Smart Multivariate Spectrophotometric Determination of Two Co-Administered Autoimmune Drugs; Sulfasalazine and Pentoxifylline; in Bulk and Spiked Human Plasma.

BACKGROUND: Sulfasalazine and pentoxifylline are co-prescribed together to treat psoriasis and pemphigus vulgaris. Sulfasalazine is an anti-inflammatory, immunosuppressant, and antibiotic drug, while pentoxifylline is a vasodilator and immunosuppressant. The spectra of the two drugs and plasma suffer from severe overlap. OBJECTIVE: This work aims to simultaneously determine sulfasalazine and pentoxifylline in their binary mixture and spiked human plasma by the assessment of their UV spectral data. METHODS: Two model updated chemometric methods were established using principal component regression and partial least-squares regression models. The two models were validated in accordance with the U.S. Food and Drug Administration guidelines for bioanalysis and were applied for the determination of both drugs in synthetic mixtures or spiked human plasma. RESULTS: Accuracy and precision were within the accepted limits. In addition, three different assessment methods were used to evaluate the environmental greenness of the proposed models. CONCLUSION: The two updated models are simple, rapid, sensitive, and precise, and could be easily applied in QC laboratories for determination of sulfasalazine and pentoxifylline, without any preliminary separation steps or interference from plasma matrix. HIGHLIGHTS: Two updated chemometric models called principlal component regression and partial least-squares regression were established for determination of sulfasalazine and pentoxifylline in spiked human plasma using UV spectrophotometric data.

Development of HPLC-UV Method for Simultaneous Determination of Corticosteroid Co-Administered Immune Therapy.

Prednisolone (PDS) has recently been utilized to treat a variety of medical disorders, including autoimmune illnesses and cancer. It is also used to treat coronavirus disease 2019 infection-related respiratory problems. Because it may induce health problems including gastrointestinal lesions and ulceration, it has to be used alongside other drugs like esomeprazole (ESM), which acts as a proton pump antagonist to reduce the probability of ulceration. As a result, the goal of this research is to create an environmentally safe and sensitive high-performance liquid chromatography (HPLC) approach for determining PDS and ESM in their binary combination and spiked human plasma. C8 column (100 × 4.6 mm, 5 µm) and gradient mobile phase elution were used to separate the studied drugs with ultraviolet recognition at 290 nm. Caffeine was utilized as an internal standard to adjust the sample variance. Plasma, caffeine, ESM and PDS all had tR values of 1.4, 3.5, 6.3 and 7.3, respectively. The suggested method's greenness features were evaluated using three greenness evaluation tools: green analytical procedure index, analytical greenness metric approach and analytical eco-scale, and the findings were approved and satisfied. Validation parameters were evaluated in accordance with US-FDA recommendations in order to meet the global desires for biological analysis technique, acceptable limits were obtained.

Green Easily Implemented Spectrophotometric Methods for Concurrent Determination of Ephedrine Hydrochloride and Naphazoline Nitrate in Nasal Preparations Containing Methylparaben.

BACKGROUND: Spectrophotometric resolution of a mixture of several drugs is considered a cheaper, simpler, and more versatile alternative compared to costly chromatographic instruments. OBJECTIVE: The work aims to resolve the interfering spectra of ephedrine hydrochloride, naphazoline nitrate, and methylparaben in nasal preparations using smart spectrophotometric methods. METHOD: In our work, derivative and dual-wavelength methods were combined to eliminate this interference, under the name of derivative dual-wavelength method. Other methods, namely successive derivative subtraction and chemometric analysis, were also able to eliminate this interference. The methods have proven their applicability as they follow the International Conference on Harmonization (ICH) requirements regarding repeatability, precision, accuracy, selectivity, and linearity. Eco-scale, GAPI, and AGREE tools were used to estimate the possible environmental effects of the methods. RESULTS: Acceptable results for repeatability, precision, accuracy, selectivity, and linearity were obtained. Limit of detection (LOD) values were 2.2 for ephedrine and 0.3 for naphazoline. The correlation coefficients were above 0.999. The methods were proven to be safe for application. CONCLUSIONS: The introduced methods are cheap and easily implemented compared to chromatographic techniques. They can be used in purity-checking of raw material and estimation of concentrations in market formulations. The replacement of the published chromatographic techniques with our developed methods is useful when needing to save money, effort, and time. HIGHLIGHTS: The three components of a decongestant nasal preparation were determined using cheap, green, and versatile spectrophotometric methods that keep the advantages of chromatographic techniques, including accuracy, reproducibility, and selectivity.

A validated ecofriendly chromatographic method for determination of myasthenia gravis combined medications in spiked human plasma.

Recently, the main interest of analytical chemistry researchers has been the development of green analytical methods to minimize harmful effects on the environment and natural life. Therefore, an RP-HPLC method was developed and assessed regarding its greenness criteria using three greenness assessment tools: an analytical eco-scale, an analytical greenness metric approach and a green analytical procedure index. This method aims to separate and quantitatively determine three co-administered drugs, namely pyridostigmine bromide (PYR), 6-mercaptopurine (MRC) and prednisolone (PRD), in their tertiary mixture and spiked human plasma. These drugs are co-administered to manage myasthenia gravis autoimmune disease. The separation was done using a C18 column and a gradient elution of a mixture of 0.1% H3 PO4 aqueous solution (pH 2.3) and methanol. The flow rate was adjusted to 1 ml/min and detection was done at 254 (for PYR and PRD) and at 330 nm (for MRC). The lower limits of quantitation were 15, 2, and 5 µg/ml for PYR, MER and PRD, respectively. Linear correlations were obtained and found to be near 1. In addition, the proposed method was validated according to the US Food and Drug Administration's instructions, and the results proved its success to determine the three studied drugs in their tertiary mixture and spiked human plasma.

Tailoring Two Chemometric Models for Determination of Three Neuromuscular Combined Medications and Application to Spiked Human Plasma.

BACKGROUND: Prednisolone, 6-mercaptopurine, and pyridostigmine bromide are co-administered together to treat a neuromuscular autoimmune disease called myasthenia gravis. Prednisolone and 6-mercaptopurine are immunosuppressant drugs. 6-Mercaptopurine is the active form of the pro-drug azathioprine. Pyridostigmine bromide is a cholinesterase enzyme inhibitor. Curently, green characteristics are taken into account by analysts when they develop new methods. OBJECTIVE: The spectra of the three compounds and plasma are highly overlapped, so this study aims to remove this overlap and determine the three components quantitatively in raw powders and spiked human plasma using green methods. METHODS: Two multivariate updated chemometric models called principle component analysis and partial least-squares were developed. Three greenness assessment tools: the eco-scale, the analytical greenness metric approach, and the green analytical method index, were used to evaluate the greenness behavior of the generated models. RESULTS: The two models were verified in accordance with Food and Drug Administration requirements, and the results were within acceptable limits. In addition they are relatively green in accordance with the abovementioned greenness evaluation tools. CONCLUSION: The developed models succeeded in determining the proposed drugs in their tertiary combinations and spiked human plasma with satisfactory precision, accuracy, and good greenness behavior. HIGHLIGHTS: Two ecologically evaluated, multivariate spectrophotometric methods were developed for the determination of pyridostigmine bromide, 6-mercaptopurine, and prednisolone in spiked human plasma.

Simultaneous spectrophotometric determination of fluphenazine HCl and nortriptyline HCl in presence of their potential impurities perphenazine and dibenzosuberone in bulk and pharmaceutical formulation.

Fluphenazine HCl (FLU) is an anxiolytic, while Nortriptyline HCl (NOR) is an anti-depressant. They are co-formulated together to treat depression and schizophrenia. Perphenazine (PER) and dibenzosuberone (DBZ) are the pharmacopeial impurities of FLU and NOR, respectively. Four spectrophotometric and multivariate chemometric methods were developed to determine the two drugs together or in presence of their two impurities in their bulk and pharmaceutical formulation. Method (A) is the triple divisor-ratio derivative (TDR) method, where the zero order spectrum of each component was divided by a mixture of the other 3 components, then the peak amplitudes of the first derivative spectra of FLU, NOR and DBZ were measured at 265, 245.4 and 283.2 nm, respectively. Method (B) is the double divisor-ratio difference-dual wavelength (DD-RD-DW) method, in which each component spectrum mixture was divided by a binary mixture of 2 of the interfering components. In the resulting ratio spectra, the amplitude difference is calculated between 2 wavelengths at which the third interfering component has zero difference. Methods (C and D) are the principle component analysis (PCA) and partial least squares (PLS) models. Methods (A and B) failed to quantify PER (FLU impurity), while (C and D) succeeded to quantify all components. The four methods have been applied for the prediction of the FLU and NOR in their pharmaceutical formulation with good accuracy and precision. The proposed methods have been validated according to the ICH guidelines and the results were within the acceptable limits.

US FDA-validated TLC method with four greenness assessment evaluations for simultaneous determination of prednisolone and esomeprazole in spiked human plasma.

Recently, prednisolone has been used in treating many medical conditions, such as autoimmune diseases and cancer. It is also prescribed to mitigate the respiratory complications caused by COVID-19 infection. It can cause some health complications, such as GIT ulcers, so it should be co-administered with proton-pump inhibitors, such as esomeprazole, to prevent the risk of ulcers. This work aims to develop an ecofriendly and sensitive TLC method for simultaneous determination of esomeprazole and prednisolone in their binary mixtures and spiked human plasma. Separation was performed using a mixture of ethyl acetate, methanol, and ammonia (9.5:0.5:0.1, v/v/v) as an eluting system with UV scanning at 245 nm. Dapoxetine was used as an internal standard to correct the variation during sampling. The resulting Rf values for plasma, esomeprazole, prednisolone, and dapoxetine were 0.03, 0.51, 0.72 and 0.85, respectively. Four greenness assessment tools-national environmental method index, eco-scale assessments, analytical greenness metric approach (AGREE), and green analytical procedure index (GAPI)-were used to evaluate the greenness characteristics of the proposed method to the environment, and the results were acceptable and satisfactory. Validation parameters were checked according to the US FDA guidelines to achieve the international requirements for bioanalytical method validation, and the results were within the accepted ranges.

Multivariate Model Update Chemometric Methods for Determination of Prednisolone and Esomeprazole in Spiked Human Plasma: A Comparative Study.

BACKGROUND: Prednisolone (PRD) is an immunosuppressant and anti-inflammatory drug, although it may cause peptic ulcers as a side effect. Esomeprazole (ESO) is used for the treatment of peptic ulcers, therefore the two drugs are co-administered in cases of organ transplantation and autoimmune diseases. OBJECTIVE: This work aims to determine the two drugs simultaneously, in bulk, and in spiked human plasma by eliminating the overlap of their spectra and the interference of the plasma matrix. METHODS: Two simple and effective updated chemometric models-principal component analysis (PCA) and partial least squares (PLS)-were established using UV spectrophotometric data. RESULTS: The two updated models have been validated according to the U.S. Food and Drug Administration guidelines with acceptable results. The results were statistically compared with those of the reported methods, where no significant difference was found, indicating the validity of the developed methods. The two updated models have been successfully applied for prediction of the proposed drugs with good accuracy and precision. CONCLUSION: The two updated models are simple, rapid, sensitive, and precise and could be easily applied in quality control laboratories for determination of PRD and ESO, without any preliminary separation steps or interference from plasma matrixes. HIGHLIGHTS: Two model updated chemometric models, PCA and PLS, were established for determination of PRD and ESO in spiked human plasma using UV spectrophotometric data.

Quantitative Determination of Anti-Migraine Quaternary Mixture in Presence of p-Aminophenol and 4-Chloroacetanilide.

A validated RP-HPLC method was developed for the estimation of paracetamol, caffeine, metoclopramide and ergotamine simultaneously in bulk and pharmaceutical formulation. The method was extended for the determination of two paracetamol genotoxic and nephrotoxic impurities and degradation products namely p-aminophenol and 4-chloroacetanilide. Separation was done on octadecyl column (15 cm × 2.1 mm, 5 µm). Gradient elution was performed using mobile phase consisting of acetonitrile and water (pH 3) in ratio of (20: 80, v/v) for the first 3 min, (50: 50, v/v) for the next 4.5 min, then (20: 80, v/v) for the final 2.5 min. The flow rate was 0.7 mL/min throughout the run which took 10 min. UV detection was done at 230 nm. The proposed method agreed with ICH guidelines of method validation. The suggested method was applied for the estimation of the proposed drugs in their dosage form. Statistical comparison was done between the results obtained from the suggested method and those obtained from reported HPLC method. The proposed method is more beneficial than the published one because of having higher sensitivity and selectivity as it allows the detection of the proposed drugs in lower concentrations in the presence of paracetamol toxic impurities, p-aminophenol and 4-chloroacetanilide.

Different chromatographic methods for determination of alogliptin benzoate, metformin hydrochloride, and metformin impurity in bulk and pharmaceutical dosage form.

Two simple, sensitive, and reproducible methods were developed for the determination of alogliptin and metformin hydrochloride in presence of metformin impurity "melamin" in pure form and in pharmaceutical formulation. Method (A) was a thin layer chromatographic method in which separation was achieved using ethyl acetate-methanol-formic acid (6:3.8:0.2, by volume) as a developing system followed by densitometric scanning at 230 nm. Method (B) was a high-performance liquid chromatography method; separation was achieved on C18 column, the mobile phase consisted of a mixture of sodium lauryl sulfate buffer 0.1% w/v, pH 3: methanol in the ratio 70:30, v/v and measurement was done at 220 nm. System suitability testing parameters were calculated to ascertain the quality performance of the developed chromatographic methods. The proposed methods have been validated regarding accuracy, precision, and selectivity, moreover they have been successfully applied to Westirizide tablets containing both alogliptin and metformin hydrochloride, results indicate that there was no interference from additives. No significance difference was found when these methods were compared to the reported one.

Three Spectrophotometric Methods for Quantitative Analysis of Duloxetine in Presence of its Toxic Impurity: 1-Naphthol.

BACKGROUND: Duloxetine hydrochloride (DUL) is a drug used to treat depression and anxiety. 1-Naphthol is a potential toxic impurity of DUL, as it causes hepatotoxicity in humans, and it is harmful to aquatic life. OBJECTIVE: Three simple, selective, rapid, accurate and precise methods were developed and validated according to International Conference on Harmonization (ICH) guidelines with respect to linearity, accuracy, precision and selectivity for analysis of duloxetine hydrochloride (DUL) in the presence of its potential toxic impurity 1-Naphthol in different laboratory-prepared mixtures and pharmaceutical formulations. METHODS: Method (A) is the first derivative of the ratio spectra spectrophotometric (1DD) method which allows determination of DUL at 251 nm and 1-Naphthol at 305.2 nm without interference from each other. Method B (dual wavelength) means that two different wavelengths were chosen to each drug, where the absorbance difference at these two wavelengths is equal to zero to the second drug. The chosen two wavelengths for DUL were 221.4 nm and 235.6, where the absorbance difference for 1-naphthol at these two wavelengths was equal to zero. While the chosen wavelengths for 1-naphthol were 247.8 nm and 297 nm, where the absorbance difference for DUL at these two wavelengths was equal to zero. Method (C) is the mean centering of ratio spectra spectrophotometric (MCR) method, which depends on measuring the mean centered values of ratio spectra of both DUL and 1-Naphthol at 226 nm. RESULTS: These methods were validated and agreed with the requirements of ICH guidelines with respect to linearity, accuracy, precision and selectivity. CONCLUSIONS: The results indicate the ability of developed methods to be used for routine quality control analysis of DUL in bulk and pharmaceutical formulations in the presence of its potential impurity 1-Naphthol.

Validated ecofriendly chromatographic method for quantitative determination of anti-migraine quaternary mixture.

A novel ecofriendly, cost and time saving high-performance thin-layer chromatographic method was developed and validated for simultaneous determination of metoclopramide, ergotamine, caffeine, and paracetamol in bulk and pharmaceutical formulation. The separation was carried out on silica gel plates, using ethyl acetate:ethanol:ammonia (9:1:0.1, v/v/v) as a developing system. Ultraviolet detection was carried out at 272 nm. The resulting retention times were 0.15, 0.36, 0.49, and 0.74 min for metoclopramide, ergotamine, caffeine, and paracetamol, respectively. The greenness profile assessment was achieved to the proposed method to evaluate its greenness characters to the environment with acceptable results. Validation parameters were checked according to International Conference of Harmonization guidelines to achieve the international requirements for quality control analysis of the proposed drugs.

HPTLC Separation of a Hepatoprotective Combination in Pharmaceutical Formulation and Human Plasma.

A binary mixture of Silymarin (SR) and Vitamin E (VE) acetate, of an antioxidant and a hepatoprotective effect, has been analyzed using a sensitive, selective and economic high performance thin layer chromatographic (HPTLC) method in their pure forms, pharmaceutical formulation and spiked human plasma. SR and VE were separated on 60F254 silica gel plates using hexane:acetone:formic acid (7:3:0.15, v/v/v) as a developing system with UV detection at 215 nm. The method was evaluated for linearity, accuracy, precision, selectivity, limit of detection (LOD) and limit of quantification (LOQ). SR and VE were detected in the linear range of 0.2-2.5 and 0.2-4.5 µg/band, respectively. Method validation was done as per ICH guidelines and acceptable results of accuracy of 99.86 ± 1.190 and 100.22 ± 1.609 for SR and VE, respectively were obtained. The method has been successfully applied for determination of the studied drugs in their pharmaceutical formulation without any interference from excipients, and in spiked plasma samples. Results obtained by the developed HPTLC-densitometric method were statistically compared to those obtained by the reported HPLC methods and no significant difference was found between them.

A Validated Green HPTLC Method for Quantitative Determination of Dapoxetine Hydrochloride and Tadalafil in Bulk and Pharmaceutical Formulations.

Dapoxetine hydrochloride (DAP) and Tadalafil (TAD) were separated and determined quantitatively using a validated green high-performance thin layer chromatographic (HPTLC) method in their binary mixtures either as raw materials or in pharmaceutical formulations. The concentration ranges were 0.1-1.6 and 0.2-2.5 µg/band for dapoxetine and tadalafil, respectively, with accuracies of 98.93% ± 0.62 and 99.26% ± 1.39, respectively. Silica gel HPTLC F254 plates were used to carry out the separation. The mobile phase used was a mixture of ethanol-ethyl acetate (1:9 by volume), which is environmentally green and harmless. Densitometric scanning with UV detector was used to detect the separated peaks at 222 nm. ICH guidelines were followed to validate the suggested method, and the results prove that they can be used for regular analysis in quality control laboratories with compatible results.

Linear Support Vector Regression and Partial Least-Squares for Determination of Dapoxetine Hydrochloride and Tadalafil in Binary Pharmaceutical Mixtures.

BACKGROUND: Dapoxetine (DAP) is a serotonin-norepinephrine reuptake inhibitor, and Tadalafil (TAD) is a phosphodiesterase type-5 inhibitor. Both are coformulated as tablets called Erectafil® for treatment of erectile ejaculation. OBJECTIVE: DAP and TAD were analyzed in their binary mixtures and pharmaceutical formulations using two multivariate calibration chemometric models. METHODS: Partial least-squares (PLS) and linear support vector regression (SVR) models were applied using two factor-four level experimental design and UV-spectrophotometric data. They were compared to each other, and their advantages and disadvantages were discussed. RESULTS: The developed methods succeeded to determine DAP and TAD in different ratios with good results regarding International Conference on Harmonization guidelines. Linearity ranges were 2-15 µg/mL and 3-30 µg/mL for DAP and TAD, respectively, with good accuracy of 100 ± 0.37 for DAP and 100 ± 0.8 for TAD regarding PLS model and 100.04 ± 0.32 for DAP and 99.89 ± 0.77 for TAD regarding SVR model. Good precision values of 0.787 for DAP and 0.793 for TAD regarding PLS model and 1.105 for DAP and 0.930 for TAD regarding SVR model were obtained. The two models were applied on the dosage forms and statistically compared with the published HPLC method with no significant difference regarding accuracy and precision. CONCLUSIONS: The two models can be utilized for routine analysis and QC of DAP and TAD in their bulk and pharmaceutical formulations. The SVR model gives better results and generalization ability than those of the PLS model regarding accuracy and prediction error, while the latter is better for being simpler and faster.

Quantitative determination of Dapoxetine Hydrochloride and Tadalafil using different validated spectrophotometric methods.

Three rapid, simple and selective spectrophotometric methods were developed for determination of Dapoxetine Hydrochloride (DAP) and Tadalafil (TAD) in bulk and pharmaceutical dosage forms. Method (A) is simultaneous first derivative (1D) spectrophotometric method in which the peak amplitudes of the first derivative spectra (1D) were measured for both DAP and TAD at 322.4 nm and 230 nm, respectively with no interference from each other. Method (B) is the area under curve (AUC) spectrophotometric method in which the areas under curve in the wavelength ranges 228-240 nm and 242-254 nm are used for determination of DAP and TAD respectively. Method (C) is ratio subtraction combined with extended ratio subtraction spectrophotometry (EXRS) in which TAD was determined by dividing the mixture spectra by the spectrum of 15 µg/mL solution of DAP, while DAP was be determined by dividing the mixture spectra by the spectrum of 30 µg/mL solution of TAD. The developed methods were applied to different laboratory prepared mixtures of DAP and TAD. These methods were validated according to the ICH guidelines with respect to linearity, accuracy, precision, selectivity and specificity, and can be used for routine quality control analysis of DAP and TAD in their dosage forms.

Kinetic study and mechanism of Niclosamide degradation.

A spectrophotometric kinetic study of Niclosamide alkaline degradation as a function of drug concentration, alkaline concentration and temperature has been established utilizing double divisor-ratio spectra spectrophotometric method. The developed method allowed determination of Niclosamide in presence of its alkaline degradation products; namely; 2-chloro-4-nitro aniline (DEG I) and 5-chloro salicylic acid (DEG II) with characterization of its degradation mechanism. It was found that degradation kinetic of Niclosamide followed pseudo-first order under the established experimental conditions with a degradation rate constant (k) of 0.0829 mol/h and half life (t1/2) of 8.35 h. The overall degradation rate constant as a function of the temperature under the given conditions obeyed Arrhenius equation where the activation energy was calculated to be 3.41 kcal/mol.

Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product.

Accurate, selective, sensitive and precise HPTLC-densitometric and RP-HPLC methods were developed and validated for determination of bumadizone calcium semi-hydrate in the presence of its alkaline-induced degradation product and in pharmaceutical formulation. Method A uses HPTLC-densitometry, depending on separation and quantitation of bumadizone and its alkaline-induced degradation product on TLC silica gel 60 F(254) plates, using hexane-ethyl acetate-glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP-HPLC separation of bumadizone and its alkaline-induced degradation product using a mobile phase consisting of methanol-water-acetonitrile (20:30:50, v/v/v) on a Phenomenex C(18) column at a flow-rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method.