Sorption of levonorgestrel on polyethylene, polystyrene and polypropylene microplastics.

Sorption studies involving microplastics (MPs) are essential to understand the mechanisms implicated in contaminant retention. In this research, a complete study of the sorption behaviour of a hormonal contraceptive -levonorgestrel- in MPs of different composition in two distinct matrices was performed, using high-performance liquid chromatography coupled to a UV detector for the determination of levonorgestrel. Characterization of the studied MPs was achieved by X-ray diffraction, differential scanning calorimetry, and Fourier-transformed infrared spectroscopy. Kinetic and isotherm studies were performed using a batch design under controlled conditions: 500 mg of MPs pellets of 3-5 mm diameter, agitation at 125 rpm, and 30 °C. The comparison of results in ultrapure water and artificial seawater, revealed changes in sorption capacity, and the predominant sorption mechanisms involved. Overall, all studied MPs showed sorption affinity towards levonorgestrel, being low-density polyethylene the one with the highest sorption capacity in ultrapure water and polystyrene in seawater.

Effects of the Gestagen Levonorgestrel in a Life Cycle Test with Zebrafish (Danio rerio).

The amount of pharmaceuticals transferred to the aquatic environment via municipal and hospital waste water is steadily increasing. The progress in medical research has resulted in the manufacture of active substances of increased stability, specificity, and potency, which can trigger adverse effects in aquatic organisms. Moreover, advanced analytical methods allow the detection of pharmaceuticals in environmental matrices at very low concentrations, which increases the number of substances to be assessed. Levonorgestrel is a synthetic gestagen commonly used in medicinal products for contraception. Because progestogenic compounds could have an impact on fish maturation processes, a life cycle test was performed to assess the effects of levonorgestrel exposure of the embryonic to the adult stages of zebrafish (Danio rerio) at mean measured concentrations of 0.06, 0.16, 0.47, 1.64, and 5.45 ng/L. Apical endpoints were survival, growth, reproduction, and sex ratio. Determination of endocrine modulation was completed by measurement of vitellogenin and 11-keto testosterone in blood plasma, as well as by histopathological analysis of gonads. For all parameters, control values were within the recommended quality range. The most prominent levonorgestrel effect was a shift toward an increased number of male fish at 1.64 and especially 5.45 ng/L, at which point all fish were histologically determined to be males and no spawning occurred; 11-keto testosterone was significantly decreased. A no-observed-effect concentration (NOEC) of 0.47 ng levonorgestrel/L was confirmed by the fertilization capability of adult fish, the male maturation stages, and female gonad histopathology. Whereas hatch and juvenile growth were not affected, posthatch survival was significantly impeded at ≥0.47 ng levonorgestrel/L, although it was not clearly related to the test concentration. For male length and weight, the same NOEC of 0.16 ng/L was obtained at study termination. Environ Toxicol Chem 2022;41:580-591. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

RP-HPLC method validation for fast extraction and quantification of Levonorgestrel drug from silicone based intrauterine device intended for in-process and finished formulation.

BACKGROUND: To develop and validate a simple and consistent reversed phase high performance liquid chromatography (RP-HPLC) method for the estimation of Levonorgestrel (LNG) drug from silicone based intrauterine device. METHODS: Sample solution was prepared using tetrahydrofuran (THF) as solvent for the drug extraction, and RP-HPLC analysis was performed using Luna C18 analytical column (150 × 4.6 mm, 5 µm, 100 Å - Phenomenex), with a mobile phase consisting of a mixture of acetonitrile and water (50:50, v/v) at a flow rate of 1.0 ml/min and injection volume of 20 µl. Detection was carried out at 241 nm in PDA detector, with a total run time of 15 min. The method was validated in accordance with ICH guidelines. Method applicability was tested for optimizing formulation using quality-by-design approach, to check the stability and content uniformity of levonorgestrel-silicone mixture (core blend), and quantifying the amount of LNG from commercially available silicone based formulation. RESULTS: The retention time for LNG drug was obtained at 8.5 min (± 0.3 min). A linear relationship was observed over the concentration range of 2.6-15.6 µg/ml with the correlation coefficient (r) value 0.9999. The method was found to be precise within the acceptable limit (RSD < 2%) and the drug recovery from the intrauterine device was found in the range 99.78-100.0%. Content uniformity for different prototypes developed was observed in the range of 91.6-101.4%, and assay of optimized core blend was in the range of 97.78-106.79% during the 10 days of retention period for stability studies. CONCLUSION: The validated method is found to be a simple, accurate, precise, reproducible, and hence can be used for the routine analysis of LNG such as in-process, quality control and stability assays of silicone based intrauterine devices by RP-HPLC.

A New Strategy for the Analysis of Steroid Hormones in Industrial Wastewaters by Paper Spray Ionization Mass Spectrometry.

A new approach using paper spray ionization mass spectrometry (PSI-MS) for the analysis of steroid hormones in wastewater samples has been demonstrated. Triangular papers containing paraffin barriers as microfluidic channels were used to direct the sample solution to the paper tip, preventing the sample from spreading over the corners of the paper. The method was used to analyze the hormones levonorgestrel and algestone acetophenide in industrial wastewaters. Analytical curves presented a correlation coefficient (R2) above 0.99. Limits of quantification were below 2.3 ppm and limits of detection below 0.7 ppm. Values of precision (coefficient of variation) and accuracy (relative error) were less than 15% for all analyses. Recovery results ranged from 82% to 102%. Levonorgestrel was also analyzed by high-performance liquid chromatography coupled to mass spectrometry in order to compare the analytical performance with PSI-MS. No statistically significant differences were found between both methods. This study demonstrates the usefulness of PSI-MS for rapid analysis of hormones in industrial wastewater samples and also indicates its potential to be employed as a simple and reliable analytical method in environmental sciences.

Breast levonorgestrel concentrations in women using a levonorgestrel-releasing intrauterine system.

OBJECTIVE: To measure breast tissue and serum LNG concentrations in women using a LNG-IUS. STUDY DESIGN: This pilot study was performed in 25 healthy women undergoing breast surgery at the Ghent University hospital. LNG concentrations were measured in serum and microdissected breast tissue samples using a validated ultra-performance liquid chromatography/tandem mass spectrometry assay. RESULT(S): The mean LNG concentration in the 18 LNG-IUS users was 0.18±0.16 ng/mL in serum and 0.26±0.28 ng/g in breast tissue. For four women without any form of hormonal contraceptive (the negative controls), the mean concentrations were below the limit of quantification, i.e., 0.15 ng/mL and 0.20 ng/g, for serum and breast tissue, respectively. For the three positive controls the concentrations in the serum (20.5 and 3.4 ng/ml) and the breast (3.74 and 1.24 ng/g) were respectively for the 20 µg EE/100 µg users and 315 pg/ml in the serum and 1.17 ng/g in the breast for the minipill user. The intracellular free fraction of LNG may be as low as 0.008 ng/g. CONCLUSION(S): The concentration of LNG in breast epithelium cells in women using the LNG-IUS is very low. IMPLICATIONS: The relationship between the serum and breast tissue levels of LNG was studied in women using a LNG-IUS or oral LNG-containing contraception. Compared to oral contraception, the tissue levels of LNG in LNG-IUS users are much lower in the breast. It is not known what level of LNG exposure in the breast would stimulate RANKL and WNT4 expression; such information is needed.

A sensitive method detecting trace levels of levonorgestrel using LC-HRMS.

OBJECTIVE: To develop a high resolution mass spectrometry (HRMS) method to quantify levonorgestrel (LNG) in serum. STUDY DESIGN: Levonorgestrel was extracted using solid phase extraction and measured using liquid chromatography (LC) HRMS. RESULTS: Low limit of quantification (LLOQ) was 25 pg/mL and low limit of detection (LLOD) was 12.5 pg/mL. Precision and accuracy bias were <10%. LNG in serum samples from Mirena® users ranged between 37 to 219 pg/mL (n=12). In eight out of 22 patients with suspected intrauterine device (IUD) expulsion LNG was detected (26-1272 pg/mL). CONCLUSION: A sensitive, fast and simple LC-HRMS method was developed to detect trace levels of LNG.

Electrospun Transdermal Patch for Contraceptive Hormone Delivery.

BACKGROUND: A transdermal patch for delivery of Levonorgestrel (LNG) can be used for long-acting contraception. OBJECTIVE: In this study, we designed and characterized a patch made of nonwoven electrospun microfibers comprised of Polycaprolactone (PCL) encapsulating LNG for slow release in a mineral oil matrix. METHODS AND RESULTS: Scanning electron microscopy showed uniform, randomly oriented PCL fibers with large interconnected voids filled with mineral oil. Thermogravimetric analysis indicated that LNG loaded into PCL fibers had thermal stability up to ~200°C. Differential Scanning Calorimetry suggested that LNG was dispersed in the electrospun fibers without interaction between the LNG and PCL, and without formation of drug crystals. Fourier Transform Infrared spectroscopy and X-ray diffraction results further supported the conclusion that there was no chemical drug-polymer interaction in LNGloaded fibers. Effective in vitro flux (i) from patches into mineral oil was 1.9 µgcm-2h-1, (ii) from mineral oil across porcine skin was 4.6 µgcm-2h-1 and (iii) from patches across porcine skin was 1.7 µgcm- 2h-1, indicating that transdermal delivery rate was controlled by a combination of the patch and the skin. CONCLUSION: The LNG-loaded patches demonstrated steady delivery of LNG across skin for up to 5 days in vitro. With additional development, LNG-loaded electrospun PCL patches could be used for long-acting contraception.

Transcriptomic and physiological changes in Eastern Mosquitofish (Gambusia holbrooki) after exposure to progestins and anti-progestagens.

Endocrine active compounds (EACs) remain an important group of chemicals that require additional evaluation to determine their environmental impacts. While estrogens and androgens were previously demonstrated to impact organisms during environmental exposures, progestagens have recently been shown to have strong impacts on aquatic organisms. To gain an understanding of the impacts of these types of chemicals on aquatic species, experiments evaluating the mechanisms of action of progestagen exposure were conducted with the Eastern Mosquitofish (Gambusia holbrooki). The objective of this study was to conduct hepatic microarray analysis of male and female G. holbrooki exposed to progestins and anti-progestagens. In addition, we evaluated the ability of levonorgestrel, a synthetic progesterone (progestin), to induce anal fin elongation and to determine how anal fin growth is modulated during co-exposures with progesterone and androgen receptor antagonists. Gene expression analyses were conducted on male and female G. holbrooki exposed for 48h to the agonist levonorgestrel, the antagonist mifepristone, or a mixture of the two chemicals. Microarray analysis revealed that mifepristone does not act as an anti-progestagen in G. holbrooki in liver tissues, and that levonorgestrel elicits strong effects on the processes of embryo development and lipid transport. Levonorgestrel was also demonstrated to induce male secondary sexual characteristic formation in females, and co-exposure of either an androgen or levonorgestrel in the presence of the anti-androgen flutamide prevented anal fin elongation. These results provide indications as to the potential impacts of progestins, including non-target effects such as secondary sexual characteristic formation, and demonstrate the importance of this class of chemicals on aquatic organisms.

Concentrations of levonorgestrel and ethinylestradiol in wastewater effluents: Is the progestin also cause for concern?

Synthetic hormones have been widely reported in treated sewage effluents, and consequently receiving aquatic environments. Ethinylestradiol (EE2) is a potent synthetic estrogen commonly used in conjunction with levonorgestrel in oral contraceptive pills. Both EE2 and levonorgestrel have been identified in the aquatic environment, but although there is a significant amount of literature on EE2, there is much less information on levonorgestrel. Using Australian prescription data as well as excretion and predicted wastewater removal rates, the concentrations of EE2 and levonorgestrel in Australian wastewater were calculated at 0.1 ng/L to 0.5 ng/L and 0.2 ng/L to 0.6 ng/L, respectively. Both compounds were analyzed in treated wastewater and surface water grab samples from 3 Southeast Queensland, Australia sites. The predicted no-effect concentration (PNEC) for EE2 of 0.1 ng/L was exceeded at most sites, with EE2 concentrations up to 2 ng/L in treated effluent, albeit quickly diluted to 0.1 ng/L to 0.2 ng/L in the receiving environment. A provisional PNEC for levonorgestrel of 0.1 ng/L derived in the present study was slightly lower than predicted effluent concentrations of 0.2 ng/L to 0.6 ng/L, indicating a potential risk of endocrine-related effects in exposed aquatic species. The detection limit for levonorgestrel in the present study was 2.5 ng/L, and all samples were below detection limit. The present study's results suggest that improvements in analytical capabilities for levonorgestrel are warranted to more accurately quantify the risk of this compound in the receiving environment. Environ Toxicol Chem 2016;35:1378-1385. © 2015 SETAC.

HPLC-MS/MS analysis of steroid hormones in environmental water samples.

Today, freshwaters, such as lakes and rivers, are subject to controlled pollution. Steroid hormones are chemically very stable highly lipophilic molecules. Their biological properties have a strong impact on the endocrine regulation of species. Steroids have estrogenic, androgenic, thyroidogenic or progestogenic effects and based on them, they could disturb the physiological mechanisms of freshwater species. We focused on progestins as they are the main active ingredients of contraceptive pharmaceuticals. Progestins have been shown to impair reproduction in fish, amphibians, and mollusks at low ng/L concentrations. Certain progestins, such as levonorgestrel (LNG) have androgenic properties also. We selected the most used active substances drospirenone (DRO), LNG, and progesterone (PRG) and then developed and optimized a liquid chromatographic-mass spectrometric method with solid-phase extraction to measure them. Using our sensitive method (LOQ 0.03-0.11 ng/L) we could measure steroids even between 0.1 and 1 ng/L. Analyzing freshwater samples from the Lake Balaton catchment area, we found influents where the concentration of these hormones was 0.26-4.30 (DRO), 0.85-3.40 (LNG), and 0.23-13.67 (PRG) ng/L. Out of 53 collecting places, 21 contained measurable progestin levels, which clearly demonstrates the applicability of our method, legitimates toxicology experiments with effected species, and indicates monitoring efforts.

The synthetic progestin levonorgestrel is a potent androgen in the three-spined stickleback (Gasterosteus aculeatus).

The use of progestins has resulted in contamination of aquatic environments and some progestins have in experimental studies been shown to impair reproduction in fish and amphibians at low ng L(-1) concentrations. The mechanisms underlying their reproductive toxicity are largely unknown. Some progestins, such as levonorgestrel (LNG), exert androgenic effects in mammals by activating the androgen receptor (AR). Male three-spined stickleback (Gasterosteus aculeatus) kidneys produce spiggin, a gluelike glycoprotein used in nest building, and its production is directly governed by androgens. Spiggin is normally absent in females but its production in female kidneys can be induced by AR agonists. Spiggin serves as the best known biomarker for androgens in fish. We exposed adult female sticklebacks to LNG at 5.5, 40, and 358 ng L(-1) for 21 days. Androgenic effects were found at LNG concentrations ≥40 ng L(-1) including induction of spiggin transcription, kidney hypertrophy, and suppressed liver vitellogenin transcription. These are the first in vivo quantitative data showing that LNG is a potent androgen in fish supporting the contention that androgenic effects of certain progestins contribute to their reproductive toxicity.

An ultra-sensitive online SPE-LC-MS/MS method for the quantification of levonorgestrel released from intrauterine devices.

A selective and sensitive liquid chromatography-tandem mass spectrometry method for the determination of very low levonorgestrel (D-(-)-norgestrel) serum levels such as those found in patients using levonorgestrel-releasing intrauterine devices (IUDs) was developed. To achieve the sub-nanomolar sensitivity needed to measure such serum levels, a diethyl ether extraction sample preparation protocol was applied prior to the online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) assay. Analyte quantification from the selected reaction monitoring experiments relied on the use of sixfold deuterated norgestrel as internal standard. The final method was linear up to 1.50 ng/ml with a lower limit of quantification (LLOQ) of 0.05 ng/ml. It was found to be precise and accurate with imprecision <8% and bias <6% assessed at three control levels. Total analyte recovery measured in patient pools at three concentration levels was found to exceed 92%. Matrix interferences were excluded by post-column analyte infusion experiments. As a proof of concept, a set of IUD patient serum samples was screened for their levonorgestrel content. A total of 97.5% (n = 94) of the samples did show serum levels exceeding the LLOQ, proving the applicability of the assay in relevant clinical cohorts. This method must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the in vitro diagnostic directive (98/79/EG) of the European community.

A study of some practical aspects of high temperature liquid chromatography in pharmaceutical applications.

In the pharmaceutical industry fast and efficient separation techniques play an increasing role among analytical methods because the samples to be investigated grow both in complexity and number, and there is an increasing time pressure to complete the analysis. Reducing the analysis time without decreasing the efficiency is possible using higher pressures, elevated temperatures, smaller particle sizes, or a combination of these approaches. Recently developed chromatographic techniques such as the UHPLC (ultra high performance liquid chromatography) and HTLC (high temperature liquid chromatography) are highly promising in meeting these demands. In this study, high temperature liquid chromatography (HTLC) with a zirconia-based column and temperatures elevated up to 150°C was used. We investigated the chromatographic behaviour of a steroid active pharmaceutical ingredient (levonorgestrel) and its structurally related impurities as model compounds. The effect of the temperature in the range of 50-150°C and the flow-rate in the range of 0.5-3.0 ml/min, and using methanol as an organic modifier, were studied for optimisation of the separation method.

Monitoring of progestins: development of immunochemical methods for purification and detection of levonorgestrel.

A polyclonal antibody (Ab) for the progestin levonorgestrel (LNG) was generated, and immunochemical assays for its detection, clean-up and concentration were developed. A highly specific microplate diagnostic assay for the detection of LNG was developed that used the enzyme linked immunosorbent assay (ELISA) method. The LNG ELISA developed was sensitive and reproducible; it exhibited I(50) and I(20) values of 3.3+/-1.8 ng mL(-1) and 0.6+/-0.4 ng mL(-1), respectively, and the Abs did not cross react with any of the tested steroid hormones. The above Abs were used to develop a sol-gel-based immunoaffinity purification (IAP) method for concentration and clean-up of LNG that is compatible with subsequent immunochemical or instrumental chemical analytical procedures, such as liquid chromatography followed by mass spectrometry (LC-MS/MS). Development of the columns included successful entrapment of Abs within a tetramethoxysilane (TMOS)-based SiO(2) polymer network. The Abs could bind the free analyte from solution, and the bound analyte could be easily eluted from the sol-gel matrix at high recoveries. The Ab selectivity towards the antigen was high, in both ELISA and the sol-gel columns, but the entrapped Abs cross-reacted with two other steroid hormones--ethynylestradiol (EE2) and nortestosterone (NT) - which share similar epitopes with LNG, despite the lack of cross reactivity in the ELISA. The validity of the method was investigated by LC-MS/MS and a good analytical correlation was obtained.

Preparation and characterization of an immunoaffinity chromatography column for the selective extraction of trace contraceptive drug levonorgestrel from water samples.

The preparation and characterization of an immunoaffinity chromatography (IAC) column for the specific extraction and enrichment of trace contraceptive drug levonorgestrel (LNG) from water samples were described. The IAC column was constructed by covalently coupling specific polyclonal antibody against LNG to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions, as well as the effect of flow rate on the extraction were carefully optimized. Pure water, 5% of methanol and 50% of methanol were respectively selected as loading, washing and eluting solutions, while the flow rates in the loading, washing and eluting steps were selected to be 1.0, 2.0 and 0.5 mL min(-1), respectively. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, extraction recovery and stability. It was found that, for IAC column packed with 0.2g of solid support immobilized with antibody, the maximum capacity for LNG was about 260 ng. The extraction recoveries of the column for LNG at three different spiked concentrations were within 95.3-106.9%. After more than 35 times repeated usage, there was not significant loss of specific recognition. Using high performance liquid chromatography (HPLC) as an analytical tool, trace amount of LNG in the range of ng L(-1) was found in river water and wastewater samples after 600-fold enrichment, demonstrated the feasibility of the prepared IAC column for LNG extraction.

Characterization of new types of stationary phases for fast liquid chromatographic applications.

The performance of a narrow bore silica based monolith column (5 cm x 2 mm) was compared to 5 cm long narrow bore (internal diameter < or = 2.1 mm) columns, packed with shell particles (2.7 microm) and totally porous sub-2 microm particles (1.5 microm, 1.7 microm and 1.9 microm) in gradient and isocratic elution separations of steroids. The highest peak capacity could be achieved with the column packed with 1.5 microm totally porous particles. The columns packed with porous 1.7 microm and shell 2.7 microm particles showed very similar capacity. The monolith column provided the lowest capacity during gradient elution. The plate height (HETP) of the 2.7 microm Ascentis Express column was very similar to the HETP obtained with 1.5 microm and 1.7 microm totally porous particles. The Chromolith monolithic column displayed an efficiency that is comparable to that of columns packed with spherical particles having their diameter between 3 microm and 4 microm. A kinetic plot analysis is presented to compare the theoretical analysis speed of different separation media. At 200 bar, the monolith column provided the highest performance when the required plate number was higher than 5000 (N>5000), however the efficiency drifted off faster in the range of N<5000 than in the case of packed columns. If the possibility of maximum performance was utilized (1000 bar for sub-2 microm particles, 600 bar for shell particles and 200 bar for monolith column) the monolith column would provide the poorest efficiency, while the column, packed with 1.5 microm particles offered the shortest impedance time.

Effects of synthetic gestagens on fish reproduction.

Although it is well known that estrogenic steroidal hormones are able to affect the sexual development and reproduction of fish at low concentrations, no data on environmental effects of the class of progestogenic hormones are available yet. Synthetic gestagens (progestins) are a component in oral contraceptives. Upon their use, a fraction of the progestins will be excreted via urine into the aquatic environment. On the basis of their pharmacological action in mammals, it is supposed that fish reproduction is the most sensitive endpoint for the progestin treatment. In order to test this assumption, the effects of two progestins currently marketed in contraceptive formulations, levonorgestrel (LNG) and drospirenone (DRSP), were investigated in adult fathead minnows (Pimephales promelas) following an Organization for Economic Cooperation and Development 21-d fish reproduction screening assay draft protocol with additional end points. Levonorgestrel was tested at measured concentrations of 0.8, 3.3, and 29.6 ng/L, and DRSP at concentrations of 0.66, 6.5, and 70 microg/L. Both tested progestins caused an inhibition of reproduction. For LNG, this occurred at concentrations of >or=0.8 ng/L, no no-observed-effect concentration (NOEC) could be defined. Higher concentrations resulted in masculinization of females with de novo synthesis of nuptial tubercles. Drospirenone treatment, however, affected the reproductive success of fathead minnow at concentrations of 6.5 microg/L and higher with a clear dose-response relationship and a NOEC of 0.66 microg/L, which is above environmentally relevant concentrations.

Simultaneous determination of mifepristone and monodemethyl-mifepristone in human plasma by liquid chromatography-tandem mass spectrometry method using levonorgestrel as an internal standard: application to a pharmacokinetic study.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine mifepristone and monodemethyl-mifepristone in human plasma using levonorgestrel as the internal standard (IS). After solid-phase extraction of the plasma samples, mifepristone, monodemethyl-mifepristone and the IS were subjected to LC-MS/MS analysis using electro-spray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an XTERRA MS C(18) column (150 x 2.1 mm i.d., 5 microm). The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration ranges of 5-2000 ng/mL for mifepristone and monodemethyl-mifepristone. The recoveries of the method were found to be 94.5-103.7% for mifepristone and 70.7-77.3% for monodemethyl-mifepristone. The method had a lower limit of quantification (LLOQ) of 5.0 ng/mL and a lower limit of detection (LOD) of 1.0 ng/mL for both mifepristone and monodemethyl-mifepristone. The intra- and inter-batch precision was less than 15% for all quality control samples at concentrations of 10, 100 and 1000 ng/mL. These results indicate that the method was efficient with a short run time (4.5 min) and acceptable accuracy, precision and sensitivity. The validated LC-MS/MS method was successfully used in a pharmacokinetic study in healthy female volunteers after oral administration of 25 mg mifepristone tablet.

High performance liquid chromatography/ion-trap mass spectrometry for separation and simultaneous determination of ethynylestradiol, gestodene, levonorgestrel, cyproterone acetate and desogestrel.

A fast and highly sensitive high performance liquid chromatographic/ion-trap mass spectrometric method (LC/MS) has been developed for simultaneous determination of ethynylestradiol (EE2), gestodene (GES), levonorgestrel (LNG), cyproterone acetate (CPA) and desogestrel (DES). Among three types of sorbents tested (C8, C18 and phenyl) from two suppliers, the best separation was achieved on reverse phase Zorbax SB-Phenyl column using aqueous methanol as a mobile phase. A linear gradient profile from 70 up to 100% (v/v) in 7th min, kept constant at 100% up to 10th min and followed by a negative gradient to 70% of methanol up to 12th min was used for elution. Applicability of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) and influence of the mobile phase composition, its flow rate, capillary/vaporizer temperature of API source and in-source fragmentor voltage ionization are discussed. The on-column limits of quantification (10S/N) were 300 pg of EE2, 14 pg of GES and LNG, 4 pg of CPA and 960 pg of DES per injection (1 microL) using APCI with data collection in selected ion monitoring (SIM) mode. The analytical performance of the method was evaluated using the determination of EE2, GES, LNG, CPA and DES in contraceptives and river water samples.

Comparative study on the application of capillary liquid chromatography and capillary electrochromatography for investigation of enantiomeric purity of the contraceptive drug levonorgestrel.

In the last few years, it has been shown that capillary electrochromatography (CEC) is a promising technique for enantioseparations. However, to date almost no studies are published on a critical comparison of CEC and its pressure-driven counterpart, capillary liquid chromatography (CLC) for real samples. In this study, the goal was to compare CLC and CEC for the determination of the enantiomeric purity of the contraceptive drug levonorgestrel and its pharmaceutical formulation. The study prevailed that not all potential advantages of CEC over CLC can easily be transformed in a real gain of detection limit of the enantiomeric impurity. However, certain advantages of CEC over CLC have been unambiguously shown.