A Green-and-White Integrative Analytical Strategy Combining Univariate and Chemometric Techniques for Quantifying Recently Approved Multi-Drug Eye Solution and Potentially Cancer-Causing Impurities: Application to the Aqueous Humor.

BACKGROUND: Drug impurities are now seen as a major threat to the production of pharmaceuticals around the world and a major part of the global contamination problem, especially when it comes to carcinogenic impurities. OBJECTIVE: We present the first spectrophotometric strategy based on a combination of univariate and multivariate methods as impurity profiling methods for the estimation of lignocaine (LIG) and fluorescein (FLS) with their carcinogenic impurities: 2,6-xylidine (XYL) and benzene-1,3-diol (BZD). METHOD: The data processing strategy depends on overcoming unresolved bands by employing five affordable, accurate, selective, and sensitive methods. The methods applied were a direct UV univariate spectrophotometric analysis (D0) and four multivariate chemometric methods, including classical least squares (CLS), principal component regression (PCR), partial least squares (PLS), and genetic algorithm (GA-PLS). FLS analysis (1-16 µg/mL) was performed using the D0 method at 478 nm; then, the application of the ratio subtraction method (RSM) allowed the removal of interference caused by the FLS spectrum. From the resulting ratio spectra, LIG, XYL, and BZD can be efficiently determined by chemometrics. The calibration set was carefully selected at five concentration levels using a partial factorial training design, resulting in 25 mixtures with central levels of 160, 40, and 3 µg/mL for LIG, XYL, and BZD, respectively. Another 13 samples were applied to validate the predictive ability. RESULTS: The statistical parameters demonstrated exceptional recoveries and smaller prediction errors, confirming the experimental model's predictive power. CONCLUSIONS: The proposed approach was effectively tested using newly FDA-approved LIG and FLS pharmaceutical preparation and aqueous humor. Additionally, it was effectively assessed for whiteness, greenness, and sustainability using five assessment tools. HIGHLIGHTS: With its remarkable analytical performance, sustainability, affordability, simplicity, and cost-efficiency, the proposed strategy is an indispensable tool for quality control and in situ analysis in little-equipped laboratories, increasing the proposed approach's surveillance ability.

Simultaneous analysis of the of levamisole with triclabendazole in pharmaceuticals through developing TLC and HPLC-PDA chromatographic techniques and their greenness assessment using GAPI and AGREE methods.

Two simple and rapid chromatographic methods were developed and validated for the analysis of levamisole and triclabendazole simultaneously in pure and pharmaceutical products. The first method is thin-layer chromatography (TLC) with densitometry, and the second method is high-performance liquid chromatography with PDA detection (HPLC-PDA). A Hypersil BDS C18 column with dimensions of 4.6 × 150 mm and a particle size of 5 µm was used in the HPLC-PDA method. An isocratic condition was used to carry out the separation, and the mobile phase was made up of acetonitrile and a 0.03 M potassium dihydrogen phosphate buffer in double-distilled water. The ratio of the mobile phase preparation was 70:30 (v/v), and the flow rate was 1 mL/min. A wavelength of 215 nm was employed for analyte detection. Precoated silica gel 60 F254 aluminium plates were used for the TLC method's separation. Mobile phase was made of ethyl acetate, hexane, methanol, and ammonia (69:15:15:1) for the separation. The detection wavelength selected was 215 nm. According to the International Council for Harmonization (ICH) guidelines, the proposed methods were validated and it was found that the two chromatographic methods are accurate, precise, and linear for both compounds in the range of 3.75-37.5 and 6-60 mg/L for the HPLC method for levamisole and triclabendazole, respectively and in the range of 2-14 µg/spot for the TLC method. The developed methods greenness profile was assessed using AGREE and ComplexGAPI tools.

Environmentally sustainable DRS-FTIR probe assisted by chemometric tools for quality control analysis of cinnarizine and piracetam having diverged concentration ranges: Validation, greenness, and whiteness studies.

A novel diffuse reflectance fourier transform infrared spectroscopic method accompanied by chemometrics was optimized to fulfill the white analytical chemistry and green analytical chemistry principles for the quantification of cinnarizine and piracetam for the first time without any prior separation in their challenging pharmaceutical preparation, which has a pretty substantial difference in the concentration of cinnarizine/piracetam (1:16). Furthermore, the suggested method was used for cinnarizine/piracetam dissolution testing as an effective alternative to traditional methods. For the cinnarizine/piracetam dissolution tests, we used a dissolution vessel with 900 mL of phosphate buffer pH 2.5 at 37 °C ± 0.5 °C, then the sampling was carried out by frequent withdrawal of 20 µl samples from the dissolution vessel at a one-minute interval, over one hour, then representative fourier transform infrared spectra were recorded. To create a partial-least-squares regression model, a fractional factorial design with 5 different levels and 2 factors was used. This led to the creation of 25 mixtures, 15 as a calibration set and 10 as a validation set, with varying concentration ranges: 1-75 and 16-1000 µg/mL for cinnarizine/piracetam, respectively. Upon optimization of the partial-least-squares regression model, in terms of latent variables and spectral region, root mean square error of cross-validation of 0.477 and 0.270, for cinnarizine/piracetam respectively, were obtained. The optimized partial-least-squares regression model was further validated, providing good results in terms of recovery% (around 98 to 102 %), root mean square error of prediction (0.436 and 3.329), relative root mean square error of prediction (1.210 and 1.245), bias-corrected mean square error of prediction (0.059 and 0.081), and limit of detection (0.125 and 2.786) for cinnarizine/piracetam respectively. Ultimately, the developed method was assessed for whiteness, greenness, and sustainability using five assessment tools. the developed method achieved a greener national environmental method index and complementary green analytical procedure index quadrants with higher eco-scale assessment scores (91), analytical greenness metric scores (0.87), and red-greenblue 12 algorithm scores (89.7) than the reported methods, showing high practical and environmental acceptance for quality control of cinnarizine/piracetam.

An innovative nanoparticle-modified carbon paste sensor for ultrasensitive detection of lignocaine and its extremely carcinogenic metabolite residues in bovine food samples: Application of NEMI, ESA, AGREE, ComplexGAPI, and RGB12 algorithms.

Nowadays, veterinary medicine residues have been viewed as a major threat to food safety worldwide, especially when dealing with carcinogenic residues. Herein, we present the first differential pulse voltammetric method for the quantification of lignocaine and its carcinogenic metabolite 2,6-xylidine residues in bovine food samples, aided by five greenness and whiteness assessment tools, including NEMI, ESA, ComplexGAPI, AGREE, and RGB12. The method depends on the electrochemical oxidation after modification of the carbon paste sensor with recycled Al2O3-NPs functionalized multi-walled carbon nanoparticles. The produced sensor (Al2O3-NPs/MWCNTs/CPE) was characterized using XRD, FT-IR, EDX, SEM, and TEM. As expected, the active surface area and electron transfer processes were accelerated by the modification, resulting in ultra-sensitive quantification with detection limits of 19.00 and 13.94 nM for lignocaine and 2,6-xylidine, respectively. In terms of greenness, whiteness, sustainability, analytical effectiveness, and economic and practical considerations, the proposed method outperforms the reported methods.

Novel Deconvoluted Synchronous Spectrofluorimetric Method For Simultaneous Determination Of Bisoprolol and Atorvastatin As Single or Co-administrated Drugs in Bulk and Plasma; Green Assessment.

Atorvastatin and bisoprolol are two medications often prescribed together for the management of cardiovascular disease and to reduce mortality. Through a simple and direct technique based on deconvolution and synchronous of the spectrofluorometric spectra, the innovative method enables simultaneous quantification of bisoprolol and atorvastatin as single or co-formulated dosage forms in bulk and plasma. The method depends on measuring the amplitudes of bisoprolol and atorvastatin at 298 nm and 363 nm directly after deconvolution where the other drug doesn't show interference. The linearity of the method was 0.02-0.5 µg/mL and 0.3-25 µg/mL for bisoprolol and atorvastatin, respectively. LOD and LOQ were 0.004, 0.085 µg/mL and 0.013, 0.259 µg/mL for bisoprolol and atorvastatin respectively. Furthermore, green assessment of the method using Eco-Scale and GAPI scale. The method was precise, economical, simple, smart, time-saving and eco-friendly, which allowed its application in quality control unit and in lab assessment.

Construction and application of highly sensitive spinel nanocrystalline zinc chromite decorated multiwalled carbon nanotube modified carbon paste electrode (ZnCr2O4@MWCNTs/CPE) for electrochemical determination of alogliptin benzoate in bulk and its dosage form: green chemistry assessment.

A new sensor for alogliptin benzoate (ALG) estimation based on a simple and sensitive method was evolved on multiwalled-carbon-nanotube modified nanocrystalline zinc chromite carbon paste electrodes (ZnCr2O4@MWCNTs/CPEs). ALG electrochemical behavior was evaluated using a cyclic voltammetry (CV), square wave voltammetry (SWV) and chronoamperometry (CA). The new electrode materials were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), energy dispersive X-ray analysis (EDX) for elemental analysis and mapping, and X-ray diffraction (XRD) and the X-ray photoelectron spectroscopy (XPS) measurements. All these measurements exhibiting enhanced activity and high conductivity compared to the bare electrode without modification. The calibration curves obtained for ALG were in the ranges of 0.1-20 µmol L-1 with a quantification and detection limits of 0.09 and 0.03 µmol L-1, respectively. The prepared sensor showed a good sensitivity and selectivity with less over potential for ALG determination. Finally, the presented method was successfully applied as a simple, precise and selective electrochemical electrode for the estimation of ALG in its pharmaceutical dosage form.

Innovative electrochemical electrode modified with Al2O3 nanoparticle decorated MWCNTs for ultra-trace determination of tamsulosin and solifenacin in human plasma and urine samples and their pharmaceutical dosage form.

A simple, cheap, sensitive, and time-saving square wave voltammetric (SWV) procedure using a carbon paste electrode modified with aluminum oxide nanoparticle decorated multi-walled carbon nanoparticles (Al2O3-NPs/MWCNTs/CPE) is presented for the ultra-sensitive determination of tamsulosin (TAM) and solifenacin (SOL), one of the most prescribed pharmaceutical combinations in urology. Characterization of the developed electrode was performed using scanning electron microscopy (SEM), X-ray diffraction (XRD) patterns, energy dispersive X-ray analysis (EDX), transmission electron microscopy (TEM) and FT-IR spectrophotometry. The voltammetric behavior of TAM/SOL was evaluated using Al2O3-NPs in different content and electrode compositions. The use of Al2O3 functionalized MWCNTs as a CPE modifier increased the process of electron transfer as well as improved the electrode active surface area therefore, ultra-sensitive results were acquired with a linear range of 10-100 and 12-125 ng ml-1 for TAM and SOL respectively, and a limit of the detection value of 2.69 and 3.25 ng ml-1 for TAM and SOL, respectively. Interestingly, the proposed method succeeded in quantifying TAM and SOL with acceptable percentage recoveries in dosage forms having diverged concentration ranges and in the biological fluids with very low peak plasma concentration (C max). Furthermore, the proposed method was validated, according to the ICH criteria, and shown to be accurate and reproducible.

A New Chemometrically Assisted UV Spectrophotometric Method for Simultaneous Determination of Tamsulosin and Dutasteride in Their Pharmaceutical Mixture.

BACKGROUND: Tamsulosin (TAM) and dutasteride (DUT) are ranked among the most frequently prescribed therapies in urology. Interestingly, studies have also been carried out on TAM/DUT in terms of their ability to protect against recent COVID-19. However, very few studies were reported for their simultaneous quantification in their combined dosage form and were mainly based on chromatographic analysis. Subsequently, it is very important to offer a simple, selective, sensitive, and rapid method for the quantification of TAM and DUT in their challenging dosage form. OBJECTIVE: In this study, a new chemometrically assisted ultraviolet (UV) spectrophotometric method has been presented for the quantification of TAM and DUT without any prior separation. METHOD: For the calibration set, a partial factorial experimental design was used, resulting in 25 mixtures with central levels of 20 and 25 µg/mL for TAM and DUT, respectively. In addition, to assess the predictive ability of the developed approaches, another central composite design of 13 samples was used as a validation set. Post-processing by chemometric analysis of the recorded zero-order UV spectra of these sets has been applied. These chemometric approaches include partial least-squares (PLS) and genetic algorithm (GA), as an effective variable selection technique, coupled with PLS. RESULTS: The models' validation criteria displayed excellent recoveries and lower errors of prediction. CONCLUSIONS: The proposed models were effectively used to determine TAM/DUT in their combined dosage form, and statistical comparison with the reported method revealed satisfactory results. HIGHLIGHTS: Overall, this work presents powerful simple, selective, sensitive, and precise methods for simultaneous quantification of TAM/DUT in their dosage form with satisfactory results. The predictive ability and accuracy of the developed methods offer the opportunity to be employed as a quality control technique for the routine analysis of TAM/DUT when chromatographic instruments are not available.

Simultaneous spectrophotometric quantitative analysis of velpatasvir and sofosbuvir in recently approved FDA pharmaceutical preparation using artificial neural networks and genetic algorithm artificial neural networks.

Two chemometric assisted spectrophotometric models were applied for the quantitative analysis of velpatasvir and sofosbuvir in their newly FDA approved pharmaceutical dosage form. The UV absorption spectra of velpatasvir and sofosbuvir showed certain degree of overlap which exhibited degree of difficulty for the choice of certain method provides simultaneous quantitative analysis of the cited drugs. Artificial neural networks and genetic algorithm artificial neural networks were the suitable model for the quantitative analysis of velpatasvir and sofosbuvir in their binary mixture. Experimental design and building the calibration set for the binary mixture were achieved to implement the described models. The proposed models were optimized with the aid of five-levels, two factors experimental design. Spectral region of 380-400 nm was rejected which resulted in 181 variables. GA reduced absorbance matrix to 72 and 36 variables for velpatasvir and sofosbuvir respectively. The models succeeded to estimate the studied drugs with acceptable values of root mean square error of calibration and root mean square error of prediction. The developed models were successfully applied to the quantitative analysis of the two drugs in Epclusa® tablets. The results were statistically compared with another published quantitative analytical method with no significant difference by applying Student t-test and variance ratio F-test.

Second derivative synchronous fluorescence determination of avanafil in the presence of its acid-induced degradation product aided by powerful Lean Six Sigma tools augmented with D-optimal design.

In this work, the quantitative determination of an erectile dysfunctional drug avanafil in the presence of its acid-induced degradation product was achieved via the application of a pre-optimized novel spectrofluorimetric method. The fluorescence emission wavelength was recorded at 370 and 407 nm, after being excited at 268 and 271 nm for avanafil and its acid-induced degradation product, respectively. Direct determination of avanafil based on its native fluorescence is restricted because the emission spectra of both components are heavily overlapped. Therefore, to overcome this constraint, a novel second derivative synchronous fluorescence method was evolved to eliminate this overlapping. The ideal determination wavelength was found to be 377 nm. Augmentation of lean six sigma (LSS) with response surface methodology (RSM) play a significant role in the development of robust specifications to ensure quality at the six sigma level with a high level of statistical confidence and targeted performance. All of the experimental conditions were optimized using D-optimal design as a RSM to select the optimal parameters. In addition, this work includes a graphical representation of the relationships between various variables that can greatly affect the results and the intensity of the synchronous fluorescence.

Application of different spectrofluorimetric methods for determination of lesinurad and allopurinol in pharmaceutical preparation and human plasma.

Lesinurad and allopurinol combination is newly FDA approved for treatment of patients suffering from hyperuricemia associated with uncontrolled gout. In the present work, two different highly sensitive, selective and accurate fluorescence spectroscopic methods were developed for quantitative analysis of lesinurad and allopurinol in their pharmaceutical dosage form without any tedious operation procedure. Lesinurad was quantitatively analyzed based on its unique native fluorescence nature. Lesinurad fluorescence emission was quantitatively determined at 343 nm after excitation at 288 nm without any interference from allopurinol. Allopurinol, has free terminal secondary amino group, reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBDCl) through nucleophilic substitution mechanism forming highly fluorescent dark yellow fluorophore. Allopurinol was quantitavely analyzed based on measurement the emission fluorescence intensity of the fluorescent dark yellow fluorophore at 535 nm after excitation at 465 nm. Different parameters which affect the described methods of the studied drugs were carefully checked and optimized. Calibration graphs were found to be linear over the concentration range of 0.25-4.0 µg/mL for lesinurad and 0.2-20 µg/mL for allopurinol. The proposed methods were successfully applied for the quantitative analysis of the two drugs in Duzallo® pharmaceutical dosage form and spiked human plasma.

Simultaneous determination of elbasvir and grazoprevir in their pharmaceutical formulation by synchronous fluorescence spectroscopy coupled to dual wavelength method.

In the present study, a sensitive, selective and accurate synchronous fluorescence spectroscopic method was utilized for simultaneous estimation of elbasvir and grazoprevir in their pharmaceutical formulation. The developed method based on measurement of the synchronous fluorescence intensity of the studied drugs at constant wavelength difference (Δλ) = 50 nm. Elbasvir can be determined directly at 312 nm without interference from grazoprevir. Grazoprevir can be determined by application of dual wavelength method by taking the difference in synchronous fluorescence intensity at 390 & 372 nm to remove interference from elbasvir. Calibration graphs were found to be linear over the concentration range of 50-700 ng/mL for elbasvir and 100-900 ng/mL for grazoprevir. The developed method was successfully applied to the quantitative analysis of the two drugs in Zepatier® tablets.

First derivative synchronous fluorescence spectroscopy for the determination of Gatifloxacin in presence of its oxidative degradation product: Application to pharmaceutical preparation.

A highly accurate and precise spectrofluorimetric method was established for quantitation of Gatifloxacin in pure material, pharmaceutical formulations and in the existence of its oxidative degradation product. The emission was recorded at 487 nm after the excitation at 290 nm. Using micelle, sodium dodecyl sulphate (SDS), enhanced fluorescence intensity of Gatifloxacin-SDS complex. The optimization of numerous experimental conditions was carried out. The improved emission showed a suitable linear correlation between derivative synchronous fluorescence power and concentration of Gatifloxacin over the range of 10 to 100 ng/mL with a determination coefficient equals 0.9996. Studying cytotoxicity and antimicrobial susceptibility for oxidative degradation product of Gatifloxacin was carried out using Gatifloxacin as a control. In comparison, the proposed method presented a superior sensitivity and enhanced stability over the reported method.

A comparative study of different aspects in manipulating ratio spectra used for the analysis of cefradine in the presence of its alkaline degradation product.

Six stability-indicating UV-spectrophotometric methods manipulating ratio spectra were utilized for the analysis of cefradine in presence of its alkaline degradate. These methods are different forms of transformations; ratio difference, mean centering, derivative ratio using numerical differentiation, derivative ratio using Savitsky-Golay filter, continuous wavelet transform and derivative continuous wavelet transform. Water was used as a solvent and the linearity ranges were 6-26 µg/mL. Determination of accuracy and precision for the suggested procedures were executed. Assessment of specificity was run through analyzing laboratory prepared mixtures containing cefradine and its alkaline degradate. The suggested methods were useful for cefradine estimation in tablets. Statistically, the outputs obtained from the recommended and published methods reveal no significant differences.

Development and validation of a copper ligand-exchange chromatographic method for the estimation of D-lactic acid in Ringer-lactate solution.

In this study, the chromatographic conditions for separation and determination of L- and D-lactic acid enantiomers by copper ligand exchange chromatography have been examined and optimized statistically using a response surface methodology (RSM). The chromatographic variables: copper sulfate, acetic acid and organic modifier were screened by operating a 2-level full factorial design (FFD). The significant effect of independent chromatographic variables was analyzed using the analysis of variance (ANOVA). Variables proved significant (p < 0.05) were cautiously tuned using RSM with a face-centered central composite design. Moreover, a D-optimality design was employed to minimize the variation in the regression coefficients of the fitted model. The proposed model represented an excellent example of fulfilling the efficiency of factorial designs in optimizing the chromatographic conditions and maximizing the output. The chromatographic separation was achieved on Supelco Astec CLC-D chiral bidentate ligand (5.0 µm, 150.0 × 4.6 mm). The isocratic mobile phase composition was 7 mM anhydrous copper sulfate in 1.0 mM acetic acid containing 4% methanol. A photodiode array detector was used to determine the optimal detection wavelength, which was at 236 nm. A linear calibration curve was obtained in the range of 30.0-3600 µg mL-1 with a high value for the coefficient of determination (R2 ≥ 0.999). The optimized method has been successfully applied to the determination of lactate in the commercial Ringer-lactate solution for injection. The results obtained were in excellent agreement with the label claim with no interference from other additives commonly co-formulated with the drug. Intra- and inter-day precision, detection and quantification limits, as well as percent coefficient of variation, have been estimated according to ICH guidelines for assessment of analytical procedures.

Simultaneous Determination of Elbasvir and Grazoprevir in Their Pharmaceutical Preparation Using High-Performance Liquid Chromatographic Method.

A high-performance liquid chromatographic method has been developed for simultaneous determination of elbasvir and grazoprevir; two new Food and Drug Administration (FDA) approved drugs. The two drugs were co-formulated for treatment of hepatitis C virus in their combined pharmaceutical dosage form. The chromatographic separation has been achieved using a reversed phase BDS Hypersil C18 column with a mobile phase consists of acetonitrile:methanol (50:50, v/v) at flow rate of 1 mL/min and UV detection at 253 nm. Computational investigation for finding the best stationary phase revealed that C18 column fits better for the simultaneous chromatographic analysis of the studied drugs. Linearity, accuracy and precision have been found to be acceptable over the concentration range of 1-20 µg/mL for the studied drugs. The described method has been successfully applied for simultaneous determination of the studied drugs in their pharmaceutical dosage form.

Spectrofluorimetric determination of acotiamide hydrochloride trihydrate in the presence of its oxidative degradation product.

Acotiamide hydrochloride trihydrate is a novel gastroprokinetic drug which has been recently approved for the treatment of patients with functional dyspepsia. This study presents the first reported to investigate the fluorimetric behavior of acotiamide hydrochloride trihydrate in the presence of its oxidative degradation product. All variables that affect fluorescence intensity were studied and optimized. The described method involved the measurement of native fluorescence of the drug in ethanol at 404 nm after excitation at 326 nm. Calibration plot was found to be linear over the concentration range 0.1-0.9 µg/ml. The specificity of the method has been tested via selective determination of the studied drug in its synthetic mixtures with its degradation product. The proposed method has been successfully applied to the analysis of the drug in its new pharmaceutical dosage form and the results have been statistically compared with the reported HPLC method showing no significant differences by applying t-test and F-test.

Simultaneous determination of rosuvastatin and propranolol in their binary mixture by synchronous spectrofluorimetry.

Simultaneous determination of rosuvastatin calcium and propranolol hydrochloride using the first derivative synchronous spectrofluorimetry was described. This method involves measuring the synchronous fluorescence of both drugs in ethanol using, ∆ λ = 60 nm then the first derivative was recorded and the peak amplitudes were measured at 350 and 374 nm for rosuvastatin calcium and propranolol hydrochloride, respectively. Under the optimum conditions, the linear ranges of rosuvastatin calcium and propranolol hydrochloride were 0.2-2 µg/mL and 0.1-1 µg/mL, respectively. The method was used for quantitative analysis of the drugs in raw materials and pharmaceutical dosage form. The validity of the proposed method was assessed according to an international conference on harmonization (ICH) guidelines.

Validated Stability Indicating High Performance Liquid Chromatographic Determination of Lesinurad.

Lesinurad is a novel selective uric acid reabsorption inhibitor which has been newly approved for the treatment of the chronic gout. The behavior of lesinurad under various stress conditions (hydrolysis, oxidation, thermal and photolysis) has been investigated as per ICH guidelines. The drug has been found to be labile to acidic hydrolysis, basic hydrolysis and oxidation but stable in neutral, thermal and photolytic conditions. A high performance liquid chromatographic method has been developed for selective determination of the studied drug in the presence of its related degradation products. Good chromatographic resolution has been achieved using a reversed phase BDS Hypersil C18 stationary phase with an isocratic elution of a mobile phase consists of acetonitrile:water (65:35, v/v) at a flow rate of 1 mL/min and UV detection at 290 nm. Two degradation products have been identified by IR and mass spectral scans. The method was validated according to ICH guidelines. The linearity range has been found to be acceptable over the concentration range of 1-20 µg/mL. The developed method has been successfully applied for the estimation of lesinurad in its pharmaceutical dosage form and could be used for the routine analysis of the studied drug in the quality control laboratories.

Simultaneous Spectrophotometric Determination of Elbasvir and Grazoprevir in a Pharmaceutical Preparation.

Three UV spectrophotometric methods have been developed for the simultaneous determination of two new Food and Drug Administration-approved drugs, elbasvir (EBV) and grazoprevir (GRV), in their combined pharmaceutical dosage form. These methods include dual wavelength (DW), classic least-squares (CLS), and principal component regression (PCR). To achieve the DW method, two wavelengths were chosen for each drug in a way to ensure the difference in absorbance was zero from one drug to the other. GRV revealed equal absorbance at 351 and 315 nm, for which the distinctions in absorbance were measured for the determination of EBV. In the same way, distinctions in absorbance at 375 and 334.5 nm were measured for the determination of GRV. Alternatively, the CLS and PCR models were applied to the spectra analysis because the synchronous inclusion of many unreal wavelengths rather than using a single wavelength greatly increased the precision and predictive ability of the methods. The proposed methods were successfully applied to the assay of these drugs in their pharmaceutical formulation. The obtained results were statistically compared with manufacturing methods. The results conclude that there was no significant difference between the proposed methods and the manufacturing method with respect to accuracy and precision.