Stability-indicating HPLC-DAD Method Research Articles

Development of a validated stability-indicating HPLC- DAD method for dasabuvir and the characterization of its degradation products using LC-QToF-MS/MS

A stability-indicating HPLC-DAD method was developed and validated for the simultaneous determination of dasabuvir and its degradation products in the pharmaceutical formulation. The proposed method utilized a Symmetry® C18 (4.6 x 75 mm, 3.5 µm) column, and the mobile phase consisted of an isocratic elution of formic acid (0.1%) and acetonitrile (55:45, v/v), at a flow of 1 mL min-1; analytes were detected at 244 nm. Dasabuvir was submitted to different stress degradation conditions, such as acidic, alkaline, neutral, thermal, oxidative and photolytic, and the structural elucidation of degradation products was performed using LC-QToF-MS/MS. The HPLC-DAD stability-indicating method was validated for selectivity, linearity, limit of detection and quantification, accuracy, precision and robustness, according to ICH guidelines. Dasabuvir produced two degradation products (DP1 and DP2) from the alkaline stress conditions, which were characterized in negative ion mode. Dasabuvir was linear in the range 9.78 to 136.92 µg mL-1, and DP and DP were linear in the range 2.9 to 20.2 µg mL-1 and 1.3 to 14.9 µg mL-1, respectively. The 1 2 recovery ranged between 99.16 and 100.86%, while precision ranged from 1.02 to 2.89%. As the method can effectively separate the dasabuvir from its degradation products and quantitate them, it may be employed as a stability-indicating method for the pharmaceutical formulation.

A novel stability-indicating HPLC-DAD method for determination of favipiravir, a potential antiviral drug for COVID-19 treatment; application to degradation kinetic studies and in-vitro dissolution profiling

Modern pharmaceutical analysis is paying a lot of attention to the stability of novel drug formulations as well as establishment of suitable stability-indicating approaches. In the current work, a comprehensive stability-indicating HPLC-DAD method has been developed and validated for determination of favipiravir (FAV) which is a novel and emerging antiviral option in COVID-19 treatment. The stability of FAV was examined under different stress conditions. FAV was found to be susceptible to acid, base hydrolysis and oxidative degradation. Structure elucidation of the forced degradation products was carried out using mass spectrometry (MS) operated in electrospray ionization mode. Effective separation of FAV and its induced degradation products was achieved using isocratic elution mode on Zorbax C18 column maintained at 30 °C. The mobile phase used was comprised of 25.0 mM phosphate buffer (pH 3.5 ± 0.05) containing 0.1% (w/v) heptane sulphonic acid sodium salt-methanol–acetonitrile (62:28:10, by volume), delivered at flow rate of 1.0 mL/min. The diode array detector signal for FAV was monitored at 321.0 nm over a concentration range of 6.25–250.00 µg/mL. The potential mechanisms for generation of degradation products were postulated through comparison of MS1 fragmentation pattern of FAV and its degradation products. Moreover, the proposed method was also extended to study the degradation kinetics. Additionally, dissolution profiling of FAV in different media was monitored. Clearly, the suggested approach is accurate, reliable, time-saving, and cost-effective. As a result, it may be utilized for regular quality control and stability assessment of FAV in its tablet dosage form.

Development and Validation of a Stability-Indicating HPLC-DAD Method for the Determination of Canagliflozin and Metformin Simultaneously in Combination Dosage Form

Development and Validation of a Stability-Indicating HPLC-DAD Method for the Determination of Canagliflozin and Metformin Simultaneously in Combination Dosage Form

Stability-indicating method for the novel antifungal compound RI76: Characterization and in vitro antifungal activity of its active degradation product.

RI76 is a novel 2-thiazolylhydrazone compound with reported antifungal activity. In preclinical drug development, it is fundamental to know the impurity profile and to understand degradation mechanisms of the molecule. In our study, RI76 was subjected to forced degradation conditions, and a stability-indicating HPLC-DAD method was developed and validated. Separation was carried out on a C18 column (150 × 4.6 mm i.d., 5 μm) maintained at 40°C using a 1 mL/min flow rate of 2 mM ammonium acetate with 0.1% formic acid (pH 3.0) and acetonitrile in gradient mode. The method was linear in the range of 0.7-91 μg/mL for RI76 and 0.7-25 μg/mL for its degradation product PD76. The formation of a major degradation product was quickly observed when RI76 was in aqueous solution. The chemical structure of this product, named PD76, was proposed based on LC-UV-MS experiments, synthesized in-house, and confirmed by NMR spectroscopy and chromatographic analysis. In vitro antifungal activity assays demonstrated that this resultant product shows a promising activity against clinically important Candida and Cryptococcus strains, matching or surpassing the activity of its precursor and of well-established antifungal drugs.

A comprehensive approach for the simultaneous analysis of all main water-soluble vitamins in multivitamin preparations by a stability-indicating HPLC-DAD method

A stability-indicating HPLC-DAD method for simultaneous determination of all nine main water-soluble vitamins, in addition to two commonly used vitamers, was developed and fully validated in analytical ranges, adjusted to their recommended dietary allowance values. An XSelect CSH C18 column with gradient elution using phosphate buffer and methanol was used for their optimal separation. The results from forced degradation studies along with peak purity tests and response ratios at dual wavelengths for the individual vitamins in all tested samples confirmed the method’s stability-indicative nature. The complete developed methodology, including a single sample preparation for the vitamins simultaneous analysis, was applied to their assay in 13 commercial multivitamin preparations, revealing mostly higher amounts than the label claims. The developed method is applicable for stability testing, multivitamin products shelf-life determination as well as routine assay analysis of all water-soluble vitamins in their most common forms, including the analytically most demanding flavin mononucleotide.

Stability-indicating HPLC-DAD method for the simultaneous determination of fluoroquinolone in combination with a non-steroidal anti-inflammatory drug in pharmaceutical formulation

We developed and validated a stability-indicating assay method for the simultaneous determination of enrofloxacin and piroxicam in combination and in the presence of degradation products. Reversephase high-performance liquid chromatography analyses were carried out on a Vertisep C18 column and acetonitrile-water (48:52 v/v, pH 3.0) mobile phase with a 1.00 mL min−1 flow rate. The efficient chromatographic separation of these drugs and their forced degradation products was achieved in less than 5 min with a peak purity match factor higher than 950. The method used showed linearity in the concentration ranges of 0.25 to 16.0 μg mL−1 for enrofloxacin (r = 0.9997) and 0.125 to 8.0 μg mL−1 for piroxicam (r = 0.9999) as well as precision (relative standard deviation lower than 2%), accuracy (mean recovery 100 ± 2%), and robustness, according to ICH (International Conference on Harmonization) and AOAC (Association of Official Analytical Chemists) guidelines. This method can simultaneously determine the combination of these drugs in a veterinary formulation and separate the drug peaks from their forced degradation products. Additionally, its optimized chromatographic conditions can contribute to the quality control of this formulation in pharmaceutical manufacturing plants and minimize waste from the organic solvent.

A Highly Selective Stability-Indicating HPLC Method for Simultaneous Estimation of Sertaconazole with Two Coformulated Preservatives in Pharmaceutical Dosage Forms

The present investigation deals with the development of a validated stability-indicating HPLC–DAD method for the simultaneous determination of sertaconazole nitrate (SN) and two coformulated preservatives: sorbic acid (SA) and methylparaben (MP). The analytes were subjected to stress conditions: acidic and alkaline hydrolysis, oxidation, and photo and thermal degradation. The suggested technique offers high resolution among the degradation products and the analytes. Moreover, the adopted method proved to be highly specific upon resolution of the three analytes from 14 different pharmaceutical compounds related to antifungals, preservatives, and antioxidants. The obtained results conform to the accelerated stability study of the cream form upon storage for 6 months at 40 °C and 75% RH. The HPLC procedure was validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. The linearity ranges for SN, MP, and SA were 1–200, 1–250, and 0.5–100 μg mL−1, respectively, with correlation coefficients > 0.9999. The developed method was successfully applied for the assay of sertaconazole cream, powder, and spray dosage forms.

Development and Validation of Stability-indicating HPLC-DAD method for simultaneous determination of Emtricitabine, Rilpivirine, and Tenofovir Alafenamide in bulk and their Pharmaceutical dosage forms

Development and Validation of Stability-indicating HPLC-DAD method for simultaneous determination of Emtricitabine, Rilpivirine, and Tenofovir Alafenamide in bulk and their Pharmaceutical dosage forms

Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir

A comprehensive stability indicating HPLC with diode array detection method was developed for the determination of the recently approved antiviral drug daclatasvirdihydrochloride (DCV) which is used for the treatment of chronic Hepatitis C Virus (HCV) genotype 3 infection. Effective chromatographic separation was achieved using Waters C8 column (4.6×250mm, 5μm particle size) with isocratic elution of the mobile phase composed of mixed phosphate buffer pH 2.5 and acetonitrile in the ratio of 75:25 (by volume). The mobile phase was pumped at a flow rate of 1.2mL/min, and quantification of DCV was based on measuring its peak areas at 306nm. DCV eluted at retention time 5.4min. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity, range, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity range was 0.6-60μg/mL with correlation coefficient>0.99999. The drug was subjected to forced degradation conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal degradation. The proposed method proved to be stability-indicating by resolution of the drug from its forced-degradation products. The validated HPLC method was successfully applied to analysis of the cited drug in its tablets.

Stability-Indicating HPLC-DAD Method for the Determination of Linagliptin in Tablet Dosage Form: Application to Degradation Kinetics.

This work aims to develop HPLC-DAD method for linagliptin (LNG) quantitation in the presence of its degradation products in tablets and to study its degradation kinetics. LNG samples were extracted from tablets and diluted. Various ICH degradation conditions were applied to study LNG degradation kinetics. LNG was assayed by a C18 column using a simple isocratic mobile phase (methanol:water containing 0.3% TEA, 40:60, pH 4.5) pumped at 1 mL/min. The drug was detected at 225 nm. The method showed high linearity (r2 > 0.999) with CV% and % error of the mean <2% over the range of 1-50 μg/mL. Limits of detection and quantitation were 0.3 and 1.0 μg/mL, respectively. LNG retention time was 11 min with a total run time of 17 min. In all degradation conditions applied, LNG was well separated from its degradation products. The method was successfully used to quantitate LNG content in its tablets and study its degradation kinetics in acidic, alkaline and oxidative forced degradation experiments. In conclusion, simple, precise and reliable method for the separation and determination of LNG in the presence of its degradation products under different stress conditions was developed and validated according to the latest ICH guidelines.

A validated stability-indicating HPLC-DAD method for simultaneous determination of econazole nitrate, triamcinolone acetonide, benzoic acid and butylated hydroxyanisole in cream dosage form

A comprehensive stability-indicating HPLC-DAD method for simultaneous determination of econazole nitrate (EN), triamcinolone acetonide (TA), benzoic acid (BA) and butylated hydroxyanisole (BHA).

Development and Validation of Stability-Indicating HPLC-DAD Method for Simultaneous Determination of Emtricitabine, Elvetegravir, Cobicistat and Tenofovir in Their Tablet Dosage Forms.

Abstract: A Simple, accurate, specific and rugged reverse phase liquid chromatographic method was developed for the simultaneous estimation of Emtricitabine, Elvetigravir, Cobicistat and Tenofovir in bulk and tablet dosage form. A reverse phase gradient program has been developed to separate the all four active ingredients. The mobile phase consisting of 0.05M Phosphate buffer pH 3.0 (adjusted with dilute phosphoric acid) and Acetonitrile in the ratio 95:5 from 0 min to 4 minutes, further increased the Acetonitrile ratio from 5 to 50 from 4 min to 10 minutes, on a reverse phase C18 column (250x4.6mm, 5 μ) with a flow rate of 1.0 ml/min, monitored at 240nm. The mean retention times of Emtricitabine, Elvetigravir, Cobicistat and Tenofovir were found to be 1.5, 5.4, 6.6 and 7.5 min respectively. The proposed method was validated in terms of Linearity, Range, Accuracy, Precision, Specificity, Robustness and Ruggedness and the method was successfully applied for the estimation of Emtricitabine, Elvetigravir, Cobicistat and Tenofovir in combined tablet dosage form. Key words: Emtricitabine, Elvetigravir, Cobicistat, Tenofovir, HPLC-DAD, Tablet.

A Stability-Indicating HPLC-DAD Method for Determination of Ferulic Acid into Microparticles: Development, Validation, Forced Degradation, and Encapsulation Efficiency.

A simple stability-indicating HPLC-DAD method was validated for the determination of ferulic acid (FA) in polymeric microparticles. Chromatographic conditions consisted of a RP C18 column (250 mm × 4.60 mm, 5 μm, 110 Å) using a mixture of methanol and water pH 3.0 (48 : 52 v/v) as mobile phase at a flow rate of 1.0 mL/min with UV detection at 320 nm. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of quantification, limit of detection, accuracy, precision, and robustness provided suitable results regarding all parameters investigated. The calibration curve was linear in the concentration range of 10.0–70.0 μg/mL with a correlation coefficient >0.999. Precision (intraday and interday) was demonstrated by a relative standard deviation lower than 2.0%. Accuracy was assessed by the recovery test of FA from polymeric microparticles (99.02% to 100.73%). Specificity showed no interference from the components of polymeric microparticles or from the degradation products derived from acidic, basic, and photolytic conditions. In conclusion, the method is suitable to be applied to assay FA as bulk drug and into polymeric microparticles and can be used for studying its stability and degradation kinetics.

Validated stability-indicating HPLC-DAD method for determination of the phosphodiesterase (PDE-4) inhibitor roflumilast

A comprehensive stability indicating HPLC with diode array detection method was developed for determination of the recently approved phosphodiesterase type 4 (PDE-4) inhibitor roflumilast (RFL). Effective chromatographic separation was achieved using Durashell C18 column (4.6×250mm, 5μm particle size) with isocratic elution of the mobile phase composed of 0.0065M ammonium acetate pH 6.3, methanol and acetonitrile in the ratio of 30:35:35 (by volume). The mobile phase was pumped at a flow rate of 1.3mL/min, and quantification of RFL was based on measuring its peak areas at 251nm. RFL eluted at retention time 6.2min. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity, range, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity range was 2.5–200μg/mL with correlation coefficient >0.9998. The drug was subjected to stress conditions of neutral, acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the drug from its forced degradation products. Moreover, specificity of the method was verified by resolution of drug from more than 20 pharmaceutical compounds of various medicinal categories. The validated HPLC method was successfully applied to the analysis of the cited drug in its tablet dosage form. The proposed method made use of DAD as a tool for peak identity and purity confirmation.

Stability-indicating HPLC-DAD method for the simultaneous determination of fluoroquinolones and corticosteroids in ophthalmic formulations

We develop and validate a stability-indicating assay method for simultaneous determination of gatifloxacin and prednisolone acetate, or of ciprofloxacin hydrochloride and dexamethasone in combination and in the presence of degradation products.

A Stability-Indicating HPLC-DAD Method for Determination of Stiripentol: Development, Validation, Kinetics, Structure Elucidation and Application to Commercial Dosage Form.

A rapid, simple, sensitive, and accurate isocratic reversed-phase stability-indicating high performance liquid chromatography method has been developed and validated for the determination of stiripentol and its degradation product in its bulk form and pharmaceutical dosage form. Chromatographic separation was achieved on a Symmetry C18 column and quantification was achieved using photodiode array detector (DAD). The method was validated in accordance with the ICH requirements showing specificity, linearity (r 2 = 0.9996, range of 1–25 μg/mL), precision (relative standard deviation lower than 2%), accuracy (mean recovery 100.08 ± 1.73), limits of detection and quantitation (LOD = 0.024 and LOQ = 0.081 μg/mL), and robustness. Stiripentol was subjected to various stress conditions and it has shown marked stability under alkaline hydrolytic stress conditions, thermal, oxidative, and photolytic conditions. Stiripentol degraded only under acidic conditions, forming a single degradation product which was well resolved from the pure drug with significantly different retention time values. This degradation product was characterized by 1H-NMR and 13C-NMR spectroscopy as well as ion trap mass spectrometry. The results demonstrated that the method would have a great value when applied in quality control and stability studies for stiripentol.

Development and application of a novel, dual-mode gradient, stability-indicating HPLC-DAD method for the simultaneous determination and purity assessment of mebeverine hydrochloride, diloxanide furoate and their corresponding major degradation products in combination with some gastrointestinal drugs in the form of oral doses

A simple, precise, rapid, stability-indicating reversed phase high performance liquid chromatographic method with photodiode array detection was developed and validated for the determination of mebeverine hydrochloride in combination with sulpiride or with diloxanide furoate and metronidazole in the presence of their corresponding degradation products. Optimum separation was achieved in less than 10min using an X-Bridge C18 column (150mm×4.6mm i.d., 3.5μm particle size); elution was accomplished via the application of a dual-mode solvent and flow rate gradient system. This elution system enables the separation of nine components within a cycle time of 15min and with a resolution greater than 2.5. Detection was conducted at 230nm, and purity assessment was performed using a photodiode array detector. The method has been validated with respect to specificity, linearity, accuracy, precision, limit of quantitation, limit of detection, robustness and ruggedness. The validation criteria were met in all cases. The developed HPLC method was successfully applied to commercial tablets. It was shown that this method is very sensitive to the determination of the degradation products, downward to 0.1w/w% levels, which is far below the limits for testing these degradation products within their corresponding intact drugs.

Validated stability-indicating HPLC-DAD method of analysis for the antihypertensive triple mixture of amlodipine besylate, valsartan and hydrochlorothiazide in their tablets

A simple and selective HPLC-DAD stability indicating method was developed for the simultaneous determination of the three antihypertensive drugs amlodipine besylate (AML), valsartan (VAL) and hydrochlorothiazide (HCT) in their combined formulation. Effective chromatographic separation was achieved using Zorbax SB-C8 column (4.6×250mm, 5μmps) with gradient elution of the mobile phase composed of 0.025M phosphoric acid and acetonitrile at a flow rate of 1mL/min. The multiple wavelength detector was set at 238nm for measurement of AML and 225nm for both VAL and HCT. Quantification was based on measuring the peak areas. The three compounds were resolved with retention times of 4.9, 6.4 and 8.3min for HCT, AML and VAL respectively. Analytical performance of the proposed HPLC procedure was statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity ranges were 5–200, 5–200 and 10–200μg/mL for AML, VAL and HCT respectively with correlation coefficients >0.9993. The three drugs were subjected to stress conditions of acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the analytes from their forced-degradation products. The validated HPLC method was applied to the analysis of the cited antihypertensive drugs in their combined pharmaceutical tablets (Exforge HCT). The proposed method made use of DAD as a tool for peak identity and purity confirmation.

Validated Stability-Indicating HPLC-DAD Method for the Simultaneous Determination of Diclofenac Sodium and Diflunisal in Their Combined Dosage Form

A simple, rapid, and highly selective HPLC-DAD method was developed for the simultaneous determination of diclofenac sodium (DIC) and diflunisal (DIF) in pure form and in their combined formulation. Effective chromatographic separation was achieved using a Zorbax SB-C8 (4.6×250 mm, 5 μm particle size) column with a mobile phase composed of 0.05 M phosphoric acid, acetonitrile, and methanol in the ratio of 40:48:12 (by volume). The mobile phase was pumped isocratically at a flow rate of 1 mL/min, and quantification of the analytes was based on measuring their peak areas at 228 nm. The retention times for diflunisal and diclofenac were about 7.9 and 9.5 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Calibration curves were linear in the ranges of 5–100 μg/mL for both drugs with correlation coefficients >0.9998. The proposed method proved to be selective and stability-indicating by the resolution of the two analytes from four of their related substances and potential impurities as well as from forced-degradation (hydrolysis, oxidation, photolysis, and dry heat) products. The validated HPLC method was successfully applied to the analysis of DIC and DIF in their combined dosage form (suppositories). The proposed method made use of the diode array detector (DAD) as a tool for peak identity and purity confirmation.