An efficient spectrofluorimetric method adopts doxazosin, terazosin and alfuzosin coupling with orthophthalaldehyde: Application in human plasma.

A new, selective and sensitive spectrofluorimetric method was designed for the quantitation of doxazosin (DOX), terazosin (TER) and alfuzosin (ALF) in their dosage forms and human plasma. The method adopts efficient derivatization of the studied drugs with ortho-phthalaldehyde (OPA), in the presence of 2-mercaptoethanol in borate buffer (pH9.7) to generate a highly fluorescent isoindole derivatives, which can strongly enhance the fluorescence intensities of the studied drugs, allowing their sensitive determination at 430nm after excitation at 337nm. The fluorescence-concentration plots were rectilinear over the ranges (10.0-400.0) ng/mL. Detection and quantification limits were found to be (0.52-3.88) and (1.59-11.76) ng/mL, respectively. The proposed method was validated according to ICH guidelines, and successfully applied for the determination of pharmaceutical preparations of the studied drugs. Moreover, the high sensitivity of the proposed method permits its successful application to the analysis of the studied drugs in spiked human plasma with % recovery (96.12±1.34-100.66±0.57, n=3). A proposal for the reaction mechanism was presented.

Highly sensitive spectrofluorimetric method for determination of doxazosin through derivatization with fluorescamine; Application to content uniformity testing.

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).

Rayleigh light scattering detection of three α1-adrenoceptor antagonists coupled with high performance liquid chromatograph.

Herein, a Rayleigh light-scattering (RLS) detection method combined with high performance liquid chromatograph (HPLC) without any post-column probe was developed for the separation and determination of three α1-adrenoceptor antagonists. The quantitative analysis is benefiting from RLS signal enhancement upon addition of methanol which induced molecular aggregation to form an hydrophobic interface between aggregates and water that produce a sort of superficial enhanced scattering effect. A good chromatographic separation among the compounds was achieved using a Gemini 5u C18 reversed phase column (250 mm × 4.6 mm; 4 µm) with a mobile phase consisting of methanol and ammonium acetate-formic acid buffer solution (25 mM; pH=3.0) at the flow rate of 0.7 mL min(-1). The RLS signal was monitored at λex=λem=354 nm. A limit of detection (LOD) of 0.065-0.70 µg L(-1) was reached and a linear range was found between peak height and concentration in the range of 0.75-15 µg L(-1) for doxazosin mesylate (DOX), 0.075-3.0 µg L(-1) for prazosin hydrochloride (PRH), and 0.25-5 µg L(-1) for terazosin hydrochloride (TEH), with linear regression coefficients all above 0.999. Recoveries from spiked urine samples were 88.4-99.0% which is within acceptable limits. The proposed method is convenient, reliable and sensitive which has been used successfully in human urine samples.

Mixed micelle cloud point-magnetic dispersive µ-solid phase extraction of doxazosin and alfuzosin.

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.

Rapid and sensitive online determination of some selective α1-blockers by flow injection analysis with micelle-enhanced fluorescence detection.

A rapid, sensitive and selective flow injection analysis (FIA) method was developed for the determination of some selective α1-blockers including; terazosin (TER), doxazosin (DOX), prazosin (PRZ), and alfuzosin (ALF). The method was based on enhancement of the native fluorescence of the studied drugs in the presence of sodium dodecyl sulfate (SDS). The method was optimized for the buffer type, concentration and pH, surfactant type and concentration, flow rate and detection wavelengths in order to achieve the maximum sensitivity. The results showed that the best sensitivity was obtained by using SDS (10 mM) in phosphate buffer (20 mM, pH = 3), flow rate was 0.5 ml/min and the detector was set at λex = 250 and λem = 389. Under these optimum conditions there was a linear relationship between the concentration and the fluorescence intensity in the range from 5-400 ng ml(-) with correlation coefficient of more than 0.998. The detection and quantitation limits for the studied drugs by the proposed method were 3.2-11.9 ng ml(-1) and 10.8-39.7 ng ml(-1), respectively. The method was validated in accordance with the requirements of ICH guidelines and shown to be suitable for intended applications. Moreover, the binding constants for α1-blockers -SDS system were determined using the adduct model. The proposed method has been applied successfully for the analysis of the pure forms for studied drugs and also their pharmaceutical formulations and the results were compared with official methods.

Transfer of doxazosin into breast milk.

To the best of our knowledge, there have been no published studies of doxazosin transfer into human milk. In rats, milk concentrations twentyfold higher than in plasma have been reported. Based on these animal data, some references advise to avoid breastfeeding during doxazosin therapy. However, the physicochemical properties of doxazosin suggest low transfer into human milk. A 37-year-old breastfeeding woman who was administered doxazosin 4 mg daily for 2 doses was studied. Doxazosin concentrations in milk and plasma were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The milk/plasma area under the concentration-time curve (AUC0-18 hours) ratio was 0.1. This finding is consistent with what could be predicted based on the physicochemical properties of doxazosin. The average and maximum milk concentrations were 2.9 and 4.2 µg/L. These values correspond to estimated relative infant doses of 0.06% and 0.09%, respectively, assuming standard infant milk intake. These values are well below the generally accepted cutoff of 10% for predicting safety during breastfeeding. A low relative infant dose of < 0.1% suggests that maternal doxazosin therapy may be compatible with breastfeeding after careful individual risk-benefit analysis.

Spectrophotometric determination of doxazosin mesylate in tablets by ion-pair and charge-transfer complexation reactions.

Two accurate, easy spectrophotometric methods for the determination of doxazosin mesylate were described. The first method was based on the formation of ion-pair complexes with the acidic sulfophthalein dyes bromocresol purple (BCP) and bromophenol blue (BPB) in pH 3.3 and 4.5 citrate-phosphate buffer, respectively. The formed complexes were extracted into dichloromethane, and their absorbance was measured at 403 and 410 nm for BCP and BPB, respectively. The second method was based on the charge transfer reaction of the drug as an n-electron donor with either 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or 7,7,8,8-tetracyanoquinodimethane (TCNQ) as pi-acceptors, to give colored radical anions. The absorbances of products were measured at 457 nm in acetonitrile and 838 nm in methanol for DDQ and TCNQ, respectively. Under the optimum reaction conditions, Beer's law was obeyed with a good correlation coefficient (r = 0.9997-0.9999) in the concentration ranges 3.0-18.0, 3.0-20.0, 15.0-95.0, and 10.0-100.0 microg/mL for the BCP, BPB, DDQ, and TCNQ methods, respectively. Limits of detection of the BCP, BPB, DDQ, and TCNQ methods were 0.314, 0.408, 1.935, and 1.610 microg/mL, respectively. The limits of quantification were 1.045, 1.360, 6.449, and 5.367 microg/mL, respectively. The parameters molar absorptivity, precision, accuracy, recovery, robustness, and stability constant were studied. The proposed methods were successfully applied for determination of the drug in tablets with good accuracy and precision. Statistical comparison of the results with those obtained by a reported method showed good agreement and indicated no significant difference in accuracy and precision.

Separation, characterization, and quantitation of process-related substances of the anti-hypertensive drug doxazosin mesylate by reversed-phase LC with PDA and ESI-MS as detectors.

A simple and rapid reversed-phase liquid chromatography (LC) method with photodiode array (PDA) and electrospray ionization (ESI)-mass spectrometry (MS) as detectors was developed and validated to separate, identify, and quantitate the related substances of Doxazosin mesylate (DXZN) for monitoring the reactions involved during process development. The high-performance liquid chromatography profiles of related-substances of DXZN are used as fingerprints to follow the procedures used in the manufacturing units. The separation is accomplished on an Inertsil ODS-3 column with acetonitrile-ammonium acetate (10 mM, pH 4.0) as the mobile phase, using a gradient elution mode and monitoring the eluents by a photodiode array detector at 265 nm at ambient temperature. LC-ESI-MS-MS is used to identify the additional impurities formed during the synthesis. The identified impurities were synthesized and characterized by UV, Fourier transform-IR, 1H NMR, and MS data. The detection limits for the impurities are 0.74 - 4.14 x 10(-9) g, and the method is found to be suitable not only for the monitoring of synthetic reactions, but also for quality assurance of DXZN in bulk drugs and formulations.

On-line simultaneous removal of human serum albumin and enrichment of doxazosin using a weak cation-exchange monolithic column.

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.

Enantiomeric resolution of doxazosin mesylate and its process-related substances on polysaccharide chiral stationary phases.

High-performance liquid chromatographic methods for separation of racemic doxazosin mesylate and its synthetic precursors on polysaccharide based stationary phases viz., amylose tris-(3,5-dimethylphenylcarbamate) (Chiralpak AD-H) and cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD-H) were developed. The base line separation with Rs>1.50 was obtained using a mobile phase containing n-hexane-alcohol-0.1% diethylamine (ethanol, 1-propanol and 2-propanol) in various proportions. The effect of concentration of the alcoholic modifiers on the resolution was studied. A good separation was achieved on amylose based Chiralpak AD-H column when compared with cellulose based Chiralcel OD-H. The effects of structural features of the solutes and solvents on discrimination between the enantiomers were examined. The detection was carried out at 240 nm with UV detector while identification by polarimetric detector connected in series. The method was suitable not only for process development of doxazosin mesylate but also determination of enantiomeric purity of bulk drugs and pharmaceuticals.

Validated specific HPLC methods for determination of prazosin, terazosin and doxazosin in the presence of degradation products formed under ICH-recommended stress conditions.

The present paper describes development of stability-indicating high-performance liquid chromatographic (HPLC) assay methods for three alpha-adrenergic-blocker drug substances, namely, prazosin, terazosin and doxazosin, in the presence of degradation products generated from forced decomposition studies. Resolution of drugs from degradation products was obtained using a reversed-phase C-18 column using water/acetonitrile/methanol/glacial acetic acid/diethylamine (25:35:40:1:0.017) as mobile phase for prazosin and terazosin and acetonitrile/water/glacial acetic acid/diethylamine (65:35:1:0.02) for doxazosin. The detection was done at 254 nm. The methods were validated with respect to linearity, precision, accuracy, specificity and robustness.

Stability-indicating methods for the determination of doxazosin mezylate and celecoxib.

Two stability-indicating methods were developed for the determination of doxazosin mesylate (I) and celecoxib (II) in the presence of their degradation products. The first method depends on the use of first derivative spectrophotometry (D(1)) at 256, 269 nm for (I) and (II), respectively. This method determines (I) and (II) in concentration ranges of 0.8-12 and 1-20 microg ml(-1) with mean percentage accuracies of 99.21+/-0.88 and 99.59+/-1.67% for (I) and (II), respectively. The second method depends on the quantitative densitometric evaluation of thin-layer chromatography of (I) and (II) in the presence of their degradation products without any interference. Methylisobutyl ketone-glacial acetic acid-water (20:10:10) was used as a mobile phase for (I) and cyclohexane-dichloromethane-diethyleamine (50:40:10) for (II). The chromatograms were scanned at 248 and 253 nm for (I) and (II), respectively. This method determines (I) and (II) in concentration ranges of l-4 microg per spot for both drugs with mean percentage accuracies of 100.19+/-0.95 and 99.91+/-1.95% for (I) and (II), respectively. The suggested methods were used to determine doxazosin mesylate and celecoxib in bulk powder, laboratory-prepared mixtures and pharmaceutical dosage forms (cardura tablet and celebrex capsule). The results obtained by applying the proposed methods were statistically analysed and compared with those obtained by the reported methods.

Flow injection analysis of doxazosin mesylate using UV-detection.

A flow injection analysis (FIA) of doxazosin mesylate (DOX) using UV detection is described in this study. The best solvent system was found to be consisting of 0.1 mol l(-1) acetate buffer at pH 4 having 10%MeOH. A flow rate of 1 ml min(-1) was pumped and active material was detected at 365 nm. The calibration equation was linear in the range of 1.3 x 10(-5) to 6.4 x 10(-5) mol l(-1). Limit of detection and limit of quantitation were calculated to be 1.6 x 10(-6) and 4 x 10(-6) mol l(-1) with a RSD 1.27 and 1.16% (n=8), respectively. The proposed method was applied to the determination of DOX in the pharmaceutical preparations. The results were compared with those obtained from UV-Spectrophotometry. The results showed that there is a good agreement between FIA method and UV-Spectrophotometry. The validation studies were realised by the related applications and the results were evaluated statistically. According to the results, insignificant difference was observed between the methods.

Voltammetric determination of doxazosin in tablets using rotating platinum electrode.

This study describes the voltammetric behaviour of doxazosin molecule based on the oxidation on the surface of platinum electrode in the stationary and rotating conditions and determine doxazosin in the tablets by differential pulse technique at only rotating condition. The experiments were carried out in the supporting electrolyte consisting of 0.2 M KCl and 0.2 M buffer solution in 10% (v/v) ethanol. The effect of initial potential was investigated and no adsorption effect was observed during use of +500 mV. The influence of pH on the peak current and peak potential was examined and the most symmetrical peaks were obtained at 0.5 M H2SO4 for rotating conditions. In the rotation range of 50-1000 rpm and up to 1.0x10(-5) M doxazosin, the factor affecting the voltammetric current was diffusional. The effect of rate of potential was tested between 2 and 20 mV for the stationary condition and the character of current was found to be diffusional up to 3x10(-5) M concentration of doxazosin solutions. The voltammetric determination of doxazosin in tablets was realised in the optimum rotating system conditions and depending on the statistical evaluations, acceptable results were obtained. Therefore, the method proposed in this study is practical, sensitive and accurate for the analysis of doxazosin in the quality control laboratories.

Pulse polarographic (constant and increasing) determinations of doxazosin in pharmaceutical tablets.

The optimum conditions using DC polarography and the determination of doxazosin employing SIAP and SCAP polarographic techniques are described in this study. All the experiments were conducted in the supporting electrolyte consisting of 20% ethanol (v/v), 0.2 M KCl and 0.2 M acetate buffer at various pH values in order to examine the optimum conditions, and pH 3.5 for the determination of doxazosin. Well-defined curves were obtained in the pH range of 1.5-7.5. The system was diffusional and irreversible at pH 3.5. The calibration studies were performed by using SIAP and SCAP polarography and satisfactory results were observed for all techniques. Since the sensitivity of SIAP and SCAP techniques were higher than the others, the determination of doxazosin was performed in filtered and unfiltered tablet solutions containing 4 mg active material. In the analysis of a tablet, the relative standard deviations (Srel %) of the techniques are in the filtered solutions +/- 0.9 (SIAP), +/- 0.8 (SCAP) and in the unfiltered solutions +/- 0.7 (SIAP), +/- 0.8 (SCAP) and no interference was observed during the analysis. The determination methods proposed in this study appear to be accurate, rapid and practicable. Therefore, these techniques may be suitable for the content uniformity tests.