RESUMEN
OBJECTIVES: The aim of the present study was to validate a simple, sensitive, HPLC method of analysis of doxazosin in human plasma with fluorescence detection. METHODS: The validated method employed one-step direct protein precipitation with acetonitrile. Chromatographic separation was attained using a reverse-phase 250mm×4.6mm 5µ Hypersil® BDS C 18 column and the mobile phase consisted of 10mm sodium dihydrogen phosphate dihydrate (pH=3.0) and acetonitrile at a ratio of (65:35 v/v). The method was evaluated in terms of linearity, precision, accuracy, selectivity and stability as per standard guidelines. The total run time was about 4.5min which make this method suitable for high throughput analyses. This method was applied to the bioequivalence study of two doxazosin tablets in healthy human volunteers. RESULTS: Good linear response was achieved over the range of 5.0-200ng/mL. The observed within- and between-day assay precision ranged from 0.64% to 14.73%; accuracy varied between 94.11% and 105%. The 90% confidence intervals for the ratio Cmax, and AUC 0-∞ of the test product over those of reference were within the acceptable range (0.8-1.25) for bioequivalence. CONCLUSION: The developed method was simple and could be applied to therapeutic drug monitoring of doxazosin.
Asunto(s)
Doxazosina/sangre , Monitoreo de Drogas/métodos , Cromatografía Líquida de Alta Presión/métodos , Doxazosina/farmacocinética , Humanos , Equivalencia TerapéuticaRESUMEN
Alpha1-adrenergic-blocking drugs, namely; alfuzosin hydrochloride (ALF), doxazosin mesylate (DOX) and terazosin hydrochloride (TER) are effective as antihypertensive agents as well as in management of benign prostatic hypertrophy. In this study, a simple, very fast, highly sensitive and cheap technique was optimized for assay of these drugs in pure states and pharmaceutical tablets. The proposed method is dependent on enhancement of the native fluorescence of investigated drugs using the polyoxyethylene 50 stearate (POE50S) micellar system. The method showed excitation at 325, 340 and 250 nm for ALF, DOX and TER, respectively and an emission maxima at 382 nm. The fluorescence intensity-concentration charts of studied drugs were attained utilizing concentration ranges (2.0-60.0 ng mL-1 ) for DOX and (4.0-100.0 ng mL-1 ) for ALF and TER with quantitation limits 2.9, 1.6 and 2.5 ng mL-1 for ALF, DOX and TER, respectively. The suggested technique was approved according to International Council for Harmonisation (ICH) standards and the United States Food and Drug Administration (US FDA) bioanalytical method validation and has been effectively applied for assay of these medications in their dosage forms as well as for content uniformity test. The developed procedure was also efficiently applied for determination of these drugs in real human plasma with high accuracy.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1/sangre , Antihipertensivos/sangre , Espectrometría de Fluorescencia/métodos , Doxazosina/sangre , Fluorescencia , Humanos , Límite de Detección , Micelas , Preparaciones Farmacéuticas/sangre , Plasma/química , Prazosina/análogos & derivados , Prazosina/sangre , Quinazolinas/sangre , Comprimidos/químicaRESUMEN
A highly sensitive, simple and selective spectrofluorimetric method has been developed and validated for determination of doxazosin mesylate in pure form, pharmaceutical formulations and human plasma. The method is based on the reaction between doxazosin mesylate and fluorescamine in Teorell buffer solution (pH 3) to give highly fluorescent derivative that can be measured at 489 nm using excitation wavelength of 385 nm. Different experimental parameters affecting the reaction were carefully studied and optimized. The calibration plot was constructed over the concentration range of 16-400 ng mL(-1) with quantitation limit of 14.3 ng mL(-1). The developed procedure was validated according to ICH guidelines and the results were satisfactory. The proposed method has been successfully applied to the analysis of the cited drug in its pharmaceutical preparations as well as for content uniformity testing. The results showed excellent agreement with the reported method with respect to precision and accuracy. In addition, the drug concentration was determined in the spiked human plasma by the suggested method with % recovery in the range of 96.2-98.3% (SD; 0.76-0.93, n=5).
Asunto(s)
Antihipertensivos/análisis , Antihipertensivos/sangre , Doxazosina/análisis , Doxazosina/sangre , Espectrometría de Fluorescencia/métodos , Fluorescamina/química , Humanos , Límite de Detección , Preparaciones Farmacéuticas/químicaRESUMEN
Previous publications showed that the value of LLOQ (lowest limit of quantification) for doxazosin and its enantiomers in biological samples were above 0.1 ng·m L(-1). The present study was designed to establish a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification at very low concentration of (-)doxazosin in rat plasma after intravenous administration of (-)doxazosinï¼3.0 mg·kg(-1)ï¼. The plasma samples containing prazosin as an internal standard were extracted by solid-phase extraction (SPE) and separated on Acquity BEH C(18) (50 mm × 2.1 mm, 1.7 µm) column under alkaline conditions of the mobile phase. (-)Doxazosin was monitored under positive ionization condition by multiple reaction monitoring (MRM) with an ESI source. The good linear range of (-)doxazosin varied from 10.4 pg·m L(-1) to 13 ng·m L(-1)ï¼r = 0.992 2), and the lowest limit of quantification was 10.4 pg·m L(-1). An assessment of the matrix effect using post-extraction spiking method and post-column infusion method demonstrated that co-eluting matrix components did not significantly influenced the ionization of (-)doxazosin and prazosin (IS). Using the new method, we accurately measured (-)doxazosin concentration at 48 h after intravenous administration in the rats, and the concentration was 0.034 4 ± 0.010 2 ng·m L(-1). The method is specific, sensitive, and suitable for determining (-)doxazosin at very low concentration in rat plasma samples.
Asunto(s)
Doxazosina/sangre , Administración Intravenosa , Animales , Cromatografía Liquida , Plasma , Prazosina , Ratas , Sensibilidad y Especificidad , Extracción en Fase Sólida , Estereoisomerismo , Espectrometría de Masas en TándemRESUMEN
Doxazosin (DOX), a long-lasting α1-adrenoceptor antagonist, is used clinically as a racemate that consists of two optical isomers. In humans and rats, following oral administration of racemic DOX [(±)-DOX], the plasma concentration of the (-)-isomer is lower than that of the (+)-isomer, but the mechanism for this interaction is not known. In this study, a chiral HPLC with fluorescence detection was used to measure the drug concentrations for analysis of the stereoselective metabolism of DOX in in vivo and in vitro experiments. We found that the plasma levels of the (-)-isomer were significantly lower than those of the (+)-enantiomer following i.v. administration of (±)-DOX to the rats and that the depletion rate constant (kdep) of (-)-DOX (0.0107±0.0007L/min) was significantly larger than that of (+)-DOX (kdep 0.0088±0.0005L/min) (p<0.05) when (±)-DOX was incubated with rat liver microsomes (RLMs). However, (-)-DOX was not depleted faster than (+)-DOX following their separate incubation with RLMs. The metabolism of (-)- or (+)-isomer in RLMs was catalysed by CYP3A because the depletion of the compounds was inhibited by ketoconazole (a potent CYP3A-selective inhibitor) similarly. More importantly, the kdep of (+)-DOX in the 1.0/2.0 and 0.5/2.5 (+)-DOX/(-)-DOX mixtures was significantly lower than that of (-)-DOX in the 1.0/2.0 and 0.5/2.5 (-)-DOX/(+)-DOX mixtures (p<0.05). In conclusion, although (-)-DOX is not depleted faster than (+)-DOX when only a single isomer of DOX is incubated with rat liver microsomes, it is depleted much faster than (+)-DOX when a mixture of the two isomers was used, suggesting a prominent and stereoselective inhibition of the (-)-isomer over the (+)-isomer at the CYP3A enzyme.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1/sangre , Citocromo P-450 CYP3A/metabolismo , Doxazosina/sangre , Antagonistas de Receptores Adrenérgicos alfa 1/administración & dosificación , Antagonistas de Receptores Adrenérgicos alfa 1/farmacocinética , Animales , Cromatografía Líquida de Alta Presión , Doxazosina/administración & dosificación , Doxazosina/farmacocinética , Ratas , Espectrometría de Fluorescencia , EstereoisomerismoRESUMEN
Mixed micelle cloud point extraction (MM-CPE) combined with magnetic dispersive µ-solid phase extraction (MD-µ-SPE) has been developed as a new approach for the extraction of doxazosin (DOX) and alfuzosin (ALF) prior to fluorescence analysis. The mixed micelle anionic surfactant sodium dodecyl sulfate and non-ionic polyoxyethylene(7.5)nonylphenylether was used as the extraction solvent in MM-CPE, and diatomite bonding Fe3O4 magnetic nanoparticles were used as the adsorbent in MD-µ-SPE. The method was based on MM-CPE of DOX and ALF in the surfactant-rich phase. Magnetic materials were used to retrieve the surfactant-rich phase, which easily separated from the aqueous phase under magnetic field. At optimum conditions, a linear relationship between DOX and ALF was obtained in the range of 5-300 ng mL(-1), and the limits of detection were 0.21 and 0.16 ng mL(-1), respectively. The proposed method was successfully applied for the determination of the drugs in pharmaceutical preparations, urine samples, and plasma samples.
Asunto(s)
Doxazosina/análisis , Doxazosina/aislamiento & purificación , Quinazolinas/análisis , Quinazolinas/aislamiento & purificación , Extracción en Fase Sólida/métodos , Calibración , Doxazosina/sangre , Humanos , Concentración de Iones de Hidrógeno , Límite de Detección , Fenómenos Magnéticos , Nanopartículas de Magnetita , Micelas , Quinazolinas/sangre , Cloruro de Sodio/química , Dodecil Sulfato de Sodio , Extracción en Fase Sólida/instrumentación , Solventes/química , Espectrofotometría Ultravioleta/métodos , Tensoactivos/química , TemperaturaRESUMEN
AIM: Doxazosin is a racemic mixture of (-)doxazosin and (+)doxazosin that is currently used as an add-on therapy for hypertension. In this study we investigated the contribution of each enantiomer to the hypotensive action of long-term administration of (±)doxazosin in conscious rats. METHODS: Blood pressure of conscious SD rats was measured using a volume pressure recording system. The rats were orally administered (-)doxazosin, (+)doxazosin, or (±)doxazosin (8 mg·kg(-1)·d(-1)) for 12 weeks. Plasma concentrations of the agents were analyzed with HPLC. The effect of the agents on α1-adrenoceptor was examined in isolated rat caudal artery preparations. RESULTS: Treatment of conscious rats with a single dose of (±)doxazosin (8 mg/kg) did not affected DBP and MBP, but significantly decreased SBP by 11.9% 4 h after the administration. Long-term treatment of conscious rats with (±)doxazosin significantly decreased SBP, DBP and MBP with a maximal decrease of SBP by 29.3% 8 h after the last administration. The rank order of the hypotensive actions caused by long-term treatment in conscious rats was (±)doxazosin>(+)doxazosin>>(-)doxazosin. However, the pKB values for inhibiting NA-induced contraction of isolated rat caudal artery were (+)doxazosin (8.995)>(±)doxazosin (8.694)>(-)doxazosin (8.032). The plasma concentrations of (-)doxazosin, (+)doxazosin, and (±)doxazosin were 18.26±3.55, 177.11±20.66, and 113.18±13.21 ng/mL, respectively, 8 h after the last administration of these agents. CONCLUSION: Long-term treatment with (±)doxazosin produces potent hypotensive action in conscious rats that seems to result from synergic interaction of the two enantiomers.
Asunto(s)
Antihipertensivos/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Estado de Conciencia/efectos de los fármacos , Doxazosina/administración & dosificación , Administración Oral , Animales , Antihipertensivos/sangre , Presión Sanguínea/fisiología , Estado de Conciencia/fisiología , Doxazosina/sangre , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
The study is to establish an HPLC method using fluorescence detector for the determination of doxazosin enantiomers and investigate their chiral inversion in vitro and in vivo. Ultron ES-OVM was taken as the chiral chromatographic column, and the column temperature was 30 degrees C. Isocratic elution using a mobile phase of phosphate buffer-acetonitrile (85 : 15, v/v) at a flow rate of 0.8 mL x min(-1) was done. The fluorescence detection was set at lambda(Ex) = 255 nm and lambda(Em) = 385 nm. Prazosin was used as the internal standard. (-) Doxazosin or (+) doxazosin added into rat plasma in vitro was determined after incubating in 37 degrees C water bath for 2, 5 and 10 days. (-) Doxazosin or (+) doxazosin was administered orally to the rats for one months. Plasma samples were taken at 8 h after the last administration. A good linear relationship was achieved when the concentration of doxazosin enantiomers was within the range of 4 - 2 000 ng x mL(-1). The average recovery for (-) doxazosin was 99.5% with RSD 3.6%, and for (+) doxazosin was 99.3% with RSD 4.3%. Chiral inversion was observed neither in vitro nor in vivo studies. The method is selective, accurate and reproducible, which is suitable for the detection of doxazosin enantiomers in rat plasma. The in vitro and in vivo studies indicate that chiral inversion occurs uneasily between (-) doxazosin and (+) doxazosin in the rat.
Asunto(s)
Doxazosina/sangre , Doxazosina/química , Animales , Análisis Químico de la Sangre/métodos , Masculino , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , EstereoisomerismoRESUMEN
A rapid and sensitive method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneous determination of doxazosin and verapamil in human serum has been developed. Trimipramine-d3 as an isotopic labelled internal standard was used for quantification. Serum samples were prepared by simple liquid-liquid extraction with mixture of tert butyl methyl ether and ethyl acetate (1:1, v:v). The analytes and internal standard were separated on C18 column using an isocratic elution with 5 mM ammonium formate with 0.02% formic acid and 0.02% formic acid in acetonitrile (55:45, v:v) at a flow rate of 1.1 mL/min. Positive TurboIonSpray mass spectrometry was used with multiple reaction monitoring of the transitions at: m/z 455.3â165.2 and 150.2 for verapamil, m/z 452.2â344.4 and 247.4 for doxazosin, m/z 298.2â103.1 for trimipramine-d3. Linearity was achieved between 1 and 500 ng/mL (R² ≥ 0.997) for both analytes. An extensive pre-study method validation was carried out in accordance with FDA guidelines. This assay was successfully applied to determine the serum concentrations of doxazosin and verapamil in suspect non-compliance patients.
Asunto(s)
Antihipertensivos/sangre , Cromatografía Liquida/métodos , Doxazosina/sangre , Hipertensión/sangre , Cooperación del Paciente , Espectrometría de Masas en Tándem/métodos , Verapamilo/sangre , Humanos , Hipertensión/tratamiento farmacológicoRESUMEN
An enantioselective and sensitive method was developed and validated for determination of doxazosin enantiomers in human plasma by liquid chromatography-tandem mass spectrometry. The enantiomers of doxazosin were extracted from plasma using ethyl ether/dichloromethane (3/2, v/v) under alkaline conditions. Baseline chiral separation was obtained within 9 min on an ovomucoid column using an isocratic mobile phase of methanol/5mM ammonium acetate/formic acid (20/80/0.016, v/v/v) at a flow rate of 0.60 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 452-->344 for doxazosin enantiomers, and m/z 384-->247 for prazosin (internal standard). The method was linear in the concentration range of 0.100-50.0 ng/mL for each enantiomer using 200 microL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.100 ng/mL. The intra- and inter-assay precision was 5.0-11.1% and 5.7-7.6% for R-(-)-doxazosin and S-(+)-doxazosin, respectively. The accuracy was 97.4-99.5% for R-(-)-doxazosin and 96.8-102.8% for S-(+)-doxazosin. No chiral inversion was observed during the plasma storage, preparation and analysis. The method proved adequate for enantioselective pharmacokinetic studies of doxazosin after oral administration of therapeutic doses of racemic doxazosin.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1/sangre , Cromatografía Liquida/métodos , Doxazosina/sangre , Ovomucina/química , Espectrometría de Masas en Tándem/métodos , Antagonistas de Receptores Adrenérgicos alfa 1/química , Antagonistas de Receptores Adrenérgicos alfa 1/farmacocinética , Doxazosina/química , Doxazosina/farmacocinética , Estabilidad de Medicamentos , Humanos , Análisis de los Mínimos Cuadrados , Prazosina/análisis , Prazosina/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , EstereoisomerismoRESUMEN
INTRODUCTION: ICH S7A and S7B guidelines recommend the use of conscious animals for assessment of non-clinical cardiovascular safety of new chemical entities prior to testing in humans. Protocol design and data analysis techniques can affect the quality of the data produced and can therefore ultimately influence the clinical management of cardiovascular risk. It is therefore essential to have an understanding of the magnitude of changes detectable and the clinical relevance of these changes. This paper describes the utilisation of "super-intervals" to analyse and interpret data obtained from our conscious telemetered dog cardiovascular safety protocol and reports the statistical power achieved to detect changes in various cardiovascular parameters. METHODS: Cardiovascular data from 18 dog telemetry studies were used to calculate the statistical power to detect changes in cardiovascular parameters. Each study followed a test compound versus vehicle cross-over experimental design with 24h monitoring (n=4). 1 min mean raw data from each individual animal was compressed into 15 min mean data for each dose group for visualisation. Larger summary periods, or "super-intervals", were then selected to best represent any observed cardiovascular effects whilst taking into account the pharmacokinetic profile of the drug e.g. intervals of 1 to 6, 7 to 14 and 14 to 22h post-dose. RESULTS: With this methodology and study design we predict, using the median percentile that our studies have 80% power to detect the following changes: HR (+/-10bpm), LV +dP/dt max (+/-375mmHg/s), MBP (+/-5mmHg) and QTc (+/-4ms). DISCUSSION: Super-intervals are a simple way to handle the high degree of natural variability seen with any ambulatory cardiovascular assessment and, in our hands, result in highly statistically powered studies. The ability of this model to detect cardiovascular changes of small, but biologically relevant, magnitude enables confident decision making around the cardiovascular safety of new chemical entities.
Asunto(s)
Antiinfecciosos/farmacología , Antihipertensivos/farmacología , Compuestos Aza/farmacología , Sistema Cardiovascular/efectos de los fármacos , Doxazosina/farmacología , Electrocardiografía/efectos de los fármacos , Quinolinas/farmacología , Animales , Antiinfecciosos/administración & dosificación , Antiinfecciosos/sangre , Antihipertensivos/administración & dosificación , Antihipertensivos/sangre , Compuestos Aza/administración & dosificación , Compuestos Aza/sangre , Presión Sanguínea/efectos de los fármacos , Estado de Conciencia , Interpretación Estadística de Datos , Perros , Relación Dosis-Respuesta a Droga , Doxazosina/administración & dosificación , Doxazosina/sangre , Evaluación Preclínica de Medicamentos , Fluoroquinolonas , Frecuencia Cardíaca/efectos de los fármacos , Síndrome de QT Prolongado/diagnóstico , Masculino , Modelos Animales , Modelos Estadísticos , Moxifloxacino , Quinolinas/administración & dosificación , Quinolinas/sangre , Telemetría , Factores de TiempoRESUMEN
A rapid and sensitive reversed phase liquid chromatography- tandem mass spectrometry (LC-MS-MS) method is developed for the determination of doxazosin in canine plasma. The samples are prepared by precipitation of proteins using a mixture of methanol and acetonitrile, followed by freezing and evaporation of the organic solvent. The remaining dry residue is redissolved in mobile phase and analyzed by LC-MS-MS with positive electrospray ionization using the selected reactions monitoring mode. An XTerra MS C(18) column, a mobile phase composed of acetonitrile and 2mM ammonium acetate with gradient elution, and a flow rate of 400 microL/min are employed. The elution times for prazosin (internal standard) and doxazosin are approximately 8 and 10 min, respectively. Calibration curves are linear in the 1-20 ng/mL concentration range. Limits of detection and quantification are 0.4 ng/mL and 1.2 ng/mL, respectively. Recovery is higher than 94%. Intra- and inter-day relative standard deviations are below 7% and 8%, respectively. The method is applied for the determination of doxazosin plasma levels following a single administration of doxazosin base and doxazosin mesylate tablets (2 mg dose) to dogs in the fed state. The results indicate possible superiority of the mesylate salt on the plasma input rates of doxazosin.
Asunto(s)
Antagonistas Adrenérgicos alfa/sangre , Cromatografía de Fase Inversa/métodos , Doxazosina/sangre , Espectrometría de Masas en Tándem/métodos , Administración Oral , Antagonistas Adrenérgicos alfa/administración & dosificación , Antagonistas Adrenérgicos alfa/farmacocinética , Animales , Calibración , Perros , Doxazosina/administración & dosificación , Doxazosina/farmacocinética , Femenino , Límite de DetecciónRESUMEN
This study attempted to characterize the influence of core and coating formulations on the release profiles to establish in vitro/in vivo correlations of pulsatile pattern for a pulsatile drug delivery system activated by membrane rupture based on three core tablet formulations (A-core: HPMC 50+4000 cps, B-core: E10M, and C-core: K100M) coated with various thicknesses of a semipermeable ethylcellulose membrane plasticized with HPMC 606 (Pharmacoat 606) at different ratios with/without adding various amounts of water to dissolve it in the coating solution. Drug release behaviors were investigated using apparatus II in four media of pH 1.2 solution, pH 6.8 buffer, deionized water, and a NaCl solution rotated at 75, 100, and 150 rpm. Pilot studies of the in vivo pharmacokinetics were conducted as well for comparison with the in vitro results. Results demonstrated that drug release from the three kinds of core tablets in deionized water increased with an increasing stirring rate, and decreased with an increasing viscosity grade of HPMC used in the core formulations. A significant promotion of drug release from core tablets was observed for the three levels of NaCl media in comparison with that in deionized water. Results further demonstrated that a slightly slower release rate in pH 1.2 solution and a faster release rate in pH 6.8 buffer than that in deionized water were observed for the A-core and B-core tablets, with the former being slower than the latter. However, similar release rates in the three kinds of media were observed for C-core tablets, but they were slower than those for the A- and B-core tablets. Dissolution of coated tablets showed that the controlling membrane was ruptured by osmotic pressure and swelling which activated drug release with a lag time. The lag time was not influenced by the pH value of the release medium or by the rotation speeds. The lag time increased with a higher coating level, but decreased with the addition of the hydrophilic plasticizer, Pharmacoat 606, and of the water amount in the coating solution. The lag time also increased with a higher concentration of NaCl in the medium. The release rate after the lag time was determined by the extent of retardation of gelation of HPMC in the core tablet based on the ionic strength of the medium. Results of the three pilot crossover studies for the exemplified pulsatile systems indicated that the lag time for the in vivo plasma profile was well correlated with that determined from the in vitro release profile in pH 1.2 solution and the in vivo release rate was better reflected by that performed in pH 6.8 buffer.
Asunto(s)
Celulosa/análogos & derivados , Doxazosina/farmacocinética , Portadores de Fármacos , Membranas Artificiales , Metilcelulosa/análogos & derivados , Administración Oral , Adulto , Celulosa/química , Química Farmacéutica , Estudios Cruzados , Preparaciones de Acción Retardada , Doxazosina/administración & dosificación , Doxazosina/sangre , Doxazosina/química , Humanos , Concentración de Iones de Hidrógeno , Derivados de la Hipromelosa , Metilcelulosa/química , Modelos Biológicos , Presión Osmótica , Permeabilidad , Proyectos Piloto , Quimioterapia por Pulso , Cloruro de Sodio/química , Solubilidad , Comprimidos , Viscosidad , Agua/químicaRESUMEN
Hydrophilic interaction LC with MS/MS (HILIC-MS/MS) was described as a rapid, sensitive, and selective method for the quantification of doxazosin in human plasma. Doxazosin and cisapride (internal standard) were extracted from human plasma with ethyl acetate at alkaline pH and analyzed on an Atlantis HILIC Silica column with the mobile phase of ACN/ammonium formate (100 mM, pH 4.5) (93:7 v/v). The analytes were detected using an ESI MS/MS in the selective-reaction-monitoring mode. The standard curve was linear (r = 0.9994) over the concentration range of 0.2-50 ng/mL. The LOQ for doxazosin was 0.2 ng/mL using 100 microL plasma sample. The CV and relative error for intra- and interassay at four QC levels were 3.7-8.7% and 0.0-9.8%, respectively. The matrix effect for doxazosin and cisapride were practically absent. The recoveries of doxazosin and cisapride were 67.4 and 61.7%, respectively. This method was successfully applied to the pharmacokinetic study of doxazosin in humans.
Asunto(s)
Cromatografía Liquida/métodos , Doxazosina/sangre , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/normas , Cromatografía Liquida/estadística & datos numéricos , Cisaprida/sangre , Cisaprida/normas , Doxazosina/farmacocinética , Doxazosina/normas , Humanos , Control de Calidad , Estándares de Referencia , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/normas , Espectrometría de Masas en Tándem/estadística & datos numéricosRESUMEN
Doxazosin mesylate is a selective alpha-adrenoreceptor antagonist for the treatment of hypertension and benign prostatic hyperplasia. A simple high performance liquid chromatographic method has been developed and validated for the quantitative determination of doxazosin in plasma. A reversed phase C18 column was used for the separation of doxazosin and prazosin (internal standard) with a mobile phase composed of water, acetonitrile, triethylamine (68:32:0.2 v/v, pH 5.0) at a flow rate of 1.2 mL/min. The fluorescence detector was operated at 246 (excitation) and 389 nm (emission). Intra- and inter-day precision and accuracy were acceptable for all quality control samples including the lower limit of quantification of 1 ng/mL. Recovery of doxazosin from human plasma was greater than 93.4%. Doxazosin was stable in human plasma under various storage conditions. This method was used successfully for a pharmacokinetic study in plasma after oral administration of multiple 4-mg dose of doxazosin gastrointestinal therapeutic system formulation to 16 healthy volunteers. At steady state the mean area under the curve for a dosing interval and elimination half-life were calculated to be 367.0 +/- 63.5 ng x hr/mL and 29.2 +/- 4.5 hr, respectively. There was no difference in pharmacokinetic parameters between male and female.
Asunto(s)
Antagonistas Adrenérgicos alfa/administración & dosificación , Antagonistas Adrenérgicos alfa/farmacocinética , Doxazosina/administración & dosificación , Doxazosina/farmacocinética , Antagonistas Adrenérgicos alfa/sangre , Adulto , Área Bajo la Curva , Pueblo Asiatico , Cromatografía Líquida de Alta Presión , Preparaciones de Acción Retardada , Doxazosina/sangre , Femenino , Semivida , Humanos , Corea (Geográfico) , Masculino , Factores SexualesRESUMEN
This study aims to develop a standard protocol for the relative bioavailability testing of doxazosin mesylate tablets. For this purpose, a simple rapid and selective LC-MS method using a single quadrupole mass spectrometer was developed and validated to determine the concentration of doxazosin mesylate in human plasma. Using this method, we carried out a study of relative bioavailability. N-Hexylane-tertiary butyl methyl ether (1:1, v/v) was used to extract doxazosin mesylate and terazosin (internal standard, I.S.) from an alkaline plasma sample. LC separation was performed on a Thermo Hypersil-Hypurity C18 (5 microm, 150 mm x 2.1mm) using aqueous solution (20 mmol/l ammonium acetate, pH 4.28), methanol and acetonitrile (55:10:35, v/v/v) as the mobile phase. The retention time of doxazosin and the internal standard was 2.7 and 1.8 min, respectively. Quadrupole MS detection was done by monitoring at m/z 388 (M+1) corresponding to doxazosin mesylate and at m/z 452 (M+1) for I.S. The assay method described above showed acceptable precision, accuracy, linearity, stability, and specificity. The bioavailability of doxazosin mesylate was evaluated in 12 healthy Chinese male volunteers. The following pharmacokinetic parameters were elucidated after administering a single dose of 4 mg doxazosin. The area under the plasma concentration versus time curve from time 0 to 72 h (AUC(0-72 h)) 743.4+/-149.5 ngh/ml; peak plasma concentration (C(max)) 47.66 ng/ml; time to C(max) (T(max)) 3.0+/-1.0 h; and elimination half-life (t(1/2)) 18-20 h. The method was successfully used to determine the relative bioavailability of doxazosin mesylate.
Asunto(s)
Antagonistas Adrenérgicos alfa/farmacocinética , Doxazosina/farmacocinética , Antagonistas Adrenérgicos alfa/administración & dosificación , Adulto , Área Bajo la Curva , Disponibilidad Biológica , Calibración , Cromatografía Liquida , Estudios Cruzados , Doxazosina/administración & dosificación , Doxazosina/sangre , Congelación , Semivida , Humanos , Masculino , Espectrometría de Masas , Estándares de Referencia , Reproducibilidad de los Resultados , Soluciones , Espectrometría de Masa por Ionización de Electrospray , Comprimidos , Equivalencia TerapéuticaRESUMEN
A weak cation-exchange monolithic column was prepared by modifying the GMA-EDMA (glycidyl methacrylate-co-ethylene glycol dimethacrylate) monoliths with ethylenediamine and monochloracetic acid. The properties of the column were investigated; the column exhibited the ability of low backpressure and fast analysis. Using this monolithic column, trace doxazosin in human serum albumin (HSA) solution and plasma samples has been on-line tested, the extraction efficiency and the maximum loading capacity of the monolithic column were obtained. The results showed that the monolithic column could realize deproteinization and trace drug enrichment simultaneously in the HSA solution and human plasma, which provided a simple, cheap, effective and friendly to environment method for assaying drugs in the blood.
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Cromatografía por Intercambio Iónico/instrumentación , Cromatografía por Intercambio Iónico/métodos , Doxazosina/análisis , Albúmina Sérica/aislamiento & purificación , Acetatos/química , Resinas de Intercambio de Catión , Doxazosina/sangre , Compuestos Epoxi/química , Etilenodiaminas/química , Humanos , Metacrilatos/química , Estructura Molecular , Reproducibilidad de los Resultados , Albúmina Sérica/química , Solventes/químicaRESUMEN
A sensitive, selective and rapid method for the analysis of doxazosine (DOX) in human plasma based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) is described. DOX and tamsulosin, used as an internal standard (IS), were extracted by liquid-liquid extraction, and the chromatography was performed on a C18 UPLC column packed with 1.7 mum particles. The total run time was 2 min. Detection was achieved by the multiple reaction monitoring of the following transitions: m/z 452-->344 and m/z 409-->228 for DOX and IS, respectively. Transitions of m/z 452-->247 and m/z 409-->271 were also collected for confirmation purposes. The calibration curve based on peak area ratio was linear up to at least 100 ng ml(-1), with a detection limit of 0.02 ng ml(-1) (a signal-to-noise ratio of 3). The method showed satisfactory reproducibility, and the short-term stability of the analyte was assessed. The method was successfully applied to the analysis of DOX in human plasma.
Asunto(s)
Doxazosina/sangre , Espectrometría de Masas/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Liquida/métodos , Humanos , Preparaciones Farmacéuticas/análisisRESUMEN
A highly sensitive high-performance liquid chromatographic quantification method with fluorescence detection was developed and validated for the determination of doxazosin in human plasma. The developed method employed one-step extraction of doxazosin from plasma matrix with ethyl acetate using propranolol as an internal standard. Chromatographic separation was obtained within 8.0 min using a reverse-phase Capcell-Pak C(18) column (150 x 4.6 mm i.d., 5 microm) and the mobile phase consisted of methanol-water containing 10 mM perchloric acid and 1.8 mM sodium heptane sulfonic acid (50:50, v/v) and was set at a flow rate of 1.5 mL/min. The calibration curve constructed was linear in the range of 0.3-50.0 ng/mL. The proposed method achieved a lower limit of quantification of 0.3 ng/mL, better than the reported HPLC methods. Average recoveries of doxazosin and the internal standard from human plasma matrix were 87.0 and 85.9%, respectively. The present method was validated by evaluating the precision and accuracy for inter- and intraday variation in the concentration range 0.3-50 ng/mL. The precision values expressed as relative standard deviations in the inter- and intraday validation were 1.17-6.29 and 0.84-5.94%, respectively. This method was successfully applied to the bioequivalence study of two doxazosin controlled release tablets in healthy, male human subjects.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Doxazosina/sangre , Preparaciones de Acción Retardada/análisis , Estabilidad de Medicamentos , Humanos , Masculino , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Equivalencia TerapéuticaRESUMEN
A simple high-performance liquid chromatographic method for the determination of doxazosin in human plasma was developed and validated. Prazosin was used as internal standard. After extraction twice with ethyl acetate, chromatographic separation of doxazosin in human plasma was carried out using a reversed-phase Apollo C18 column (250 x 4.6 mm, 5 microm) with mobile phase of methanol-acetonitrile-0.04 m disodium hydrogen orthophosphate (22:22:56, v/v/v) adjusted to pH 4.9 with 0.9 m phosphoric acid and quantified by fluorescence detection operated with an excitation wavelength of 246 nm and an emission wavelength of 389 nm. The lower limit of quantification (LLOQ) of this assay was 1 ng/mL using 500 microL human plasma. Linearity was established over the range 1-25 ng/mL (r2 > 0.9994). The intra- and inter-day accuracy ranged from 90.5 to 104.4% and the coefficient of variation were not more than 8.6% for both intra- and inter-day precision, over the range of the calibration curve. The absolute recoveries of doxazosin and prazosin from human plasma were more than 91%. Doxazosin demonstrated acceptable short-term, long-term and freeze-thaw stability in human plasma. The assay has been successfully applied to plasma sample ana-lysis for pharmacokinetic study.