representing estrogenic pollutants, dropped to the "no effect" range. A model for a SCWO plant treating secondary-municipal-effluent-RO-retentate for a city of 100,000 capita-equivalent was developed, based on a shell & tube SCWO flow reactor, showing > 75% energy-efficiency. The model yielded that for the extreme case of a zero caloric-value feed-solution, the total OPEX and CAPEX would be < $6.0 ± 2.5 per m3 of secondary effluents, i.e., two orders of magnitude lower than the reported environmental shadow-price associated with CECs (contaminants of emerging concern). Further work is required on the continuous and efficient separation of the salt-matrix, which can lead to higher overall heat transfer coefficients and enable further reduction in capital costs.
... As previously mentioned, Estrone (E1) and Acridine (ACR) were chosen as the decomposition intermediate model compounds in the E2 and CBZ SCWO experiments, respectively. E1 was chosen because it is a known endocrine disrupting pollutant, and the only monitored E2 degradation product, according to pharmacopoeias regulations (Havlíková et al., 2006). It was identified as a possible E2 degradation ...
Continuous subcritical and supercritical water oxidation experiments were conducted on dilute carbamazepine- and estradiol-containing synthetic solutions used to simulate the removal of model emerging pollutants from secondary municipal effluents. The operating conditions comprised 340-500°C, retention time of 24 to 453 s and a stoichiometric oxidant ratio (O.C.) between 4 and 64. The transformation of the various species was determined at the outlet and by modeling a segmented non-isothermal reaction system. Four empirical power law kinetic models were established to represent both the pollutants' degradation and TOC removal efficiencies, using nonlinear multiple regression coupled with bootstrapping and K-fold cross-validation. The mineralization and degradation models for both pollutants yielded a R² of 67-80.5% vs. the experimental results. Discussion on the various model assumptions revealed that attributing full model deviations to the constant oxygen concentration or to the laminar reactor flow, yielded a deviation of 6% and 15% in the removal efficiencies, respectively. However, the expected deviation of the models was lower than 0.32% at conditions leading to (almost) full mineralization (45-60 s, 480-500°C and O.C.s of 5-10). The methodologies developed in the study are useful for interpreting future results obtained from SCWO of actual secondary effluent solutions.
... A literature survey revealed that only few research studies are available on the simultaneous determination of EDA and EE in different formulations (Amin & Hassenbach, 1979;Babják, Balogh, Gazdag, & Görög, 2002;Carignan, Lodge, & Skakum, 1982;Gluck & Shek, 1980;Havlíková, Nováková, Matysová, Šícha, & Solich, 2006;Koneru, Kimbahune, Mubeen, Lalitha, & Chakraborty, 2015;Kotra, Ramisetti, & Konidala, 2017;Maziarz, 2013;Nair et al., 2018;Patel, Rathod, & Patel, 2013;Prasad, Reddy, Prasad, & Mukkanti, 2001;Qi Gui & Nieuweboer, 1989;Strusiak, Hoogerheide, & Gardner, 1982;Suhendi, 2016;Summers & Fountain, 2013;Yamada, Kuwahara, Takamatsu, & Hayase, 2000;Zorica, 2006 ...
Ethynodiol diacetate and Ethinyl estradiol tablets are indicated to prevent pregnancy in women who use oral contraceptives as a contraception method. Ethynodiol Diacetate (EDA) / Ethinyl Estradiol (EE) were separated using the RP‐HPLC with Agilent zorbax SB‐Phenyl, 4.6 mm × 15 cm, 5 μm, and mobile phase as a gradient mixture with acetonitrile and milli‐Q water. For Ethynodiol Diacetate (EDA) /Ethinyl Estradiol (EE), the linearity and recovery were found in the rage of 0.025mg/mL‐0.25mg/mL for EDA, 0.001mg/mL‐0.01mg/mL of EE and 0.05mg/mL‐0.18mg/mL of EDA, 0.002mg/mL‐0.007mg/mL of EE. The method is validated according to the regulatory guidelines concerning system suitability, specificity, repeatability, recovery, linearity, robustness, and stability of the sample solution.
... Because estrone and estradiol are not water-soluble, they are eliminated from the body mainly as their sulfate and glucuronide metabolites [56]. Numerous methods have been developed and used for the detection of these hormones including gas chromatography [57,58], high performance liquid chromatography [59,60], voltammetry [61,62], electrochemical immunosensing [63][64][65][66][67], chronoamperometry [68,69], enzyme biosensors [70], DNA biosensors [71,72], radioimmunoassays [73,74], colorimetric assays [75], chemiluminescent assays [76,77] and fluoroimmunoassays [78]. For example, the synthesis of an estradiol-tetraphenylporphyrin (E2-TPP) conjugate has been described [79]. ...
Small-molecule detection is important for many applications including clinical diagnostics, drug discovery, environmental screening, and food technology. Current techniques suffer from various limitations including cost, complex sample processing, massive instrumentation, and need for expertise. To overcome these limitations, a new optical immunosensing assay for the detection of small molecules was developed and assessed with the targets estrone (E1) and estradiol (E2). For this purpose, phosphorescent indicators were designed based on the tetrabenzoporphyrin skeleton directly linked to E1 or E2, or attached through a linker, with phosphorescence lifetimes in the range of ~100–~300 μs. The assay is an indicator displacement assay (IDA). The best performances of our optical immunosensor were obtained with the indicators E1-L-Por and E2-L-Por. As they bound to specific polyclonal antibodies, their phosphorescence (τ ~200 μs) was quenched. When an endogenous competitor was added, the indicator was displaced, and the phosphorescence was immediately recovered. These effects were measured with a new optical device, described here, and able to detect picograms of luminescent molecules emitting in the NIR range, simply by measuring phosphorescence decay. This radical switch-off/switch on process demonstrates that E1-L-Por and E2-L-Por are good candidates for in vivo and in vitro immunosensing of E1 and E2. Importantly, the present immunosensing assay can be easily adapted to other small molecules such as other hormones and drugs.
... 17-beta estradiol (E2) is one of the most commonly known steroid hormones that are produced naturally in humans and animals [6]. This hormone is responsible for sexual reproduction in women and it is found in the highest concentration in pregnant women [7,8]. This hormone can also be taken as a supplement for women with hormone imbalance. ...
In this work, Fe3O4-Al2O3@CNFs nanocomposite was synthesised and used as a nanosorbent in the ultrasound-assisted dispersive magnetic solid phase extraction (UA-DMSPE) of 17-beta estradiol (E2) in wastewater samples. The quantification of E2 was achieved using high performance liquid chromatography coupled with diode array detector (HPLC-DAD). Various parameters affecting the efficiency of this sample preparation technique were optimised to achieve excellent sensitivity and high recoveries of E2. Response surface methodology was utilised for optimisation of these parameters. Using the optimised conditions, the linear dynamic range was achieved in the range of 0.1–1000 μg L⁻¹ and the correlation coefficient was found to be 0.9981. The preconcentration factor, enrichment factor, limit of detection (LOD) and limit of quantification (LOQ) were 67, 169, 0.025 μg L⁻¹ and 0.083 μg L⁻¹, respectively. The relative standard deviation (%RSD) for the intraday (n = 10) and interday (n = 5 working days) were 1.8% and 3.3%, respectively. The developed UA-DMSPE/HPLC-DAD method was applied for the preconcentration and determination of E2 in wastewater samples. The obtained results indicated that E2 was present in the wastewater samples.
... Hom-Diaz et al. (2015) have detected several transformation products of 17β-E2 (e.g., E1, 2-OH-17β-E2, 4-OH-17β-E2) during wastewater microalgae cultivation. In addition, during E1 and 17β-E2 degradation process, the generation of intermediate products dehydroestrone (6,7-DHE1, Δ-8,9 -DHE1, and Δ-9,11 -DHE1) (Ma and Yates 2017) and Δ-9,11 -dehydro-17β-estradiol (Δ-9,11 -DH-17β-E2) (Havlíková et al. 2006) with unsaturated B-ring as minor metabolites might occur (see Fig. 2). In addition, as the precursor of E2, E1 can also transform back to both E2 isomers as the intermediate accompanied by preferential formation of 17β-E2 with reducing the ketone group of E1 to alcoholic hydroxyl group under anaerobic condition (Zheng et al. 2012;Mashtare et al. 2013b), leading to the co-occurrence of 17α-and 17β-E2. ...
Natural steroid estrogens (NSEs), including free estrogens (FEs) and conjugated estrogens (CEs), are of emerging concern globally among public and scientific community due to their recognized adverse effects on human and wildlife endocrine systems in recent years. In this review, the properties, occurrence, sorption process, and transformation pathways of NSEs are clarified in the environment. The work comprehensively summarizes the occurrence of both free and conjugated estrogens in different natural and built environments (e.g., river, WWTPs, CAFOs, soil, and sediment). The sorption process of NSEs can be impacted by organic compounds, colloids, composition of clay minerals, specific surface area (SSA), cation exchange capacity (CEC), and pH value. The degradation and transformation of free and conjugated estrogens in the environment primarily involves oxidation, reduction, deconjugation, and esterification reactions. Elaboration about the major, subordinate, and minor transformation pathways of both biotic and abiotic processes among NSEs is highlighted. The moiety types and binding sites also would affect deconjugation degree and preferential transformation pathways of CEs. Notably, some intermediate products of NSEs still remain estrogenic potency during transformation process; the elimination of total estrogenic activity needs to be addressed in further studies. The in-depth researches regarding the behavior of both free and conjugated estrogens are further required to tackle their contamination problem in the ecosystem.
Graphical abstractᅟ
... Part of this acetyl-CoA is used for the biosynthesis of cholesterol which, in turn, is the precursor of steroid hormones (Berg et al., 2002). Steroids are involved in the differentiation, growth and physiology of reproductive organs (Havlíková et al., 2006;Pearce and Jordan, 2004). ...
Early pregnancy probability (P16) in heifers is a trait of high economic value for beef cattle production. Early pregnancy is defined based on the conception and calving of a heifer given that the animal had entered the breeding season at about 16 months of age and is considered a strong predictor of puberty in Nellore cattle. The aim of this study was to identify polymorphisms in the protein phosphatase 3 – catalytic subunit, alpha isozyme (PPP3CA) and fatty acid-binding proteins 4 (FABP4) genes, which have been associated with P16. The exon regions of the candidate genes were sequenced in 380 heifers. Two polymorphisms were detected in PPP3CA and 13 in FABP4. A deletion (rs134413439) was identified in the FABP4 gene in all samples, which could be a variation that occurs across breeds since the reference genome is from a Hereford cow. Two SNPs in the FABP4 gene were associated with P16 after Bonferroni correction. However, none of the haplotypes exerted a significant effect (P>0.05). This study showed that the PPP3CA and FABP4 genes are polymorphic in Nellore cattle. Furthermore, the FABP4 gene was found to be associated with early pregnancy in heifers, reinforcing the contribution of lipid metabolism to reproduction.
... Separation on Zorbax SB-CN/LC-UV (Havlíková et al., 2006) SPE with Oasis/(LC-(HPLC fluorescence (Panter et al., 2006) ...
p>Endocrine disruptors are defined as exogenous agents that alter the function of endocrine system, which in turn, causes adverse health effects in an intact organism or its progeny. Of these compounds, 17Beta-estradiol is of primary importance, since it is physiologically present in both men and women, as well as, being produced synthetically as a component in some pharmaceutical products. Once it reaches the aquatic environment through domestic sewage, ground water and streams, it makes a serious threat to the aquatic life. The review tackles the biological significance of these compounds as well as the danger that they present to the surrounding environment, areas at which these compounds have been detected worldwide, the methods used in detection and fundamentally significant solutions to get rid of this hazard using different methods such as; the bioremediation process.</p
... 17b-estradiol was analyzed using an HP Agilent 1100 highperformance liquid chromatography (HPLC) system equipped with a ZORBAX SB-C18 column (4.6 Â 150 mm, 3.5 mm) and by modifying the method by Havlikova et al. (2006) . The mobile phase was a mixture of 40:60 (v/v) of acetonitrile and phosphoric acid (0.085%) at a flow rate of 1 mL min À1 . ...
Emerging Organic Contaminants (EOCs) such as steroidal estrogen hormones are of growing concern in recent years, as trace concentrations of these hormones can cause adverse effects on the environmental and human health. While these hormones have been widely detected in soil and groundwater, effective technology has been lacking for in-situ degradation of these contaminants. This chapter illustrates a new class of stabilized MnO2 nanoparticles and a new in-situ technology for oxidative degradation of EOCs in soil and groundwater. The stabilized nanoparticles were prepared using a low-cost, food-grade Carboxymethyl Cellulose (CMC) as a stabilizer. The nanoparticles were then characterized and tested for their effectiveness for degradation of both aqueous and soil-sorbed E2 (17ß-estradiol). Column tests confirmed the effectiveness of the nanoparticles for in-situ remediation of soil sorbed E2. The nanoparticle treatment decreased both water leachable and soil-sorbed E2, offering a useful alternative for in-situ remediation of EOCs in the subsurface.
... The major methodologies employed in 17b-estradiol determination in environmental samples are based on chromatographic methods, such as the HPLC [8], LC [9], LC/MS [10], and ELISA tests [11]. Chromatographic methods, although highly accurate, do not easily allow continuous in situ analysis and often require several previous sample preparation steps, including extraction and cleanup procedures, to obtain a final extract fully compatible with chromatographic determination. ...
... This monitoring allows to diagnose menstrual dysfunctions, and thus, to detect the states of hypoestrogenism Scheme 1. Schematic representation of the 17-E electrochemical immunosensor. a mass spectrometer (GC-MS and HPLC-MS, respectively) [8][9][10][11][12][13][14]. These methods have the disadvantages that the samples require pretreatment and derivatization. ...
... 17b-estradiol was analyzed using an HP Agilent 1100 highperformance liquid chromatography (HPLC) system equipped with a ZORBAX SB-C18 column (4.6 Â 150 mm, 3.5 mm) and by modifying the method by Havlikova et al. (2006) . The mobile phase was a mixture of 40:60 (v/v) of acetonitrile and phosphoric acid (0.085%) at a flow rate of 1 mL min À1 . ...
Manganese oxide (MnO2) was reported to be effective for degrading aqueous pharmaceutical chemicals. However, little is known about its potential use for degrading soil-sorbed contaminants. To bridge this knowledge gap, we synthesized, for the first time, a class of stabilized MnO2 nanoparticles using carboxymethyl celluloses (CMC) as a stabilizer, and tested their effectiveness for degrading aqueous and soil-sorbed estradiol. The most desired particles (highest reactivity and soil deliverability) were obtained at a CMC/MnO2 molar ratio of 1.39 × 10(-3), which yielded a mean hydrodynamic size of 39.5 nm and a narrow size distribution (SD = 0.8 nm). While non-stabilized MnO2 particles rapidly aggregated and were not transportable through a soil column, CMC-stabilized nanoparticles remained fully dispersed in water and were soil deliverable. At typical aquatic pH (6-7), CMC-stabilized MnO2 exhibited faster degradation kinetics for oxidation of 17β-estradiol than non-stabilized MnO2. The reactivity advantage becomes more evident when used for treating soil-sorbed estradiol owing to the ability of CMC to complex with metal ions and prevent the reactive sites from binding with inhibitive soil components. A retarded first-order rate model was able to interpret the oxidation kinetics for CMC-stabilized MnO2. When used for degrading soil-sorbed estradiol, several factors may inhibit the oxidation effectiveness, including desorption rate, soil-MnO2 interactions, and soil-released metals and reductants. CMC-stabilized MnO2 nanoparticles hold the potential for facilitating in situ oxidative degradation of various emerging contaminants in soil and groundwater.
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... On the contrary, liquid chromatography (LC) has only been employed in a few occasions (12,13) regardless of its advantages with respect to the already mentioned techniques. Thus, unlike GC-MS, LC enables the determination of steroid without derivatization and it is not limited by such factors as properties of the substances (non volatile) and high molecular weight. ...
The aim of this research was to develop a sensitive, accurate and rapid method for simultaneous determination of steroid hormones, ethinylestradiol (EED) and drospirenone (DROSP) in commercially available oral contraceptive tablets. Tested pharmaceutical formulation contains an estrogen in a small amount with 100-times bigger amount of a synthetic progestin. The combination of EED and DROSP was analyzed using a Purospher® STAR RP-18e reversed-phase column (150 X 4.0 mm I.D.; particle size 5 µm) with a mobile phase constituted of 47% acetonitrile: 53% water (V/V). The elution was carried out at a flow rate of 1.50 ml /min. All analyses were performed at room temperature (24 ± 2°C). A diode array detector connected in series with fluorescence detector measured the UV absorbance of DROSP at 265 nm and fluorescence of EED at 310 nm (excitation at 285 nm). The proposed method was validated by determination of linearity, precision, accuracy and sensitivity. Calibration curves for ETE and DROSP were obtained using standard solutions of EED with concentrations ranged from 0.6 to 3.0 g/ml and standard solutions of DROSP with concentrations ranged from 60.0 to 300.0 g/ml. Correlation coefficients were 1.0 and 0.9998 for EED and DROSP, respectively. The precision of the method was confirmed by assessment of repeatability and reproducibility. Relative standard deviations obtained in the investigation of repeatability were: 0.52 % and 0.05 % for EED and DROSP, respectively. Relative standard deviations obtained in the investigation of reproducibility were: 1.22 % and 0.74 % for EED and DROSP, respectively. The average recovery for samples containing EED and DROSP were 98.99 % and 99.85 % for EED and DROSP, respectively. The limits of detection for EED and DROSP were 0.65 ng/ml and 0.0774 g/ml, respectively, which indicates an excellent sensitivity of the proposed method. The method was applied for Uniformity of dosage units testing in commercially available oral contraceptive tablets. In conclusion, we recommended HPLC method with UV and fluorescence detection as a method of choice for determination of ethinylestradiol, present at a very low dosage level in low-dose oral contraceptives that contain bigger amount of synthetic progestin.
... The analytical methods normally used for 17b-estradiol determination are based on chromatographic techniques such as high-performance liquid chromatography (HPLC) [35] and liquid chromatography (LC). [36] Electrochemical determination of 17bestradiol is based on the irreversible oxidation of the phenol group present in the molecule. However, it has been reported that this electrochemical process presents a low current response over surfaces [37] such as platinum, glassy carbon and boron-doped diamonds. ...
A novel material for the electrochemical determination of endocrine disruptors using a composite based on graphene oxide modified with cadmium telluride quantum dots has been evaluated. The morphology, structure and electrochemical performance of the composite electrodes were characterised by transmission electron microscopy, dynamic light scattering, UV-visible absorption spectra, fluorescence spectra, Raman spectra and cyclic voltammetry. The dynamic light scattering, transmission electronic microscopy and spectrophotometric measurements all showed good distribution of the quantum dots with a small particle size. The electrochemical measurements demonstrated the high performance of the composite response in the presence of a light source. Differential pulse voltammetry allowed the development of a method to determine 17β-estradiol levels in the range from 0.2 to 4.0 μmol L−1 with a detection limit of 2.8 nmol L−1 (0.76 μg L−1).
... Ozonolysis experiments on progesterone have been successful, resulting in degradation products which need to be characterised more precisely Barron et al., 2006). UV experiments have mainly been carried out in liquid with other EDCs, hormones and micro-organisms (Doll and Frimmel, 2003;Havlikova et al., 2006;Ku et al., 2006;Rosenfeldt and Linden, 2004;Wolfe, 1990). ...
Atomic force microscopy (AFM) has been used to monitor the appearance and behaviour of the hormone progesterone on mineral surfaces, including mica and highly oriented pyrolytic graphite (HOPG). Different solvents have been used resulting in various features on the two substrates. After the application of 254 nm ultra-violet (UV) light, changes in structure of the adsorbed hormone have been observed. To understand the reactions at the progesterone-mica interface in aqueous solution, adsorption has been studied in situ with AFM.
... It may also be used as replacement therapy for female hypogonadism or primary ovarian failure. The decrease of 17 β-estradiol at menopause is often accompanied by vascular instability and an increasing risk of osteoporosis [2] . ...
This article describes gas chromatography-flame ionization detection method for determination of 17 β-estradiol in rabbit plasma. 17 β-estradiol and internal standard progesterone were extracted from plasma using liquid-liquid extraction method. Linearity was found between 0.25 and 20 μg/ml (r(2)=0.994) for plasma samples. Intra-day and inter-day precision, expressed as the relative standard deviation were less than 5.5%, and accuracy (relative error) was less than 3.5%. The mean recovery of 17 β-estradiol samples was 94.4%. The limits of detection and quantification of method for plasma samples were 0.10 μg/ml and 0.15 μg/ml, respectively. Also, clinically used other 10 drugs were investigated to check for potential interferences and the method was successfully applied to the determination of 17 β-estradiol in New Zealand white rabbits.
... Cholesterol bonded stationary phases may be also applied for separation of steroid hormones such as estradiol, estrone, estriol, etc., which modulate the differentiation, growth and physiology of reproductive organs [23,24]. In the human body steroids are generated from cholesterol and they are transformed into other steroids. ...
Chromatographic properties of four cholesterol bonded phases with different structures were studied. The columns used were packed with a stationary phase containing a cholesterol molecule attached to the silica surface using different types of linkage molecules. Columns were compared according to the retention and separation selectivity of steroid hormones. The measurements were done using binary hydro-organic mobile phases with methanol, ethanol and acetonitrile as an organic modifier. The presented results show that the coverage density of the bonded ligands and the type of organic modifier strongly influence the retention mechanism and separation selectivity of steroid hormones on cholesterol-containing adsorbents.
... Estradiol (1,3,5(10)-estratrien-3,17-diol), estrone (1,3,5(10)estratrien-3-ol-17-one) and estriol (1,3,5(10)-estratrien-3,16a-17-triol) are endogenous estrogenic steroids (Scheme 1) that modulate the differentiation, growth and physiology of reproductive organs [1,2]. Through the estrogenic receptor ␣ (ER␣) and estrogenic receptor  (ER), which are members of the nuclear receptor superfamily of transcription factors, estradiol also affects bone tissue, liver, brain and the cardiovascular system. ...
A new LC-MS/MS method for the separation, identification and quantification of residues of 17alpha-estradiol (17alpha-E2) and 17beta-estradiol (17beta-E2) in bovine serum is reported. Deuterium-labelled 17beta-estradiol was used as internal standard. The method was in-house validated in accordance with European Union criteria and adopted in a proficiency study organised by the Community Reference Laboratory (CRL-RIVM, Bilthoven, The Netherlands). The analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a C18 LC column. They were then ionized in a heated nebulizer (HN) interface operating in negative ion mode, where only intact deprotonated molecules, [M-H](-), were generated at m/z 271 and 274 for 17alpha/17beta-E2 and 17beta-E2-d(3), respectively. The decision limits obtained (CCalpha, i.e., critical concentration alpha) were 0.06 ng/mL and 0.03 ng/mL, respectively for 17alpha-E2 and 17beta-E2. Detection capability (CCbeta, i.e., critical concentration beta) values were 0.08 ng/mL and 0.04 ng/mL, respectively, for 17alpha-E2 and 17beta-E2. Precision, accuracy and specificity were satisfactory, recovery ranged from 86.3% to 93.2% and the method resulted sensitive for the required purposes. This method is currently in use for Official Control purposes.
This project investigated the abilities of Pleurotus ostreatus to sequester and break down the naturally occurring steroidal hormone 17-B estradiol through bioremediation in an aquatic environment. Pure fungal cultures were transferred to straw and wood substrate and colonized substrate was transferred to aquaria containing concentrations of estradiol in moving water. Samples were analyzed using High Performance Liquid Chromatography (HPLC-UV). Results were inconclusive due to detection limitation of the HPLC-UV system utilized and the adsorption of 17B-estradiol onto components in the aquarium. In a separate experiment, water samples of the Flint River Watershed, were sent to Arizona Laboratory for Emerging Contaminants for analysis through GC/LC/MS. This analysis showed levels of 17a-ethynyl estradiol well above those that caused morphological and behavioral changes (40 ng/L) in fish during in vitro experiments. The results of the water sampling also included BPA, a chemical which mimics steriodal estrogens. Another experiment measured the dry weight of P. ostreatus when exposed to 17B-estradiol (mixed in acetone) for 18 days. There was not a significant difference in dry weights of P. ostreatus grown with or without estradiol. The final experiement involved uptake and adsorption of 17B-estradiol using a human serum ELISA kit after 8 days of exposure to estradiol. Results were inconclusive due to organic contaminants. Growth of mycelium was observed in all in vitro experiments. This could indicate P. ostreatus was not inhibited by 17B-estradiol and may be able to use it as a carbon source.
The analysis of 17β‐estradiol with high sensitivity and selectivity is extremely relevant to control the impacts that this compound can cause on health and the environment. Thus, we describe the development and application of a magneto carbon paste electrode based on magnetic molecularly imprinted polymer (MCPE‐MMIP) for determination of 17β‐estradiol. The analyte adsorbed on the MMIP was immobilized on the electrode surface by magnetic capture. The morphological and structural characterization of the obtained MMIP suggests that the material was effectively synthesized. MCPE‐MMIP showed an improvement in the sensitivity for 17β‐estradiol detection when compared to electrode configurations in the absence of this material. The optimum conditions (0.10 mol L⁻¹ phosphate buffer pH 7.0) were reached by differential pulse adsorptive stripping voltammetry (DPAdSV), in which the method presented linearity ranged from 0.06 to 175 μmol L⁻¹ with limits of detection and quantification of 0.02 and 0.06 μmol L⁻¹, respectively. The proposed sensor was applied effectively in the analysis of 17β‐estradiol in river water and raw milk samples, exhibiting excellent recovery values (between 96.20 and 104 %), which were confirmed by HPLC analysis.
An efficient, fast and sensitive method for the determination of 17β-estradiol, (E2) and 17α-ethinylestradiol (EE2) in pharmaceutical formulations and in urine was developed and validated using a hanging mercury drop electrode (HMDE), screen printed carbon electrodes (SPCE), and screen printed carbon nanotube electrodes (SPCNTE). Both analytes are adsorbed on the working electrodes. To obtain sensitive and selective methods, the effects of various parameters such as pH, adsorption potential, and time (Eads, tads) were optimized. The optimum experimental conditions chosen for the two analytes were pH: 10.0; Eads: −0.60 and tads: 30 s, when HMDE was used. Under these conditions, one reduction signal was found at −1.31 V for E2 and two reduction signals at −0.23 V and −1.20 V for EE2. The detection limits (DLs) were found to be 0.3 μg L⁻¹ for E2, 14.8 μg L⁻¹ for EE2 at −0.23 V, and 9.7 μg L⁻¹ for EE2 at −1.20 V. On the other hand, in screen printed electrodes E2 and EE2 present oxidation of the phenolic hydroxyl groups at 0.30, 0.31, 0.32, and 0.33 V (pH:10) with DLs of 242, 277; 182, and 191 μg L⁻¹ for SPCE and SPCNTE, respectively. The method was successfully applied to the determination of these analytes in Primaquin® (E2), Gynera® (EE2), spiked urine (with EE2), and urine samples of women who used Tinelle® (EE2) as contraceptive drug.
In this study, a novel approach for effective liquid-liquid microextraction based on deep eutectic solvent (DES) decomposition was suggested for the first time. It was established that DESs synthesized from tetrabutylammonium bromide and long-chain alcohols decomposed in aqueous phase resulting in in situ dispersion of organic phase and extraction of hydrophobic analyte(s). It this process long-chain alcohol acted as an extraction solvent and tetrabutylammonium bromide acted as a dispersive agent and promoted mass transfer between aqueous and organic phases as a salting out agent. Phenomenon of DES decomposition was studied in detail and applied for separation and preconcentration in chemical analysis for the first time. The developed approach was applied for 17β-estradiol microextraction from transdermal gel samples as a proof-of-concept example. The results showed that the in situ dispersed organic phase obtained can provide efficient extraction of 17β-estradiol with good extraction recovery (95 ± 5%) and excellent reproducibility (6%). The reported approach proves to be fast, simple, and inexpensive.
Aptasensing mechanism for femtomolar detection of estradiol [E2], an endocrine disruptor.
Estrogen conjugates are precursors of free estrogens such as 17ß-estradiol (E2) and estrone (E1), which cause potent endocrine disrupting effects on aquatic organisms. In this study, microcosm laboratory experiments were conducted at 25 °C in an agricultural soil to investigate the aerobic degradation and metabolite formation kinetics of 17ß-estradiol-3-glucuronide (E2-3G) and 17ß-estradiol-3-sulphate (E2-3S). The aerobic degradation of E2-3G and E2-3S followed first-order kinetics and the degradation rates were inversely related to their initial concentrations. The degradation of E2-3G and E2-3S was extraordinarily rapid with half of mass lost within hours. Considerable quantities of E2-3G (7.68 ng/g) and E2-3S (4.84 ng/g) were detected at the end of the 20-d experiment, particularly for high initial concentrations. The major degradation pathway of E2-3G and E2-3S was oxidation, yielding the primary metabolites 17ß-estrone-3-glucuronide and 17ß-estrone-3-sulphate, respectively. Common metabolites were E2, the second primary metabolite, and E1, the secondary metabolite. Additionally, ring B unsaturated estrogens and their sulfate conjugates were tentatively proposed as minor metabolites. The persistence of E2-3G and E2-3S (up to 20 d) suggests that the high rate of application of conjugated estrogen-containing substances could be responsible for the frequent detection of free estrogens in surface and subsurface water.
Study objective was to assess skin-to-skin drug transfer potential that may occur due to drug retention in human epidermis (DRE) pretreated with application of estradiol transdermal drug delivery systems (TDDS) and other estradiol transdermal dosage forms (gels and sprays). TDDS (products-A, B, and C) with varying formulation design and composition, and other estradiol transdermal products (gel and spray) were applied to heat separated human epidermis (HSE) and subjected to in vitro drug permeation study. Amounts of DRE were quantified after 24 h. The DRE with product-B was significantly (P < 0.001) higher than that with product-C, product-A, gel, and spray. However, products-A and C, gel, and spray showed almost the same (P > 0.05) amounts of DRE. A separate in vitro permeation study was carried out to determine amounts of drug transferred from drug-retaining epidermis to untreated HSE. The amounts of drug transferred, due to DRE after 8 h, with product-C were significantly (P < 0.001) higher than those with products-A and B, gel, and spray. The in vitro study results indicate a high potential of skin-to-skin drug transfer due to the DRE after labeled period of using estradiol TDDS, though the clinical relevance of these findings is yet to be determined.
The aim of current study was the application of validated TLC-densitometric method for
identification and determination of Estradiol hemihydrate in dosage forms. The applied TLC
conditions were: Silicagel G60F254 glass plates; mobile phase: chloroform : acetone = 90 : 10 v/v,
migration distance of mobile phase: 120 mm, UV-detection at = 254 nm. All of the
experimental results for the content of Estradiol hemihydrate correspond to the respective
confidence interval: Estrofem table: 1.78 mg ÷ 2.12 mg; Femoston F1 table: 1.88 mg ÷ 2.2 mg;
Femoston F2 table: 1.99 mg ÷ 2.19 mg; Trisequens T1 table: 1.78 mg ÷ 2.18 mg;
Trisequens T2 table: 1.92 mg ÷ 2.12 mg; Trisequens T3 table: 0.97 mg ÷ 1.17 mg.
The proposed validated TLC-densitometric method is appropriate for quality control of
Estradiol hemihydrate in commercially available tablets.
Key words: Estradiol hemihydrate, TLC, Densitometry, Tablets, analysis, Determination.
Norelgestromin/ethinyl estradiol is a progestin/estrogen combination hormonal contraceptive indicated for the prevention of pregnancy in women. The very poor solubility and wettability of these drugs, along with their high potency (adsorption issues), give rise to difficulties in designing intravenous (IV) formulations to assess absolute bioavailability of products containing both drugs. The purpose of this study was to develop an IV formulation, evaluate its stability under different conditions and evaluate its compatibility with IV sets for potential use in absolute bioavailability studies in humans. Also, a selective high-performance liquid chromatography (HPLC) method for quantification of ethinyl estradiol and norelgestromin in polysorbate 80 matrix was developed and validated.
Norelgestromin/ethinyl estradiol IV solution was prepared using sterile water for injection with 2.5% ethanol and 2.5% polysorbate 80 as a cosolvent/surfactant system to obtain a final drug solution of 25 μg ethinyl estradiol and 252 μg norelgestromin from a concentrated stock drug solution. The stabilities of the concentrated stock and IV solutions were assessed after storing them in the refrigerator (3.7 ± 0.6 °C) and at room temperature (19.5 ± 0.5 °C), respectively. Additional studies were conducted to examine the stability of the IV solution using an Alarias® low sorbing IV administration set with and without an inline filter. The solution was allowed to drip at 1 mL/min over a 60 min period. Samples were obtained at the beginning, middle and end of the 60 min duration. The chemical stability was evaluated for up to 10 days. Norelgestromin and ethinyl estradiol concentration, purity, and degradant levels were determined using the HPLC method. The norelgestromin/ethinyl estradiol IV formulation met the chemical stability criteria when tested on day 1 through day 9 (216 h). Norelgestromin concentrations assayed in stock and IV solutions were in the range of 90.0–98.5% and 90.9–98.8% after 9 days, respectively. As for ethinyl estradiol, the assayed concentrations were in the range of 91.8–100.9% and 92.7–100.8% for the stock and IV solutions, respectively. The administration set was found to be compatible with both drugs; the assayed concentrations were in the range of 99.2–100.3% for norelgestromin and 96.3–102.7% for ethinyl estradiol, but the inline filter showed some adsorption of ethinyl estradiol; where the assayed concentrations were in the range of 98.1–99.8% for norelgestromin and 95.9–97.4% for ethinyl estradiol.
The present study provided evidence supporting the suitability of an intravenous formulation for norelgestromin/ethinyl estradiol using ethanol/polysorbate 80 as a cosolvent/surfactant system. Both IV and concentrated stock solutions when stored at room temperature and refrigeration, respectively, were found to be chemically stable up to 9 days. These results indicated that this formulation is chemically stable and can be used over the time period tested. This IV formulation can be used to evaluate the absolute bioavailability of products containing norelgestromin and ethinyl estradiol provided that microbial testing of the IV formulation is performed.
Magnetic mesoporous nanoparticles of Fe3O4@mSiO2 were prepared through co-precipitation and tetraethoxysilane hydrolysis at 60 °C and pH 10 with hexadecyl trimethyl ammonium bromide as the template. Hydroxypropyl β-cyclodextrin was modified onto Fe3O4@mSiO2 under sodium citrate and N2 protection via an ultrasound process to increase the catalytic efficiency. The prepared nanoparticles were characterized by Fourier transform infrared spectrometry, X-ray diffraction, scanning electron microscopy and a peroxidase-like activity assay. The results showed that the prepared nanoparticles have magnetic properties, peroxidase-like activity and a loose spherical cluster structure. Compared with Fe3O4@mSiO2 nanoparticles, Fe3O4@mSiO2@HP-β-CD nanoparticles exhibit higher catalytic ability toward both H2O2 and 3,3′,5,5′-tetramethylbenzidine. Fe3O4@mSiO2@HP-β-CD nanoparticles can catalyse β-estradiol (β-E2) oxidation in the presence of H2O2 and be used as a colourimetric sensor for indirect detection of β-E2. A good linear relationship was obtained from 0.8 μM to 16 μM. The limit of detection of the proposed method was 0.2 μM. The visual method was successfully used in the analysis of β-E2 in commercial tablets and animal feeds, with recovery ranging from 92.6% to 110%.
This report describes the development and validation of a reverse phase high-performance liquid chromatography (HPLC) method with UV detection for the determination of the hormones estriol, estradiol, estrone, and progesterone in topically applied products. The developed method was then used to conduct a postmarket survey of consumer products for these hormones. Each product was first mixed with Celite and then extracted with methanol. Extracts were cleaned on a Waters Oasis HLB solid phase extraction cartridge, and then analyzed using reversed phase HPLC. The analytes were separated using an Agilent Zorbax Eclipse XDB C8 (5 μm, 250 mm by 4.6 mm) analytical column and detected by their absorbance at 230 nm. Chromatographic separation was achieved by a 1.0-ml/min linear gradient from 30% acetonitrile and 70% water to 80% acetonitrile and 20% water over 30 min. A final 5 min hold time and a re-equilibration time of 10 min were used to prepare the column for subsequent analysis. Recovery from two different brand lotions spiked with three different levels of estriol, estradiol, estrone, and progesterone ranged from 81.8% to 101%. In this study, a total of 70 cosmetic products were surveyed. Twenty two (63%) of the 35 products were labeled as containing an estrogen and/ or progesterone and also provided quantitative label information about the hormone ingredient. The most frequently labeled hormones were progesterone (66%), estriol (46%), estradiol (11%), and estrone (6%). Six products labeled as containing estriol were found to contain estradiol. An estrogen and/or progesterone were found in 34 products at concentrations ranging from 86.0 to 26,800 μg/g. Progesterone was not found in one product labeled as containing this hormone. An additional 35 products, which did not list hormones on their labels, were analyzed and estrogen or progesterone was not detected in these products.
The electrochemical behaviors of estrone in the presence of various surfactants were examined with great details. It is found that a cationic surfactant, cetyltrimethylammonium bromide (CTAB), obviously facilitates the electro-oxidation of estrone at carbon paste electrode (CPE) from the significant peak current enhancement and the negative shift of peak potential. Additionally, chronocoulometry and electrochemical impedance spectroscopy (EIS) were also used for further investigation of the electrode process of estrone, indicating that low concentration of CTAB exhibits excellent enhancement effects on the electrochemical oxidation of estrone, greatly enhances the diffusion coefficient and the electron transfer rate. Based on this, an electrochemical method was proposed for the determination of estrone. The oxidation peak current is proportional to the concentration of estrone in the ranges over 9.0 × 10-8 - 8.0 × 10-6 mol/L, and a low detection limit of 4.0 × 10-8 mol/L was obtained for 180s accumulation at open circuit (S/N = 3). Finally, this proposed method was demonstrated using estrone tablets with good satisfaction.
The objective was to quantify drug loss due to cold flow (CF) in marketed estradiol transdermal drug delivery systems (TDDS), and study its influence on the in vitro flux and drug transfer across contacting skin. TDDS samples (products-A and B) were induced with CF at 25 and 32°C/60%RH by applying 1-kg force for 72h. CF was measured as percent dimensional change and amount of drug loss/migration in CF region. In vitro drug permeation studies were conducted across human epidermis from TDDS excluding CF region, and CF region alone against control (without CF). In both products, significantly higher percentage of CF (dimensional change and drug migration) was observed at 32°C compared to 25°C. In vitro flux from both products excluding CF region either at 25 or 32°C was the same, but significantly lower compared to control. Drug transferred from CF region of product-A after 8h was the same at 25 and 32°C, but significantly higher in product-B. Flux from both products together with CF region at 32°C was significantly lower than that observed at 25°C. Results showed that excessive CF at storage (25°C) and clinical usage (32°C) conditions may have implications on product performance and safety of estradiol TDDS.
For estradiol (E2) the separation of the degradation products 6- and 9,11-E2 is especially challenging due to their structural similarity. There is no method described in the literature yet which adequately addresses this problem. The present study describes a HPLC method for the separation and quantitation of E2 and its degradation products 6α-hydroxy-E2, 6β-hydroxy-E2, 6-keto-E2, Δ9,11-E2, β-equilenol and Δ6-E2. The method employs a KinetexTM PFP analytical column, using methanol and deionized water as mobile phases. Different UV- and fluorescence detection modes were used for maximal sensitivity and specificity. The applicability and capability of the method was demonstrated for Vagifem® tablets. Finally, the method was validated with respect to selectivity, sensitivity, linearity, precision and accuracy.
Desenvolveu-se um procedimento de microextração por emulsificação assistida por ultrassom e aperfeiçoada por surfactante (UASEME) combinado com cromatografia líquida de alta eficiência com detector de arranjo de diodos para a determinação de 17b-estradiol (βE2), estrona (E1) e dietilestilbestrol (DES) em água. Ultrassom foi aplicado para auxiliar a emulsificação e Triton X-100 foi usado como dispersor e emulsificante. Tetracloreto de carbono foi usado como solvente extrator. Empregando otimização global, o procedimento foi caracterizado por um intervalo linear aceitável de 10 a 1000 ng mL-1 para βE2, E1 e DES (r > 0,997), exatidão e precisão validadas (RSD 0,85-1,28% (n = 5)) e alta sensibilidade com limites de detecção de 0,200, 0,100 e 0,125 ng mL-1 para βE2, E1 e DES, respectivamente. O procedimento foi aplicado para a análise de amostras típicas de água, com fatores de enriquecimento de 85,29, 173,45 e 97,05 para βE2, E1 e DES, respectivamente, e boas recuperações (≥ 89,82%). De maneira geral, o procedimento desenvolvido foi simples e confiável, com potencial de aplicação em larga escala para análises da água.
A simple, rapid and cost-effective NP-TLC method combined with densitometry was developed for determination of estradiol hemihydrate in tablet dosage form. TLC-densitometric analysis was conducted on silica gel 60F254 plates. Benzene + methanol in volume composition 9:1 was used as a mobile phase. The detection was carried out at 200 nm. Applied chromatographic conditions enabled complete separation of estradiol hemihydrate (EL) from its related substance, namely estrone (EN) with simple procedure of sample preparation. Linearity range for estradiol hemihydrate was from 0.50 to 1.50 μg/spot. The detection limit (LOD) and quantitative limit (LOQ) values of estradiol hemihydrate were 0.14 μg/spot and 0.41 μg/spot, respectively. The developed method was found to be precise, accurate, and robust. The assay value of estradiol hemihydrate was consistent with the limit recommended by Pharmacopoeia. The elaborated TLC-densitometric method can be used as an alternative method for the accurate assay of estradiol hemihydrate in tablet dosage form because it fulfills the validation criteria of an analytical method designated for quantity control of pharmaceutical preparations.
Estrogen steroids, represented by estradiol and its related substances, include both structurally very close and simultaneously different analogs. Their separation still remains an analytical challenge. Subcritical fluid chromatography (SbFC) on sub-2-micron particles was found to be an appropriate tool to obtain fast and efficient separation of nine target analytes. Among the four tested stationary phases charged hybrid modified with PFP (pentafluorophenyl) moiety was found to be the most convenient providing the fastest separation within 1.6 min using quick gradient elution with carbon dioxide and methanol as an organic modifier. However, complete separation was obtained also on other tested phases including bare hybrid stationary phase, hybrid stationary phase modified with 2-EP (2-ethylpyridine) and also C18, which is less typical in SbFC. The baseline separation on the latter columns was achieved by means of a temperature increase, a change in organic modifier type and gradient time increase respectively.Quantitative performance was evaluated at optimized conditions and method validation was accomplished. Excellent repeatability of both retention times (RSD<0.15%) and peak areas (RSD<1%) was observed. The method was linear in the range of 1.0–1000.0 μg/ml for all steroids with the lowest calibration point being an LOQ, except for Δ-derivatives, that provided better sensitivity and thus LOQ of 0.5 μg/ml. The sensitivity was sufficient for the analysis of real samples although it was still five times lower compared to UHPLC-UV experiments.
A derivative UV spectrophotometric method for determination of estradiol valerate in tablets was validated. The parameters specificity, linearity, precision, accuracy, limit of detection and limit of quantitation were studied according to validation guidelines. The first-order derivative spectra were obtained at N = 5, Δλ = 4.0 nm, and determinations were made at 270 nm. The method showed specificity and linearity in the concentration range of 0.20 to 0.40 mg mL-1. The intra and interday precision data demonstrated the method has good reproducibility. Accuracy was also evaluated and results were satisfactory. The proposed method was successfully applied to a pharmaceutical formulation.
A thin film of poly(l-serine) was prepared via electropolymerization for the determination of trace levels of estradiol. In pH 5.0 phosphate buffer,
l-serine was oxidized during the cyclic potential sweeps between −0.60 and 2.0 V, forming a thin film at the electrode surface.
The electrochemical behavior of estradiol was investigated. The oxidation peak potential of estradiol shifts negatively at
the poly(l-serine) film-coated glassy carbon electrode (GCE) compared with that at the bare GCE. Otherwise, the oxidation peak current
greatly increases at the poly(l-serine) film-modified GCE. These phenomena suggest that the poly(l-serine) film exhibits catalytic activity towards the electrochemical oxidation of estradiol. Based on this, a sensitive,
rapid and simple electrochemical method was proposed for the determination of estradiol. The limit of detection is evaluated
to be 2.0 × 10−8 mol L−1. Finally, this method was successfully used to determine estradiol in blood serum.
Caprolactone (Mn∼3000Da, melting point ∼46°C) oligomer microspheres were prepared by an organic solvent-free process, and were investigated as a delivery system of a hydrophobic model drug (estradiol). The drug was dissolved in a polycaprolactone/poly(ethylene glycol-b-caprolactone) mixture at 50°C, which was above the melting points of both polymers. The mixture was homogenized in water for 10min at 50°C, then it was quenched in the ice bath for 10min to harden the microsphere. The polycaprolactone of the poly(ethylene glycol-b-caprolactone) coaggregated into the microsphere, and poly(ethylene glycol) formed a shell layer of the microsphere that protects the microsphere from interparticle aggregation during the hardening process of the microsphere in water. The size of microsphere could be controlled by the amount of polycaprolactone relative to poly(ethylene glycol-b-caprolactone). Estradiol release from the microsphere was investigated.
We have developed a synergic micro-extraction procedure based on ionic liquid for the preconcentration and determination of glucocorticoids in water samples. Using non-ionic surfactant Triton X-100 (TX-100) as synergic reagent, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF(6) ) accomplished extraction rapidly without heating in water bath. One key property of ionic liquids that highlights their potential is their wide liquid temperature range. The improved extraction was named as ionic liquid supported vortex-assisted synergic micro-extraction (ILSVA-SME). Compared with the traditional liquid-liquid extraction (LLE) and cloud point extraction (CPE), ILSVA-SME was accomplished in 8 min with considerably high recovery. The proposed method greatly improved the sensitivity of high performance liquid chromatography (HPLC) for the determination of glucocorticoids. The results obtained indicated a good linearity with the correlation coefficient of 0.997 over the range of 0.6-300 ng mL(-1) ，and high sensitivity with limits of detection of 4.11, 9.19 and 7.50 ng mL(-1) for HB, BD and NPP, respectively. The relative standard deviation (RSD) of the method was 1.57-1.81% (n = 6) with enrichment factor (EF) of 99.85, and good recoveries (≥97.24%). The method was successfully applied to the determination of glucocorticoids in mineral, water of Dianchi lake and tap water samples.
Purity assessment for high-purity organic substances is regarded as one of the core competences of national metrology institutes.
For this reason, Consultative Committee for Amount of Substance—Metrology in Chemistry (CCQM) regularly organized intercomparison
studies to compare and demonstrate the degree of equivalence of national measurement capabilities for purity determination.
This paper reports the method development work for the participation in a recent CCQM key comparison on purity assessment
for estradiol. An indirect consecutive estimation approach was adopted to determine the purity of the estradiol sample, i.e.,
through the identification and quantification of all possible impurities present in the sample. In the course of assessment,
a variety of analytical techniques were applied. The most challenging part was to identify and estimate the amount of unknown
organic-related substance present in the testing material. For the moisture determination, a gas chromatography with thermal
conductivity detection (GC–TCD) method was employed to supplement the results obtained using coulometric Karl Fischer titration
technique. Inorganic impurities and volatile organic compounds residual were analyzed using inductively coupled plasma mass
spectrometry (ICP–MS) and gas chromatography–mass spectrometry (GC–MS) techniques, respectively, and their contributions were
found to be comparatively insignificant.
Keywords17β-estradiol–Purity assessment–CCQM study
A sensitive method for the determination of estradiol is presented that is based on an electrode modified with multi-walled
carbon nanotubes (MWNTs) along with an ionic liquid. The resulting electrode showed more sensitive voltammetric response to
estradiol than other electrodes modified with MWNTs. The peak current for estradiol is linearly related to concentration over
two decades, and the limit of detection is 5nM. The method was applied to the determination of estradiol in rabbit blood
serum and in environmental waters. The recoveries were 95.3–104.0% and 97.0–104.7%, respectively.
A novel flow injection nanocrystals (NCs) chemiluminescence (CL) analysis method has been established for the determination of estradiol, estriol and estrone based on the enhancement of CdTe NCs-KMnO(4) CL reaction catalyzed by sodium hexametaphosphate. Glutathione (GSH)-capped CdTe nanocrystals were synthesized in aqueous medium, and the CdTe NCs emitted at around 555 nm was selected as the light emitter in CdTe NCs-KMnO(4) chemiluminescence (CL) system. It has been found that sodium hexametaphosphate (SHMP) enhanced the CL of the CdTe NCs-KMnO(4) system and estrogens increased these CL signals again in near neutral solution. UV-visible spectra, photoluminescence (PL) spectra, transmission electron microscopy (TEM) and CL spectra were used to characterize CdTe nanoparticles and investigate the mechanism of the CL reaction. On the basis of the enhancement, a novel flow-injection CL method has been established for the determination of estrogens. Under the optimum experimental conditions, three linear relationships were obtained. The method described is simple, sensitive, and has been successfully utilized for the determination of estrogens in tap water samples.
A simple and rapid method for determination of 17 β-estradiol in pharmaceutical preparation was developed and validated using gas chromatography with flame ionization detection (GC-FID). The solutions of standard and the sample were prepared in methanol. GC separation was performed in about 7.7 min using a 30 m x 0.32 mm I.D. (film thickness 0.25 μm) HP-5 capilary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min -1. After injection of the sample at inlet temperature 250°C, the temperature of the GC oven was as follows: initial temperature was 150°C, held for 1.5 min, increased to 260°C at a rate of 50°C min -1 held for 5 min, and finally to 270°C at a rate of 10°C min -1 and held for 3.3 min. Detector temperature is 300°C. 2 μl was injected in splitless mode. Calibration curves were linear between the concentration range 0.25-50 μg ml -1. The method was validated for specificity, linearity, precision, accuracy and limit of quantitation. Also, the method was applied to directly and easily to the analysis of the pharmaceutical preparation (Estrofem tablet).
A porous monolithic capillary column based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) (poly (MAA-co-EGDMA)) was prepared using methanol and polyethylene glycol 6000 as mixed porogens. The monolith has the characteristics of good permeability, high extraction efficiency and long lifetime. Improved permeability of the monolith could realize sample loading with high flow rate. A simple and convenient construction that employed valve-switch technique was designed for online coupling of the monolithic capillary column to high performance liquid chromatography. In order to obtain optimum extraction efficiency, the extraction conditions including sample pH, sample volume, extraction and desorption flow rate were investigated. Under the optimum conditions, the enrichment factors were 180-362 for five estrogens, indicating remarkable preconcentration ability of the monolithic capillary column. The dynamic binding capacity (DBC) was estimated to be 3.73 mg mL⁻¹ via frontal analysis. Finally the monolithic capillary column was successfully applied to online enrichment of estrogens from urine and milk samples followed by high performance chromatography. Low detection limits (S/N=3) of the proposed method were achieved in the range of 0.04-0.35 μg L⁻¹. The recoveries were 95.6-106.1% and 76.5-116.8% for the spiked urine and milk samples respectively, with the RSDs of 1.7-9.9%.
To assess the potential risks associated with the environmental exposure of steroid estrogens, a novel highly efficient and selective estrogen enrichment procedure based on the use of molecularly imprinted polymer has been developed and evaluated. Herein, analogue of estrogens, namely 17-ethyl estradiol (EE(2)) was used as the pseudo template, to avoid the leakage of a trace amount of the target analytes. The resulting pseudo molecularly imprinted polymers (PMIPs) showed large sorption capacity, high recognition ability and fast binding kinetics for estrogens. Moreover, using these imprinted particles as dispersive solid-phase extraction (DSPE) materials, the amounts of three estrogens (E(1), E(2) and E(3)) which were detected by HPLC-UV from the chicken tissue samples were 0.28, 0.31 and 0.17 μg g(-1), and the recoveries were 72.5-78.7%, 90.3-95.2% and 80.5-83.6% in spiked chicken tissue samples with RSD <7%, respectively. All these results reveal that EE(2)-PMIPs as DSPE materials coupled with HPLC-UV could be applied to the highly selective separation and sensitive determination of trace estrogens in chicken tissue samples.
A high performance liquid chromatographic method for the determination of 17α-estradiol and 17β-estradiol with fluorimetric detection after precolumn derivatization with p-nitrobenzoyl chloride was developed. Estradiols in urine were hydrolyzed with 0.5 mol/L HCl, and then enriched and cleaned-up by ENV-18 C18 solid phase extraction column. Phenomenex™ C 18 (250 mm x 4.6 mm) column was used. Mobile phase consisted of acetonitrile-water (55:45, V/V) with a flow rate of 1.0 mL/min. Fluorescence detection was done with the excitation at 282 nm and emission at 315 nm. The linear ranges of 17α-estradiol and 17β-estradiol were found to be 7. 0 × 10-3 mg/L - 10 mg/L, and the mean recoveries of 17α-estradiol and 17β-estradiol were 83.5% and 88.2% respectively. Their limits of detection both were 7.0 × 10-3 mg/L. This method has been applied to the determination of 17α-estradiol and 17β-estradiol in urine with satisfactory results.
A sensitive method for the determination of 17β-estradiol,estrone and estriol in river water has been developed, based on SPE cartridge extraction and HPLC coupled with negative electron ion spray tandem mass spectrometry(LC/MS/MS) in the multiple reaction monitoring mode(MRM). An internal standard calibration method using a stable isotopic labeled 17β-estradiol was employed for quantitation. The limit of detection (LOD) for these estrogens in river water was 0.3 ng/1. Good linearities of the calibration curve were obtained in the concentration range from 0.4 ng/1 to 80 ng/1. The correlation coefficients were > 0.995 for all estrogens. The recoveries of these estrogens were 65.9 ∼ 96.9%. The relative standard deviations were 5.8 ∼ 11.1% for 0.4 ng/1, 8.1∼ 11.8% for 0.8 ng/1, 4.2 ∼ 12.6 ng/1 for 1.6 ng/1. Using this method, the behavior and occurrence of estrogens in river water and the effluent of sewage treatment plants were investigated in Chikugo-river(Kyusyu, Japan). A few estrogens were detected in the river water by LC/MS/MS, and the levels were ND-0.6 ng/1. However, in all samples of sewage-treatment plants, 17β-estradiol and estrone were detected, and the levels were 0.5 ∼ 4.2 ng/1(17β-estradiol) and 5.3 ∼ 35.1 ng/1 (estrone). On the other hand, 17β-estradiol was detected in most samples, including river water, by ELISA(17/18); the levels in sewage-treatment plants were 6.0 ∼ 13.5 times higher than those by LC/MS/MS. The differences in these results might be due to the cross reaction of ELISA.
A multiresidue analytical method for the determination of the most common and biologically active natural and synthetic steroids (four estrogens: estriol, 17β-estradiol, 17α-ethynylestradiol, estrone; one progestagen: progesterone and six androgens: trenbolone, boldenone, nandrolone, testosterone, 17α-methyltestosterone, stanozolol) in environmental waters was developed. The analytes were isolated from water samples by solid phase extraction (SPE) utilizing a graphitized carbon black adsorbent (Carbograph-1). The final samples were analyzed by reversed-phase high performance liquid chromatography with tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS-MS). Ionization was performed in a heated nebulizer (HN) interface operating in the positive ion mode. The protonated ions [M+H] and the dehydrated ions [M+H-H2O] (for estriol, 17α-estradiol, 17β-estradiol, and 17α-ethynylestradiol) were used as precursor ion for collision-induced dissociation (CID), and two diagnostic product ions for each analyte were identified for the unambiguous steroid confirmation by multiple reaction monitoring (MRM) mode. Method performance, for this analytical procedure, was validated by analyzing groundwater and river water samples fortified at level of 20 ng/L. The average recovery for each analyte exceeded 82%. Good method precision was demonstrated with percent relative standard deviation of less than 7.2% for all analytes.
A high performance liquid chromatographic method (HPLC) for the simultaneous determination of 4-nonylphenol, bisphenol A, 17α-ethinylestradiol and three endogenic estrogens including 17α-estradiol, 17β-estradiol, estriol in urine sample, based on precolumn derivatization with p-nitrobenzoyl chloride, is presented in this paper. The estrogens mentioned above in urine were firstly hydrolyzed with 0.6 mol/l HCl, and then enriched and cleaned-up by ENV-18 C18 solid phase extraction (SPE) column. The estrogens on column were eluted with dichloromethane, and the eluent was evaporated to dryness under gentle nitrogen flow. The residue was allowed to react with p-nitrobenzoyl chloride at 25 °C for 30 min. Separation was performed on a C18 column with gradient elution using acetonitrile and water as mobile phase. A fluorescence detection system was used to detect the fluorescent derivatization products. The detection limit of the method was 2.7 μg/l for bisphenol A and 17β-estradiol, 2.9 μg/l for 4-nonylphenol, 4.6 μg/l for 17α-estradiol and 17α-ethinylestradiol and 8.3 μg/l for estriol, respectively. The relative standard deviations (R.S.D.) ranged from 1.29 to 4.52% and the recoveries ranged from 85.5 to 99.9%. The method was applied to the determination of those six estrogens mentioned above in human urine samples collected from 20 healthy volunteers (aged 21–29). Bisphenol A (BPA) and 4-nonylphenol (NP) were detected with average contents of 1.22 ± 1.38 mg/l and 0.38 ± 0.77 mg/l in 10 male urine samples and 1.29 ± 1.22 mg/l and 0.05 ± 0.05 mg/l in 10 female urine samples, respectively. 17α-ethinylestradiol (α-EE2) was also detected with average contents of 0.13 ± 0.41 mg/l and 0.06 ± 0.15 mg/l in male and female urine samples, respectively.
The influence of temperature on retention and separation of estrogens, progesterone derivatives and beta-cyclodextrin in reversed-phase high-performance liquid chromatography has been studied. Steroids were detected using direct UV detection at 240 and 280 nm. Detection of beta-cyclodextrin was achieved using a post-column indirect photometric method. Chromatographic experiments were performed using an acetonitrile-water mobile phase (30%, v/v) and a wide range of column temperatures from 0 to 80 degrees C with 20 degrees C steps. Linear Van't Hoff plots were observed for steroids and beta-cyclodextrin when an unmodified binary mobile phase was applied. The retention of steroids was strongly influenced by temperature when the mobile phase was modified with beta-cyclodextrin at a concentration of 12 mM. Particularly, for 17beta-estradiol and 20alpha-hydroxyprogesterone a strong deviation from the linear Van't Hoff plots and a remarkable affinity for beta-cyclodextrin was observed. Polynomial regression calculations were performed to fit the set of experimental data points. Using third-order polynomial equations, minimum separation factor values (alphamin) were calculated for temperatures from -10 to + 100 degrees C with 1 degrees C steps. The best chromatographic conditions for separation of multicomponent samples were chosen. A possible retention mechanism for solutes in the presence of macrocyclic additives is discussed. The results presented describe the role of temperature in high-performance liquid chromatography systems in which the mobile phase is modified with an inclusion agent.
This article describes the proper selection of extraction solvents to eliminate interference from a polymer matrix to the quantitation of estradiol degradation products in a transdermal formulation by reversed-phase liquid chromatography. The separation is performed by gradient elution with acetonitrile and water as the mobile phase on Inertsil ODS columns. Severe band distortion and insufficient recovery are observed for two geometric degradation products (or impurities) when the sample is prepared by acetonitrile. It is anticipated that the poor resolution and recovery are caused by multiple retention processes due to the reversible binding of degradation products to the polymer matrix (or the impurity-polymer interaction). This interaction is eliminated by adding formamide, a solvent that possesses similar properties to the matrix, in the extraction solvent. It is believed that the favorable interaction between formamide and the polymer matrix releases the impurity molecules, and they can then be separated by a single retention mode. It has been confirmed experimentally that the use of formamide in the extraction solvent not only sharpens the peaks tremendously, but also recovers the degradation products completely.
This paper presents the development of an analytical procedure for the determination of two sexual steroid hormones: 17beta-estradiol and estrone, and the synthetic contraceptive estrogen, 17alpha-ethynylestradiol in effluents of wastewater treatment plants. Samples are extracted via solid-phase extraction using C18 cartridges. Extracts in ethyl acetate are then purified with a liquid-liquid separation with aqueous sodium chloride, then with a clean-up on a Florisil cartridge. Steroids are analyzed using an LC-MS-MS ion trap system. Internal quantification with the corresponding deuterated steroids leads to limits of quantification at 5 ng/l for estrone and 10 ng/l for estradiol and ethynylestradiol. In mineral spiked water, recoveries are 91% for 17beta-estradiol, 97% for estrone and 87% for 17alpha-ethynylestradiol and RSDs are 15% for 17beta-estradiol, 11% for estrone and 23% for 17alpha-ethynylestradiol.
A multi-residue analytical method has been developed for the determination of various classes of selected endocrine disruptors. This method allows the simultaneous extraction and quantification of different estrogens (estradiol, estrone, estriol, estradiol-17-glucuronide, estradiol diacetate, estrone-3-sulfate, ethynyl estradiol and diethylstilbestrol), pesticides (atrazine, simazine, desethylatrazine, isoproturon and diuron), and bisphenol A in natural waters. In the method developed, 500 ml of water are preconcentrated on LiChrolut RP-18 cartridges. Further analysis is carried out by liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure chemical ionisation (APCI) in the positive ion mode for determination of pesticides and electrospray in the negative ionisation mode for determination of estrogens and bisphenol A. Recoveries for most compounds were between 90 and 119%, except for bisphenol A (81%) and diethylstilbestrol (70%), with relative standard deviations below 20%. Limits of detection ranged between 2 and 15 ng/l. The method was used to study the occurrence of the selected pollutants in surface and groundwater used for abstraction of drinking water in a waterworks and to evaluate the removal efficiency of the different water treatments applied. Water samples from the river, the aquifer, and after each treatment stage (sand filtration, ozonation, activated carbon filtration and post-chlorination) were taken monthly from February to August of 2002. The presence in river water of atrazine, simazine, diuron and bisphenol A were relatively frequent at concentrations usually below 0.1 microg/l. Lower levels, below 0.02 microg/l, were usual for isoproturon. Estrone-3-sulfate and estrone were detected occasionally in the river. Most of the compounds were completely removed during the water treatment, especially after activated carbon filtration.
A rapid, sensitive, and specific high-performance liquid chromatography-electrospray ionization-multistage mass spectrometry (MS) method for measuring endogenous ketolic estrogen metabolites in human urine has been developed. The method requires a single hydrolysis/extraction/derivatization step and only 2.5 mL of urine, yet is able to simultaneously quantify estrone and its 2-methoxy and 2-, 4-, and 16alpha-hydroxy derivatives, 16-ketoestradiol, and 2-hydroxyestrone-3-methyl ether metabolites. The combination of a simple hydrazone derivatization step with multistage MS greatly enhances the sensitivity and specificity of the analysis of endogenous estrogen within human urine. Standard curves are linear over a 100-fold concentration range with linear regression correlation coefficients typically greater than 0.99. The lower limit of quantitation for each ketolic estrogen is 0.2 ng/2.5-mL urine sample (10 pg on column), with an accuracy of 93-103% and an overall precision, including the hydrolysis, extraction, and derivatization steps, of 1-13% relative standard derivation (RSD) for samples prepared concurrently and 8-16% RSD for samples prepared in separate batches. This method also allows for the identification of 2-hydroxyestrone-3-methyl ether in urine obtained from both pre- and postmenopausal women. This potentially protective estrogen metabolite has been previously reported only in the urine of pregnant women. Since individual patterns of estrogen metabolism may influence the risk of breast cancer, accurate and specific measurement of estrogen metabolites in biological matrixes will facilitate future research on breast cancer prevention, screening, and treatment.
The pathological steroid biosynthesis of a virilizing ovarian tumor was examined via high performance liquid chromatography-radioimmunoassay (HPLC-RIA) determination of the intratissular steroid concentrations. Sex cord-stromal tumor of the ovary was obtained surgically from an 18-year-old female patient with extremely high androst-4-ene-3,17-dione (4-en-dione) and testosterone (Test) blood serum levels. The tissue specimen was extracted with ethyl acetate and the extract was then purified on a C18 mini-column with methanol-water eluents. Steroids were isolated by reversed-phase HPLC on a C18 silica gel column with 51%, 55% and 64% v/v methanol-water eluents. Steroids in the collected eluent fractions were detected by the radioactivity of tritiated internal standards and then quantified by specific RIAs. In the tumor specimen, very high 17alpha-hydroxyprogesterone (17-OH-Prog; 6300 fmol/g), dehydro-epiandrosterone (2870 fmol/g), androst-4-ene-3,17-dione (3000 fmol/g), testosterone (5700 fmol/g) concentrations, and less progesterone (PROG; 320 fmol/g) and androst-5-ene-3beta,17beta-diol (5-en-diol; 320 fmol/g), were determined. Tissue levels of 5alpha-dihydrotestosterone (DHT), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), 5alpha-androstane-3beta,17beta-diol (3beta-diol), and 17beta-estradiol were found to be 71, 20, 28, and 12 fmol/g, respectively. Steroid profile analysis verified a pathological steroid biosynthesis in the ovarian tumor and suggested that the 17alpha-hydroxylase (17alpha-H), 17,20-lyase (17,20-L), and 3beta-hydroxysteroid dehydrogenase/Delta5-4-isomerase (Delta5-3beta-HSD) activities were particularly elevated in this tumorous tissue. Present data demonstrate that the analysis of intratissular steroid profile by a HPLC-RIA method may valuably contribute to the steroidal pathophysiology of endocrine tumors.
A stability-indicating liquid chromatographic method for the determination of degradation products and impurities in Vagifem, estradiol vaginal tablets has been developed and validated. Vagifem is a low dose preparation containing only 25microg 17beta-estradiol in a tablet matrix of 80mg (a drug to excipient ratio of 1:3200). This paper presents the rationale for the optimization of the sample preparation in order to minimize placebo interference as well as validation data for linearity, accuracy, precision, ruggedness, specificity and limits of detection and quantification. Data shows that the method is suitable for routine analysis of minute amounts of estradiol impurities.
The specific and non-specific assay methods in the European and US Pharmacopoeias are critically evaluated. Emphasis is made on the discussion of the increasing role of impurity tests and decreasing, moreover questionable role of assay methods in characterising the quality of bulk drug materials. Various possibilities are also discussed for calculating the active ingredient content from the results of the assay and impurity tests. Only bulk drug materials are dealt with excluding from this study pharmaceutical formulations.
An efficient method for analyzing seven sexual hormones in cosmetics, namely, estriol, estradiol-17beta, estrone, testosterone, methyl-testosterone, progesterone and diethylstilbestrol by reversed-phase high performance liquid chromatography was developed. Samples were saponified with 20 g/L sodium hydroxide. The sexual hormones were then extracted with dichloromethane-acetic acetate (40:1, v/v) under acidic conditions (pH 3, adjusted with 1 mol/L HCl). An XTerra RP18 column was employed and a mixture of water-methanol-acetonitrile (50:32:18, v/v) was used as mobile phase. The seven sexual hormones were detected at 230 nm. The average spiked recoveries for the seven sexual hormones ranged from 75.6% to 97.8% with relative standard deviations of 1.9% to 7.2%. The linear ranges of determination were from 5 to 50 mg/L with correlation coefficients of 0.9999, and the limits of detection were from 3.7 to 12 ng. The method is suitable to simultaneously assay the seven sexual hormones in cosmetics with simplicity and accuracy in a single injection.
This paper deals with the development of a novel method for simultaneous determination of estradiol, its degradation product estrone, and two preservatives, methylparaben and propylparaben, in the topical preparation Estradiol HBF. After optimization of the analytical conditions the method was validated and applied in studies of the stability of the topical preparation Estrogel HBF. Separation of all these compounds was performed on a Supelco Discovery C18 (250 mm x 3.0 mm, 5 microm) analytical column. A mixture of acetonitrile, methanol, and water (23:24:53 v/ v) was chosen as mobile phase. UV absorbance at 225 nm was used for detection and quantitation of analytes. The total analysis time was less than 12 min at a flow rate of 0.9 mL min(-1). All the compounds were isolated from the topical gel by simple extraction with an acetonitrile solution of hydrocortisone, as internal standard, and using sonication and centrifugation thereafter. The supernatant was injected directly on to the analytical column. The recovery of the procedure was from 96.9 to 100.4%. Validation of method according international guidelines was successfully performed.