A validated stability-indicating reversed-phase-HPLC method for dipyridamole in the presence of degradation products and its process-related impurities in pharmaceutical dosage forms.

In this study, we developed and validated a method to determine dipyridamole-related impurities in pharmaceutical dosage forms using the reversed-phase-HPLC technique. All impurities were separated on a YMC pack C8 (150 mm × 4.6 mm, 3.0 µm) analytical column using a suitable mobile phase. Mobile phase A was 10 mM concentration of phosphate buffer (pH adjusted to 4.7 by adding diluted orthophosphoric acid) and mobile phase B was buffer:acetonitrile:methanol (at the ratio of 30:40:30 v/v). The optimized chromatographic conditions used in the experiment were as follows: flow rate, 1.0 mL/min; injection volume, 10 µL and column temperature, 35°C. Chromatographic detection was performed at 295 nm. The stressed samples were analyzed for degradation under acidic, basic, peroxide, water hydrolysis, and physical degradation conditions. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines, and found to be specific, linear, accurate and have a robust stability-indicating nature. The method showed excellent linearity from limit of quantification (LOQ) to 150% level of concentrations for all impurities. The correlation coefficient (r2 ) for all impurities was between 0.995 and 0.999. The recovery study was performed from LOQ to 150% level concentrations, with mean recovery values between 92.9% and 103.2%, respectively. The developed method can be used to determine dipyridamole and its relative impurities. The degradation and validated study results indicate its stability-indicating nature. Therefore, the method can be used in pharmaceutical research and development and quality control departments.

Chemometrics Tools in Detection and Quantitation of the Main Impurities Present in Aspirin/Dipyridamole Extended-Release Capsules.

Aspirin (ASP) and dipyridamole (DIP) in combination is widely used in the prevention of secondary events after stroke and transient ischemic attack. Salicylic acid is a well-known impurity of ASP, and the DIP extended-release formulation may contain ester impurities originating from the reaction with tartaric acid. UV spectral data analysis of the active ingredients in the presence of their main impurities is presented using multivariate approaches. Four chemometric-assisted spectrophotometric methods, namely, partial least-squares, concentration residuals augmented classical least-squares (CRACLS), multivariate curve resolution (MCR) alternating least-squares (ALS), and artificial neural networks, were developed and validated. The quantitative analyses of all the proposed calibrations were compared by percentage recoveries, root mean square error of prediction, and standard error of prediction. In addition, r(2) values between the pure and estimated spectral profiles were used to evaluate the qualitative analysis of CRACLS and MCR-ALS. The lowest error was obtained by the CRACLS model, whereas the best correlation was achieved using MCR-ALS. The four multivariate calibration methods could successfully be applied for the extended-release formulation analysis. The application results were also validated by analysis of the stored dosage-form solution, which showed a susceptibility of DIP esterification in the extended-release formulation. Statistical comparison between the proposed and official methods showed no significant difference.

Formulation and evaluation of gastroretentive floating drug delivery system of dipyridamole.

A multiple-unit floating alginate bead drug delivery system with prolonged stomach retention time was developed in this study. The floating alginate beads were prepared by ionic cross-linking method, using CaCO3 as the gas-forming agent. Over 92% of the beads remained floating after 9 h. In order to prepare sustained-release dosage forms of dipyridamole, the solid dispersion technique was applied using a blend of Eudragit L100 and Eudragit RLPO. Afterwards, the solid dispersions of dipyridamole were incorporated into the floating alginate beads. The drug release was modified by changing the ratio of Eudragit RLPO and Eudragit L100 in the solid dispersions. The in vivo results showed that the relative bioavailability of alginate beads was enhanced by approximately 2.52-fold compared with that of the commercial tablet. Therefore, our study illustrated the potential use of floating alginate beads combined with the solid dispersion technique for the delivery of acid-soluble compounds, such as dipyridamole.

Determination of dipyridamole using TCPO-H2O2 chemiluminescence in the presence of silver nanoparticles.

The method is based on the fact that dipyridamole can enhance the chemiluminescence (CL) emission from the redox reaction of bis (2,4,6-tricholorophenyl) oxalate (TCPO) with H(2)O(2) in the presence of silver nanoparticles (AgNPs). The CL reaction mechanism was discussed. The effect of concentrations of TCPO, H(2)O(2), AgNPs and pH value on the CL reaction were investigated. Under the optimum conditions, the linear dynamic range was 1.0-1000 × 10(-9) g/mL and the detection limit (3σ) was 9 × 10(-10) g/mL. The relative standard deviation (RSD) was 4.8% for 1.0 × 10(-9) g/mL dipyridamole (n = 7). The proposed method has been successfully applied to the determination of dipyridamole tablets and the recovery was 99-103%.

A fluorescent polarization-based assay for the identification of disruptors of the RCAN1-calcineurin A protein complex.

Calcineurin is a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programs, including immune response. One of the most studied substrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most commonly used immunosuppressants in transplantation therapies. Unfortunately, their mechanism of action, completely blocking the calcineurin phosphatase activity while also requiring continuous administration, bears severe side effects. During recent years, the family of regulators of calcineurin (RCAN) has been described and studied extensively as modulators of calcineurin signaling pathways. The RCAN1 region, spanning amino acids 198 to 218 and responsible for inhibiting the calcineurin-NFAT signaling pathway in vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin-NFAT interaction without affecting the general calcineurin phosphatase activity. Here we report the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1(198-218)-CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction. Moreover, three small molecules with a potential immunosuppressive effect were also identified.

Electrochemical determination of dipyridamole at a carbon paste electrode using cetyltrimethyl ammonium bromide as enhancing element.

Electrochemical behaviors of dipyridamole (DIP) at a carbon paste electrode in the presence of cetyltrimethyl ammonium bromide (CTAB) were investigated by voltammetry, chronocoulometry and electrochemical impedance spectroscopy (EIS). The results indicated that the electrochemical responses of DIP are apparently improved by CTAB, due to the enhanced accumulation of dipyridamole at the carbon paste electrode surface. Electrochemical parameters for the oxidation of DIP were explored by chronocoulometry. Under optimal conditions, the oxidation peak current at 0.53 V was proportional to DIP concentration in the range of 0.03-12 microg mL-1, with a detection limit of 0.01 microg mL-1 for 120 s accumulation by different pulse voltammetry (DPV). This method was applied to the determination of DIP in the tablet samples. The results were satisfying compared with that obtained by standardized method of high performance liquid chromatography (HPLC).

Determination of dipyridamole in pharmaceutical preparations using square wave voltammetry.

An analytical methodology using square wave voltammetry (SWV) at a hanging mercury drop electrode (HMDE) was developed for the quantitative determination of dipyridamole (DIP), a drug used for the treatment of several cardiovascular diseases, in pharmaceutical tablets and injections of Persantin in phosphate buffer (pH 3.0; 0.1M). After optimization of the parameters for SWV, analytical curves were obtained for application in the range of 1.28 x 10(-6)M to 7.02 x 10(-6)M. It was found a detection limit (DL) of 1.88 x 10(-8)M (9.50 ng/ml). The repeatability and the reproducibility of the method were determinated by successive measurements of DIP solutions on the range of the analytical curve with a coefficient variation of 0.97% (n=5) and 1.15%, respectively. The apparent recoveries were obtained by the IUPAC recommended procedure using the second reduction peak. Recoveries obtained by SWV were compared with the UV-vis spectrophotometric method. It was found that the determination of DIP in Persantin tablets gave a mean value of 75.6+/-0.4 mg (100.8%) and 68.9+/-0.3 mg (91.8%) for SWV and UV-vis spectrophotometry, respectively. In the case of injections, it was found 10.4+/-0.1 mg (103.4%) and 9.9+/-0.2 mg (99.9%) for SWV and UV-vis spectrophotometry. Both apparent recoveries for the two types of formulations are in good accordance with the declared value of 75 mg (tablets) and 10 mg (injections).

[Chemiluminescence method for determination of dipyridamole].

A new system for the determination of dipyridamole with flow injection chemiluminescence was described. It is based on the chemiluminescence reaction of dipyridamole-potassium permanganate with rhodamine B. Tween-80 was found to be an enhancer of the chemiluminescence reaction. A method based on the enhanced chemiluminescence for dipyridamole determination has been developed. The method has high sensitivity, selectivity and good repeatability with a linear concentration range of 5.0 x 10(-8) - 5.0 x 10(-5) g x mL(-1), a detection limit of 1.7 x 10(-8) g x mL(-1) and a RSD of 1.1% (n = 11, cs = 1.0 x 10(-6) g x mL(-1)).

Sensitive and simple determination of the vasodilator agent dipyridamole in pharmaceutical preparations by phosphorimetry.

The applicability of heavy atom-induced room-temperature phosphorescence to pharmaceutical samples is demonstrated in this work. Thus a new, simple, rapid, and selective phosphorimetric method for dipyridamole determination is proposed. The phosphorescence signals are a consequence of intermolecular protection when analytes are exclusively in the presence of heavy atom salts and sodium sulfite as an oxygen scavenger to minimize RTP quenching. The determination was performed in 0.1 mol L(-1) thallium(I) nitrate and 8 mmol L(-1) sodium sulfite at a measurement temperature of 20 degrees C. The phosphorescence intensity was measured at 635 nm, with excitation at 305 nm. Phosphorescence was easily developed; a linear concentration range was obtained between 0 and 100 ng mL(-1) with a detection limit of 940 ng L(-1), an analytical sensitivity of 2.5 ng mL(-1), and a standard deviation of 2.7% at 60 ng mL(-1) concentration. The method has been successfully applied to the analysis of dipyridamole in a unique Spanish commercial formulation containing 100 ng mL(-1) per capsule. The recovery was 101.6% with 6.5% standard deviation of analytical measurement. The method using the standard addition methodology has been validated.

Spectrofluorometric resolution of closely overlapping drug mixtures using chemometrics methods.

A modified parallel factors analysis (PARAFAC) algorithm with a penalty diagonalization error (PDE), newly proposed by the present authors, was utilized to simultaneously resolve drug mixtures of propranolol (PRO), dipyridamole (DIP) and amiloride (AMI) without any loss of sensitivity. The analyses were performed in aqueous solution. The experimental results demonstrated that the profiles of the spectra and the concentrations could be accurately resolved using the PDE algorithm with a high sensitivity and stable repeatability. That is to say, the closely overlapping problem of the spectra could be easily solved. Furthermore, simultaneous determinations of three kinds of tablets, which contain PRO, AMI and DIP, respectively, were successfully performed with satisfactory results.

[Applications of phosphorimetry in pharmaceutical analysis].

Applications of phosphorimetry including solid substrate phosphorescence, liquid medium phosphorescence, low temperature phosphorescence and phosphorescence sensors were reviewed in pharmaceutical analysis. The drugs involved here included the varieties of alkaloid, Chinese traditional medicine, tetracyclines, quinolone, riboflavin, anticancer medicine, naphazoline, naproxen, nafronyl dipyridamole and so on. Solid surface phosphorimetry is characterized by sample volume of microliter grade, simple and fast operation procedures in pharmaceutical analysis. The combination of liquid phosphorescence with flow injection analysis and chemosensing technique has good advantages in fast, continuous and on-line monitoring of medicines. Modified low temperature phosphorimetry still remains its high sensitivity and overcomes some disadvantages in the procedures. Phosphorimetry will be more widely applied to pharmaceutical analysis as the development of sensitive and quenching, energy transfer, derivative and immunization luminescence.

Spectrofluorometric estimation of aspirin and dipyridamole in pure admixtures and in dosage forms.

Aspirin and dipyridamole in pure admixtures and in dosage forms have been estimated by spectrofluorometry. Aspirin (2-12 mcg ml-1) was estimated in 1% v/v glacial acetic acid in chloroform using 246 and 345 nm for excitation and emission respectively. Dipyridamole (2-12 mcg ml-1) has been estimated in chloroform using 420 nm for excitation and 475 nm for emission. The non-interference of the excipients as well as the drugs in the estimation of each other, as evidenced by the results, indicate that this method may be used for the routine estimation of aspirin and dipyridamole in tablet preparations.

Recurrence of biliary drug lithiasis due to dipyridamole.

The occurrence of drug-containing gallstones is not often observed. To our knowledge, three drugs have so far been incriminated-ceftriaxone, glaphenine, and dipyridamole. This report presents the case of an 85-year-old woman who developed a recurrent drug-containing gallstone caused by dipyridamole eighteen months after a previous stone had been removed endoscopically.

Phosphorimetric determination of dipyridamole in pharmaceutical preparations.

Room temperature phosphorescence was applied to the determination of dipyridamole in pharmaceutical preparations. The response was linear in the concentration range 100-1600 ng ml-1. The use of phosphorescence enhancers such as thallium(I) nitrate (external heavy atom), sodium dodecyl sulfate (microemulsion stabilizer) and sodium sulfite (deoxygenation agent) was studied and optimized to obtain maximum sensitivity and adequate selectivity. The determination was performed in 0.026 M sodium dodecyl sulfate, 0.0156 M thallium nitrate and 0.02 M sodium sulfite. The pH value was 11.5, adjusted by adding sodium hydroxide. The phosphorescence was totally developed in 15 min, after that the intensity was measured at lambda ex = 303 nm and lambda em = 616 nm. The recovery of the method was tested on commercial formulations containing dipyridamole. The recoveries obtained were 94.67 +/- 0.58% for Persantin and 96.75 +/- 1.37% for Asasantin. The overall least squares regression method was applied to find the most exact straight line that fits the experimental data. The detection limit according to the error propagation theory was 16.4 ng ml-1. The repeatability and relative standard deviation were also determined according to this theory.

Determination of dipyridamole by modified extraction-gravimetry with a surface acoustic wave resonator sensor.

A simple and sensitive extraction-gravimetric method for the determination of dipyridamole is presented. The method is based on the extraction of free dipyridamole with chloroform, after neutralization with a basic agent, followed by measurement of the frequency shift response of the specially designed surface acoustic wave resonator sensor after evaporation of the extractant from the surface of the resonator. The frequency shift response was proportional to the amount of dipyridamole in the range 0.065-1.12 micrograms. Experimental parameters and the effect of interfering substances on the assay of dipyridamole were also examined in this study. The method was applied to the determination of dipyridamole in tablets.