SERS determination of the antihypertensive drugs prazosin and losartan by using silver nanoparticles coated with ß-cyclodextrin.

A surface-enhanced Raman scattering (SERS) method is described for the determination of prazosin (PRH) and losartan (LOS). Silver nanoparticles modified with ß-cyclodextrin (CD-S-Ag NPs) were prepared and serve as a sensitive SERS substrate. ß-CD is both a reductant for silver ions and a host molecule that binds the analytes which leads to strong SERS enhancement. The method has distinct features: (a) The linear response extends from 0.1 to 60 µM for PRH, and from 1.0 to 100 µM for LOS; (b) the respective limits of detection are as low as 15 nM and 0.92 µM; and (c) the specific SERS bands of PRH and LOS are located at 703 and 1298 cm-1 respectively. The method was successfully applied to the determination of PRH and LOS illegally added to healthcare products. The recovery of PRH and LOS from spiked samples ranges between 91.3 and 109.3%, and from 87.4 to 105.2%, respectively, both with relative standard deviation of <5%. Graphical abstractSchematic representation of a SERS method involving ß-CD-S-Ag nanoparticles for determination of prazosin and losartan via formation of an inclusion complex.

Utility of Hantzsch reaction for development of highly sensitive spectrofluorimetric method for determination of alfuzosin and terazosin in bulk, dosage forms and human plasma.

A highly sensitive, cheap, simple and accurate spectrofluorimetric method has been developed and validated for the determination of alfuzosin hydrochloride and terazosin hydrochloride in their pharmaceutical dosage forms and in human plasma. The developed method is based on the reaction of the primary amine moiety in the studied drugs with acetylacetone and formaldehyde according to the Hantzsch reaction, producing yellow fluorescent products that can be measured spectrofluorimetrically at 480 nm after excitation at 415 nm. Different experimental parameters affecting the development and stability of the reaction products were carefully studied and optimized. The fluorescence-concentration plots of alfuzosin and terazosin were rectilinear over a concentration range of 70-900 ng ml-1 , with quantitation limits 27.1 and 32.2 ng ml-1 for alfuzosin and terazosin, respectively. The proposed method was validated according to ICH guidelines and successfully applied to the analysis of the investigated drugs in dosage forms, content uniformity test and spiked human plasma with high accuracy.

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.

Electrochemical Oxidation of Different Therapeutic Classes of Pharmaceuticals Using Graphite-PVC Composite Electrode.

This study reports electrochemical treatment of different therapeutic classes of pharmaceuticals (caffeine, prazosin, enalapril, carbamazepine, nifedipine, levonorgestrel, and simvastatin) in a mixture. The electrochemical process was investigated using graphite-PVC anode at different applied voltages (3, 5, and 12 V), initial concentrations of studied pharmaceuticals in aqueous solution (5 and 10 mg/L), and concentrations of sodium chloride (1 and 2 g/L). The % removal of pharmaceuticals increased with the applied voltage, and was found higher than 98% after 50 min of electrolysis at 5 V. Energy consumption ranged between 0.760 and 3.300 Wh/mg using 12 V being the highest value compared to 3 and 5 V. The formation of chlorinated by-products from four selected pharmaceuticals, simvastatin (C11H13Cl3O5, and C10H12Cl4O3), prazosin (C13H12Cl3N5O3 and C10H11Cl4N2O2), carbamazepine and caffeine (C15H11N2O2Cl and C8H9N4O2Cl) was identified and elucidated using liquid chromatography-time of flight mass spectrometry (LC-TOF/MS).

Updating the European Pharmacopoeia impurity profiling method for terazosin and suggesting alternative columns.

This work was motivated by the demand of European Directorate for the Quality of Medicines and HealthCare (EDQM). A new liquid chromatographic (LC) method was developed for terazosin impurity profiling to replace the old European Pharmacopoeia (Ph. Eur.) method. This new method is published as part of the new Ph. Eur. monograph proposal of terazosin in Pharmeuropa issue 32.2. The aim of the method renewal was to cut the analysis time from 90 min (2 × 45 min) down to below 20 min. The Ph. Eur. monograph method is based on two different chromatographic separations to analyze the specified impurities of terazosin. The reason for the two methods is that two of the impurities are not sufficiently retained in reversed phase (RP) conditions, not even with 100% water as eluent. Therefore, next to RP, an ion-pair (IP) chromatographic method has to be applied to analyze those two impurities. With our new proposed method it was possible to appropriately increase the retention of the two critical compounds using alternative stationary phases (instead of a C18 phase which is suggested by the Ph. Eur. method). Applying a pentafluoro-phenyl (PFP) stationary phase, it was feasible to separate and adequately retain all the impurities. The detection wavelength was also changed compared to the Ph. Eur. method and is now appropriate for the detection and quantification of all impurities using perchloric acid in the mobile phase at low pH. Another goal of the present study was to develop a generic workflow and to evaluate the chromatographic resolution in a wide range of method variables and suggest some replacement columns for terazosin impurity profiling. Retention modeling was applied to study the chromatographic behavior of the compounds of interest and visualize resolution for the different columns, where a given criterion is fulfilled. A zone (set of chromatographic conditions) of a robust space could be then quickly identified by the overlay of the individual response surfaces (resolution maps). It was also demonstrated that two columns from different providers (Kinetex F5 and SpeedCore PFP) can be used as replacement columns, providing sufficient resolution at the same working point and a high degree of robustness.

Determination of terazosin in the presence of prazosin: Different state-of-the-art machine learning algorithms with UV spectroscopy.

Counterfeit drugs have adverse effects on public health; chromatographic methods can be used but they are costly. In this study, we developed cost-effective and environmentally friendly methodology for the analysis of terazosin HCl (TZ) in the presence prazosin hydrochloride (PZ) using UV spectroscopy in conjunction with machine learning (ML) models. Variable selection algorithms were applied to select most informative spectral variables. Thirty-five ML models were assessed and their performances were compared. The models covered a wide range of prediction mechanisms, such as tree-based, linear, self-organizing maps, neural network, Gaussian process, boosting, bagging, Bayesian models, kernel methods, and quantile regression. The values of the root mean square error (RMSE), coefficient of determination (R2), and absolute mean error (MAE) were obtained for the evaluation of the developed models. According to the results of these performance indices, linear model showed the highest prediction capacity among all other models. RMSE, R2 and MAE values of (0.159, 0.997 and 0.131) and (0.196, 0.99 and 0.161) were obtained for train and test datasets, respectively. The predictive models in this study can be useful for the researchers who are interested to work on the determination of active ingredients in pharmaceutical dosage forms in the presence of interference using UV spectroscopy; therefore, it was used to determine TZ without interference of PZ.

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.

Palladium nanoparticles in electrochemical sensing of trace terazosin in human serum and pharmaceutical preparations.

In this approach, palladium nanoparticle film was simply fabricated on the surface of carbon paste electrode by electrochemical deposition method. The film was characterized using scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The prepared electrode exhibited an excellent electrocatalytic activity toward detection of trace amounts of terazosin, which is an antihypertensive drug. Under the optimum experimental conditions, a linear range of 1.0×10-8-1.0×10-3molL-1 with a detection limit of 1.9×10-9molL-1 was obtained for determination of terazosin using differential pulse voltammetry as a sensitive method. The efficiency of palladium nanoparticle film on the surface of carbon paste electrode successfully proved for determination of terazosin in pharmaceutical sample and human serum sample with promising recovery results. The effect of some foreign species has been studied.

Renewal of an old European Pharmacopoeia method for Terazosin using modeling with mass spectrometric peak tracking.

An older method for terazosin was reworked in order to reduce the analysis time from 90min (2×45min) to below 5min. The method in European Pharmacopoeia (Ph.Eur.) investigates the specified impurities separately. The reason of the different methods is that the retention of two impurities is not adequate in reversed phase, not even with 100% water. Therefore ion-pair-chromatography has to be applied and since that two impurities absorb at low UV-wavelength they had to be analyzed by different method than the other specified impurities. In our new method we could improve the retention with pH elevation using a new type of stationary phases available for high pH applications. Also a detection wavelength could be selected that is appropriate for the detection and quantification of all impurities. The method development is the bottleneck of liquid chromatography even today, when more and more fast chromatographic systems are used. Expert knowledge with intelligent programs is available to reduce the time of method development and offer extra information about the robustness of the separation. Design of Experiments (DoE) for simultaneous optimization of gradient time (tG), temperature (T) and ternary eluent composition (tC) requires 12 experiments. A good alternative way to identify a certain peak in different chromatograms is the molecular mass of the compound, due to its high specificity. Liquid Chromatography-Mass Spectrometry (LC-MS) is now a routine technique and increasingly available in laboratories. In our experiment for the resolution- and retention modeling the DryLab4 method development software (Version 4.2) was used. In recent versions of the software the use of (m/z)-MS-data is possible along the UV-peak-area-tracking technology. The modelled and measured chromatograms showed excellent correlations. The average retention time deviations were ca. 0.5s and there was no difference between the predicted and measured Rs,crit -values.

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.

Determinations of tiodazosin and levulinic acid from tablets by high-performance liquid chromatography.

Specific assays of tiodazosin and levulinic acid from tablet preparations were developed using high-performance liquid chromatography. Tiodazosin was determined with acetonitrile 50% (v/v) in pH 5.7 acetate buffer on a 300 X 3.9-mm i.d. microparticulate C-18 column using 254 nm detection with butylparaben as the internal standard. Levulinic acid was determined at 280 nm with 1 N acetic acid on a 300 X 3.9-mm i.d. microparticulate phenyl column using an external standard. The assays were run sequentially. The relative standard deviations of sample variability (2 sigma, n = 6) and recoveries from synthetic preparation were +/- 1.6 and approximately 95%, respectively, for both assays.

Spectrophotometric determination of some pharmaceutical piperazine derivatives through charge-transfer and ion-pair complexation reactions.

Simple and sensitive spectrophotometric methods are described for the assay of three piperazine derivatives; ketoconazole, piribedil and prazosin hydrochloride based on charge-transfer and ion-pair complexation reactions. The first method is based on the reaction of the basic drug with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) in acetonitrile. The orange-red colour formed due to the formation of charge-transfer complex showed maximum absorbance at 460 nm. The second method is based upon the interaction of the basic drug in dry chloroform with bromophenol blue (BPB) in the same solvent to produce a stable yellow ion-pair complex which absorbs at 410 nm. Beer's law was obeyed for both methods and the relative standard deviations were found to be less than 1%. The two methods can be applied to the analysis of tablets, with no evidence of interference from excipients. A more detailed investigation of the complex was made with respect to its composition, association constant and free energy change.

Ion-selective electrode for the determination of prazosin in tablets.

A poly (vinylchloride) (PVC) membrane electrode selective for prazosinium cation based on prazosinium-phosphotungstate (PZ)(3)PT ion-associate is prepared. The electrode exhibits a linear response with an approximate Nernstian slope over the range of 2.7 x 10(-6)-10(-2) M. Calibration graph slope of the electrode is 58 mV PZ(-1) concentration decade when preconditioned by soaking in distilled water for 1 h-20 days. The working pH of the electrode ranged from 1.5 to 6.4 and exhibits very good selectivity for the PZ with respect to a large number of inorganic cations and organic substances of biological importance. The standard additions method and potentiometric titration are used to determine the PZ in pure solution and in pharmaceutical preparations with satisfactory results.

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.

Determination of alpha 1-adrenoceptor antagonists in plasma by radioreceptor assay.

A simple, rapid and sensitive radioreceptor assay (RRA) for the quantification of alpha 1-adrenoceptor antagonists such as prazosin in plasma is described. The method involves the use of an RRA based on [3H]prazosin displacement in rat cerebral cortical membranes. The method is reliable, with intra-assay and inter-assay RSDs ranging from 5.9 to 9.2%. The limit of detection is 0.2 (prazosin hydrochloride), 0.05 (tamsulosin hydrochloride) and 0.3 (bunazosin hydrochloride) pmol per assay. Using this method the plasma levels of prazosin hydrochloride were determined in beagle dogs administered orally 2.39 mumol kg-1 of this drug. The plasma levels of prazosin in beagle dogs are in good agreement with those obtained using a high-performance liquid chromatography (HPLC). This RRA proved to be applicable to the monitoring of plasma prazosin levels in patients with essential hypertension and/or benign prostatic hypertrophy receiving therapy with this drug with the therapeutic dosage schedule. Thus, the concentrations of alpha 1-adrenoceptor antagonists in plasma can be adequately monitored by RRA as well as by HPLC.

Voltammetric and spectrophotometric techniques for the determination of the antihypertensive drug Prazosin in urine and formulations.

A sensitive method was developed to determine Prazosin using a nafion modified carbon paste electrode (NMCPE). Prazosin was accumulated at a potential of 750 mV in Britton-Robinson buffer (pH 6.0) and then a negative sweep was made obtaining a cathodic peak close to 0 V. Cyclic voltammetric studies indicated that the process was quasi-reversible, and fundamentally controlled by adsorption. To obtain a good sensitivity, the instrumental and accumulation variables were studied using differential pulse voltammetry (DPV). Adsorptive voltammetric peak currents showed a linear response for Prazosin concentrations in the range between 4.0 x 10(-11) and 4.0 x 10(-8) M with two different slopes, and a detection limit (LOD) of 3.1 x 10(-11)M was obtained. The variation coefficient (CV) for a 8.0 x 10(-10) M solution (n = 10) was 4.08%. A spectrophotometric study of Prazosin was also carried out and two absorption bands were obtained at 246 and 329 nm (pH 1.8). The band at 329 nm was pH-dependent and its height and position changed with the pH values, so this allowed the pK'a determination (7.14 +/- 0.20) using different methods. The detection limit reached by means of UV-spectrophotometry was 0.9 x 10(-7) M, and the variation coefficient for 1.5 x 10(-5) M Prazosin solutions was 1.14% (n = 10). Although the sensitivity of the UV-spectrophotometric method was lower than that obtained using adsorptive stripping-differential pulse voltammetry (AdS-DPV), it could be applied to the determination of Prazosin in Minipres tablets. The voltammetric method was used for the determination of the drug in human urine samples at trace levels with good recoveries.

Sensitive fluorimetric method based on sequential injection analysis technique used for dissolution studies and quality control of prazosin hydrochloride in tablets.

This report introduces a fully automated flow system for drug-dissolution studies based on the coupling of the sequential injection analysis (SIA) technique with a conventional dissolution apparatus. The methodology described was used for monitoring of dissolution profiles of prazosin hydrochloride (PRH) in pharmaceutical formulation. The very sensitive fluorimetric detection of PRH was performed at lambda(ex)=244 nm (lambda(em)>or=389 nm). Under the optimal conditions, the calibration curve was linear over the range 0.02-2.43 mg x l(-1) of PRH with R.S.D. 1.89, 1.23, and 1.80% (n=10) when determining 0.02, 1.22, and 2.43 mg x l(-1) of PRH in standard solutions, respectively. Equation of the calibration curve was calculated giving the following values: F=4.108 c-3.9 (n=6), r=0.9996. Detection limit was calculated 0.007 mg x l(-1) of PRH. The dissolution test of Deprazolin tablets was programmed for 60 min, with a continuous sampling rate of 70 h(-1) under conditions required by USP 26. Results obtained by SIA technique compared well with HPLC standard method.

Visual and quantitative screening of α1-adrenoceptor antagonists in living cells using quantum dots.

The performance of α1-adrenoceptor antagonists in living cells was assessed using quantum dots conjugated to a derivative of the α1-adrenoceptor antagonist prazosin. The optimum receptor binding condition and apparent Kd of prazosin-conjugated quantum dots was first determined, followed by application of these structures to drug screening. Total internal reflection fluorescence microscopy and flow cytometry were used to visually and quantitatively measure the affinity of five candidate drugs. The observed affinity order and the affinity coefficient Ki were consistent with previously reported values. These results suggest that this method is suitable for specific drug screening in living cells and is able to realize the displacement assay over the large ranges of dissociation constants.

Dual effects of α2 -adrenoceptors in modulating myogenic tone in sheep isolated internal anal sphincter.

BACKGROUND: The role of α-adrenoceptors in promoting continence through modulation of sphincter tone has focused primarily on the effects of α1 -adrenoceptors. We have used three clinically available agents, which are selective for α2 -adrenoceptors, to investigate their role in contractile and neurogenic responses on the internal anal sphincter (IAS). METHODS: IAS strips, which had spontaneously generated tone, were used to investigate the contractile effect of lofexidine, brimonidine, and dexmedetomidine on muscle tone in the presence or absence of subtype selective antagonists. The effect of brimonidine on the magnitude and time course of neurogenic responses generated by electrical field stimulation (EFS) was also examined. The affinity of test compounds at α1 - and α2 -adrenoceptors was established by competition binding with [3H]-prazosin and [3H]-RX821002. KEY RESULTS: All agonists caused concentration-dependent contraction of the IAS and lofexidine demonstrated an enantiomeric difference in potency with a 10-fold difference between the (-) and (+) isomers. Responses to lofexidine and dexmedetomidine were inhibited in the presence of the α1 -adrenoceptor selective antagonist prazosin, but not in the presence of RX811059 (α2 -adrenoceptor selective antagonist); brimonidine responses were inhibited by RX811059 and, to a lesser extent, by prazosin. Brimonidine affected both magnitude and duration of neurogenic responses, which was reversed in the presence of RX811059. CONCLUSIONS & INFERENCES: We conclude that α2 -adrenoceptors can mediate contraction of IAS, although this effect is most evident with efficacious imidazoline agonists rather than the most selective ligand. In addition, this receptor subtype can directly inhibit noradrenergic contractile responses to EFS and, indirectly, enhance nitrergic relaxatory responses.

Preparation and physicochemical characterization of prazosin conjugated PLGA nanoparticles for drug delivery of flutamide

In the current work, a sustained drug delivery system of flutamide (FLT) was developed using Poly(D,L-lactide-co-glycolide) (PLGA) decorated bypoly(ethylene glycol) (PEG) grafted prazosin (PLGA-PEG-Praz) as a targeting moiety. In a multi-step reaction, PLGA was linked to PEG and prazosin. The structure of the synthesized polymers was confirmed by FTIR and 1H-NMR. Flutamide-loaded nanoparticles were prepared by quasi-emulsion solvent diffusion technique. The nanoparticles were evaluated for size, zeta potential, polydispersity index, drug crystallinity, loading efficiency, and release properties. Also, the physicochemical properties of the nanoparticles were analyzed using Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry, and Powder X-Ray Diffractometry (XRD). The particle size of nanoparticles was ranged between 191 and 249 nm. Loading efficiency of nanoparticles was about 43%-69%. Results showed a steady release rate for nanoparticles compared to that of a pure drug powder. SEM characterization confirmed that particles were in nanosize range. DSC and XRPD results verified a decrease in drug crystallinity in the prepared formulations. In conclusion, the results of this study showed that PLGA-PEG-Praz nanoparticles could be a good choice to improve the physicochemical properties of the drug and these formulations can increase Flutamide efficacy.