Polypyrrole nanotubes for electrochemically controlled extraction of atrazine, caffeine and progesterone.

Polypyrrole (PPy) was electrochemically synthesized with charge control on the surface of a steel mesh. Two different morphologies (globular and nanotubular) were created and characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The modified electrodes were used as extraction phases in solid-phase extraction (SPE) and electrochemically controlled solid-phase extraction (EC-SPE) of atrazine, caffeine and progesterone. Raman spectroscopy was employed for the structural characterization of PPy after long exposure to the analytes. The electrochemical behavior was studied by cyclic voltammetry which revealed the higher capacitive behavior of polypyrrole nanotubes because of the huge superficial area, also no electrocatalytical behavior was observed evidencing the strong adsorption of the analytes on the PPy surface. The effects of the PPy oxidation state on the extraction performance were evaluated by in-situ electrochemical sorption experiments. The sorption capacity was evaluated by gas chromatography coupled to mass spectrometry (GC-MS). The method displays good stability, repeatability and reproducibility. The limits of detection range between 1.7-16.7 µg L-1. Following the extraction of river water samples, it was possible to identify the presence of other endogenous organic compounds besides the analytes of interest. This indicates the potential of the method and material developed in this work. Graphical abstract Schematic representation of a steel mesh electrode covered with polypyrrole nanotubes used as extraction phase for separation of contaminants from aqueous samples. The oxidation level of polypyrrole was electrochemically tuned by which the adsorption of analytes is deeply affected.

Nanostructured Bismuth Film Electrode for Detection of Progesterone.

Progesterone is an important hormone responsible, among others, for maintaining pregnancy via inhibition of uterus muscles activity; thus, following its concentration levels in pregnant women is of immense importance in the endeavor to prevent premature birth. In this work, the nanostructured bismuth film electrode (nsBiFE) was studied for detection of progesterone in neutral medium. Due to the ability to accumulate progesterone at the nsBiFE, the adsorptive cathodic stripping voltammetry was beneficially exploited. The nsBiFE was prepared on the surface of a glassy carbon supporting electrode and several parameters influencing the detection of progesterone were investigated. The nsBiFE exhibited superior electroanalytical characteristics in comparison to other bismuth-based electrodes and unmodified glassy carbon electrode together with satisfactory response toward low concentrations of progesterone, which are consistent with clinically significant levels.

Inter-assay variation and reproducibility of progesterone measurements during ovarian stimulation for IVF.

In recent years there is increasing evidence that elevated progesterone levels during ovarian stimulation for IVF / ICSI have a negative impact on the ART-outcome. However, different progesterone assays were used in the previous studies and different assays might produce varying results. This retrospective study evaluated the reproducibility and reliability of different progesterone assays with a special focus on progesterone levels below 1.5 ng/ml, as this range is crucial for early detection of progesterone rise during ovarian stimulation for IVF. A total of 413 blood samples were categorized in different progesterone ranges and whether they were retrieved on the day of final oocyte maturation and the results were compared regarding their reproducibility and reliability. To compare the reproducibility between the different progesterone assays, the Intraclass Correlation Coefficient (ICC) was calculated and interpretation of the ICC results was done according to Cicchetti, ranging from poor to excellent. The correlation of the assays was excellent when all samples were compared including samples retrieved on day of final oocyte maturation, however in the ranges of progesterone levels 1.0 ng/ml to < 1.5 ng/ml, 0.8 ng/ml to < 1.0 ng/ml and < 0.8 ng/ml, the ICC varied between poor and excellent. The assays "gen III" and "Architect" showed an excellent reproducibility of progesterone results throughout all ranges of progesterone levels. This analysis demonstrates, that different progesterone assays have a limited reproducibility and that the results depend on the assay used and the range of progesterone level. This fact leads to two important conclusions. Firstly the limited reproducibility might lead to substantially different treatment decisions in ovarian stimulation treatment for IVF and secondly critical interpretation of thresholds, provided by meta-analysis, is crucial despite the risk that the so far gained clinical experience might become irrelevant and has to be adjusted to the results, obtained by each assay.

Nonenzymatic electrochemical sensor based on imidazole-functionalized graphene oxide for progesterone detection.

The modification of electrode surfaces has been the target of study for many researchers in order to improve the analytical performance of electrochemical sensors. Herein, the use of an imidazole-functionalized graphene oxide (GO-IMZ) as an artificial enzymatic active site for voltammetric determination of progesterone (P4) is described for the first time. The morphology and electrochemical performance of electrode modified with GO-IMZ were characterized by scanning electron microscopy and cyclic voltammetry, respectively. Under optimized conditions, the proposed sensor showed a synergistic effect of the GO sheets and the imidazole groups anchored on its backbone, which promoted a significant enhancement on electrochemical reduction of P4. Figures of merits such as linear dynamic response for P4 concentration ranging from 0.22 to 14.0 µmol L-1, limit of detection of 68 nmol L-1 and limit of quantification and 210 nmol L-1 were found. In addition, presented a higher sensitivity, 426 nA L µmol-1, when compared to the unmodified electrode. Overall, the proposed device showed to be a promising platform for a simple, rapid, and direct analysis of progesterone.

Countercurrent separation assisted identification of two mammalian steroid hormones in Vitex negundo.

Countercurrent separation (CCS) has been widely used for the separation of high abundance compounds. However, the identification of low abundance compounds, such as mammalian steroid hormones, from natural sources is still a challenging task. A mixture of 14 human steroid hormone reference compounds was prepared for the development of a CCS enrichment strategy. The TLC-based GUESS (Generally Useful Estimate of Solvent Systems) method along with partitioning experiments were implemented to develop a process for the enrichment of these low abundance compounds with CCS. The application of CCS to the steroid hormone enrichment of Vitex negundo extracts was demonstrated by the identification of progesterone and estriol. This method provides a CCS-driven strategy to mine plant sources for low abundance compounds.

Biotransformation of Progesterone by the Ascomycete Aspergillus niger N402.

The ability of the ascomycete Aspergillus niger N402 to transform exogenous progesterone was investigated. We found that this strain has steroid-hydroxylating activity and can introduce a hydroxyl group into the progesterone molecule mainly at positions C11(α) and C21 with predominant formation of 21-hydroxyprogesterone (deoxycortone). In addition, formation of 6ß,11α-dihydroxyprogesterone was also observed. Studying the effects of the growth medium composition and temperature on progesterone conversion by A. niger N402 showed that the most intense accumulation of 21-hydroxyprogesterone occurred in minimal synthetic medium at 28°C. Increasing the cultivation temperature to 37°C resulted in almost complete inhibition of the hydroxylase activity in the minimal medium. In the complete medium, a similar increase in temperature inhibited 11α-hydroxylase activity and completely suppressed 6ß-hydroxylase activity, but it produced no effect on 21-hydroxylating activity.

The relationship between fat and progesterone, estradiol, and chorionic gonadotropin levels in Quebec cow's milk.

PURPOSE: The majority of milk in industrialized countries is obtained from pregnant cows, which contains increased levels of estrogen and progesterone compared to non-pregnant cows. The aim of this study was to quantify the amount of hormones present in milk with different fat content because previous studies on humans have shown potential effects of increased milk consumption on serum and urine hormone levels as well as on sperm parameters. However, it is unclear whether consumption of milk at the currently recommended levels would lead to systemic effects. METHODS: Samples of cow's milk of varying fat concentrations (0, 1, 2, 3.25, 10, and 35%) were analyzed via competitive ELISA assays. RESULTS: Progesterone concentrations were significantly correlated to increasing fat content of milk (r = 0.8251, p = 0.04). CONCLUSIONS: Research on conditions in which additional progesterone may have an effect on human health should consider inclusion of limitation of milk intake and its effects. Further studies are needed to determine the concentration of progesterone in milk of different fat content in other regions and countries and to quantify the potential pathophysiologic role.

Impact of sample extraction on the accurate measurement of progesterone in human serum by liquid chromatography tandem mass spectrometry.

In the present study, the impact of the extraction solvent on the accuracy of endogenous progesterone assay in human serum has been investigated using two selective reaction monitoring (SRM) transitions (315>97 & 315>109). Higher levels of noise and more interference were observed when more polar solvents were used for extraction, thus resulting in serious bias of the measured values of progesterone in serum. This is confirmed by monitoring the ion ratio of 315>97-315>109. This issue could not be easily resolved by changes in MS/MS transitions or chromatography conditions. More bias was observed with the SRM transition 315>109 for the polar solvent extraction. Hexane and 1-chlorobutane (polarity index of 0 and 1, respectively) did provide the cleanest samples with a lower noise level in the chromatograms. Moreover, the measured values of progesterone were not changed with different SRM transitions or longer retention time in search of an improved separation. Recovery tests of progesterone have been performed with 1-chlorobutane in matrices with phosphate buffered saline (PBS) 1x, PBS 1×3% bovine serum albumin (BSA), stripped serum/H2O (1:1) and unstripped serum. The recovery (70%∼80%) consistency is observed not only at different levels but also in different matrices. The equivalent recovery between PBS 1x, PBS 1×3% BSA and unstripped serum shows that the impact of progesterone binding to serum proteins on the measurement accuracy can be avoided with this sample preparation procedure. No significant matrix effect on the determination of progesterone was observed with 1-chlorobutane. Within the range of 12.5-2000pg/mL, a good linearity is observed with R>0.99 and weighting factor 1/X. Bias and covariance efficiency of QCs are within 10%. With 1-chlorobutane as the extraction solvent, the concentration of progesterone was measured where the range for postmenopausal serum is 5.74∼91.7pg/mL, which is well below the reported concentrations of 314 pg/mL∼942pg/mL in postmenopausal serum by immunoassay-based techniques, while the range in premenopausal serum is 12.8 pg/mL∼18.6ng/mL.

Validation of Electrochemiluminiscence Immunoassay for Ovarian Steroid Determination in Rhinella arenarum.

In this work, we describe the validation of an electrochemiluminescence immunoassay (ECLIA) that allowed us for the first time to determine the levels of progesterone (P4 ) and testosterone (T) secreted by Rhinella arenarum follicles during the preovulatory (POP) and reproductive (RP) periods. We also verified the relation between P4 and T levels and oocyte maturation. Moreover, we demonstrated that the extraction protocol developed for the determinations of P4 and T by ECLIA proved to be efficient and reproducible since the efficacy of the extraction was above 95% in all assays conducted. The results indicate that in the validation process the variation coefficient (CV) between assays is compatible with the analytical procedures based on automated immunoassays (CV < 8%) and that the adaptation proposed for the samples allows the determination of T and P4 with the Cobas e-411 analyzer. Our results indicate that in basal conditions the levels of T released by R. arenarum follicles were higher than those of P4 during POP and RP. In these conditions, steroid secretion failed to induce germinal vesicle break down (GVBD) in the follicles. Under gonadotropin stimulation, steroidogenesis showed a remarkable increase in both periods, especially during POP. This increase was correlated with a high maturation percentage in the follicles incubated in vitro (GVBD = 72 ± 16%) during POP. During RP, human Chorionic Gonadotropin (hCG) induced 81.75 ± 9.1% GVBD. This study is the first report of the seasonal steroidogenic activity in the ovary of R. arenarum in situ using an ECLIA-modified protocol developed in our laboratory.

Evaluation of storage and evaporation in the removal efficiency of D-norgestrel and progesterone in human urine.

Pharmaceuticals are emerging contaminants and it must be noted that approximately 70 % of them are excreted via urine. Therefore, urine usage implies the risk of transfer of pharmaceutical residues to agricultural fields and environment contamination. Thus, this study aimed on the development and validation of a LC-MS/MS method for D-norgestrel (D-NOR) and progesterone (PRO) determination in human urine, as well as the evaluation of the removal efficiency of two methods (storage and evaporation), and the effects of acidification with sulfuric acid. The storage process was evaluated for 6 weeks, while the evaporation was assessed at three different temperatures (50, 75, and 100 °C). All experiments were done with normal urine (pH = 6.0) and acidified urine (pH = 2.0, with sulfuric acid). The results of validation showed good method efficiency. In the second week of storage, higher hormone degradation was observed. In the evaporation method, both D-NOR and PRO were almost completely degraded when the volume was reduced to the lowermost level. Results also indicate that acidification did not affect degradation. Overall, the results showed that combination of two methods can be employed for more efficient hormone removal in urine.

Divinyl sulfone cross-linked cyclodextrin-based polymeric materials: synthesis and applications as sorbents and encapsulating agents.

The aim of this study was to evaluate the crosslinking abilities of divinyl sulfone (DVS) for the preparation of novel water-insoluble cyclodextrin-based polymers (CDPs) capable of forming inclusion complexes with different guest molecules. Reaction of DVS with native α-cyclodextrin (α-CD), ß-cyclodextrin (ß-CD) and/or starch generates a variety of homo- and hetero-CDPs with different degrees of crosslinking as a function of the reactants' stoichiometric ratio. The novel materials were characterized by powder X-ray diffraction, electron microscopy and for their sorption of phenol and 4-nitrophenol. They were further evaluated as sorbents with phenolic pollutants (bisphenol A and ß-naphthol) and bioactive compounds (the hormone progesterone and curcumin). Data obtained from the inclusion experiments show that the degree of cross-linking has a minor influence on the yield of inclusion complex formation and highlight the important role of the CDs, supporting a sorption process based on the formation of inclusion complexes. In general, the inclusion processes are better described by a Freundlich isotherm although an important number of them can also be fitted to the Langmuir isotherm with R2 ≥ 0.9, suggesting a sorption onto a monolayer of homogeneous sites.

Evaluation of a molecularly imprinted polymer for determination of steroids in goat milk by matrix solid phase dispersion.

A molecularly imprinted polymer-matrix solid-phase dispersion methodology for simultaneous determination of five steroids in goat milk samples was proposed. Factors affecting the extraction recovery such as sample/dispersant ratio and washing and elution solvents were investigated. The molecularly imprinted polymer used as dispersant in the matrix solid-phase dispersion procedure showed high affinity to steroids, and the obtained extracts were sufficiently cleaned to be directly analyzed. Analytical separation was performed by micellar electrokinetic chromatography using a capillary electrophoresis system equipped with a diode array detector. A background electrolyte composed of borate buffer (25mM, pH 9.3), sodium dodecyl sulfate (10mM) and acetonitrile (20%) was used. The developed MIP-MSPD methodology was applied for direct determination of testosterone (T), estrone (E1), 17ß-estradiol (17ß-E2), 17α-ethinylestradiol (EE2) and progesterone (P) in different goat milk samples. Mean recoveries obtained ranged from 81% to 110%, with relative standard deviations (RSD)≤12%. The molecularly imprinted polymer-matrix solid-phase dispersion method is fast, selective, cost-effective and environment-friendly compared with other pretreatment methods used for extraction of steroids in milk.

Removal of endocrine disrupting compounds in a lab-scale anaerobic/aerobic sequencing batch reactor unit.

The fate and removal of six selected endocrine disrupting compounds in a lab-scale anaerobic/aerobic (A/O) sequencing batch reactor (SBR), operating at 5 days, solids retention time (SRT) were investigated. A carbamazepine (CBZ), acetaminophen (ATP), diltiazem (DTZ), butyl benzyl phthalate (BBP), estrone and progesterone mix was spiked as model endocrine disrupting compounds (EDC) into domestic wastewater obtained from a nearby sewage treatment plant. The influent, effluent and sludge samples from the SBR unit were analysed by using an LC/MS/MS instrument equipped with electrospray ionization. More than 80% removal was observed for all the EDCs tested. It was found that biodegradation is the most important mechanism for BBP, ATP and progesterone. Biodegradation constants were calculated according to the simplified Monod model for these compounds. The DTZ seemed to have lower rate of biodegradation. The CBZ appeared totally resistant to biodegradation. However, it presented a high rate of sorption onto the sludge and was thereby treated. This contradicts with the literature studies.

Chemometric evaluation of urinary steroid hormone levels as potential biomarkers of neuroendocrine tumors.

Neuroendocrine tumors (NETs) are uncommon tumors which can secrete specific hormone products such as peptides, biogenic amines and hormones. So far, the diagnosis of NETs has been difficult because most NET markers are not specific for a given tumor and none of the NET markers can be used to fulfil the criteria of high specificity and high sensitivity for the screening procedure. However, by combining the measurements of different NET markers, they become highly sensitive and specific diagnostic tests. The aim of the work was to identify whether urinary steroid hormones can be identified as potential new biomarkers of NETs, which could be used as prognostic and clinical course monitoring factors. Thus, a rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection has been developed for the determination of cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone in human urine. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification were 0.5 and 1 ng mL-1 for each steroid hormone, respectively. Linearity was confirmed within a range of 1-300 ng mL-1 with a correlation coefficient greater than 0.9995 for all analytes. The described method was successfully applied for the quantification of six endogenous steroid levels in human urine. Studies were performed on 20 healthy volunteers and 19 patients with NETs. Next, for better understanding of tumor biology in NETs and for checking whether steroid hormones can be used as potential biomarkers of NETs, a chemometric analysis of urinary steroid hormone levels in both data sets was performed.

Simultaneous determination of veterinary antibiotics and hormone in broiler manure, soil and manure compost by liquid chromatography-tandem mass spectrometry.

A multi-residue analytical method was developed to quantify nine antibiotics and one hormone in soil, broiler manure and manure compost. The developed method was based on ultrasonic extraction with MeOH:ACN:EDTA:McIlvaine buffer, solid phase extraction (SPE) using HLB (3 cc/60 mg) cartridge, followed by instrumental analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with 25 min total run time. It was validated and tested on soil, broiler manure and manure compost samples and showed that the method is able to simultaneously detect and quantify the target analytes with good selectivity and sensitivity. The developed method was linear in a concentration range from its instrumental quantification limit (IQL) to 500 ng/mL, with correlation coefficients higher than 0.999. The overall method performance was good for the majority of the analytes, with recoveries range from 63% to 121% in all the sample matrices. The method quantification limit (MQL) for the 10 target analytes in the soil, broiler manure and manure compost samples were 2-10, 3-16 and 5-15 µg/kg dry weight (DW), respectively. The method has also included tilmicosin, an antibiotic known to be reported in the environment for the first time. The developed method was then applied on broiler manure samples and its relative manure amended agricultural soil samples to identify and quantify veterinary antibiotic and hormone residues in the environment. These analytes were detected in broiler manure and soil samples, with maximum concentrations reaching up to 78516.1 µg/kg DW (doxycycline) and 1331.4 µg/kg DW (flumequine), respectively. The results showed that the method can potentially be adopted for the analysis of veterinary antibiotic and hormone wastes in solid environmental matrices.

Occurrence of progesterone and related animal steroids in two higher plants.

Previously, the presence of a wide variety of chemically diverse steroids has been identified in both flora and fauna. Despite the relatively small differences in chemical structures and large differences in physiological function of steroids, new discoveries indicate that plants and animals are more closely related than previously thought. In this regard, the present study gathers supporting evidence for shared phylogenetic roots of structurally similar steroids produced by these two eukaryotic taxa. Definitive proof for the presence of progesterone (3) in a vascular plant, Juglans regia, is provided. Additional evidence is gleaned from the characterization of five new plant steroids from Adonis aleppica: three 3-O-sulfated pregnenolones (6a/ b, 7), a sulfated H-5beta cardenolide, strophanthidin-3-O-sulfate (8), and spirophanthigenin (10), a novel C-18 oxygenated spirocyclic derivative of strophanthidin. The ab initio isolation and structure elucidation (NMR, MS) of these genuine minor plant steroids offers information on preparative metabolomic profiling at the ppm level and provides striking evidence for the conserved structural space of pregnanes and its congeners across the phylogenetic tree.

[Influence factors of the separation of steroids using oil-in-water microemulsion liquid chromatography].

The effect of the varying operating parameters on the separation of steroids was studied by oil-in-water microemulsion liquid chromatography (MELC). The parameters included the surfactant concentration, the type of oil phase, the nature of organic solvent additives, and the pore size of the stationary-phase. The optimized conditions for the separation of 6 steroids were as follows: a Venusil ASB C18 (T) column (5 microm, 30 nm, 250 mm x 4.6 mm) was used at 35 degrees C, the microemulsion mobile phase consisted of 30 g/L sodium dodecylsulfate (SDS), 0.8% (w/w) n-octane, 6.6% (w/w) n-butanol. The optimized method can be used for the separation, identification and simultaneous determination of steroids in bulk drugs and in pharmaceutical dose forms.

Ozone oxidation of pharmaceuticals, endocrine disruptors and pesticides during drinking water treatment.

This study investigates the oxidation of pharmaceuticals, endocrine disrupting compounds and pesticides during ozonation applied in drinking water treatment. In the first step, second-order rate constants for the reactions of selected compounds with molecular ozone (k(O3)) were determined in bench-scale experiments at pH 8.10: caffeine (650+/-22M(-1)s(-1)), progesterone (601+/-9M(-1)s(-1)), medroxyprogesterone (558+/-9M(-1)s(-1)), norethindrone (2215+/-76M(-1)s(-1)) and levonorgestrel (1427+/-62M(-1)s(-1)). Compared to phenolic estrogens (estrone, 17beta-estradiol, estriol and 17alpha-ethinylestradiol), the selected progestogen endocrine disruptors reacted far slower with ozone. In the second part of the study, bench-scale experiments were conducted with surface waters spiked with 16 target compounds to assess their oxidative removal using ozone and determine if bench-scale results would accurately predict full-scale removal data. Overall, the data provided evidence that ozone is effective for removing trace organic contaminants from water with ozone doses typically applied in drinking water treatment. Ozonation removed over 80% of caffeine, pharmaceuticals and endocrine disruptors within the CT value of about 2 mg min L(-1). As expected, pesticides were found to be the most recalcitrant compounds to oxidize. Caffeine can be used as an indicator compound to gauge the efficacy of ozone treatment.

Simultaneous determination of 15 steroidal oral contraceptives in water using solid-phase disk extraction followed by high performance liquid chromatography-tandem mass spectrometry.

A rapid, accurate and sensitive method for simultaneous determination of 15 steroidal hormones including four estrogens (estrone, 17beta-estradiol, 17alpha-ethynylestradiol, estriol) and eleven progestogens (17beta-estradiol-3-benzoate, 19-norethindrone, gestodene, levonorgestrel, medroxyprogesterone, cyproterone acetate, megestrol-17-acetate, progesterone, norethindrone acetate, chlormadinone-17-acetate, and hydroxy progesterone caproate) in environmental waters was developed by coupling solid-phase disk extraction (SPDE) to ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with electrospray ionization. Among three types of extraction tested (C(8) SPDE, C(18) SPDE and C(18) SPE), the most satisfactory result was achieved using C(18) SPDE for its satisfactory recovery (75.6 to 101.4%) and short extraction time (15 min for 1L deionised water). The validity of this method was investigated and good analytical performance for all the analytes was obtained, including low limits of method detection (0.5-3.4 ng/L) and excellent linear dynamic range (1.0-50.0 ng/L). The method was applied to determine the steroidal hormones in 10 environmental waters including tap water, river water, lake water and waste water in Beijing. No progestogen was detected in all samples and estrone, estriol, 17alpha-ethynylestradiol were found in most samples at levels between 1.8 and 127.9 ng/L.