Nehal F. Farid

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.

Validated HPLC-DAD method for stability study of sulbutiamine HCl

Sulbutiamine (SUL) is a synthetic thiamine derivative widely used for treatment of memory disorders. In this study, a newly developed gradient HPLC-DAD method demonstrating no interference from the different degradation products of SUL has been optimized and validated. The drug was subjected to variable stress conditions including hydrolysis (at different pH values), oxidation, photolysis and dry heat. The drug was found to be labile to hydrolysis, oxidation and photolysis but stable in thermal and neutral hydrolytic degradation. Successful chromatographic separation of SUL from all degradation products with significantly different tR values was achieved on a ZORBAX Eclipse Plus C18 column using a mobile phase containing a gradient mixture of solvent A (50 mM KH2PO4 (pH 3.6 ± 0.2)) and solvent B (methanol). UV detection was performed at 254 nm using a photodiode array detector (DAD). The reliability of the method was assessed by evaluation of accuracy, precision, specificity, robustness and ruggedness according to USP guidelines. The linear regression analysis data for the calibration curve showed a good relationship in the range of 2–40 μg mL−1. System suitability tests were performed, and selectivity (α) and resolution (Rs) factors were found to be greater than 1.5 and 2, respectively. The assay method was successfully used to estimate SUL in Arcalion Forte® tablets and good percentage recoveries were obtained. The developed method compared favorably with the reported spectrophotometric method.

Stability-Indicating HPTLC Method for Studying Stress Degradation Behavior of Sulbutiamine HCl

Sulbutiamine (SUL) is an ester of thiazides with neurotropic action. A new stability indicating HPTLC method has been developed and validated for the determination of SUL in the presence of different degradation products. The drug was subjected to different stress conditions following ICH strategy such as hydrolytic degradation (neutral, alkaline and acidic hydrolysis), oxidation, photodegradation and dry heat degradation. The drug demonstrated degradation under all decomposition conditions except neutral hydrolysis and dry heat, where the drug was completely degraded with 0.1 N NaOH, 1 N HCl and 30% H2O2 while it was partially degradaed by 0.1 N HCl, 3% H2O2 and UV light. Structure elucidation of the resulting degradation products was performed using ESI-Q-MS-MS. A well-defined peak for SUL was obtained at Rf = 0.46 and was completely separated from all obtained degradation products. Chromatographic separation was carried out on HPTLC aluminum plates precoated with silica gel 60 F254 using acetone-methylene chloride-ammonia buffer (pH 8.5 ± 0.2) (7:3:0.5, v/v) as a developing system. Densitometric scanning of the separated peaks was performed at 254 nm. System suitability testing parameters were calculated to ascertain the quality performance of the developed method. The method was validated with respect to USP guidelines regarding accuracy, precision, specificity, robustness and ruggedness. Good correlation coefficients were achieved in the range of 0.4-5.0 µg/band, and the limit of detection and limit of quantitation were found to be 0.11 and 0.33 µg/band, respectively. The utility of the suggested method was verified by application to Arcalion forte® tablets where no interference from additives was found.

Determination of flutamide and two major metabolites using HPLC–DAD and HPTLC methods

Flutamide is a potential antineoplastic drug classified as an anti-androgen. It is a therapy for men with advanced prostate cancer, administered orally after which it undergoes extensively first pass metabolism in the liver with the production of several metabolites. These metabolites are predominantly excreted in urine. One of the important metabolites in plasma is 4-nitro-3-(trifluoromethyl)phenylamine (Flu-1), while the main metabolite in urine is 2-amino-5-nitro-4-(trifluoromethyl)phenol (Flu-3). In this work the two metabolites, Flu-1 and Flu-3, have been synthesized, and then structural confirmation has been carried out by HNMR analysis. Efforts were exerted to develop chromatographic methods for resolving Flutamide and its metabolites with the use of acceptable solvents without affecting the efficiency of the methods. The drug along with its metabolites were quantitatively analyzed in pure form, human urine, and plasma samples using two chromatographic methods, HPTLC and HPLC–DAD methods. FDA guidelines for bio-analytical method validation were followed and USP recommendations were used for analytical method validation. Interference from excipients has been tested by application of the methods to pharmaceutical tablets. No significant difference was found between the proposed methods and the official one when they were statistically compared at p value of 0.05%.

Chromatographic analysis of ledipasvir and sofosbuvir: New treatment for chronic hepatitis C infection with application to human plasma

ABSTRACT Sofosbuvir (SOF) and ledipasvir (LED) are recently approved and coformulated as directly acting antiviral agents used for treatment of hepatitis C virus (HCV). A reversed phase high performance liquid chromatography - diode array detector (RP-HPLC/DAD) method was developed and validated for the first time for the analysis of newly formulated anti-HCV combination, in pure form, pharmaceutical formulation and in human plasma. In the developed method, separation was performed on Zorbax® Eclipse C18 column using a gradient mixture of acetonitrile–water as a mobile phase and scanning was performed at 260 nm (for SOF) and 330 nm (for LED). The two drugs were completely separated from each other and from plasma, where plasma peak appeared at 2.76 ± 0.05 min, SOF at 4.25 ± 0.05, and LED at 7.35 ± 0.05. The developed method showed high sensitivity, the drugs showed linearity in the range of 1–45 µg/mL for both pure form and spiked human plasma. Three freeze–thaw cycles were performed separately at two different temperatures, −8 and −20°C. No significant loss of the studied drugs were observed during repeated thawing and freezing. Validation parameters such as accuracy, precision, robustness, and ruggedness were tested in compliance with USP recommendations, where acceptable results were obtained. Applying to pharmaceutical formulation showed no interference from tablet excipients. GRAPHICAL ABSTRACT

Efficient UPLC and CE methods for the simultaneous determination of azelastine hydrochloride and its genotoxic impurity

A novel stability-indicating UPLC and CE method was established and validated for the determination of azelastine hydrochloride (AZL) and its genotoxic impurity, benzohydrazide, in the presence of benzalkonium chloride. The developed UPLC method was based on chromatographic separation using a C18 column as a stationary phase and acetonitrile-(0.1% w/v) aqueous sodium lauryl sulfate (55:45, v/v, pH 5 with phosphoric acid) as a mobile phase with a flow rate of 1.2 mL/min and UV detection at 215 nm. The chromatographic run time was ~2 min. The developed CE method depended on using a stationary phase of Standard Bare Fused Silica Capillaries (75 μm i.d. × 59 cm and 50 cm detection length) and the applied voltage was 30 kV using 40 mm phosphate buffer (pH 2 with aqueous H3 PO4 ); the detection wavelength was 225 nm. The analysis time was about 6 min. The suggested methods were successfully applied for the analysis of AZL in a pharmaceutical preparation. The validity of the developed methods was assessed by applying the standard addition technique and no interference from excipients was observed. The results obtained by the proposed methods were statistically analyzed and compared with the manufacturers method and no significant difference was found between the compared methods.

TLC-Densitometric and RP-HPLC Methods for Simultaneous Determination of Dexamethasone and Chlorpheniramine Maleate in the Presence of Methylparaben and Propylparaben

Validated simple, sensitive, and highly selective methods are applied for the quantitative determination of dexamethasone and chlorpheniramine maleate in the presence of their reported preservatives (methylparaben and propylparaben), whether in pure forms or in pharmaceutical formulation. TLC is the first method, in which dexamethasone, chlorpheniramine maleate, methylparaben, and propylparaben are separated on silica gel TLC F254 plates using hexane-acetone-ammonia (5.5 + 4.5 + 0.5, v/v/v) as the developing phase. Separated bands are scanned at 254 nm over a concentration range of 0.1-1.7 and 0.4-2.8 μg/band, with mean ± SD recoveries of 99.12 ± 0.964 and 100.14 ± 0.962%, for dexamethasone and chlorpheniramine maleate, respectively. Reversed-phase HPLC is the second method, in which a mixture of dexamethasone and chlorpheniramine maleate, methylparaben, and propylparaben is separated on a reversed-phase silica C18 (5 μm particle size, 250 mm, 4.6 mm id) column using 0.1 M ammonium acetate buffer-acetonitrile (60 + 40, v/v, pH 3) as the mobile phase. The drugs were detected at 220 nm over a concentration range of 5-50 μg/mL, 2-90 μg/mL, 4-100 μg/mL, and 7-50 μg/mL, with mean ± SD recoveries of 100.85 ± 0.905, 99.67 ± 1.281, 100.20 ± 0.906, and 99.81 ± 0.954%, for dexamethasone, chlorpheniramine maleate, methylparaben paraben, and propylparaben, respectively. The advantages of the suggested methods over previously reported methods are the ability to detect lower concentrations of the main drugs and to show better resolution of interfering preservatives; hence, these methods could be more reliable for routine QC analyses.