Laboratory-prepared Mixtures Research Articles

Synchronous Fluorescence Method for Determination of Bevacizumab and Fluorouracil in Laboratory Prepared Mixture, Pharmaceutical Dosage Forms and Spiked Plasma

Bevacizumab (BVC) is commonly co-administered with 5-fluorouracil (FLO) as a highly successful treatment for colon cancer. In this study, two spectrofluorimetric techniques were combined to provide an approach for concurrent quantification of BVC and FLO that is very sensitive, quick, easy and accurate. The inclusion of Tween-80 micellar system increased the native fluorescence intensity values of BVC and FLO, while the second derivative of the synchronous fluorescence intensity of the cited drugs at Δλ=100 nm allowed the concurrent estimation of cited drugs. The effect of various experimental conditions on the synchronous fluorescence of cited drugs were extensively examined to optimize them. The second derivative synchronous fluorimetry peak amplitudes for BVC and FLO were recorded at 450 nm and 440 nm, respectively. At a range of 100–1100 and 100–600 ng mL−1, the fluorescence-concentration graphs were built, with smaller quantification limits of 60.0 and 70.0 ng mL−1 and detection limits of 20.0 and 23.0 ng mL−1 for BVC and FLO, respectively. Without any major interference, the technique was effectively used to determine the cited drugs in raw material along with pharmaceutical formulations. The proposed technique was shown to be extremely precise and accurate when related to the reported techniques statistically.

Quantitative Analysis of Two Pharmaceutical Combinations Containing Amlodipine with Either Bisoprolol or Candesartan Using Different UV Spectrophotometric Methods.

Two fixed-dose combination tablets of amlodipine (AML) with bisoprolol (BIS) and amlodipine with candesartan (CAN) were recently approved for the treatment of hypertension. Three UV spectrophotometric analytical methods were developed for the estimation of the above drugs in their combination mixtures. For the AML and BIS mixture, AML was quantified directly by a zero-order spectrophotometric method at 360 nm without interference from BIS, whereas BIS was quantified by the ratio difference (RD) spectrophotometric method by calculating the difference between the amplitude values of the ratio spectra at 226 and 215 nm. For the AML and CAN mixture, AML was quantified directly by a zero-order spectrophotometric method at 360 nm without interference from CAN, whereas CAN was quantified by the first derivative of ratio spectra method by measuring the amplitude values of the derived spectra at 259 nm.BIS). The proposed methods were successfully applied for quantitative analysis of AML with BIS and AML with CAN in their synthetic laboratory-prepared mixtures and pharmaceutical tablets with acceptable results in terms of accuracy and precision. Simple, accurate, and environmentally friendly spectrophotometric methods were described for selective determination of the investigated drugs in their fixed-dose combinations.

Different approaches for the assessment of greenness of spectrophotometric methodologies utilized for resolving the spectral overlap of newly approved binary hypoglycemic pharmaceutical mixture

Simultaneous measurement of saxagliptin hydrochloride (SAG) and dapagliflozin propanediol monohydrate (DAG) in bulk powder, laboratory-prepared mixtures, and pharmaceutical dosage form were applied by utilizing three precise and sensitive spectrophotometric techniques which were developed and validated. The first method was the induced dual-wavelength approach (IDW), which relied primarily on the use of alternative equality factors (F) to abolish the effect of DAG when determining SAG and vice versa. The ratio difference method (RDM) was the second method, which used 25 μg/ml of DAG and 20 μg/ml of SAG as divisors to determine the amplitude difference on the ratio spectrum of SAG and DAG, respectively. SAG was determined at λmax 221 nm after plateau subtraction followed by multiplication by the divisor of DAG 25 μg/ml using the third method, ratio subtraction coupled with extended ratio subtraction method (RSER). Subsequently, using an extension ratio subtraction of the spectra, DAG was determined at λmax 225 nm was determined. The developed methods were effectively used to estimate SAG and DAG in laboratory-prepared mixtures and pharmaceutical dosage forms, with satisfactory recoveries. The methodologies were assessed for their environmental friendliness using the analytical eco-scale, analytical GREEnness calculator, and green analytical procedureindex (GAPI). These methodologies were validated following the International Conference on Harmonisation (ICH) requirements. A statistical comparison of the obtained findings to those of the published method revealed no significant differences in precision and accuracy. Because of their high precision and cost-effectiveness, the developed methods can be used in quality control laboratories to determine the binary mixture.

Spectrophotometric methods for determination of glimepiride and pioglitazone hydrochloride mixture and application in their pharmaceutical formulation

Simple, accurate, and precise four spectrophotometric methods were developed and validated for simultaneous determination of glimepiride and pioglitazone hydrochloride in their pharmaceutical formulation. The first spectrophotometric method was the dual-wavelength which determined glimepiride at 219.0 and 228.0 nm and pioglitazone hydrochloride at 268.0 nm. The second one is the first derivative of ratio spectra (DD1) spectrophotometry in which the peak amplitudes were used at 238.0 nm and 268.0 nm for glimepiride and pioglitazone hydrochloride, respectively. The third method is ratio subtraction in which glimepiride was determined at 228.0 nm in the presence of pioglitazone hydrochloride which was determined by extended ratio subtraction at 268.0 nm. The fourth method was the ratio difference to determine glimepiride and pioglitazone hydrochloride. Beer’s law was confirmed in the concentration range 2.50–15.00 µg mL-1, and 10.00–50.00 µg mL-1 for glimepiride and pioglitazone respectively for the four methods. The proposed methods were used to determine both drugs in their pure powdered form with mean percentage recoveries of 99.91 ± 1.117% for glimepiride and 99.76 ± 0.911% for pioglitazone hydrochloride in method (A). In method (B), the mean percentage recoveries were 100.12 ± 0.89% for glimepiride and 100.02 ± 1.06% for pioglitazone hydrochloride. In method (C) glimepiride was 100.01 ± 0.592% and 99.85 ± 0.845% for pioglitazone hydrochloride by extended ratio subtraction. And finally, in method (D) the mean percentage recoveries were 100.66 ± 0.670% for glimepiride and 99.92 ± 0.988% for pioglitazone hydrochloride. The developed methods were successfully applied for the determination of glimepiride and pioglitazone hydrochloride in pure powder and dosage form. The suggested methods were also used to determine both compounds in laboratory-prepared mixtures. The accuracy, precision, and linearity ranges of the developed methods were determined. The results obtained were compared statistically with the official method, and there was no significant difference between the proposed methods and the official method for accuracy and precision.

Green quantitative spectrofluorometric analysis of rupatadine and montelukast at nanogram scale using direct and synchronous techniques.

Two green-sensitive spectrofluorometric methods were investigated for assay of rupatadine (RUP) [method I] and its binary mixture with montelukast (MKT) [method II]. Method I depends on measuring native fluorescence of RUP in the presence of 0.10 M H2SO4 and 0.10%w/v sodium dodecyl sulfate at 455 nm after excitation at 277 nm. The range of the first method was 0.20–2.00 µg ml−1 with detection and quantitation limits of 59.00 and 179.00 ng ml−1, respectively. Method II depends on the first derivative synchronous spectrofluorometry. The derivative intensities were measured for the two drugs in an aqueous solution containing Mcllvaine's buffer pH 2.60 at fixed Δλ of 140 nm. Each drug was estimated at zero-contribution of the other. The intensity was measured at 261 and 371 nm for RUP and MKT, respectively. The method was linear over 0.10–4.00 and 0.20–1.60 µg ml−1 with limits of detection 31.00 and 66.00 ng ml−1 and limits of quantitation 94.00 and 200.00 ng ml−1 for RUP and MKT, respectively. The method was extended to determine this mixture in laboratory-prepared mixtures and combined tablets. Method validation was performed according to ICH guidelines. Statistical interpretation of data revealed good agreement with the comparison method. Method greenness was confirmed by applying three different assessment tools.

Second-derivative synchronous fluorimetry and time-programmed HPLC-fluorescence detection for simultaneous estimation of flibanserin and sitagliptin phosphate in synthetic mixtures and human plasma samples

Diabetes Mellitus is directly related to female anaphrodesia. Female Viagra or Flibanserin (FLB), U.S. FDA approved in 2015, is specifically indicated for premenopausal Hypoactive Sexual Desire Disorder, HSDD, which is one of the primary consequences of Diabetes Mellitus. Simultaneous analysis of the concomitantly administered, FLB and oral antidiabetics, as Sitagliptin phosphate (STG), is a crucial demand to investigate mutual drug-drug interaction. The latter is responsible for uncontrolled glycaemia and higher risk of sudden hypoglycemia. Two simple, sensitive, economical and direct analytical methods, namely, Second-Derivative Synchronous Fluorimetric Spectroscopy, D2-SFS, and High Performance Liquid Chromatography with fluorimetric detection, HPLC-FD, are established for simultaneous determination of FLB and STG in their binary mixtures. First method relies on measuring D2-SFS spectra of both drugs, at Δλ = 25 nm, along linearity ranges of 0.05–1 μg/mL for both drugs. The second method is a chromatographic one with gradient elution of FLB and STG on RP-ZORBAX Eclipse C18 column (5 µm, 4.6 × 150 mm). Mobile phase; phosphate buffer: acetonitrile, pH 4.5, with a flow rate of 1 mL/min at room temperature has been used. Time programmed fluorimetric detection is optimized at λem = 305 nm for STG (0.0–5.9 min), at λem = 375 nm for FLB (6–9 min) after both excitation at λex = 257 nm, in the linear ranges of 1–40 μg/mL and 5–60 μg/mL for FLB and STG, respectively. Proposed methods have been validated according to ICH guidelines, then applied for simultaneous quantitation of FLB and STG in their laboratory-prepared mixtures and in spiked human plasma samples. Satisfactory Student’s t-value and F-variance ratio have been obtained upon comparing the results of both methods.

Different spectrophotometric methods manipulating ratio spectra for the assay of hydrocortisone acetate and clioquinol in their topical preparation

Simple and precise spectrophotometric methods for quantitative assay of a mixture of hydrocortisone acetate (HCA) and clioquinol (CL) were developed and validated through different mathematical manipulation pathways. The developed methods utilized ratio spectra for resolving binary mixtures including absorbance subtraction, ratio subtraction coupled with spectrum subtraction, constant multiplication, constant value, and derivative ratio. The proposed methods were proved to be specific by analysing the laboratory-prepared mixtures and were applied for the assay of topical preparation successfully. The methods were validated using ICH guidelines where accuracy, repeatability and intermediate precision were within the acceptable limits. The linearity range was found to be 2-22 for HCA and 1.5-7 µg/mL for CL in all proposed methods and 2-7 µg/mL for HCA and CL in absorbance subtraction method through using a unified regression equation. The findings were statistically evaluated with respect to the official and reported methods, demonstrating that there was no significant difference.

Different greenness assessment perspectives for stability-indicating RP-HPLC method used for the assay of isoxsuprine hydrochloride and four nephrotoxic and hepatotoxic photothermal degradation products

Nowadays, the analytical community focuses on applying principles of green analytical chemistry (GAC) to develop new analytical methods to reduce harmful effects on the environment and human beings. In this work, we developed, validated, and assessed the greenness profile of a RP-HPLC method for stability and purity assay of isoxsuprine hydrochloride (ISX) and four of its nephrotoxic/hepatotoxic photothermal degradation products; p-hydroxyacetophenone (AC), 1-methyl-3-phenylpropylamine (AM), and p-hydroxybenzaldehyde (HB) in pure form, laboratory-prepared mixtures, and in the pharmaceutical dosage form. A C18 Waters XTerra (3.5 μm, 4.6 × 100 mm) separated the five analytes using a mobile phase composed of 20 mM potassium dihydrogen phosphate (adjusted to pH 2.3 with ortho-phosphoric acid) and methanol. We performed gradient elution at a constant flow rate of 0.8 mL/min while monitoring the effluent absorbance at 216 nm. The method shows high sensitivity with limits of quantifications equal to 0.557, 0.304, 5.791, 0.446, and 0.497 μg/mL for ISX, AC, AM, BZ, and HB, moreover, ISX low limit of detection (0.184 µg/mL) enabled its detection in spiked human plasma at its therapeutic level of concentration (0.4 µg/mL). We used four greenness assessment tools to assess the method greenness, including the “National Environmental Method Index” (NEMI), the “Eco-Scale Assessment” (ESA), the “Green Analytical Procedure Index” (GAPI), and Analytical Greenness metric (AGREE); additionally, we used the tools as a comparative greenness study between the proposed and the reported HPLC methods. The comparison proved that the proposed method was the best regarding the principles and guidelines of GAC. Our method proved superior eco-friendly qualitative and quantitative scores relative to the reported methods, where the method achieved more green NEMI and GAPI quadrants, scored the higher ESA score (85) than reported (81,81, and 77), and the higher AGREE score (0.71) than reported (0.53,0.56,0.58).

Fate of bentonite in slag–cement–bentonite slurry trench cut-off walls for polluted sites

Soil–bentonite (SB) and slag–cement–bentonite (slag-CB) slurry trench cut-off walls are widely used low-permeability subsurface barriers against groundwater or contaminants. The bentonite in SB walls is instrumental to the performance of the barrier. However, when mixed with Portland cement and slag in slag-CB walls, bentonite participates in ‘secondary’ chemical reactions throughout cement hydration. Using mineralogical methods, the goals of this paper are to examine the fate of bentonite in slag-CB mixtures and to discuss the implications of the associated chemical reactions for the slag-CB wall performance. Laboratory-prepared mixtures of dry and hydrated slag-CB were analysed using X-ray diffraction over 3 months to investigate the evolution of mixtures during curing. Results confirm that bentonite clay minerals dissolve in slag-CB slurries and that bentonite is virtually absent in the hardened slag-CB material. Furthermore, carbonation and pozzolanic chemical reactions also take place on slag-CB hydration and curing, which form secondary minerals over time. Hence, unlike SB mixtures, the bentonite in slag-CB mixtures no longer retains its original crystalline structure, as it participates chemically in hydration processes, although it becomes a precursor of pozzolanic products. Nonetheless, bentonite is instrumental to the workability and initial set of the mixture. It is concluded that the concepts of compatibility traditionally accepted for SB walls are inappropriate for slag-CB walls. Possible implications include that due to the onset of carbonation, the barrier could degrade if the contaminants are acidic; also, the formation of pozzolanic hydrates may improve the contaminant sorption properties of the barrier.

Investigation of some univariate and multivariate spectrophotometric methods for concurrent estimation of Vancomycin and Ciprofloxacin in their laboratory prepared mixture and application to biological fluids

Four simple, rapid, accurate and precise spectrophotometric methods were established and validated in accordance with ICH Q2 (R1) guidelines for the simultaneous determination of Vancomycin (VNC) and Ciprofloxacin (CPR) in their raw materials, laboratory prepared mixtures and pharmaceutics. Method A depends on using first derivative spectrophotometry (D1) where VNC and CPR were resolved at 243.6 and 262.0 nm, respectively. Concerning method B, it is based on utilizing first derivative of ratio spectra (DD1) where determination was performed at the peak maxima at 244.0 nm and 258.0 nm for VNC and CPR, respectively. Two chemometric models were applied for the quantitative analysis of both drugs in their laboratory prepared mixtures, namely, partial least squares (PLS) (method C) and artificial neural network (ANN) (method D). For univariate methods linearity range for both drugs was in the range of 3–30 and 1–10 μg/mL for VNC and CPR, respectively. Multivariate calibration methods using five level, two factor calibration model for the development of 25 mixtures were also applied for the simultaneous estimation of the two drugs in their laboratory prepared mixture using spectral region from 200.0 to 300.0 nm using interval 1 nm. The suggested methods have been successfully extended to the assay of the two studied drugs in laboratory-prepared mixtures and pharmaceuticals with excellent recovery. First derivative spectrophotometry (D1) was also applied for the assay of both analytes in spiked human plasma with good recovery. No interaction with common pharmaceutical additives has been observed which indicate the selectivity of the method. The results obtained were favourably compared with those obtained applying the reported methods. The methods are validated in compliance with the ICH Q2 (R1) guidelines and the measured accuracy and precision are assessed to be within the accepted limits.

A Thorough HPLC Assay of Quaternary Veterinary Formulation coupled with Environmental Assessment Tool.

A new and accurate reversed phase HPLC method with UV detection has been established for any veterinarian analyst for simultaneous determination of a veterinary quaternary mixture of sulphadimidine sodium (SDS), sulphaquinoxaline sodium (SQS), diaveridine (DVD) and vitamin K3 (VTK3) in their formulation. The stationary phase was SEA C18 column (250 × 4.6mm i.d., 5μm particle size) at 25°C with an isocratic mode, using a mobile phase containing a mixture of methanol:acetonitrile:distilled water in the ratio of (20:20:60, by volume). The flow rate was 0.8mLmin-1, and UV detection was performed at 230nm. The HPLC assay was coupled with Environmental Assessment Tool (EAT), which represents a simple and proficient approach for profiling the greenness of the method. This takes into consideration the environmental, health and safety issues for all solvents that involved in the chromatographic method and calculates a total score that can be used for comparison of the greenness of different methods. The method was found to be linear over (0.5-30) μg/mL for all cited drugs with mean percentage recoveries (99.56±1.141) for VTK3, (99.56±1.056) for DVD, (99.62±1.482) for SDS and (99.52±1.205) for SQS. The results were statistically compared with those of the official and reported methods; using Student's t-test and F-test, showing no significant difference with respect to accuracy. Specificity of the applied method was assessed by analyzing the laboratory-prepared mixtures. The developed method was validated according to ICH guidelines. The proposed methodology can be applied for rapid routine assay of this combination.

Micelle-enhanced spectrofluorimetric determination of diphenhydramine: application to human plasma and its simultaneous determination with naproxen in pharmaceutical tablets.

Two simple and sensitive spectrofluorimetric methods were developed and validated for determination of diphenhydramine. The use of sodium dodecyl sulfate surfactant at pH 7 enhances the fluorescence intensity of diphenhydramine at 286 nm (method I) enabling its nanodetermination in biological samples with mean per cent recovery ± SD of 100.33 ± 1.519. Method I was validated according to ICH-Q2R1 guidelines and was successfully applied for determination of diphenhydramine in pharmaceutical dosage form and spiked human plasma in the concentration ranges 0.1-4.0 μg/mL and 0.2-1.0 μg/mL, respectively. Method I acted as a basis for the development of a first derivative synchronous spectrofluorimetry (method II) for simultaneous analysis of diphenhydramine and naproxen using a zero-crossing approach. Method II determines both drugs with linearity ranges of 0.05-3.0 μg/mL and 0.1-0.9 μg/mL for diphenhydramine and naproxen, respectively. The developed method was applied for the simultaneous determination of both drugs in their laboratory-prepared mixtures containing all expected excipients. Method II determines both drugs with a mean percent recovery ± SD of 100.56 ± 0.891 and 100.20 ± 1.125 for diphenhydramine and naproxen, respectively. The method was statistically compared with a reported method using Student's t- and F- tests, and no significant differences were observed.

Comprehensive comparative study of eco-friendly Univariate and multivariate methodological approaches on processing multi-component formulation quality

This study presents comprehensive comparative study of different eco-friendly spectrophotometric approaches without any sample treatment on processing quaternary mixture of sulphadimidine sodium (SDS), sulphaquinoxaline sodium (SQS), diaveridine (DVD) and vitamin K3 (VTK3). The different univariate complementary resolutions according to the response used for the assay of the cited drugs after applying the processing steps were implemented using successive ratio subtraction coupled with constant multiplication (SRS-CM), absorbance subtraction (AS) and amplitude modulation (AM). On the other hand, multivariate spectrophotometric models were developed and validated for simultaneous determination of the cited mixture. Resolution was accomplished by using two multivariate calibration greener models, including principal component regression (PCR) and partial least-squares (PLS). The proposed approaches are considered environmentally friendly since they use only water as reagent, which is cheap and safe for the operator. The calibration graphs are linear over the range of (4.0-13.0) μg/mL for (SDS), (1.0-10.0) μg/mL for (SQS), (1.0-11.0) μg/mL for (DVD) and (1.0-8.0) μg/mL for (VTK3). Specificity of the applied procedures was assessed by analyzing the laboratory-prepared mixtures and their combined dosage form. The outcomes of the developed methods were statistically compared with those of the official and reported methods; using Student's t-test and F-test, showing no significant difference. The proposed methodologies can be used for the routine analysis of the cited drugs in quality control laboratories.

A Bayesian approach to the deconvolution of 40Ar/39Ar data from mineral mixtures

40Ar/39Ar geochronology is a powerful technique for dating geological events and processes on timescales from hundreds to billions of years. Many 40Ar/39Ar datasets are collected from analysis of single mineral phases or phenocryst-free groundmass that cooled rapidly following a volcanic eruption, which can allow for straightforward interpretation of 40Ar/39Ar age spectra. However, 40Ar/39Ar age spectra from mixtures of multiple minerals and/or multiple age components are often complex. In such situations, interpretations commonly used for single mineral phases are inappropriate and will result in geologically spurious conclusions. Here, we present a Bayesian method for the analysis and interpretation of 40Ar/39Ar step-heating spectra that result from mixing of multiple components, where a component is defined by both its age and mineral composition. We test the efficacy of this Bayesian approach using a suite of case studies. Two of these case studies utilize 40Ar/39Ar data from laboratory-prepared mixtures, which we use to explore how the composition, age, and number of components in a mixture, as well as our prior knowledge of these parameters, influence the model results. We also present an application-based case study in which we use plausible compositions and ages from a past Mars landing site to generate a synthetic 40Ar/39Ar dataset, which we then deconvolve using our Bayesian approach. We discuss modifications to our method that could improve the model's precision and outline geologic applications of our Bayesian approach in terrestrial and extraterrestrial settings that would permit the extraction of a greater amount of temporal information.

A:Determination of Novel Promising Combination of Linagliptin and Pioglitazone HCl in Bulk and Laboratory Synthetic Mixture by Earth-Friendly Three Spectrophotometric Methods

The combination between peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonists and dipeptidyl peptidase-IV (DPP-IV) inhibitors is proven to be a more effective management of type-II diabetes mellitus (T2DM), compared to either each as single therapy. One of the novelist promising combination is linagliptin (LIN) and pioglitazone HCl (PIO) within clinical trials phase-III. Three simple, precise, accurate, reliable, and earth-friendly spectrophotometric techniques were defined and validated for resolving this novel pharmaceutical combination within laboratory synthetic mixtures and bulk form without the need of previous separation. Method-I was first-derivative (D1) spectrophotometry based on estimating LIN at 247.80 nm absorbance (PIO's zero-crossing point) and PIO at 258.40 nm absorbance (LIN's zero-crossing point) exhibiting mean percentage recoveries of 100.55% and 99.40%, respectively. Method-II was area under curve (AUC) technique involving area measurements at two designated wavelength ranges; 292-305 nm and 265-279 nm for respective LIN and PIO determination and with respective mean percentage recoveries 99.65% and 99.67%. Method-III was isosbestic point spectrophotometry where collective concentrations of both analytes were estimated at isosbestic point. 273.05 nm. Concerning PIO, the concentrations were furnished through simple deduction of LIN concentration, previously determined by AUC technique, and a mean % recovery of 99.88 % was obtained. All three proposed techniques were linear over concentration range 6-30 μg/mL and 5-50 μg/mL for LIN and PIO, respectively. Moreover, such techniques were validated as through the laboratory-prepared mixtures as refer to the International Council for Harmonization (ICH) guidelines.

The use of green spectroscopy with multivariate calibration models for simultaneous analysis of ternary drug mixture in combined oral antidiabetic pill.

In this work, different chemometric calibration models were developed and validated for the purpose of determining of ternary mixture of oral antidiabetic drugs; vildagliptin (VDG), saxagliptin (SAX) and sitagliptin phosphate (STG). The used models were Partial least squares (PLS) and Artificial Neural Networks (ANN). However, on these various models the impact of genetic algorithm (GA) as a form of variable selection was also investigated. The UV spectral data was used as basis in the quantitative study of the drugs analyzed in bulk and product formulations. The concentration range of the calibration curves of VDG, SAX and STG were 10-22μgmL-1, 24-40μgmL-1 and 82-130μgmL-1, respectively. The calibration set included nineteen mixtures and the others six were used as a validation set to test the predictability of the developed multivariate models. The validation parameters of the evaluated methods were statistically determined. For the analysis of drugs studied in laboratory-prepared mixtures and their dosage forms, PLS-1, GA-PLS-1, ANN, and GA-ENN were successfully employed. The results obtained by the developed methods were compared to those given by a reported method and there were no statistically significant differences regarding accuracy and precision.

Uv Spectrophotometric Methods for Determination of Sofosbuvir in Pure Form and Pharmaceutical Dosage Forms in Presence of Its Alkaline Degradate

Aims: Two spectrophotometric methods were developed and validated for the determination of sofosbuvir in presence of its alkaline degradate.
 Study Design: Ratio difference and ratio derivative methods were assisted for determination of sofosbuvir in presence of its alkaline degradate, laboratory-prepared mixtures and in tablet dosage forms.
 Place and Duration of Study: Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy (Girls), Al - Azhar University, between December 2019 and January 2020.
 Methodology: Two analytical methods were achieved and validated for the quantitative determination of Sofosbuvir in presence of its alkaline degradate. The first method was ratio difference (RD) method, where the UV absorption spectra of different concentrations of sofosbuvir were divided by the spectrum of a certain concentration (15 µg mL-1) as a devisor of its alkaline degradate to get the ratio difference spectra. Afterwards, the peak amplitudes difference between 253.7 and 243.5 nm were measured. The second method was the ratio derivative (1DR) method, where the first derivative of the ratio spectra (1DR) was obtained and its amplitude was measured at 247 and 268 nm. Good linearity was obtained over the concentration range of 3-15 µg mL-1 for the proposed methods. The proposed procedures were adopted for the selective determination of intact Sofosbuvir in presence of up to 80% of its degradation product. Sofosbuvir was exposed to different conditions as alkaline, acidic and oxidative degradation.
 Results: The proposed methods were developed and validated with good linearity range of 3-15 µg mL-1 for both methods, and also with good accuracy and precision. And the obtained results were statistically compared to those obtained by the reported method.
 Conclusion: Sofosbuvir was successfully determined by the proposed ratio difference and ratio derivative methods in bulk powder, laboratory prepared mixtures and tablet dosage form with good accuracy and precision. The methods were validated according to ICH guidelines. The results obtained were compared with those of the reported method and were found to be in good agreement.

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

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. 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. 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. These methods were validated and agreed with the requirements of ICH guidelines with respect to linearity, accuracy, precision and selectivity. 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 Eco-Friendly Spectrophotometric Methods for the Determination of Acefylline Piperazine and Bromhexine Hydrochloride in the Presence of Dosage Form Additives

Four simple, precise, accurate, and eco-friendly spectrophotometric methods manipulating ratio spectra were used for the determination of acefylline piperazine (AC) and bromhexine hydrochloride (BR) in the presence of two dosage form additives — methylparaben (MP) and propylparaben (PP). The dual wavelength in ratio spectra (DWRS) and derivative of double divisor of ratio spectra (DD-RS) methods were used for the simultaneous determination of AC and BR. The first derivative ratio zero crossing method (1DD) was used for the determination of BR. AC can be determined using sequential spectrophotometry, where the ratio subtraction method was applied to remove the extended spectrum of BR followed by applying the dual wave length method. The linearity of the proposed methods was investigated in the ranges 5.00–60.00 and 4.00–60.00 μg/mL for AC and BR, respectively. The selectivity of the developed methods was investigated by analyzing laboratory-prepared mixtures containing different ratios of the cited drugs and additives in addition to the analysis of their pharmaceutical dosage form. The validity of the proposed methods was further assessed using the standard addition technique. The obtained results were statistically compared with those obtained by the reported method, showing no significant difference with respect to their accuracy and precision at p = 0.05.

Validated Stability Indicating HPTLC and UV-Spectrophotometric Techniques for the Determination of Avanafil

Aims: To develop methods with complete validation according to ICH guidelines and to be applied for the determination of avanafil in pure form and in pharmaceutical formulation in the presence of its degradation products.
 Study Design: High performance thin layer chromatography (HPTLC) and different spectrophotometric methods (dual wavelength, first derivative, first derivative of ratio spectra and ratio difference are developed for simultaneous determination of avanafil in laboratory-prepared mixtures of avanafil with its degradation products and in pharmaceutical formulation.
 Place and Duration of Study: Sample: Department of Analytical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt, between May 2019 and September 2019.
 Methodology: Two techniques have been developed for the determination of avanafil in the presence of its degradation products. The first was HPTLC where separation was performed on silica gel 60 F254 plates, with chloroform: toluene: methanol: conc. ammonia (6:5:3:0.1, by volume) as a developing system and UV detection at 230 nm. The second one was UV- spectrophotometry which included dual wavelength between 267 and 292 nm, first derivative determination of the drug at 261 nm, first derivative of ratio of peak amplitudes at 275.6, 305.4 and 329 nm and the ratio difference with the amplitude difference between (266 and 250 nm).
 Results: HPTLC method was applied over the concentration range of 0.5-5. μg/spot, while spectrophotometric methods were linear over the concentration range 5-50 μg/mL for avanafil.
 Conclusion: Novel, simple and accurate method for the determination of avanafil in laboratory-prepared mixtures of avanafil with its degradation products and in pharmaceutical formulation.