Fluorescence imaging approaches for eco-friendly determination of perampanel in human plasma and application for therapeutic drug monitoring.

Antiepileptic drugs are among the most common medications that require therapeutic drug monitoring (TDM). Indeed, TDM provides a realistic approach to adjust drug doses for epilepsy based on plasma concentrations to optimize its clinical outcome. The most common technique for TDM is high-performance liquid chromatography, which has a very low green profile among analytical techniques. Perampanel (PER) is an inherently fluorescent compound that its fluorophore readily allows sensitive and quantitative measurements. This paper describes the development and validation of a sensitive, specific, and eco-friendly spectrofluorimetric method for the determination of PER. Experimental parameters affecting fluorescence intensity of the compound, including solvent dilution, temperature, and excitation wavelength, were studied and optimized. The developed spectrofluorimetric method was established in acetonitrile at λex  = 295 nm and λem  = 431 nm over a concentration range of 5-60 ng/ml. The adopted method was applied for the determination of PER in human plasma; it was effective in the range of 15-50 ng/ml. The proposed method was found to be sensitive and specific for PER and can be applied successfully in TDM of PER and in quality control laboratories.

Selective spectrofluorimetric determination of two corticosteroids along with their co-formulated drugs and degradation products in ophthalmic solution and aqueous humour.

Prednisolone acetate (PNO) and fluorometholone (FRT) are corticosteroids, co-formulated with moxifloxacin HCl (MFX) and cromolyn sodium (CML), respectively. PNO has a negligible quantum yield and its hydrolytic degradation products have enhanced fluorescence, which is 250-fold greater. FRT is a nonfluorescent drug, but its hydrolytic degradation products show reasonable fluorescence; MFX and CML have native fluorescence. Two methods were proposed based on the determination of PNO and FRT via their hydrolytic degradation products in the presence of other degradation products. Method (A) was developed for simultaneous determination of PNO and MFX in the presence of PNO degradation products by measuring peak amplitudes of the first derivative (1 D) of its enhanced fluorescence; PNO and MFX were measured at 345 and 473 nm, respectively. Method (B) is a synchronous fluorescence spectroscopic method for simultaneous determination of FRT and its co-formulated drug CML in the presence of its degradation products. Fluorescence intensities were measured at λem 283 and 347 nm for FRT and CML, respectively, using Δλ = 99.20 nm. Validation of the proposed methods was conducted as per International Council for Harmonisation (ICH) guidelines. The proposed methods were successfully applied for the determination of the proposed drugs in bulk powder, ophthalmic solution, and rabbit's aqueous humour.

A novel HPLC-DAD method for simultaneous determination of alfuzosin and solifenacin along with their official impurities induced via a stress stability study; investigation of their degradation kinetics.

Stability and impurity profiling are in high demand to guarantee the potency, safety and efficacy of new formulations along with their shelf-life. In this study, stability testing of alfuzosin (ALF) and solifenacin (SOL) in their newly co-formulated capsules was conducted under different stress conditions. The obtained degradation products were structurally elucidated and found to be their official impurities, namely; ALF impurity-D and SOL impurities-A, E & I. A selective and reliable stability-indicating HPLC method was developed for assaying the cited drugs along with three of those official impurities. Chromatographic separation was accomplished within 8 minutes using a XBridge® C18 column as the stationary phase and acetonitrile : phosphate buffer (pH 8) : triethylamine (60 : 40 : 0.02, by volume) as the mobile phase at a flow rate of 1.3 mL min-1. Quantification of the analytes was performed at 210 nm using a diode array detector through which peak purity was assessed. The proposed method was validated as per ICH guidelines and it was successfully applied for the determination of the cited drugs in their combined pharmaceutical formulation with percent recoveries of 100.47 and 100.15 for ALF and SOL, respectively. Moreover, the proposed method was exploited for the assessment of the two drugs' stability in Solitral® capsules under accelerated storage conditions. The method was further extended for studying the degradation kinetics of the two drugs.

Synchronous UPLC Resolution of Aceclofenac and Diacerin in Their Powdered Forms and Matrix Formulation: Stability Study.

Accurate, rapid and selective reversed phase ultra-performance liquid chromatography method with UV detection has been established and validated for the synchronous determination of aceclofenac (ACE) and diacerin (DIA) in the occurrence of diclofenac sodium and rhein, their main degradation products, respectively. Chromatographic separation was accomplished using Inertsil C-18 column (50 × 2.1 mm i.d., 1.7 µm particle size) in isocratic mode, with mobile phase consisting of 20 mM ammonium acetate buffer:acetonitrile in the ratio of 42:58 (v/v), pH adjusted to 3.00 by using 10% acetic acid, the flow rate of 0.25 mL/min and UV detection was performed at 265 nm. The retention times were 2.00 +/- 0.24, 2.69 +/- 0.19, 4.00 +/- 0.23 and 5.24 +/- 0.25 min for DIA, rhein, ACE and diclofenac sodium, respectively. Excellent linearity was shown over a range of 1.0-150.0 µg/mL and 0.5-87.5 µg/mL with mean percentage recoveries of 98.87 ± 1.19 and 98.84 ± 1.08 for ACE and DIA, respectively. Parameters of precision and accuracy of the method meet the established criteria. The obtained RSD values were quite low and indicate good reproducibility of the method. Thus, the developed method can be used for the combined dosage form analysis and its chemical stability studies.

Novel HPTLC densitometric methods for determination of tamsulosin HCl and tadalafil in their newly formulated dosage form: Comparative study and green profile assessment.

A promising combination of tamsulosin HCl and tadalafil has recently been introduced for treating two prevalent and associated urological disorders: benign prostate hyperplasia and erectile dysfunction. Novel HPTLC methods were designed and validated for assaying the cited drugs in their challenging combined formulation. Separation was achieved using HPTLC silica gel 60 F254 plates as a stationary phase with a densitometric measurement at 280 nm. The proposed methods with two different chromatographic systems were successfully applied: a conventional mixture (method I) of ethyl acetate-toluene-methanol-ammonia (5:3:2:0.5, by volume) and a greener one (method II) with ethyl acetate-ethanol-ammonia (8:2:0.1, by volume). The two methods were evaluated through a comparative study in terms of selectivity, tailing factor, developing time and concentration ranges. The greenness profile for each method was then appraised with several green guides, namely GlaxoSmithKline solvent sustainability guide, Environmental, Health and Safety (EHS) tool, National Environmental Method Index (NEMI) and Eco-scale. Moreover, method specificity and peak homogeneity were evaluated by peak purity assessment using the winCATS® software spectral correlation tool. The methods have potential for being simple, fast, economic and selective, and the greener one could be a good option for sustainable analysis of the drugs.

Smart spectrophotometric assessment of tamsulosin hydrochloride and tadalafil in their new pharmaceutical formulation for treatment of benign prostatic hyperplasia and erectile dysfunction.

A novel combination of tamsulosin hydrochloride and tadalafil is recently available for treatment of benign prostatic hyperplasia and erectile dysfunction. For the first time, four simple, accurate, smart and robust spectrophotometric methods have been suggested for their simultaneous quantification. The methods, namely; first derivative, ratio difference, derivative ratio and mean centering of ratio spectra, successfully resolved the spectral overlap of their challenging binary mixture. Calibration curves were linear at 2.0-40.0 and 2.0-55.0 µg/mL for tamsulosin hydrochloride and tadalafil, respectively. The methods were validated according to ICH guidelines and statistically compared with the official ones, revealing no considerable difference with respect to accuracy and precision. Specificity of the developed methods was assessed by evaluating various laboratory prepared mixtures. Furthermore, the methods were successfully applied for the quantification of the two drugs in their combined dosage form.

Smart Spectral Processing of Data for the estimation of Commonly Used Over-the-counter (OTC) Co-formulated drug; Pseudoephedrine hydrochloride and Ibuprofen.

Oral pharmaceutical preparation containing pseudoephedrine hydrochloride (PSE) and ibuprofen (IBU) is widely prescribed as over- the- counter (OTC) for treatment of common cold-sinus. Development of four precise and accurate spectrophotometric methods are established for the concurrent determination of (PSE) and (IBU)in this preparation exploiting zero and/or ratio spectra. Method I is a dual wavelength method (DW). method II is a ratio difference method (RD), method III is a constant multiplication coupled with spectrum subtraction method (CM-SS) and method IV is a constant center coupled with spectrum subtraction method (CC-SS). While, absorbance correction method (AC) is successfully established for the determination of (IBU) only exploiting zero order absorption spectra. The calibration curves are linear over the concentration range of 100.0-900.0 µg/mL for (PSE) and 200.0-1000.0 µg/mL for (IBU). No separation steps are required for the spectrophotometric procedures which augments their simplicity. Analyzing synthetic mixtures of the cited drugs evaluated the specificity of the applied methods. Validation of the analysis results have been statistically performed confirming the accuracy and reproducibility of the proposed method through recovery studies which were carried out by following ICH guidelines. Thus, the developed methods can be successfully applied routinely in quality control laboratory.

Selective and Sensitive Chromatographic Methods for Determination of a Co-Formulated Binary Mixture in Antibacterial Eye Drops and Aqueous Humor in the Presence of Their Degradation Products and Potential Impurities.

Prednisolone acetate (PDN) is a corticosteroid anti-inflammatory liable to degradation under different conditions and used with antibiotics in eye drops. Two selective stability-indicating separation techniques were developed for simultaneous determination of PDN and moxifloxacin HCl (MXF) binary mixture in pure forms, ophthalmic formulation, in the presence of PDN impurities and in the presence of their degradation products. The first method was based on HPTLC separation using silica gel 60 F254 HPTLC plates, and a developing system of toluene: ethyl acetate: methanol: ammonia (5.0: 6: 2.0: 0.05, v/v/v/v) is used with detection at 254 nm. The second method was HPLC using a mobile phase of acetonitrile: methanol: deionized water, pH 2.8 (25.0: 35.0: 40.0, v/v/v), at 254 nm. A kinetic study utilizing the developed HPLC method for PDN degradation under different stress conditions was performed. Furthermore, the method was applied for determination in rabbit aqueous humor. Validation was conducted as per ICH guidelines, and system suitability was ascertained. The calibration curves were constructed in the range 0.10-25.00 and 0.20-50.00 µg band-1, for PDN and MXF by HPTLC, while for HPLC, it was 0.02-50.00 and 0.10-50.00 µg mL-1 for both drugs, in order.

Resolution and Quantitation of Triamcinolone Acetonide and Its Coformulated Drug in the Presence of Its Impurities and Degradation Products by HPTLC and HPLC.

Two specific, sensitive, and precise stability-indicating chromatographic methods have been developed for the determination of triamcinolone acetonide (TMC) and its coformulated drug, econazole nitrate (ECZ), in the presence of TMC impurities and degradation products. The first method was based on HPTLC-spectrodensitometry in which resolution and quantitation was achieved by using silica gel 60 F254 HPTLC plates and an ethyl acetate-tetrahydrofuran-ammonia mobile phase (10.0 + 7.0 + 0.1, v/v/v). The second method was a reversed-phase HPLC method in which separation was achieved using an acetonitrile-methanol-0.05 M potassium dihydrogen phosphate mobile phase, pH 3.0 (25.0 + 15.0 + 60.0, v/v/v). In both methods, the separated components were detected at 225 nm. Validation of both methods was conducted in compliance with International Conference on Harmonization (ICH) guidelines, and system suitability was confirmed. The linearity ranges were 0.20-28.00 and 0.50-55.00 µg/band for TMC and ECZ by HPTLC, whereas for HPLC, the range was 0.05-30.00 and 1.00-40.00 µg/mL for both drugs, respectively. The methods were successfully applied for the analysis of a pharmaceutical formulation and were compared with the reported method with no significant difference.

Stability-indicating spectrofluorometric method for the determination of some cephalosporin drugs via their degradation products.

A stability-indicating spectrofluorometric method was investigated for the determination of three cephalosporin drugs, namely, cefpodoxime proxetil (CPD), cefixime trihydrate (CFX), and cefepime hydrochloride (CPM), via their acid and alkali degradation products. The three drugs were determined via their acid degradation at 432, 422, and 435 nm using an excitation wavelength of 310, 330, and 307 nm for CPD, CFX, and CPM determination, respectively, and via their alkali degradation at 407, 411, and 405 nm using an excitation wavelength of 310, 305, and 297 nm for CPD, CFX, and CPM determination, respectively. Linearity was achieved in the ranges of 0.35-3.50, 0.4-4.0, and 0.3-3.0 µg/mL for the acid degradation products of CPD, CFX, and CPM, respectively, and in ranges of 0.05-0.5, 0.1-1.0, and 0.08-0.80 µg/mL for the alkali degradation products of CPD, CFX, and CPM, respectively. The method was validated for various parameters according to International Conference on Harmonization guidelines. The method was successfully applied for the determination of these cephalosporin drugs in pharmaceutical dosage forms with good accuracy and precision. The results obtained by the proposed spectrofluorometric method were compared with good agreement to the official HPLC method.

Validated stability-indicating spectrophotometric methods for the determination of cefixime trihydrate in the presence of its acid and alkali degradation products.

Five simple, accurate, precise, and economical spectrophotometric methods have been developed for the determination of cefixime trihydrate (CFX) in the presence of its acid and alkali degradation products without prior separation. In the first method, second derivative (2D) and first derivative (1D) spectrophotometry was applied to the absorption spectra of CFX and its acid (2D) or alkali (1D) degradation products by measuring the amplitude at 289 and 308 nm, respectively. The second method was a first derivative (1DD) ratio spectrophotometric method where the peak amplitudes were measured at 311 nm in presence of the acid degradation product, and 273 and 306 nm in presence of its alkali degradation product. The third method was ratio subtraction spectrophotometry where the drug is determined at 286 nm in laboratory-prepared mixtures of CFX and its acid or alkali degradation product. The fourth method was based on dual wavelength analysis; two wavelengths were selected at which the absorbances of one component were the same, so wavelengths 209 and 252 nm were used to determine CFX in presence of its acid degradation product and 310 and 321 nm in presence of its alkali degradation product. The fifth method was bivariate spectrophotometric calibration based on four linear regression equations obtained at the wavelengths 231 and 290 nm, and 231 and 285 nm for the binary mixture of CFX with either its acid or alkali degradation product, respectively. The developed methods were successfully applied to the analysis of CFX in laboratory-prepared mixtures and pharmaceutical formulations with good recoveries, and their validation was carried out following the International Conference on Harmonization guidelines. The results obtained were statistically compared with each other and showed no significant difference with respect to accuracy and precision.

Spectrophotometric and TLC-densitometric methods for the simultaneous determination of Ezetimibe and Atorvastatin calcium.

Three sensitive methods were developed for simultaneous determination of Ezetimibe (EZB) and Atorvastatin calcium (ATVC) in binary mixtures. First derivative (D(1)) spectrophotometry was employed for simultaneous determination of EZB (223.8 nm) and ATVC (233.0 nm) with a mean percentage recovery of 100.23 ± 1.62 and 99.58 ± 0.84, respectively. Linearity ranges were 10.00-30.00 µg mL(-1) and 10.00-35.00 µg mL(-1), respectively. Isosbestic point (IS) spectrophotometry, in conjunction with second derivative (D(2)) spectrophotometry was employed for analysis of the same mixture. Total concentration was determined at IS, 224.6 nm and 238.6 nm over a concentration range of 10.00-35.00 µg mL(-1) and 5.00-30.00 µg mL(-1), respectively. ATVC concentration was determined using D(2) at 313.0 nm (10.00-35.00 µg mL(-1)) with a mean recovery percentage of 99.72 ± 1.36, while EZB was determined mathematically at 224.6 nm (99.75 ± 1.43) and 238.6 nm (99.80 ± 0.95). TLC-densitometry was employed for the determination of the same mixture; 0.10-0.60 µg band(-1) for both drugs. Separation was carried out on silica gel plates using diethyl ether-ethyl acetate (7:3 v/v). EZB and ATVC were resolved with Rf values of 0.78 and 0.13. Determination was carried out at 254.0 nm with a mean percentage recovery of 99.77 ± 1.30 and 99.86 ± 0.97, respectively. Methods were validated according to ICH guidelines and successfully applied for analysis of bulk powder and pharmaceutical formulations. Results were statistically compared to a reported method and no significant difference was noticed regarding accuracy and precision.

Separation and determination of clopidogrel and its impurities by capillary electrophoresis.

Clopidogrel bisulphate, an anti-platelet drug, has been separated from its impurities, namely impurity A, B and C by capillary zone electrophoresis (CZE) using uncoated fused-silica capillary (50.0 microm internal diameter, 31.2cm total length). Four factors affected the separation: buffer concentration, pH of the buffer, concentration of the chiral selector and the applied voltage. Optimization and robustness studies were performed with the aid of reduced central composite experimental design. The buffer used was triethylamine-phosphoric acid and the chosen chiral selector was sulphated beta-cyclodextrin (SCD). The best separation was achieved by using 10mM buffer, pH 2.3, containing 5% (mass/volume (m/v)) SCD. Reversed polarity mode was used with an applied voltage of -12kV and the capillary temperature was maintained at 20 degrees C. The method was validated for quantitative determination of the drug. It offered a limit of detection (LOD) of 0.13 microg/ml, a limit of quantitation (LOQ) of 0.4 microg/ml, and a linearity range of 0.4-300 microg/ml. Commercial bulk samples were analyzed using the developed method.

Validated stability-indicating methods for determination of cilostazol in the presence of its degradation products according to the ICH guidelines.

Sensitive and selective stability-indicating assay methods (SIAMs) are suggested for the determination of cilostazol (CIL) in the presence of its acid, alkaline and oxidative degradation products. Developing SIAMs is necessary to carry out any stability study. Stress testing of CIL was performed according to the International Conference on Harmonization (ICH) guidelines in order to validate the stability-indicating power of the analytical procedures. Stress testing showed that CIL underwent acid, alkaline and oxidative degradation; on the other hand, it showed stability towards photo- and thermal degradation. Two chromatographic SIAMs were developed, namely HPLC and HPTLC methods. The concentration range and the mean percentage recovery were 1.0-31.0 microg/ml and 99.96+/-0.46 and 0.6-14.0 microg/spot and 99.88+/-1.10 for HPLC and HPTLC methods, respectively. In addition, derivative spectrophotometric methods were developed in order to determine CIL in the presence of its acid degradation product; these were performed by using the third derivative spectra (3D) and the first derivative of the ratio spectra (1DD) methods. The linearity range and the mean percentage recovery were 2.0-34.0 microg/ml and 100.27+/-1.20 for the (3D) method, while they were 2.0-30.0 microg/ml and 99.94+/-1.18 for the (1DD) method. Also, two chemometric-assisted spectrophotometric methods, based on using partial least squares (PLS) and concentration residual augmented classical least squares method (CRACLS), for the determination of CIL were developed. Both methods were applied on zero order spectra of the mixtures of CIL and its acid degradation product, the mean percentage recovery was 100.03+/-1.09 and 99.91+/-1.27 for PLS and CRACLS, respectively. All methods were validated according to the International Conference on Harmonization (ICH) guidelines and applied on bulk powder and pharmaceutical formulations.