Two synthetic progestins and natural progesterone are responsible for most of the progestagenic activities in municipal wastewater treatment plant effluents in the Czech and Slovak republics.

Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and progesterone, which contributed 65-96% of such activity in samples where no antagonistic activity was found. The progestins medroxyprogesterone acetate, megestrol acetate and progesterone contributed most to the progestagenic activity detected in municipal effluents. Anti-progestagenic activities were found in some municipal effluents, but no causative agents were revealed because two analysed selective progesterone receptor modulators (SPRMs) with anti-progestagenic activities, mifepristone and ulipristal acetate, were not present in the effluents.

Direct coupling of packed column supercritical fluid chromatography to continuous corona discharge ion mobility spectrometry.

In this study, packed column supercritical fluid chromatography (SFC) was directly coupled to a continuous corona discharge (CD) ion mobility spectrometer (IMS) with several modifications. The main advantage of the developed detector is its capability to introduce full column effluent up to 2000 mL min(-1) CO(2) gas directly into the IMS cell relative to 40 mL min(-1) CO(2) gas as a maximum tolerance, reported for the previous IMS detectors. This achievement was made possible because of using corona discharge instead of (63)Ni as an ionization source and locating the inlet and outlet of the CO(2) gas in the counter electrode of the CD in opposite direction. In addition, a heated interface was placed between back pressure regulator (BPR) and the IMS cell to heat the output of the BPR for introducing sample as the gas phase into the IMS cell. Furthermore, a make-up methanol flow was introduced between the column outlet and BPR to provide a more uniform flow through the BPR and also to prevent freezing and deposition of the analytes in the BPR. The performance of the SFC-CD-IMS was evaluated by analysis of testosterone, medroxyprogesterone, caffeine, and theophylline as test compounds and figures of merit for these compounds have been calculated.

Development and validation of a subcritical fluid extraction and high performance liquid chromatography assay for medroxyprogesterone in aquatic products.

A simple, rapid and sensitive method was developed for the determination of medroxyprogesterone in aquatic products by extraction with subcritical 1,1,1,2-tetrafluoroethane (R134a) and high performance liquid chromatography (HPLC). A response surface methodology (RSM) was adopted to optimise extraction pressure, temperature and co-solvent volume. The optimum extraction conditions predicted within the experimental ranges were as follows: pressure, 3 MPa; temperature, 25 °C; and co-solvent volume, 6 ml. The analysis was carried out on Zorbax SB-C18 column (4.6 mm × 150 mm, 5 µm) with the mobile phase acetonitrile-water (55:45, v/v), flow rate 1.0 ml/min, temperature 30 °C and wavelength 240 nm. Good linearity of detection was obtained for medroxyprogesterone between concentrations of 50-250 ng/ml, r²=0.999. The method was validated using samples fortified with medroxyprogesterone at levels of 10, 30 and 50 ng/g, the mean recovery exceeds 90%, and the RSD values were less than 10%.

Enhancing sensitivity and precision on NIR reflectance determination of an API at low concentration: Application to an hormonal preparation.

The use of a mixed calibration sample set (intact production tablets and powdered doped samples used to enlarge calibration range) is a usual procedure for the NIR reflectance determination of the API content of a pharmaceutical solid preparation. However, the high difference in scattering properties and the intrinsic low sensitivity of NIR make difficult the achievement of a good precision when API is at a low mass proportion (≈1%, w/w). The compression of the calibration powdered samples has been studied as a very simple procedure to enhance the sensitivity of NIR reflectance measurements and, consequently, to improve precision. Different pretreatments (SNV, 1D, 2D and their combinations) have been applied to reduce the spectral difference between powdered and compressed samples. Although none eliminates completely this difference, the combined pretreatment SNV+2D has proved to be the one with a better performance. Results obtained by using both calibration sample sets (powdered and compacted) in the quantification of estradiol valerate (VE, 2mg/tablet, ≈1.6%, w/w) and medroxyprogesterone (MPA, 10mg, ≈8%, w/w) in intact tablets of the hormonal preparation show that a slight but significant improvement in precision is obtained when using compacted samples for calibration. A HPLC procedure was developed to be used as reference method.

[Study on simultaneous determination method for banded synthesis hormones residues in egg products].

OBJECTIVE: To develop method of 7 banded synthesis sex hormones residues in egg products determined by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). METHODS: The sample were enzymolied and target compounds were extracted with methanol. ZnCl2 was added to the extract solution to remove lipid and then analytes were purified by LC-C18 and LC-NH2 solid phase extraction cartridge and further determined by UPLC-MS/MS under positive ionization and multiple reaction monitoring (MRM) modes. RESULTS: The limits of detection (LOD) of UPLC-MS/MS method used for testing Chlormadione acetate (CDA), Medroxypogesterone acetate (MPA), Megestrol Acetate (MA), Testosterone propionate (TSP), Norgestrel (NG), Methyltestosterone (MTS) and Nandrolone (NT) in egg products ranged from 0.012 to 0.23 microg/kg, and the limits of quantification (LOQ) were from 0.04 to 0.76 microg/kg. Experiments on spiked samples of egg products showed that at addition level of 2.0 microg/kg, the average recoveries of the sex hormones ranged from 80.2% to 114%, and coefficients of variation from 6.7% to 14.3%; while at addition level of 4.0 microg/kg, the average recoveries ranged from 75% to 119%, and coefficient of variation from 2.9% to 7.3%. CONCLUSION: The method could be able to identify and quantify banded synthesis hormones residues in eggs and egg products. It could be simple and sensitive, suitable for statutory residue testing.

A stability-indicating HPLC method for medroxyprogesterone acetate in bulk drug and injection formulation.

A stability-indicating HPLC assay method has been developed and validated for medroxyprogesterone acetate (MPA) in bulk drug and injectable suspension. An isocratic RP-HPLC was achieved on a Hichrom C(18) column (150 mm x 4.6mm i.d., 5 microm) utilizing a mobile phase of methanol 0.020 M acetate buffer pH 5 (65:35, v/v) and a photodiode array detector at 245 nm. The stress testing of MPA was carried out under acidic and alkaline hydrolysis, and oxidation conditions. MPA was well resolved from its degradation products, a main related substance (megestrol acetate) and two preservatives (methyl paraben and propyl paraben) with the resolution >or=2. The proposed method was validated for selectivity, linearity, accuracy, precision and solution stability. The method was found to be suitable for the quality control of MPA in bulk drug and injections as well as the stability-indicating studies.

In vitro dissolution profile of water-insoluble drug dosage forms in the presence of surfactants.

The determination of the in vitro release profile of water-insoluble drug products requires dissolution media different from those used for water-soluble drug products. Since the relevance of drug dissolution in organic solvents is questionable, we investigated the use of surfactants to determine the dissolution profiles of water-insoluble drug products. In most cases, the drug dissolution rate and extent increased as the surfactant concentration in the aqueous dissolution medium increased. Suitable dissolution profiles were obtained in the presence of sodium lauryl sulfate (SLS) for water-insoluble drug products, such as griseofulvin, carbamazepine, clofibrate, medroxyprogesterone, and cortisone acetate. These findings recommend the use of surfactants for determining the aqueous dissolution of water-insoluble drug products rather than adding organic solvents to the dissolution medium.

Control of illegal medroxyprogesterone acetate-application in veal calves by residue analysis in adipose tissue using HPLC/RIA methods.

Procedures for the determination of medroxyprogesterone acetate (MPA) in adipose tissue collected at time of slaughter allowing the control of MPA application to veal calves are described. Screening radioimmunoassays after sample clean-up were sufficient for a first survey of MPA treatments in livestock herds. Validation of all positive samples was performed by two-dimensional (silica gel diol phase and RP-18 phase) HPLC/RIA immunograms. Megestrol acetate and melengestrol acetate with cross-reactivities of 31% and 0.3% respectively were clearly separated by the RP HPLC. With an absolute detection limit of 4 pg MPA/tube (90% relative binding) negative control samples did not exceed 6 pg/tube, equivalent to 6 pg/g fat in the validating method. Seventeen days after intramuscular (i.m.) injection of 24 mg MPA only 32 pg MPA/g fat were found, while i.m. injection of 60 mg MPA and a waiting period of 19 days resulted in 2700 pg MPA/g fat. After feeding two calves 20 micrograms MPA per head daily for 1 week followed by 200 micrograms MPA per head daily for 2 weeks 359 and 468 pg MPA/g fat were measured. In plasma as well as in adipose tissue more than 80% of the whole immunoreactive material was MPA itself, without indications for the presence of cross-reacting MPA metabolites as confirmed by HPLC/RIA immunograms. Based on day of slaughter ratios of accumulation of MPA from plasma into fat of MPA-fed veal calves were 52 and 72 respectively. In urine MPA was only detectable a few days after injection; as compared to a plasma concentration of 950 pg MPA/ml the amount in urine was only 37 pg MPA/ml and also 325 pg unidentified MPA-equivalents/ml.

Liquid chromatographic determination of medroxyprogesterone acetate in tablets.

A reverse-phase liquid chromatographic method is described for the assay of medroxyprogesterone acetate in tablets. An octadecylsilane (C18) column with a mobile phase of methanol-0.01M dibasic ammonium phosphate (80 + 20 v/v, pH 7.2 +/- 0.1) and photometric detection at 254 nm separates medroxyprogesterone acetate from excipients. Detector responses were linear to concentrations of medroxyprogesterone acetate over the range 50-150 micrograms/mL (r = 0.999). Mean recovery of medroxyprogesterone acetate added to tablet excipients was 100.8%. Mean assay results were 101.3% (n = 3). The assay results are comparable to those obtained by the compendial liquid chromatographic method.

Medroxyprogesterone acetate- and ethinylestradiol-induced changes in biliary bile acids of the rat studied by high-performance liquid chromatography.

The effects of subcutaneous administration (5 mg/kg per day) for seven days of medroxyprogesterone acetate (MPA) or 17 alpha-ethinylestradiol (EE) on bile flow, total bile acid output and individual biliary acids have been studied in adult male Wistar rats. Biliary bile acid composition was quantitated by a simple isocratic high-performance liquid chromatographic technique using a C18 reversed-phase radial compression column and refractive index detection. This method revealed that muricholic acids, analysed as taurine and glycine conjugates, constituted a higher proportion of biliary bile acids in the rat than previously observed with gas chromatographic techniques. Marked cholestasis was produced by EE while MPA had little effect on bile flow or total bile acid output. Despite this, both steroids significantly increased the proportion of taurine-conjugated muricholic acids relative to taurocholic acid, although the estrogen had the more pronounced effect. Further study of the hepatobiliary consequences of high doses of MPA would seem warranted in view of the important use of this progestogen for cancer therapy.

[Anabolic steroid residues in administration sites in slaughtering cattle. October 1983 - January 1985]. / Residuen van anabolica in toedieningsplaatsen bij slachtrunderen. Periode oktober 1983-januari 1985.

Samples from 573 sites of injection or implantation in carcasses of slaughtered cattle and veal calves in the Netherlands were studied by high performance liquid chromatography associated with on-line detection of the total UV spectrum (diode array detector). Anabolic substances were identified in 462 (100%) out of 573 samples. Nortestosterone was most frequently detected (84%) during the period from October 1983 to January 1985. Other xenobiotic steroids observed were medroxyprogesterone (14%), methyltestosterone (2%) and trenbolone (2%). The 'natural' steroids testosterone, oestradiol and progesterone were found in 29%, 66% and 3% of the samples respectively. The stilbene derivatives diethylstilboestrol (DES), dienestrol and hexoestrol were found in 2%, 3% and 0% of the samples. After re-introduction of DES control in the urine of cattle early in 1984, DES residues were no longer detected in the samples from sites of administration. Zeranol was not observed at all. If applicable, all anabolic agents were found to be present in a variety of esterified dosage forms.

Monitoring and identification of residues of anabolic preparations in slaughtered cattle by HPLC with diode array detection.

The new combination of isocratic high performance liquid chromatography (HPLC) with on line UV spectrum detection via a diode array configuration has been applied to the detection and identification of anabolics present in application sites of cattle. Combination of the characteristic retention time in the HPLC chromatogram and a comparison of the full spectrum between 190-400 nm of the anabolic components with that of a standard resulted in a very reliable identification. By means of this method 117 samples of application sites were investigated for the presence of anabolic residues. Of the xenobiotic anabolics , 19-nortestosterone (NT) was found most frequently (in 96 cases), whereas diethylstilbestrol (DES) was found in only 11 cases. In all samples the identification of NT and DES was confirmed by high resolution gas chromatography-mass spectrometry (GCMS).

Quantitation of hydroxyprogesterone caproate, medroxyprogesterone acetate, and progesterone by reversed-phase high-pressure liquid chromatography.

A high-pressure liquid chromatography method for the quantitation of hydroxyprogesterone caproate, medroxyprogesterone acetate, and progesterone in pharmaceutical dosage forms was developed. The method gave accurate, precise, and reproducible results. The excipients present in the dosage forms did not interfere with the assay procedure except benzyl benzoate in progesterone injection. The percent relative standard deviations based on six injections were 1.6, 2.5, 2.7% for hydroxyprogesterone caproate, medroxyprogesterone acetate, and progesterone, respectively. The stability of progesterone in ethanol--propylene glycol--water (10:50:40) was studied. The loss in potency of progesterone, even after 487 days of storage at 50 degrees, was less than 10%.

Hormone-releasing silastic intrauterine devices: effect or provera-releasing intrauterine devices on two species of primates.

Silastic intrauterine contraceptive devices (IUDs) 13 mm long and 1.07 mm in diameter could be inserted easily into patas monkey uteri which, like human uteri, expelled them. Addition of 10% Provera to these devices did not reduce the expulsion rate significantly in our study. Control devices had no effect on cycle length in rhesus monkeys. After insertion, the active IUDs frequently caused a delay in onset of menstruation; however, cycles did occur with the device in situ, and normal-length cycles were resumed following removal of the device. A short period of rapid release (almost 35% of the total amount) of Provera from the device was followed by a longer period of sustained release of low levels of the hormone. Even 9 mug/day were sufficient to maintain a decidual reaction in the endometrium of the rhesus monkey. The drug could not be detected in the blood stream at 3,6, or 12 hours in patas monkeys or at 1 or 2 months in rhesus monkeys and so may never have reached the systemic circulation. Devices currently under study in baboons catain Provera or one of three other steroids to determine whether these compounds improve retention rates as well as meet the other two criteria set for the ideal IUD incorporation, for unless we meet this first criterion we can never achieve, let alone test, the others.

Induced prolactin release in women under long-term medroxyprogesterone acetate treatment.

Medroxyprogesterone acetate (MPA) was administered, as a contraceptive, by intramuscular injection in doses of 150 mg every 90 days, or 300-450 mg every 180 days, to 5 women, over a period of 27 to 42 months. No major adverse reactions were reported, and the minor side effects were only as frequent as those reported for oral contraceptives. The LH basal levels were depressed in all subjects;; FSH and prolactin serum concentrations were within the range found in normally cycling women. Synthetic TRH released prolactin in 4 subjects, and the maximal response was within 10-20 minutes after TRH injection. In the fifth case there was no prolactin release.