Expanding the applicability of magnetic ionic liquids for multiclass determination in biological matrices based on dispersive liquid-liquid microextraction and HPLC with diode array detector analysis.

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

Magnetic ionic liquids as versatile extraction phases for the rapid determination of estrogens in human urine by dispersive liquid-liquid microextraction coupled with high-performance liquid chromatography-diode array detection.

In this study, a rapid and straightforward approach based on magnetic ionic liquids (MIL) as extraction phases and dispersive liquid-liquid microextraction (DLLME) was developed to analyze the hormones estriol, 17-ß-estradiol, 17-α-ethynylestradiol, and estrone in human urine samples. This is the first report of an application of manganese-based MILs compatible with HPLC to extract compounds of biological interest from urine samples. The hydrophobic MILs trihexyltetradecylphosphonium tetrachloromanganate (II) ([P6,6,6,14+]2[MnCl42-]) and aliquat tetrachloromanganate (II) ([Aliquat+]2[MnCl42-]) were employed and the optimized extraction conditions were comprised of 5 mg of MIL ([P6,6,6,14+]2[MnCl42-]), 5 µL of methanol (MeOH) as disperser solvent, and an extraction time of 90 s at sample pH 6. The analytical parameters of merit were determined under optimized conditions and very satisfactory results were achieved, with LODs of 2 ng mL-1 for all analytes, determination coefficients (R2) ranging from 0.9949 for 17-ß-estradiol to 0.9998 for estrone. In addition, good results of method precision were achieved with the intraday (n = 3) varying from 4.7% for 17-ß-estradiol to 19.5% for estriol (both at 5 ng mL-1) and interday precision (evaluated at 100 ng mL-1) ranging from 11.4% for estrone to 17.7% for 17-α-ethynylestradiol and analyte relative recovery evaluated in three real samples ranged from 67.5 to 115.6%. The proposed DLLME/MIL-based approach allowed for a reliable, environmentally friendly and high-throughput methodology with no need for a centrifugation step. Graphical abstract An overview of the rapid and straightforward extraction procedure using DLLME/MIL-based approach.

Electrochemical immunosensor for ethinylestradiol using diazonium salt grafting onto silver nanoparticles-silica-graphene oxide hybrids.

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

Dynamic liquid-liquid-solid microextraction based on molecularly imprinted polymer filaments on-line coupling to high performance liquid chromatography for direct analysis of estrogens in complex samples.

A novel sample preparation technique termed dynamic liquid-liquid-solid microextraction (DLLSME) was developed and on-line coupled to high performance liquid chromatography (HPLC) for direct extraction, desorption, and analysis of trace estrogens in complex samples. The DLLSME consists of the aqueous donor phase, the organic medium phase and the molecularly imprinted polymer filaments (MIPFs) as solid acceptor phase. The organic solvent with lesser density was directly added on top of the aqueous sample, and the dynamic extraction was performed by circulating the organic solvent through the MIPFs inserted into a PEEK tube which served as an extraction and desorption chamber. Afterwards, the extracted analytes on the MIPFs were on-line desorbed and then introduced into the HPLC for analysis. To evaluate the feasibility of the on-line system, a new DLLSME-HPLC method was developed for the analysis of five estrogens in aqueous samples by using 17ß-estradiol MIPFs as the solid phase. Under the optimized conditions, the enrichment factors of 51-70, limits of detection of 0.08-0.25 µg/L and precision within 4.5-6.9% were achieved. Furthermore, the proposed method was applied to the analysis of real samples including urine, milk and skin toner, satisfactory recovery (81.9-99.8%) and reproducibility (4.1-7.9%) were obtained. Especially, 0.59 µg/L of 17ß-estradiol was determined in female urine sample. The DLLSME offers an attractive alternative for direct analysis of trace analytes in aqueous samples and could potentially be extended to other adsorptive materials.

Dynamics of steroid estrogen daily concentrations in hospital effluent and connected waste water treatment plant.

Hospital effluent and connected waste water treatment plant (WWTP) influent and effluent were sampled daily to determine the levels and inter-day variations of three naturally occurring steroid estrogens: estrone, 17ß-estradiol, estriol, and synthetic 17α-ethinylestradiol. After solid phase extraction, interferences were removed with a silica gel clean-up step and the samples analysed using gas chromatography with mass selective detection (GC-MSD). The determined inter-day concentrations in hospital effluent were between 8.6 to 31.3 ng L(-1) for estrone,

A model to estimate influent and effluent concentrations of estradiol, estrone, and ethinylestradiol at sewage treatment works.

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

Effect of two oral contraceptives with different ethinyl estradiol and levonorgestrel concentrations on the urinary excretion of biochemical vasoactive markers.

In the present study the effect on the urinary excretion of vasoactive markers of two oral contraceptives (OCs), i.e., Leios, containing 0.02 mg ethinyl estradiol and 0.1 mg levonorgestrel, and Stediril 30, containing 0.03 mg ethinyl estradiol and 0.15 mg levonorgestrel, was investigated. cGMP, prostacyclin and its antagonist thromboxane, serotonin, and urodilatin, a natriuretic and diuretic peptide formed in the kidney, were measured as markers. In a comparative, double-blind, randomized, parallel group study, 34 women received Leios and 33 women Stediril 30. Nocturnal urine was collected before treatment and during cyclic treatment after 3 and 12 cycles. Both contraceptives significantly enhanced cGMP excretion after 12 cycles. The prostacyclin metabolite remained unchanged for both formulations, but the excretion of the thromboxane metabolite was significantly decreased after 12 cycles. Thus, the ratio of prostacyclin to thromboxane, crucial for the resulting effect on vascular tone, increased significantly. For the serotonin metabolite, no changes were observed for both contraceptives. The excretion of urodilatin significantly increased for both preparations after 12 cycles compared to the pretreatment values. These results indicate that the low-dose OCs Leios and Stediril 30 may stimulate the production of some vasoactive markers, at least after 12 cycles of treatment. The positive influence of these contraceptives on the various markers investigated may improve vascular tone, impede development of atherosclerosis and arterial thrombosis, and improve water and electrolyte homeostasis. These effects most likely can be attributed to the estrogenic component. Levonorgestrel may elicit no impact on these estrogen-induced changes that, however, seem only to be manifested after a longer treatment period.

Probenecid markedly reduces urinary excretion of ethinylestradiol and trimethoprim slightly reduces urinary excretion of clenbuterol.

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

Effects of drugs which influence renal transport systems on the urinary excretion of the beta 2-adrenoceptor agonist clenbuterol and the anabolic steroids ethinylestradiol and methyltestosterone.

The aim of this study was to determine whether the illegal application of clenbuterol, ethinylestradiol and methyltestosterone in cattle as growth promoters can be concealed by co-treatment with drugs that affect urinary excretion. Six male veal calves were fed with 0.8 micrograms clenbuterol kg-1 of body weight (BW), 3.5 micrograms ethinylestradiol kg-1 BW and 35 micrograms methyltestosterone kg-1 BW together twice daily for 28 days. At the eighth day of clenbuterol, ethinylestradiol and methyltestosterone treatment each calf was additionally fed either with probenecid, para-aminohippuric acid, trimethoprim, famotidine or cimetidine at three different doses which were increased in weekly intervals. During the treatment 24 h-urine and blood samples (once daily) were obtained and analysed for clenbuterol, ethinylestradiol and methyltestosterone by specific enzyme immunoassay. By high performance liquid chromatography/enzyme immunoassay it was determined whether these drugs or their metabolites interfered with the immunological detection of the growth promoters. Clenbuterol, ethinylestradiol and methyltestosterone could be detected in plasma and urine throughout the whole experiment. Co-treatment with probenecid led to a five-fold reduction in urinary excretion of ethinylestradiol and co-treatment with trimethoprim led to a three-fold reduction in urinary excretion of clenbuterol. None of the drugs reduced urinary excretion of the growth promoters to concentrations below the limit of detection. The detection of these three growth promoters in urine samples from calves which were co-treated with the drugs tested in this study can thus not be prevented.

Detection of the illegal use of ethinylestradiol in cattle urine by gas chromatography-mass spectrometry.

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

In vivo conversion of norethisterone to ethynyloestradiol in perimenopausal women.

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

Rapid and simple dansylation of phenolic steroids using a two-phase system and phase transfer catalysis.

Dansylation of phenolic steroids was carried out in chloroform-water and hexane-water two-phase systems with a tetrabutylammonium salt as phase transfer catalyst. Derivatization was complete within a few minutes on shaking at room temperature. Direct injection of part of the organic phase into a normal-phase liquid chromatography system was possible. The calibration graph of ethinyl estradiol, dansylated in a chloroform-water two-phase system, was linear over three orders of magnitude with a correlation coefficient of 0.993 (n = 8). The detection limit of dansylated ethinyl estradiol was 100 pg (signal-to-noise ratio = 2). The reproducibility of the derivatization at an analyte concentration of 200 ng/ml in chloroform was 4.1% (relative standard deviation; n = 5). A mechanism is proposed for the phase transfer catalysed dansylation of phenolic compounds.

Metabolic profiles of endogenous and ethynyl steroids in plasma and urine from women during administration of oral contraceptives.

Conjugated ethynyl and endogenous steroids in plasma and urine from two women taking an oral contraceptive (Conlumin) containing 1 mg norethindrone and 50 micrograms mestranol have been analyzed by methods based on anion and ligand exchange chromatography and gas chromatography-mass spectrometry. Conjugated norethindrone and its reduced metabolites with 3 alpha,5 alpha, 3 alpha,5 beta, 3 beta,5 beta and 3 beta,5 alpha configurations were identified in the fluids. The quantitatively major metabolites in plasma were a disulphate of the 3 alpha,5 alpha isomer and a monosulphate of the 3 alpha,5 beta isomer. The renal clearance of the former compound was low. The major urinary metabolite of norethindrone was the 3 alpha,5 beta isomer conjugated with glucuronic or sulphuric acid. Disulphates constituted only a small portion of urinary ethynyl steroids. Metabolic profiles of endogenous neutral steroids in plasma and urine during the contraceptive cycle were compared with profiles during a physiological menstrual cycle. The concentrations of steroids in plasma during contraception were similar to those during the follicular and mid phases of the menstrual cycle, whereas levels of progesterone metabolites were higher in the luteal phase. The urinary excretion of steroids was 15-30% lower during the contraceptive cycle, due to a decrease in excretion of C21O5 steroids, 11-oxygenated androgens and etiocholanolone. The increase of urinary progesterone metabolites seen during the luteal phase was not observed during contraception, but the excretion of 5 beta-pregnane-3 alpha,20 alpha-diol glucuronide was higher than during the follicular and mid phases of the menstrual cycle.

A comparative study of biliary and urinary 2-hydroxylated metabolites of [6,7-3H]17 alpha-ethynylestradiol in women.

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

Changes in estrogen metabolism after chronic oral contraceptive administration in the rhesus monkey.

The metabolism and elimination of estradiol (E2) and ethynylestradiol (EE2) were examined in adult female rhesus monkeys treated with two different combinational oral contraceptive agents at 10x the human equivalent dose. Ethynerone/mestranol (20:1), anagestone/mestranol (10:1), or vehicle was administered by gavage over a 10-year period on a cycling schedule of 21 days of dosing followed by 7 days without. At the end of the 28-day cycle, six monkeys in each group were anesthetized and administered a 14C-E2/3H-EE2 dose iv. Serial blood samples collected before and up to 6 hr after dosing were analyzed for total radioactivity and the percentage of parent compound and each metabolite was determined by HPLC. Radioimmunoassay of the baseline samples revealed that the plasma concentration of endogenous E2 was lower in the ethynerone/mestranol-treated group as compared to the vehicle control group. The total radioactivity derived from 14C-E2 was more rapidly eliminated from the plasma of the ethynerone/mestranol group than the control group. In addition, the percentages of HPLC-resolved E2 and EE2 were less in the treated group while the percentages of estrone, estrone glucuronide, and EE2 3-sulfate were enhanced as compared to the control group. The anagestone/mestranol group exhibited the same trends as the ethynerone/mestranol group but the data were not generally statistically different from the control group. These data indicate that chronically administered progestin/estrogen oral contraceptive agents reduce endogenous plasma E2 concentrations at least in part by enhancing the biotransformation of E2 to rapidly eliminated metabolites.

Pharmacokinetics and metabolism of moxestrol in animals--rat, dog and monkey.

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

The biliary and urinary metabolites of [3H]17 alpha-ethynylestradiol in women.

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

Ethynylestradiol-17 beta D-ring glucuronide conjugates are potent cholestatic agents in the rat.

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

Analysis of isomeric ethynylestradiol glucuronides in urine.

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