Development and validation of colorimetric-assisted chemometrics methods based on the localized gold nanoparticles surface plasmon resonance for fast simultaneous estimation of anti-hepatitis C virus drugs in their combined dosage form: A comparative study with HPLC method.

The present work describes a developed analytical method based on a colorimetric assay using gold nanoparticles (AuNPs) along with chemometric techniques for the simultaneous estimation of sofosbuvir (SOF) and ledipasvir (LED) in their synthetic mixtures and tablet dosage form. The applied chemometric approaches were continuous wavelet transform (CWT) and least squares support vector machine (LS-SVM). Characterization of AuNPs and AuNPs in combination with the drug was performed by UV-vis spectrophotometer, transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared (FTIR) spectroscopy. In the CWT method, the zero amplitudes were determined at 427 nm with Daubechies wavelet family for SOF (zero crossing point of LED) and 440 nm with Symlet wavelet family for LED (zero crossing point of SOF) over the concentration range of 7.5-90.0 µg/L and 40.0-100.0 µg/L with coefficients of determination (R2) of 0.9974 and 0.9907 for SOF and LED, respectively. The limit of detection (LOD) and limit of quantification (LOQ) of this method were found to be 7.92, 9.96 µg/L and 12.02, 30.2 µg/L for SOF and LED, respectively. In the LS-SVM model, the mean percentage recovery of SOF and LED in synthetic mixtures was 98.29 % and 99.25 % with root mean square error of 2.392 and 1.034, which were obtained by the optimization of regularization parameter (γ) and width of the function (σ) based on the cross-validation method. The proposed methods were also applied for the determination concentration of SOF and LED in the combined dosage form, recoveries were higher than 95 %, and relative standard deviation (RSD) values were lower than 0.4 %. The achieved results were statistically compared with those obtained from the high-performance liquid chromatography (HPLC) technique for the concurrent estimation of components through one-way analysis of variance (ANOVA), and no significant difference was found between the suggested approaches and the reference one. According to these results, simplicity, high speed, lack of time-consuming process, and cost savings are considerable benefits of colorimetry along with chemometrics methods compared to other ways.

HPLC-UV and TLC-Densitometry Methods for Simultaneous Determination of Sofosbuvir and Daclatasvir: Application to Darvoni® Tablet.

Sofosbuvir and daclatasvir are co-formulated as directly acting antiviral agents used for treatment of hepatitis C virus. Two chromatographic methods were developed for their determination; the first one is an Reversed phase-high performance liquid chromatography (RP-HPLC) method, in which the separation was performed on C8 Zorbax® SB column (4.6 × 250 mm, 5 µm) using acetonitrile:water:0.05 M phosphate buffer, pH = 8 (50:45:5, v/v/v) as a mobile phase, and ultraviolet detection was performed at 280 nm. Good resolution was obtained, and linearity was confirmed in the range of 10-100 µg/mL for both drugs. The second method is Thin layer chromatography (TLC)-densitometric one, in which sofosbuvir and daclatasvir were separated on silica gel plates using ethyl acetate:hexane:methanol (9:0.5:0.5, v/v/v) as a developing system and the scanning wavelength was 280 nm. Linearity was confirmed over a concentration range of 0.4-25.4 µg/band for sofosbuvir, whereas for daclatasvir linearity scanning was in the range of 0.4-12.8 µg/band. Both antiviral agents can be quantified simultaneously in one analytical run, which is a great time- and cost-saving valor of the developed methods. This valor is even more important in the case of the combined dosage form (Darvoni® tablets) to the pharmaceutical market.

Feasible TLC-Spectro-Densitometry Technique for Simultaneous Determination of Two Hepatitis C Antiviral Drugs, Sofosbuvir and Simeprevir: Application to Combined Pharmaceutical Dosage Forms and Human Plasma.

A high-performance thin-layer chromatographic method was developed and validated for the concurrent determination of simeprevir (SMV) and sofosbuvir (SOF). The chromatographic separation was attained on silica gel 60 F254 as stationary phase and ethyl acetate-hexane-methanol (5.0:4.0:1.0, v/v/v) as developing solvent with UV detection at 273 nm. The RF values were 0.67±0.02 and 0.43±0.02 for SMV and SOF, respectively. The method has been validated in respect to the guidelines of the International Conference on Harmonization. Linearity was maintained between 60-1,000 and 70-1,200 ng/band for SMV and SOF, respectively, with good correlation coefficients (0.9993-0.9997) for both drugs. The suggested method was highly sensitive as the calculated detection limits were 15 and 22 ng/band, while the quantitation limits were 44 and 66 ng/ band for SMV and SOF, respectively. The suggested methodology has been effectively employed for the determination of the mentioned drugs in their pure forms and their pharmaceutical dosage forms as well as human plasma without significant interference of the pharmaceutical excipients or plasma components.

Micellar high performance liquid chromatographic method for separation and validation of two anti-hepatitis C- virus drugs in pure form, human plasma and human urine.

OBJECTIVES: In the present study, an eco- friendly micellar liquid chromatographic technique was validated for separation and quantification of two drugs; namely ribavirin (RIV), and sofosbuvir (SBV) in pure form, pharmaceuticals containing them, human plasma and human urine. These drugs are administered co-administered for treatment of Hepatitis C virus (HCV) that causes hepatitis C in humans. MATERIAL AND METHODS: These drugs were separated using Nucleosil 100-5 phenyl column. Sodium dodecyl sulphate (SDS) solution (0.05M, pH 7.0) containing triethylamine (0.3%) and n-butanol (10%) was used as a mobile phase with 1.2 mLmin-1 flow rate and 215nm detection wavelength. Nine minutes were required for resolving the two drugs from the matrix. RESULTS: The method showed good linearity for RIV and SBV with correlation coefficients (r2) more than 0.9996 within the concentration ranges of (20-400) and (40-400) ngmL-1 in pure form, (30-300) and (50-300) ngmL-1 in human plasma and (20-400) and (40-400) ngmL-1 in human urine, respectively. CONCLUSION: The recommended method was applied for examination of RIV and SBV in pure and pharmaceuticals. The obtained results were statistically matched with reported methods with no significant differences. Also, the recommended method was effectively applied for estimation of both drugs in spiked human urine and plasma without purification or extraction steps and real samples of plasma and urine of humans having therapy of RIV and SBV, as well as, performing tablets dissolution-rate tests with satisfactory results.

Optimization of a sensitive and robust strategy for micellar electrokinetic chromatographic analysis of sofosbuvir in combination with its co-formulated hepatitis C antiviral drugs.

Based on our previous work with "pseudostationary-ion exchanger sweeping", we use this strategy to develop a sensitive, reliable and robust method for the analysis of the newly-FDA approved hepatitis C antiviral drugs namely; sofosbuvir (SOV), daclatasvir (DAC), ledipasvir (LED) and velpatasvir (VEP) in their pure forms and co-formulated pharmaceutical dosage forms using micellar electrokinetic chromatography (MEKC) as a separation method. For the first time, a successful separation of all the investigated compounds was achieved in less than 8 min using a basic background electrolyte (BGE) composed of 25 mmol L-1 SDS + 20% (v/v) ACN (acetonitrile) in 10 mmol L-1 disodium tetraborate buffer (final apparent pH is 9.90). A special focus was given to optimize the composition of the sample matrix to maintain the solubility of the analytes within the sample zone while gaining additional benefits regarding analyte zone focusing. It was found that replacing phosphoric acid (as a sample matrix) with a zwitterionic/isoelectric buffering compound (L-glutamic acid) has a substantial positive impact on the obtained enrichment efficiency. The interplay of other enrichment principles such as the retention factor gradient effect (RFGE) is also discussed. A full validation study is performed based on the pharmacopeial and ICH guidelines. The obtained limits of detection and quantitation are as low as 0.63 and 1.3 µg mL-1; respectively for SOV and DAC and 1.3 and 2.5 µg mL-1; respectively for LED and VEP using UV-DAD as a detection method. The selectivity of the developed method for determination of the studied compounds in their pharmaceutical dosage forms or in the presence of ribavirin (RIB) or elbasvir (ELB), which are other prescribed medications in the treatment regimen of patients with hepatitis C virus infection, is demonstrated. It is shown that with acidic sample matrix and basic BGE, an efficient and precise approach was designed in which analyte adsorption on the capillary wall was minimized while keeping repeatable peak height, peak area and migration time together with the highest possible enrichment efficiency.

Chemometrically Assisted Development of Ultra-High-Performance Liquid Chromatography Method for the Simultaneous Quantification of Sofosbuvir, Daclatasvir and Ledipasvir in Pharmaceutical Dosage Forms.

A new ultra-high-performance liquid chromatography method for the simultaneous quantification of sofosbuvir, daclatasvir and ledipasvir was developed. Two combinations of these direct-acting antivirals are used in hepatitis C virus infection therapy and show high efficacy and safety. Fractional factorial design was used for screening the most influential factors on separation and time analysis. These significant factors were optimized using a central composite design. The optimum resolution was carried out by using a Waters XBridge C18 column (150 mm, 4.6 mm ID, 5 µm) at a temperature of 35°C ± 2°C and acetonitrile/sodium perchlorate buffer (10 mM, pH = 3.2) (40: 60 v/v) as mobile phase at a flow rate of 1.5 mL min-1. UV detection was set at λ = 210 nm. A short chromatographic separation time was achieved. The developed method was validated according to the accuracy profile approach and was found specific, precise, faithful and accurate. The detection limits were between 0.07 and 0.13 µg mL-1. Hence, this novel method can be employed for the routine quality control analysis and in dissolution profile studies of generics containing these products.

Modification of N,S co-doped graphene quantum dots with p-aminothiophenol-functionalized gold nanoparticles for molecular imprint-based voltammetric determination of the antiviral drug sofosbuvir.

A molecularly imprinted polymer (MIP) was developed for the electrochemical determination of the antiviral drug sofosbuvir (SOF). The MIP was obtained by polymerization of p-aminothiophenol (p-ATP) on N,S co-doped graphene quantum dots (N,S@GQDs) in the presence of gold nanoparticles to form gold-sulfur covalent network. The presence of quantum dots improves the electron transfer rate, enhances surface activity and amplifies the signal. The nanocomposites were characterized by FTIR, TEM, EDX, and SEM. The electrochemical performance of the electrode was investigated by differential pulse voltammetry and cyclic voltammetry. The sensor uses hexacyanoferrate as the redox probe and is best operated at a potential of around 0.36 V vs. Ag/AgCl. It has a linear response over the concentration range of 1-400 nM SOF, with a detection limit of 0.36 nM. Other features include high selectivity, good reproducibility and temporal stability. The sensor was applied to the determination of SOF in spiked human plasma. Graphical abstract Novel sofosbuvir imprinted p-ATP polymer was synthesized by the aid of gold nanoparticles on N,S co-doped graphene quantum dots as a good conductive support. The imprinted polymer was used for detection of sofosbuvir in real samples by using the ferri/ferrocyanide redox probe.

Spectrophotometric assessment of the brand new antiviral combination: Sofosbuvir and velpatasvir in their pure forms and pharmaceutical formulation.

Sofosbuvir (SOF) and velpatasvir (VEL) are recently co-formulated together for the treatment of hepatitis C virus. Smart and robust spectrophotometric methods were first developed and validated for quantification of SOF and VEL in their pure forms and in their combined pharmaceutical formulation without preliminary separation. VEL has two UV maxima at 302.5 and 337.0 nm that allow its direct determination by zero-order spectrophotometric method (D°) without any interference from SOF in a linear range of 2.0-30.0 µg/mL. On the other hand, determination of SOF in presence of VEL was carried out by four smart spectrophotometric methods, developed for resolving the overlaid spectra of these binary mixture. These methods are dual wavelength (DW), ratio subtraction (RS), ratio difference (RD) and first derivative of ratio spectra method (1DD). Linearity was checked and found to be in the range of 5.0-90.0 µg/mL for SOF by all of the aforementioned spectrophotometric methods. The developed methods were optimized and validated in accordance to the ICH guidelines. They were successfully utilized for estimating both SOF and VEL in their pure forms, laboratory prepared mixtures and in their pharmaceutical formulations with good recoveries. The methods can be easily applied for the routine analysis in quality control laboratories.

Simultaneous quantification of simeprevir sodium: A hepatitis C protease inhibitor in binary and ternary mixtures with sofosbuvir and/or ledipasvir utilizing direct and H-point standard addition strategies.

Simeprevir sodium (SMV); a novel hepatitis C inhibitor, quells hepatitis C viral replication by binding to and repressing the protease, hepatitis C infection (HCV) NS3/4A. In this way, it is known as a prompt acting antiviral agent. Calibration curves of SMV were built in various solvents; ethanol, methanol, acetonitrile, chloroform and dichloromethane. It is obeyed up to 60.0 µg/mL; in all solvents at two maximum wavelengths (280 and 327 nm). Several investigations show that, SMV might be present in a mixture of Sofosbuvir (SOF) and/or Ledipasvir (LDP). So far as that is concerned, H-point standard addition strategy (HPSAS) is made to identify it in binary or ternary mixtures. Recovery studies are in the prevalent range (93.0-107.0%) with relative standard deviation <1.5%. A correlation between the developed techniques is carried out and it demonstrates that these strategies are effectively applied for the simultaneous analysis of SMV, SOF and LDP in several synthetic samples and pharmaceutics. Statistical treatment of the acquired data is carried out against a newly published HPLC technique using F- and t-treatments.

Earth-friendly spectrophotometric methods for simultaneous determination of ledipasvir and sofosbuvir: Application to average content and uniformity of dosage unit testing.

Simple, rapid, sensitive, accurate, precise and earth-friendly spectrophotometric methods were developed for the simultaneous analysis of ledipasvir (LED) and sofosbuvir (SOF) without interference of both sunset yellow dye and copovidone excipients (the most probable interferents) in their combined dosage form. These proposed methods were based on measurement of LED in synthetic mixtures and combined dosage form by first derivative (1D) spectrophotometry at 314 nm over the concentration range of 2-50 µg mL-1 with coefficient of determination (R2) > 0.9999, mean percentage recovery of 99.98 ±â€¯0.62. On the other hand, SOF in synthetic mixtures and combined dosage form was determined by five methods. Method I is based on the use of 1D spectrophotometry at 274.2 nm (zero crossing point of LED). Method II involves the application of conventional dual wavelength method (DW) at the absolute difference between SOF zero order amplitudes at 261 nm (λmax of SOF) and 364.7 nm. At these wavelengths, the absolute difference between LED zero order amplitudes was observed to equal zero. Method III depends on isosbestic point method (ISP) in which the total concentration of both drugs was measured at isosbestic point at 262.7 nm. Concentration of SOF could be obtained by subtraction of LED concentration. While, method IV depends on absorbance correction method (absorption factor method), which is based on determination of SOF concentration at 262.7 nm (λISP) and LED at 333 nm (λmax of LED). Finally, method V depends on absorbance ratio method (Q-analysis) in which 262.7 nm (λISP) and 261 nm (λmax of SOF) were selected to determine SOF concentration. The linearity range for all methods for SOF determination was 2-50 µg mL-1 with coefficient of determination (R2) > 0.9999. Methods I, II & III were also applied for determination of SOF concentration in single dosage form. Their mean percentage recoveries were 100.35 ±â€¯1.85, 99.97 ±â€¯0.54 and 100.03 ±â€¯0.49, for the three methods respectively. The proposed methods were validated according to international conference of harmonization (ICH) requirements and statistically compared to published reference methods. The ANOVA test confirmed that there is no significant differences between the proposed methods, and can be used for routine analysis of LED and SOF in commercial tablets. These developed methods were applied to estimate the average content and uniformity of dosage unit for LED/SOF combined dosage form and SOF single dosage form according to British pharmacopeia (BP) requirements.

Development of a highly sensitive high-performance thin-layer chromatography method for the screening and simultaneous determination of sofosbuvir, daclatasvir, and ledipasvir in their pure forms and their different pharmaceutical formulations.

The combination of sofosbuvir and daclatasvir or sofosbuvir and ledipasvir is now widely used as an ideal treatment for hepatitis C virus infection. For this purpose, a simple, sensitive, accurate, economic, and precise high-performance thin-layer chromatography was developed and validated for the determination of sofosbuvir, daclatasvir, and ledipasvir in their pure form as well as their different pharmaceutical products. The method used Merck high-performance thin-layer chromatography aluminum plates precoated with silica gel 60 F254 as a stationary phase and mobile phase consisting of methylene chloride/methanol/ethyl acetate/ammonia (25%) (6:1:4:1, v/v/v/v). This system was found to give compact symmetric peaks of sofosbuvir, daclatasvir, and ledipasvir with retardation factors of 0.27 ± 0.01, 0.50 ± 0.007, and 0.68 ± 0.008, respectively. The densitometric scanner was set at 275 nm using a deuterium lamp. The calibration curves were linear over the range of 100-3000 ng/spot for sofosbuvir, and daclatasvir, and range of 50-3000 ng/spot for ledipasvir. The detection limits were 22.5, 31.90, and 15.80 for sofosbuvir, daclatasvir, and ledipasvir. The quantitation limits were 67.50, 95.60, and 47.50 for sofosbuvir, daclatasvir, and ledipasvir. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines and the results were acceptable.

Efficient HPTLC-dual wavelength spectrodensitometric method for simultaneous determination of sofosbuvir and daclatasvir: Biological and pharmaceutical analysis.

Sofosbuvir (SOF) and daclatasvir (DCS) are newly discovered anti-hepatitis C drugs that have direct antiviral activity. A novel and simple high-performance thin-layer chromatography (HPTLC) method was designed for simultaneous determination of SOF and DCS in miscellaneous matrices. The method adopts coupling HPTLC with dual wavelength spectrodensitometry. Consequently, this enabled sensitive, specific and cost-effective determination of the SOF-DCS mixture. The developed HPTLC procedure is based on a simple liquid-liquid extraction, enrichment of the analytes and subsequent chromatographic separation with UV detection. Separations were performed on HPTLC silica gel 60 F254 aluminum plates with a mobile phase consisting of ethyl acetate-isopropanol (85:15, v/v). Dual wavelength scanning was carried out in the absorbance mode at 265 and 311 nm for SOF and DCS, respectively. The linear ranges were 40-640 and 20-320 ng band-1 for SOF and DCS, respectively with correlation coefficients of ≥0.9997. The detection limits were 11.3 and 6.5 ng band-1 for SOF and DCS, respectively indicating high sensitivity of the proposed method. Consequently, this permits in vitro and in vivo application of the proposed method in human plasma with good percentage recovery (94.1-103.5%). Validation parameters were assessed according to ICH guidelines and US-FDA guidelines. Furthermore, the application was extended to analysis of SOF and DCS in their pharmaceutical formulations.

Investigation of anti-Hepatitis C virus, sofosbuvir and daclatasvir, in pure form, human plasma and human urine using micellar monolithic HPLC-UV method and application to pharmacokinetic study.

Simultaneous determination of sofosbuvir (SOF), and daclatasvir (DAC) in their dosage forms, human urine and human plasma using simple and rapid micellar high performance liquid chromatographic method coupled with UV detection (HPLC-UV) had been developed and validated. These drugs are described as co-administered for treatment of Hepatitis C virus (HCV). HCV is the cause of Hepatitis C and some cancers such as liver cancer (hepatocellular carcinoma) and lymphomas in humans. Separation and quantitation were carried out on anonyx™ C8 monolithic (100 × 4.6 mm (i.d.) analytical column maintained at 25 °C. The mobile phase consisted of 0.1 M sodium dodecyl sulfate (SDS) solution containing 20% (V/V) n-propanolol and 0.3% (V/V) triethylamine and pH was adjusted to 6.5 using 0.02 M phosphoric acid, respectively. The retention times of SOF and DAC were 4.8 min, and 6.5 min, respectively. Measurements were made at flow rate of 0.5 mL/min with injection volume of 20 µL and ultraviolet (UV) detection at 226 nm. Linearity of SOF and DAC was obtained over concentration ranges of 50-400, and 40-400 ng/mL, respectively in pure form, 60-300 and 50-300 ng/mL, respectively for human plasma and over 50-400, and 40-400 ng/mL, respectively for human urine with correlation coefficient >0.999. The proposed method demonstrated excellent intra- and inter-day precision and accuracy. The suggested method was applied for determination of the drugs in pure, dosage form, and in real human plasma, real human urine and drug-dissolution test of their tablets. The obtained results have been statistically compared to reported method to give a conclusion that there is no significant differences.

Comparison between core-shell and totally porous particle stationary phases for fast and green LC determination of five hepatitis-C antiviral drugs.

The performances of core-shell 2.7 µm and fully porous sub-2 µm particles packed in narrow diameter columns were compared under the same chromatographic conditions. The stationary phases were compared for fast separation and determination of five new antiviral drugs; daclatasvir, sofosbuvir, velpatasvir, simeprevir, and ledipasvir. The gradient elution was done using ethanol as green organic modifier, which is more environmentally friendly. Although both columns provided very good resolution of the five drugs, core-shell particles had proven to be of better efficiency. Under gradient elution conditions, core-shell particles exhibited faster elution, better peak shape, and enhanced resolution adding to lower system backpressure. The column backpressure on sub-2 µm particles was more than twice that on core-shell particles. This gives a chance to use conventional high-performance liquid chromatography conditions without needing special instrumentation as that required for ultra-high performance liquid chromatography. The method was validated for determination of the five drugs by gradient elution using mobile phase composed of organic modifier ethanol and aqueous part containing 0.75 g sodium octane sufonate and 3.0 g sodium dihydrogen phosphate per liter at pH of 6.15. Detection was done using UV-detector set at 210 nm. The linearity, accuracy, and precision were found very good within the concentration range of 2-200 µg/mL.

Chemometric simultaneous determination of Sofosbuvir and Ledipasvir in pharmaceutical dosage form.

Partial least squares (PLS), different families of continuous wavelet transform (CWT), and first derivative spectrophotometry (DS) techniques were studied for quantification of Sofosbuvir (SFB) and Ledipasvir (LDV) simultaneously without separation step. The components were dissolved in Acetonitrile and the spectral behaviors were evaluated in the range of 200 to 400nm. The ultraviolet (UV) absorbance of LDV exhibits no interferences between 300 and 400nm and it was decided to predict the LDV amount through the classic spectrophotometry (CS) method in this spectral region as well. Data matrix of concentrations and calibrated models were developed, and then by applying a validation set the accuracy and precision of each model were studied. Actual concentrations versus predicted concentrations plotted and good correlation coefficients by each method resulted. Pharmaceutical dosage form was quantified by developed methods and the results were compared with the High Performance Liquid Chromatography (HPLC) reference method. Analysis Of Variance (ANOVA) in 95% confidence level showed no significant differences among methods.

A new innovative spectrophotometric method for the simultaneous determination of sofosbuvir and ledipasvir.

A new innovative spectrophotometric method is developed to determine the concentration ratios in binary mixtures by determining the zero crossing point in the first derivative of the gross curve. This relationship can be applied if the part of the UV spectrum of substance Y, that intersects with the overlaid spectra of substance X is straight. By plotting the intersection wavelength against the concentration ratio (CxCy), a straight line was obtained with a co-efficient of determination equals 0.9999. As an application, simultaneous determination of sofosbuvir and ledipasvir in their binary mixtures was performed using two methods; a direct UV spectrophotometric method for determination of ledipasvir at 333nm, and the new "wavelength-intersection ratio" method for determination of sofosbuvir. In the wavelength-intersection ratio method, different mixtures of sofosbuvir and ledipasvir containing different concentration ratios were prepared; the zero crossing point of the first derivative curve in the range 285 to 295nm were determined for each mixture. An absorbance shift in the intersection was obtained with the change in the concentration ratio (sofosbuvir/ledipasvir). When the concentration ratio was plotted against the intersection wavelength, a straight line was obtained with a coefficient of determination of 0.9992. The direct method was linear in the range 3 to 18µg/mL while the wavelength-intersection ratio method was linear in the range 11-110µg/mL. The limits of detection were determined and found to be 0.5 and 3µg/mL for ledipasvir and sofosbuvir, respectively. The accuracy and repeatability of the two methods were tested. The mean %recovery was found to be 100.19% and 100.75% for ledipasvir and sofosbuvir, respectively. The relative standard deviation was 0.57 for ledipasvir and 1.79 for sofosbuvir. The intermediate precision was also checked, the coefficients of variation for sofosbuvir and ledipasvir measurements between days did not exceed 1.88%.

Validated spectrophotometric and chromatographic methods for analysis of the recently approved hepatitis C antiviral combination ledipasvir and sofosbuvir.

This work describes five simple and reliable spectrophotometric and chromatographic methods for analysis of hepatitis C antiviral binary mixture of ledipasvir (LPV) and sofosbuvir (SBV). Method I is based on the use of Amax and derivative spectrophotometry with the zero-crossing technique where LPV was determined using its Amax and 1D amplitudes at 324 and 338nm respectively, while SBV was determined by measuring the 1D amplitudes at 276nm. Method II involves the application of the ratio spectra derivative spectrophotometry. For LPV, 12µg/mL SBV was used as divisor and the 1DD amplitudes at 239.8nm were plotted against LPV concentrations; while by using 10µg/mL LPV, the amplitudes at 279.2nm were found proportional to SBV concentrations. Method III depends on ratio-difference measurement where the peak to trough amplitudes between 229.2 and 268.4nm were measured and correlated to LPV concentration. Similarly, the amplitudes between 268.6 and 229.2nm in the SBV ratio spectra were recorded. For method IV, the two compounds were separated using HPTLC sheets of silica gel and a mobile phase composed of chloroform-methanol (94:6) followed by densitometric measurement of LPV and SBV spots at 331 and 267nm respectively. Method V depends on HPLC-DAD. Effective chromatographic separation was achieved using Thermohypersil C8 column (4.6×250mm, 5µm) with a mobile phase consisting of 0.01M sodium dihydrogen phosphate (pH 2.5) and methanol (20:80) at a flow rate 1.2mL/min and detection at 332 and 262nm for LPV and SBV respectively. Analytical performance of the developed methods was validated according to the ICH guidelines with respect to linearity, ranges, precision, accuracy, detection and quantification limits. The validated methods were successfully applied to the simultaneous analysis of LPV and SBV in mixtures of different proportions and their combined tablet dosage form.

Spectroflurimetric estimation of the new antiviral agent ledipasvir in presence of sofosbuvir.

A spectroflurimetric method has been developed and validated for the selective quantitative determination of ledipasvir in presence of sofosbuvir. In this method the native fluorescence of ledipasvir in ethanol at 405nm was measured after excitation at 340nm. The proposed method was validated according to ICH guidelines and show high sensitivity, accuracy and precision. Furthermore this method was successfully applied to the analysis of ledipasvir in pharmaceutical dosage form without interference from sofosbuvir and other additives and the results were statistically compared to a reported method and found no significant difference.

Comparative Validation of the Determination of Sofosbuvir in Pharmaceuticals by Several Inexpensive Ecofriendly Chromatographic, Electrophoretic, and Spectrophotometric Methods.

Sofosbuvir (SOFO) was approved by the U.S. Food and Drug Administration in 2013 for the treatment of hepatitis C virus infection with enhanced antiviral potency compared with earlier analogs. Notwithstanding, all current editions of the pharmacopeias still do not present any analytical methods for the quantification of SOFO. Thus, rapid, simple, and ecofriendly methods for the routine analysis of commercial formulations of SOFO are desirable. In this study, five accurate methods for the determination of SOFO in pharmaceutical tablets were developed and validated. These methods include HPLC, capillary zone electrophoresis, HPTLC, and UV spectrophotometric and derivative spectrometry methods. The proposed methods proved to be rapid, simple, sensitive, selective, and accurate analytical procedures that were suitable for the reliable determination of SOFO in pharmaceutical tablets. An analysis of variance test with P-value > 0.05 confirmed that there were no significant differences between the proposed assays. Thus, any of these methods can be used for the routine analysis of SOFO in commercial tablets.

Chemical Analysis of Counterfeit Hepatitis C Drug Found in Japan.

In January 2017, counterfeits of the hepatitis C drug 'HARVONI® Combination Tablets' (HARVONI®) were found at a pharmacy chain through unlicensed suppliers in Japan. A total of five lots of counterfeit HARVONI® (samples 1-5) bottles were found, and the ingredients of the bottles were all in tablet form. Among them, two differently shaped tablets were present in two of the bottles (categorized as samples 2A, 2B, 4A, and 4B). We analyzed the total of seven samples by high-resolution LC-MS, GC-MS and NMR. In samples 2A, 3 and 4B, sofosbuvir, the active component of another hepatitis C drug, SOVALDI® Tablets 400 mg (SOVALDI®), was detected. In sample 4A, sofosbuvir and ledipasvir, the active components of HARVONI®, were found. A direct comparison of the four samples and genuine products showed that three samples (2A, 3, 4B) are apparently SOVALDI® and that sample 2A is HARVONI®. In samples 1 and 5, several vitamins but none of the active compounds usually found in HARVONI® (i.e., sofosbuvir and ledipasvir) were detected. Our additional investigation indicates that these two samples are likely to be a commercial vitamin supplement distributed in Japan. Sample 2B, looked entirely different from HARVONI® and contained several herbal constitutents (such as ephedrine and glycyrrhizin) that are used in Japanese Kampo formulations. A further analysis indicated that sample 2B is likely to be a Kampo extract tablet of Shoseiryuto which is distributed in Japan. Considering this case, it is important to be vigilant to prevent a recurrence of distribution of counterfeit drugs.