Nada S. Abdelwahab

Validated stability indicating RP-HPLC method for determination of paracetamol, methocarbamol and their related substances

Paracetamol (PAR) and methocarbamol (MET) are co-formulated together in Methorelax® tablets which are widely used as a muscle relaxant and in the treatment of muscle-skeletal pain. On the other hand, 4-aminophenol (4-AP) and guaifenesine (GU) have been reported to be related substances and degradation products of PAR and MET, respectively. The target of this work was to develop and validate a simple, sensitive and selective stability indicating RP-HPLC method for the determination of PAR, MET, 4-AP and GU in their bulk powders and laboratory prepared mixtures. Chromatographic separation was achieved within 10 minutes with the required asymmetry, accuracy and precision on ODS column using 0.05 M KH2PO4 buffer : acetonitrile (72.5 : 27.5, v/v, pH = 6) as the mobile phase at a flow rate of 1 mL min−1 with UV detection at 225 nm. The developed method has been validated as per ICH guidelines and the calibration plots were linear over the concentration ranges of 3–20, 4–25, 0.6–8 and 0.6–8 μg mL−1 for PAR, MET, 4-AP and GU, respectively. The method has been successfully applied in the analysis of Methorelax® tablets and good results were obtained. Moreover, its results have been compared to a previously reported RP-HPLC method and no significant difference was found between the two methods.

Electroanalytical sensing of the antimicrobial drug linezolid utilising an electrochemical sensing platform based upon a multiwalled carbon nanotubes/bromocresol green modified carbon paste electrode

The electroanalytical sensing of linezolid (LIN) is explored utilising an electrochemical sensing platform based upon a multiwalled carbon nanotubes (MWCNTs)/bromocresol green (BCG) modified carbon paste electrode (MWCNT/BCG/CPE). Cyclic voltammetry, differential pulse voltammetry, chronoamperometry, electrochemical impedance spectroscopy (EIS) and scanning electron microscopy techniques are all used to characterise the properties of the electrochemical sensor. The synergetic effects of BCG and MWCNTs result in an electrocatalytic-type response providing a significantly improved electroanalytical response compared to a bare unmodified CPE. Additionally, the MWCNT/BCG/CPE sensing platform outperforms electrochemical sensors previously reported in the literature with a larger linear response and lower limit of detection. The MWCNT/BCG/CPE electrochemical sensing platform was evaluated towards the sensing of LIN where a linear range from 5.00 × 10−8 to 1.45 × 10−4 mol L−1 with a good linearity and high correlation (0.9968) was observed with the limits of detection and quantification found to correspond to 7.57 × 10−9 and 2.50 × 10−8 mol L−1 respectively. The proposed sensor was applied to the determination of LIN in the presence of cefixime trihydrate (CEF) which are both found in combined dose tablet formulations. The sensor was shown to be successfully applied to the determination of LIN in a pharmaceutical formulation and human urine and saliva samples with satisfactory recoveries. The proposed electrochemical sensing platform is simple and inexpensive and has the potential to be applied to clinical analysis, quality control and the routine determination of drugs in pharmaceutical formulations.

Stability-Indicating TLC -Densitometric Method for Simultaneous Determination of Paracetamol and Chlorzoxazone and their Toxic Impurities

A highly sensitive, selective and accurate thin-layer chromatographic (TLC)-densitometric method has been developed and validated for the simultaneous determination of paracetamol (PAR) and chlorzoxazone (CZ) and their toxic impurities, 4-amino phenol (4AP) and 2-amino-4-chlorophenol (2ACP), respectively, which are also considered to be the hydrolytic degradation products and related substances of the studied drugs. A developing system consisting of chloroform-methanol-glacial acetic acid (9.5:0.5:0.25, by volume) was found to be sufficient for chromatographic separation among the four studied components using pre-activated silica gel 60 F254 TLC plates with ultraviolet detection at 225 nm. Calibration curves were constructed in the ranges of 0.3-3, 1-10, 0.06-3 and 0.04-3 µg/band for PAR, CZ, 4AP and 2ACP, respectively, using polynomial equations. The developed method was validated according to International Conference on Harmonization guidelines and demonstrated good accuracy and precision. Moreover, the method was successfully applied for the determination of PAR and CZ in different marketed samples and the results were statistically compared to those obtained by the reported reversed-phase high-performance liquid chromatography method using F-test and Students-t test. The low detection and quantitation limits of the developed method make it suitable for quality control and stability studies of PAR and CZ in different pharmaceutical formulations.

Two spectrophotometric methods for simultaneous determination of some antihyperlipidemic drugs

Two simple, accurate, precise and economic spectrophotometric methods have been developed for simultaneous determination of Atorvastatin calcium (ATR) and Ezetimibe (EZ) in their bulk powder and pharmaceutical dosage form. Method (I) is based on dual wavelength analysis while method (II) is the mean centering of ratio spectra spectrophotometric (MCR) method. In method (I), two wavelengths were selected for each drug in such a way that the difference in absorbance was zero for the second drug. At wavelengths 226.6 and 244 nm EZ had equal absorbance values; therefore, these two wavelengths have been used to determine ATR; on a similar basis 228.6 and 262.8 nm were selected to determine EZ in their binary mixtures. In method II, the absorption spectra of both ATR and EZ with different concentrations were recorded over the range 200–350, divided by the spectrum of suitable divisor of both ATR and EZ and then the obtained ratio spectra were mean centered. The concentrations of active components were then determined from the calibration graphs obtained by measuring the amplitudes at 215–260 nm (peak to peak) for both ATR and EZ. Accuracy and precision of the developed methods have been tested; in addition recovery studies have been carried out in order to confirm their accuracy. On the other hand, selectivities of the methods were tested by application for determination of different synthetic mixtures containing different ratios of the studied drugs. The developed methods have been successfully used for determination of ATR and EZ in their combined dosage form and statistical comparison of the developed methods with the reported spectrophotometric one using F and Students t-tests showed no significant difference regarding both accuracy and precision.

Determination of Thiomersal, Lidocaine and Phenylepherine in their Ternary Mixture

This work is concerned with the development and validation of two accurate, precise and specific spectrophotometric and chromatographic methods for analysis of Phenylepherine (PE), Lidocaine (LC) and Thiomersal (TM) in their ternary mixture and in Chromoderm® solution. The developed spectrophotometric method is the mean centering of ratio spectra (MCR) spectrophotometric method that depends on using the mean centered ratio spectra in two successive steps which eliminates the derivative steps and therefore the signal to noise ratio is enhanced. On the other hand, the developed chromatographic method isocratic RP-HPLC method that depends chromatographic separation on Zorbax C18 column using a mobile phase consisting phosphate buffer: acetonitrile: triethylamine (40:60:0.1, by volume pH=6 with orthophosphoric acid). The flow rate was 0.6 mL min-1 and the eluent was monitored at 245 nm. Factors affecting the developed methods were studied and optimized. Moreover, they have been validated as per ICH guidelines. The developed methods have been successfully applied for determination of PE, LC and TM in different laboratory mixtures and in solution dosage form. Also, Statistical analysis of the results of the two developed methods with each other using F and student’s t tests showed no significant difference

Spectrophotometric and chemometric determination of hydrochlorothiazide and spironolactone in binary mixture in the presence of their impurities and degradants

Hydrochlorothiazide (HCT) and spironolactone (SPR) are mostly co-formulated in antihypertensive formulations. Several methods have been developed and validated for their determination; these methods include spectrophotometric and chemometric-assisted spectrophotometric methods. The developed spectrophotometric methods were isosbestic point (ISO) and ratio subtraction (RS) methods. The absorbance values at 232.4 (lambda(iso1)) and 257.6 nm (lambda(iso2)) were used for determination of the total mixture concentration, while HCT could be directly determined at 317.2 nm (lambda(max)) and by subtraction SPR concentration could be obtained. Also SPR concentration could be calculated by RS method using the absorbance at 243.8 nm (lambda(max)). A wavelength selection method based on genetic algorithm (GAs) was developed and compared to the conventional partial least squares method (PLS). In this method, several parameters were adjusted and the optimum parameter settings were determined using experimental design. The developed chemometric methods were successfully applied for the determination of the HCT and SPR, as well as for determination of their impurities and degradation products. The proposed methods were successfully applied for determination of HCT and SPR in commercial tablets and they were statistically compared to each other and to the reported method. No significant difference was found, providing their accuracy and precision.

Innovative Spectrophotometric Methods for Determination of Newly Discovered Combination for Hepatitis C Treatment

Abstract Ledipasvir (LED) and Sofosbuvir (SOF) are newly approved antiviral agents co-formulated for treatment of hepatitis C virus (HCV). Up till now, no analytical methods have been published for determination of this pharmaceutically important combination. In this work novel spectrophotometric methods were developed for resolving the partially overlapped spectra of LED and SOF with simple data manipulation and without preliminary separation steps. In method (I), LED was directly determined using its extended spectra at 325 nm where no interference from the co-formulated SOF while the absorption at the isoabsorptive point (λ= 262.4 nm) was used for measuring concentrations of both. By subtraction, concentration of SOF could be obtained. Method (II) is the absorbance subtraction method (AS) at which a mathematically estimated factor representing the absorbance ratio (A262.4 /A325 ) for pure LED was used for simultaneous quantitation of LED and SOF using a unique equation computed at λiso (262.4 n m). Method (III) depended on using ratio spectra and then measuring the amplitude of the constant at 325 nm for LED while using ratio subtraction spectrophotometric method to quantify SOF at 262 nm. Finally, method (IV) was area under the curve correction method (AUCC) at which the areas from 245-265 and 315-335 nm and a mathematically calculated factor for pure LED were used. The methods were validated in compliance to USP guidelines and were successfully applied to available dosage form.

Two different spectrophotometric determinations of potential anticancer drug and its toxic metabolite.

Flutamide is a hormone therapy used for men with advanced prostate cancer. Flutamide is highly susceptible to hydrolysis with the production of 3-(trifluoromethyl)aniline, which is reported to be one of its toxic metabolites, impurities and related substances according to BP and USP. Flutamide was found to be stable when exposed to oxidation by 30% hydrogen peroxide and direct sunlight for up to 4h. Two accurate and sensitive spectrophotometric methods were used for determination of flutamide in bulk and in pharmaceutical formulations. Method (I) is the area under curve (AUC) spectrophotometric method that depends on measuring the AUC in the wavelength ranges of 275-305 nm and 350-380nm and using Cramers rule. The linearity range was found to be 1-35 μg/mL and 0.5-16 μg/mL for the drug and the degradate, respectively. In method (II), combination of the isoabsorptive and dual wavelength spectrophotometric methods was used for resolving the binary mixture. The absorbance at 249.2 nm (λiso) was used for determination of total mixture concentration, while the difference in absorbance between 232 nm and 341.2 nm was used for measuring the drug concentration. By subtraction, the degradate concentration was obtained. Beers law was obeyed in the range of 2-35 μg/mL and 0.5-20 μg/mL for the drug and its degradate, respectively. The two methods were validated according to USP guidelines and were applied for determination of the drug in its pharmaceutical dosage form. Moreover AUC method was used for the kinetic study of the hydrolytic degradation of flutamide. The kinetic degradation of flutamide was found to follow pseudo-first order kinetics and is pH and temperature dependent. Activation energy, kinetic rate constants and t1/2 at different temperatures and pH values were calculated.

A novel spectral resolution and simultaneous determination of multicomponent mixture of Vitamins B1, B6, B12, Benfotiamine and Diclofenac in tablets and capsules by derivative and MCR–ALS

A novel method was developed for spectral resolution and further determination of five-component mixture including Vitamin B complex (B1, B6, B12 and Benfotiamine) along with the commonly co-formulated Diclofenac. The method is simple, sensitive, precise and could efficiently determine the five components by a complementary application of two different techniques. The first is univariate second derivative method that was successfully applied for determination of Vitamin B12. The second is Multivariate Curve Resolution using the Alternating Least Squares method (MCR-ALS) by which an efficient resolution and quantitation of the quaternary spectrally overlapped Vitamin B1, Vitamin B6, Benfotiamine and Diclofenac sodium were achieved. The effect of different constraints was studied and the correlation between the true spectra and the estimated spectral profiles were found to be 0.9998, 0.9983, 0.9993 and 0.9933 for B1, B6, Benfotiamine and Diclofenac, respectively. All components were successfully determined in tablets and capsules and the results were compared to HPLC methods and they were found to be statistically non-significant.

Validated Stability Indicating TLC-Densitometric Method for the Determination of Diacerein

This work presents an accurate, sensitive and selective thin-layer chromatography-densitometry method for the simultaneous determination of diacerein in the presence of rhein, the active metabolite and hydrolytic degradation product of diacerein, and emodin, the diacerein impurity, in bulk powder and different pharmaceutical formulations. Chromatographic separation was performed on aluminum plates precoated with 60 F254 silica gel using hexane-ethyl acetate-acetic acid (60:40:0.8, by volume) as a developing system and with detection at 230 nm. The retention factor values of diacerein, rhein and emodin were 0.12, 0.44 and 0.6, respectively. The method was successfully applied for the determination of these compounds with high sensitivity; the linearity ranges were found to be 0.5-10 µg/band (for diacerein and rhein) and 0.5-7 µg/band (for emodin). The developed method was validated according to International Conference on Harmonization guidelines and was applied for the determination of diacerein in different pharmaceutical formulations. Moreover, a statistical comparison between the results of the developed method and those of the reported reversed-phase high-performance liquid chromatography method showed no significant differences. This method can be used for the routine analysis of diacerein, rhein and emodin in quality control laboratories.