Novel generation of deep eutectic solvent as an acceptor phase in three-phase hollow fiber liquid phase microextraction for extraction and preconcentration of steroidal hormones from biological fluids.

In this study, a novel generation of deep eutectic solvents (DESs) was used as an acceptor phase in three-phase hollow fiber liquid phase microextraction (HF-LPME) based on two immiscible organic phases. It was compared with other common DESs for extraction and preconcentration of dydrogesterone (DYD) and cyproterone acetate (CPA) from urine and plasma samples. The extracted analytes were analyzed by high performance liquid chromatography with UV-vis detector (HPLC-UV). This phosphonium based DES due to low volatility, low price and multifunctionality introduced itself as worthy next generation of acceptor phase in HF-LPME. The factors affected on extraction efficiency of the analytes were investigated and optimized. The performance of the proposed method was studied in terms of linear ranges (LRs from 1 to 500µgL-1 with R2 ≥ 0.9946), precision (RSD% ≤ 6.3) and limits of detection (LODs in the range of 0.5-2µgL-1). Under the optimized conditions, preconcentration factors in the range of 187-428 were obtained. Finally, the method was applied to the analysis of DYD and CPA in human urine and plasma samples and desirable results were obtained.

Cyproterone acetate quantification in human plasma by high-performance liquid chromatography coupled to atmospheric pressure photoionization tandem mass spectrometry. Application to a comparative pharmacokinetics study.

A specific, fast and sensitive high performance liquid chromatography (HPLC) coupled to atmospheric pressure photoionization (APPI) tandem mass spectrometric (LC-MS/MS) assay was developed for the determination of cyproterone (CYP) acetate (CAS 427-51-0) in human plasma. The retention times were 3.26 and 2.90 min for CYP acetate and its internal standard (I. S.) finasteride (FIN), respectively. The overall mean recovery, using liquid/liquid extraction, was found to be 109.0, 107.7 and 100.3%, for low, medium and high concentrations, respectively. Calibration curves were linear in the concentration range of 0.1-50.0 ng/ml, and the lower limit of quantification (LLOQ) was 0.1 ng/ml. The LLOQ, 0.1 ng/ml, was sensitive enough for detecting terminal phase concentrations of the drug. Inter-batch precision of the method ranged from 2.2 to 5.55%, while Inter-batch accuracy ranged from 95.5 to 100.0%. Intra-batch precision ranged from 1.8 to 5.6%, while Intra-batch accuracy ranged from 92.0 to 99.4% at concentrations of 0.3 ng/ml, 20.0 and 40.0 ng/ml. The developed method was applied to a bioequivalenc study of CYP acetate in a group of 44 female volunteers at a single oral dose of a 2 mg tablet, in a combination of ethinylestradiol/CYP acetate (0.25/2 mg). The plasma concentration of CYP acetate did not differ significantly after administration of both formulations (test formulation and the reference one). The geometric mean and respective 90% CI of CYP acetate test/reference percent ratios were 90.66% (84.39-97.40%) for Cmax and 96.20% (90.45-102.33%) for AUC0-t.

Atmospheric pressure photoionization applied to quantitation of cyproterone acetate in human plasma.

Cyproterone acetate [6-chloro-1beta,2beta-dihydro-17alpha-hydroxy- 3'H-cyclopropa(1,2)-pregna-1,4,6-triene-3,20-dione acetate] is a powerful antiandrogen used in the treatment of women suffering from disorders associated with androgenization such as hirsutism and acne. A fast, sensitive, and robustness method is developed for the determination and quantitation of cyproterone acetate in human blood plasma by liquid chromatography coupled with tandem mass spectrometry. Cyproterone acetate is extracted from 0.2 mL human plasma by liquid-liquid extraction. The method has a chromatographic run of 4.5 min, using a C18 analytical column (100- yen 2.1-mm i.d.), and the linear calibration curve over the range is linear from 1 to 500 ng/mL (r2 > 0.994). The between-run precision, based on the relative standard deviation replicate quality controls, is 96.2% (3 ng/mL), 97.5% (120 ng/mL), and 99.1% (400 ng/mL). The between-run accuracy was +/- 2.7%, 3.1%, and 4.8% for the previously mentioned concentrations, respectively. The method is employed in a bioequivalence study of two tablet formulations of cyproterone acetate (100 mg).

Fully automated method for the liquid chromatographic-tandem mass spectrometric determination of cyproterone acetate in human plasma using restricted access material for on-line sample clean-up.

A new automated method for the quantitative analysis of cyproterone acetate (CPA) in human plasma has been developed using on-line solid phase extraction (SPE) prior to the LC-MS/MS determination. The method was based on the use of a pre-column packed with internal-surface reversed-phase material (LiChrospher RP-4 ADS, 25 mm x 2 mm) for sample clean-up coupled to LC separation on an octadecyl silica stationary phase by means of a column switching system. A 30 microl plasma sample volume was injected directly onto the pre-column using a mixture of water, acetonitrile and formic acid (90:10:0.1 (v/v/v)) adjusted to pH 4.0 with diluted ammonia as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase consisting of water, methanol and formic acid (10:90:0.1 (v/v/v)). The dispensing flow rates of the washing liquid and the LC mobile phase were 300 microl min(-1). Medroxyprogesterone acetate (MPA) was used as internal standard. The MS ionization of the analytes was achieved using electrospray (ESI) in the positive ion mode. The pseudomolecular ionic species of CPA and MPA (417.4 and 387.5) were selected to generate daughter ions at 357.4 and 327.5, respectively. Finally, the developed method was validated according to a new approach using accuracy profiles as a decision tool. Very good results with respect to accuracy, detectability, repeatability, intermediate precision and selectivity were obtained. The LOQ of cyproterone acetate was 300 pg ml(-1).

Determination of cyproterone acetate in plasma samples by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic method for the determination of cyproterone acetate in human plasma has been developed. The chromatographic separation was performed on an analytical mbondapak C8 column (125 yen 4.6 mm, i.d) with an isocratic mobile phase consisting of methanol-water (62.38 v/v). Using ultra violet detection at 282 nm, the detection limit for cyproterone acetate in plasma was 10 ng/ml. The calibration curve was linear over the concentration range 50-160. 0 ng/ml. Cyproterone was isolated from plasma by liquid-liquid extraction and the recovery was about 90% for plasma. The inter-day and intra-day assay coefficients of variation were found to be less than 10%.

Fully automated method for the liquid chromatographic determination of cyproterone acetate in plasma using restricted access material for sample pre-treatment.

A new fully automated method for the quantitative analysis of an antiandrogenic substance, cyproterone acetate (CPA), in plasma samples has been developed using on-line solid-phase extraction (SPE) prior to the determination by reversed-phase liquid chromatography (LC). The automated method was based on the use of a precolumn packed with an internal-surface reversed-phase packing material (LiChrospher RP-4 ADS) for sample clean-up coupled to LC analysis on an octadecyl stationary phase using a column-switching system. A 200-microL volume of plasma sample was injected directly on the precolumn packed with restricted access material using a mixture of water-acetonitrile (90:10, v/v) as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase which consisted of a mixture of phosphate buffer, pH 7.0-acetonitrile (54:46, v/v). The elution profiles of CPA and blank plasma samples on the precolumn and the time needed for analyte transfer from the precolumn to the analytical column were determined. Different compositions of washing liquid and mobile phase were tested to reduce the interference of plasma endogenous components. UV detection was achieved at 280 nm. Finally, the developed method was validated using a new approach, namely the application of the accuracy profile based on the interval confidence at 90% of the total measurement error (bias+standard deviation). The limit of quantification of cyproterone acetate in plasma was determined at 15 ng mL(-1). The validated method should be applicable to the determination of CPA in patients treated by at least 50 mg day(-1).

Highly sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of cyproterone acetate and free cyproterone.

We have developed a non-isotopic TR-FIA for Cyproterone acetate and Cyproterone in plasma. Synthesis of the biotinylated tracers, biotinylated Cyproterone acetate, and Cyproterone, as well as the preparation of anti-Cyproterone acetate and anti-Cyproterone antisera are reported. The specificity of anti-Cyproterone acetate antiserum resulting from the coupling of link bridge (link bridge between steroid and BSA), on the 3-position on the steroid skeleton, allowed to carry out the Cyproterone acetate assay directly on extracted plasma (without chromatography). On the other hand Cyproterone assays require a purification step, including extraction plus chromatography, because the plasma Cyproterone acetate concentrations in Cyproterone acetate-treated women are 200 times higher than for Cyproterone. Theses plasma TR-FIA of Cyproterone acetate and Cyproterone presented the advantage of needing only small doses of radioactivity for recovery controls, and better practicability related to the only existing RIA described to date.

Effects of hormone replacement therapy on hemostatic cardiovascular risk factors.

OBJECTIVES: From observational studies, there is evidence that hormone replacement therapy in postmenopausal women causes a decrease in cardiovascular events. It remains unknown, however, precisely by which mechanisms this reduction is achieved. The primary aim of this work was to study the effects of hormone replacement therapy on established hemostatic risk factors during 1-year treatment of healthy postmenopausal women. The secondary aim was to investigate whether there was any significant difference in these risk factors between hormone replacement therapy administered as a cyclic estrogen/sequential progestogen or continuous estrogen/sequential progestogen regimen. STUDY DESIGN: Sixty postmenopausal women were randomized to treatment with estradiol valerate 2 mg/day either continuously or cyclic (days 1 to 21; placebo on days 21 to 28). Both groups received cyproterone acetate 1 mg/day on days 12 to 21. Blood samples were collected before treatment and on cycle days 17 to 22 in cycles 3, 6, and 12. Thirty women with basic characteristics identical to the women included in the treatment group were included as a reference group. Blood samples were collected after 0, 6, and 12 months of observation. RESULTS: Hormone replacement therapy during 1 year caused a marginal but significant increase in plasma concentration of factor VIIc after 12 months of treatment (P <.05), a significant decrease in fibrinogen, and a significant decrease in the protein concentrations of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and lipoprotein(a) after 3, 6, and 12 months of treatment (P <.05). Possible differences in the integrated response between the reference group and the hormone replacement therapy group were evaluated by comparison of the area under the curve as estimated in each individual on the basis of each analyte in the sampling periods. The area under the curve of fibrinogen was significantly lower in the hormone replacement therapy group than in the reference group (P <.03), whereas other variables did not deviate significantly between the groups. The areas under the curve did not deviate significantly between the group that received cyclic estrogen/sequential progestogen and the group that received continuous estrogen/sequential progestogen. CONCLUSIONS: One-year treatment with hormone replacement therapy influenced favorably a number of prognostic cardiovascular risk factors in healthy women. The most important effect was the lowering of fibrinogen. Furthermore, in this study the effect of hormone replacement therapy on hemostasis did not deviate between a cyclic estrogen/sequential progestogen regimen and a continuous estrogen/sequential progestogen regimen.

Chronopharmacokinetic behaviour of cyproterone acetate in rabbits.

Cyproterone acetate (CPA) is an antiandrogenic compound that shows a rhythmic toxicologic behaviour. The purpose of this study was to determine whether CPA pharmacokinetics in the rabbit were influenced by the administration time of day. Previously synchronised rabbits received a single intravenous dose of 4 mg kg(-1) of CPA at two hours after light onset (2 HALO) and 14 hours (14 HALO) after light onset. The drug concentration in plasma samples was determined by high performance liquid chromatography. The mean concentrations in plasma were significantly higher (P<0.05) in 2 HALO than in 14 HALO animals at five, 15 and 30 minutes after dosing. Both plasma concentration profiles were fitted to two-compartment open models. Mean A, Vc and Vss differed significantly between 2 and 14 HALO dosage (P<0.005). Temporal variations in plasma protein binding, drug distribution and in drug elimination may play an important role in explaining these results.

Topical cyproterone acetate treatment in women with acne: a placebo-controlled trial.

OBJECTIVE: To evaluate the clinical and hormonal response of topically applied cyproterone acetate, oral cyproterone acetate, and placebo lotion in women with acne. DESIGN: Placebo-controlled, randomized study. SETTING: Patients were recruited from the Institute of Endocrine Cosmetics, Vienna, Austria. PATIENTS: Forty women with acne. INTERVENTIONS: Treatment with oral medication consisting of 0.035 mg of ethinyl estradiol and 2 mg of cyproterone acetate (n=12), 20 mg of topical cyproterone acetate lotion (n=12), and placebo lotion (n=16) was offered. Patients were assessed monthly for 3 months. MAIN OUTCOME MEASURES: Clinical grading according to acne severity and lesion counts as well as determinations of serum cyproterone acetate concentrations. RESULTS: After 3 months of therapy with topical cyproterone acetate, the decrease of mean facial acne grade from 1.57 to 0.67 was significantly better (P<.05) compared with placebo (which showed a change from 1.57 to 1.25), but not compared with oral medication (1.56 to 0.75) (P>.05). Lesion counts also decreased from 35.9 to 9.1 in the topical cyproterone acetate group compared with oral medication (45.4 to 15.5) (P>.05) and placebo (38.2 to 23.1) (P<.05). After topical cyproterone acetate treatment, serum cyproterone acetate concentrations were 10 times lower than those found after oral cyproterone acetate intake. CONCLUSIONS: The therapeutic effect of topically applied cyproterone acetate for acne treatment was clearly demonstrated. Topically applied sexual steroids in combination with liposomes are as effective as oral antiandrogen medication in acne treatment, while reducing the risk of adverse effects and avoiding high serum cyproterone acetate concentrations.

Tissue distribution of sex steroids: concentration of 17beta-oestradiol and cyproterone acetate in selected organs of female Wistar rats.

The tissue distribution of 17beta-oestradiol and cyproterone acetate was investigated after intravenous and intragastric administration to female Wistar rats by measuring the time course of the concentration of the sex steroids in plasma, liver, kidney, brain, and heart by radioimmunoassay. Test substances were administered intravenously in doses of 0.1 mg/kg each and intragastrically in doses of 10 mg/kg (17beta-oestradiol) and 0.1 mg/kg (cyproterone acetate) corresponding to the expected oral bioavailability. Tissue distribution was assessed within each mode of administration by AUCorgan/AUCplasma-quotients (Q-values), and between both routes of administration by F-values representing (bio- and organ availability) and R-values, which express the organ load after intragastric compared to intravenous administration if the same amount of drug has been made bioavailable in the plasma after both routes (for explanation see next page). The absolute bioavailability of 17beta-oestradiol after intragastric administration of 10 mg/kg was ca. 8%. The oestradiol liver load after intragastric administration was about 20 times higher than after intravenous administration, whereas the drug load of other organs was independent of the administration route. Cyproterone acetate was completely bioavailable after intragastric administration in a dose of 0.1 mg/kg. Cyproterone acetate levels and AUC-values in all organs investigated were higher when compared to the plasma with highest levels in the liver. The organ distribution of cyproterone acetate including the drug liver load was independent of the route of administration.

Clinical, endocrinologic, and ultrasonographic features of polycystic ovary syndrome in Singaporean adolescents.

The study sought to evaluate the clinical, endocrinologic, and ultrasonographic features in 150 Singaporean adolescent girls with polycystic ovary syndrome (PCOS) before and after treatment, which was composed primarily of a combined hormone therapy of estrogen and cyproterone acetate. The patients' ages ranged from 12 to 22 years with the majority between 15 and 18 years of age. Most of these girls were seen between their third and fifth gynecologic year. A considerable proportion of them had matured early, experiencing menarche between 9 and 12 years of age. Tanner staging was normal except for a greater proportion at higher stages for pubic and axillary hair, most likely a reflection of the substantial degree of androgenization commonly found in subjects with PCOS. All 150 patients presented with menstrual disorders including secondary amenorrhea, menarche only, anovulatory uterine bleeding, oligomenorrhea, and primary amenorrhea. The majority had normal body weight; 10% to 27% were either underweight or overweight, respectively. On ultrasound, patients presented with enlarged ovaries; enlargement was more pronounced in the right ovary with dense stroma and multiple subcapsular cysts. Many subjects had elevated androgen, luteinizing hormone (LH), and LH/ follicle-stimulating hormone (FSH) levels. Although characteristic of PCOS, FSH levels were either low or normal. Prolactin, estradiol, dehydroepiandrosterone sulfate (DHEAS), and androstenedione were generally normal. A substantial proportion of the study group had elevated cortisol levels. It was noted that adolescent girls with PCOS responded well to treatment; more than 60% showed improvement in cycle profiles following at least 1 year of treatment. Our current opinion is that adolescents with PCOS should be managed early, and that treatment should include medical correction of any hormonal or body-weight imbalance and include psychologic intervention when necessary.

Radioimmunological analysis of cyproterone acetate in human serum. Comparison with a gas chromatographic/mass spectrometric method and influence of each method on the outcome of a bioequivalence trial.

Cyproterone acetate (CAS 427-51-0, CPA) is a steroid hormone with antiandrogenic and progestogenic properties, which has been used in the therapy of prostate carcinoma in men, and for the treatment of severe acne and hirsutism in women. The aim of the present study was to compare two equally sensitive analytical methods, a radioimmunoassay (RIA) and a gas chromatographic/mass spectrometric method (GC/MS), for the quantitative determination of CPA in human serum samples and to assess their suitability for bioequivalence trials. To this end, serum samples which had been collected during a study on the bioequivalence oaf two CPA-containing formulations (Androcur 100 and Androcur 50) were analysed using both methods. Basic pharmacokinetic parameters, like Cmax, tmax, t1/2 and AUC were calculated from each data set and corresponding parameters were compared by statistical methods. A comparison of the drug concentration-time curves obtained with both analytical methods revealed that in particular at later sampling times (48 to 120 h post dose) concentration values generated by RIA were slightly higher by about 20-40% than those measured with GC/MS. This indicates the presence of cross-reacting metabolite(s), most likely the 15 beta-hydroxy-cyproterone acetate. Accordingly, the values of Cmax, AUC(0-120 h) and AUC were overestimated by the RIA method by about 10-20%, 5% and 10%, respectively. However, there was a high correlation between corresponding parameters derived from RIA and GC/MS analysis. Although AUC values were slightly overestimated with the RIA method was used, this had no influence on the decision about bioequivalence because the mean ratio of the target variables Cmax and AUC was not affected. The variance of the pharmacokinetic parameters which is relevant for the bioequivalence test were even lower for RIA based values than those calculated after GC/MS analysis. In conclusion, it was found that although the GC/MS method is superior to RIA in terms of specificity, both methods were equally suited to demonstrate the bioequivalence of the two CPA-containing formulations. Thus, in future studies of this kind, the more simple RIA may be used instead of GC/MS.