Determination of exemestane, a new aromatase inhibitor, in plasma by high-performance liquid chromatography with ultraviolet detection

doi:10.1016/0378-4347(93)80008-R

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 620, Issue 2, 29 October 1993, Pages 225-231

https://doi.org/10.1016/0378-4347(93)80008-R Get rights and content

Abstract

A sensitive and selective high-performance liquid chromatographic method for the determination of 6-methylen-androsta-1,4-diene-3,17-dione (exemestane) and its 17-dihydro metabolite in human plasma has been developed. The analytes and internal standard (Norgestrel) were extracted from plasma samples with a methylene chloride—iso—octane mixture; the organic phase was dried and the residue was reconstituted with an acetonitrile—water mixture, then analyzed by reversed-phase liquid chromatography. Quantification was achieved by ultraviolet detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human blank plasma was observed. The lower limit of quantification was 10 ng/ml plasma. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from healthy male volunteers who had received a 200-mg single oral dose of the test compound.

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Cited by (24)

Development and validation of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of exemestane in human plasma
2011, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
A number of different chromatographic columns were tested, including Zorbax SB-Phenyl 150 mm × 2.1 mm, 5 μm (Agilent Technologies); Gemini C6-Phenyl 50 mm × 2.1 mm, 3 μm (Phenomenex) and Symmetry C18 50 mm × 2.1 mm, 3.5 μm (Waters). The mixture of acetonitrile and 0.1% aqueous acetic acid was used as the initial mobile phase [3,7,8,11,12]. The results suggested that required sensitivity could not be achieved in these conditions.
Exemestane, irreversible steroidal aromatase inhibitor, acts as a false substrate for aromatase enzyme and significantly lowers circulating estrogen concentrations in postmenopausal women with hormone-sensitive breast cancer. A sensitive bioanalytical method was developed and validated to study pharmacokinetics of exemestane. The method was based on liquid–liquid extraction of exemestane with methyl t-butyl ether followed by reversed-phase liquid chromatography. Positive electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode was applied for detection of exemestane. Anastrozole was used as internal standard. Calibration curve, fitted to 1/x² weighted linear regression model, was linear in the range of 0.1–40.0 ng/mL. Intra-run precision and accuracy were 1.80–3.17% and 103.4–111.5%, respectively. Inter-run precision and accuracy measured within 3 days were 3.37–4.19% and 101.8–109.6%, respectively. Extraction recoveries of exemestane and internal standard were 79.7–86.2% and 82.9–83.6%, respectively. The method was fully validated and may be applied to pharmacokinetic studies in humans after a single dose administration of 25 mg exemestane tablets.
Development and validation of a stability indicating LC method for the assay and related substances determination of Exemestane, an aromatase inhibitor
2009, Journal of Pharmaceutical and Biomedical Analysis
A selective stability indicating HPLC method was developed and validated for quantification of impurities (process related and degradants) and assay determination of Exemestane. Stability indicating power of the method was established by forced degradation experiments and mass balance study. The chromatographic separation was achieved with Hypersil BDS-C-18 using gradient elution. The developed method is validated for parameters like accuracy, linearity, LOD, LOQ, ruggedness. Box–Behnken experimental design was applied to check the robustness of the method.
Proliposomes of exemestane for improved oral delivery: Formulation and in vitro evaluation using PAMPA, Caco-2 and rat intestine
2009, International Journal of Pharmaceutics
The aim of the present study was to develop proliposomal formulations to enhance the oral bioavailability of exemestane by improving solubility, dissolution and/or intestinal permeability. Proliposomal powder formulations were prepared using different ratios of drug (exemestane), distearoyl–phosphatidylcholine (DSPC), cholesterol and dimyristoyl–phosphatidylglycerol (DMPG) by solvent evaporation method. The effect of phospholipid composition and drug:lipid ratio on in vitro performance of proliposomes was studied. Proliposomes were characterized for their particle size distribution, thermal characteristics by differential scanning calorimetry (DSC) and dissolution behavior. Further, the formulated proliposomes were subjected to in vitro permeation or transport studies using different models such as rat intestine, parallel artificial membrane permeability assay (PAMPA) and Caco-2 cell line. Proliposomes provided enhanced exemestane dissolution due to incorporation into the phospholipid bilayers and change in the physical state from crystalline to amorphous. The in vitro transport studies in rat intestine, PAMPA and Caco-2 models revealed that the proliposomes were successful in enhancing the permeation of exemestane. These proliposomal formulations of exemestane could provide improved oral bioavailability due to enhanced solubility, permeability and hence absorption.
Synthesis of exemestane labelled with <sup>13</sup>C
2008, Steroids
LC-MS-MS determination of exemestane in human plasma with heated nebulizer interface following solid-phase extraction in the 96 well plate format
2000, Journal of Pharmaceutical and Biomedical Analysis
A sensitive, specific and rapid analytical method for the quantitation of exemestane (EXE) in human plasma has been developed. EXE, 6-methylen-androsta-1,4-diene-3,17-dione, is an orally active irreversible steroidal aromatase inhibitor used for the therapy of metastatic postmenopausal breast cancer, with estrogen-dependent pathological conditions. The method involves extraction of EXE from human plasma by solid phase extraction using C2 endcapped sorbent in the 96 well plate format (50 mg/2 ml). After conditioning of the sorbent with 1 ml of acetonitrile (x2) the plates were rinsed with 1 ml of water (x2). The prepared samples (0.5 ml plasma, spiked with [¹³C₃] EXE as internal standard (IS) and diluted with 0.5 ml water) were loaded and drawn through the plate with a minimum of vacuum. The plates were then washed with 1 ml acetonitrile:water (10:90) followed by a drying step for 30 min at full vacuum. Elution was by 0.15 ml of 0.1% trifluoracetic acid in acetonitrile (x2) under a minimum of vacuum. Aliquots of 80 μl were finally injected into the LC–MS–MS system. A Zorbax SB C8 column (4.6×150 mm, 5 μm) was used to perform the chromatographic separation; the mobile phase was 100% acetonitrile. MS detection used the heated nebulizer interface, with multiple reaction monitoring (MRM) (297→121 m/z for EXE and 300→123 m/z for IS) operated in positive ion mode. A weighed linear regression analysis (weighing factor 1/x²) was used to calculate EXE concentration in standard and unknown samples. The method was fully validated in the concentration range 0.05–25 ng ml⁻¹.
Determination of the new aromatase inhibitor exemestane in biological fluids by automated high-performance liquid chromatography followed by radioimmunoassay
1996, European Journal of Pharmaceutical Sciences
A procedure for the determination of exemestane, a new aromatase inhibitor, in biological fluids is described in this paper. Exemestane is extracted from human plasma and urine by solid-phase and liquid-liquid extraction, respectively. The test compound is then isolated from endogenous steroids, its metabolites and/or degradation products by HPLC. The exemestane-containing fraction is collected and its exemestane content measured by radioimmunoassay (RIA). The automated HPLC system allowed a high specificity and reproducibility of retention times, and eliminated almost all manual operations. The RIA allowed the accurate and precise measurement of 12 pg of exemestane/ml in plasma (inter- and intra-assay RSD=17.7 and 13.4%, respectively) and 25 pg/ml in urine (inter- and intra-assay RSD=14.5% and 8.7%, respectively). The recovery of the whole procedure was evaluated by comparison of the RIA calibration curve obtained in plasma or urine (after extraction and HPLC) with that obtained directly in RIA buffer (without extraction and HPLC). The calibration curves were practically superimposable, indicating that the recovery of the whole procedure was excellent. The method was validated in terms of reproducibility, recovery and precision in the range 10–500 pg of exemestane/ml of plasma and 20–1000 pg/ml of urine. Finally the plasma levels of exemestane in a postmenopausal healthy volunteer treated daily for 7 days with oral exemestane at a dose of 1 mg (the lowest dose administered in clinical trials) were monitored using the method here described. Exemestane was detectable in all plasma samples collected (up to 24 h after drug intake). Therefore the analytical method described here should be sufficiently sensitive and specific for the determination of exemestane in plasma and urine from clinical trials in which therapeutic doses of the drug (10–25 mg/day) are administered.

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Determination of exemestane, a new aromatase inhibitor, in plasma by high-performance liquid chromatography with ultraviolet detection

Abstract

J. Biol. Chem.

Mol. Cell. Endocrinol.

J. Steroid Biochem.

Steroids

Steroids

J. Steroid Biochem.

J. Steroid Biochem.

J. Chromatogr.