Stability-indicating RP-HPLC Method Research Articles

Advanced stability-indicating gradient elution reverse phase-high performance liquid chromatography method development and validation for simultaneous estimation of rutin and curcumin using an analytical quality by design approach

This research delves into a systematic Analytical Quality by Design (AQbD) approach to develop and validate a stability-indicating robust RP-HPLC method for the simultaneous determination of Rutin and Curcumin, compounds known for their anti-inflammatory, antioxidant, and wound-healing properties. Utilizing QbD principles, critical method parameters (CMPs) were identified and screened using design of experiments (DoE) and optimized via response surface methodology (RSM) with Central Composite Design (CCD). The optimized chromatographic separation used a Phenomenex Luna C18 column with gradient elution, using methanol and 0.1% formic acid, a flow rate of 1 ml/min, and detection at 345 nm (isosbestic point). The retention times were 5.63 mins for Rutin and 17.01 mins for Curcumin. The stability indication of the developed HPLC method was performed, and validation parameters were meticulously conducted following the ICH Q2(R1) guidelines, demonstrating linearity over 2-10 μg/ml for Rutin and 5-25 μg/ml for Curcumin, with correlation coefficients of 0.9999 and 0.9998, respectively. Precision studies showed %RSD within acceptable limits, and accuracies ranged between 97-100%. This validated HPLC method offers a reliable, sensitive approach for simultaneous quantifying Rutin and Curcumin, suitable for routine analysis in pharmaceutical laboratories and quality control.

Application of Stability-indicating RP-HPLC Method for quantification of Ticagrelor In Oral dispersible tablet dosage form

Application of Stability-indicating RP-HPLC Method for quantification of Ticagrelor In Oral dispersible tablet dosage form

DoE-Aided Optimization of RP-HPLC Method for Simultaneous Estimation of Amoxicillin and Tinidazole Loaded Mucoadhesive GRDDS Formulation for the Treatment of H. pylori

AbstractHelicobacter pylori (H. pylori) infection is one of the primary risk factors of peptic ulcer disease worldwide. Treatment of H. pylori with the conventional dosage form is often challenging due to the ineffective reach of the antibiotics to the inner layers of gastric mucosa, where the organism resides. This study developed an eco-friendly, stability-indicating RP-HPLC method to simultaneously estimate amoxicillin and tinidazole from mucoadhesive formulation targeting H. pylori infection. The mucoadhesive GRDDS formulation of antibiotics was developed with a goal of improving bioavailability at the gastric mucosa. The multivariate Box–Behnken design (BBD) was utilized to optimize chromatographic parameters. Independent variable such as ratio of mobile phase, flow rate, pH and injections volume were optimized using DoE, and analyzed using perturbation plots. A desirability of 0.981 was achieved for the optimized variables. The optimized method utilized methanol and phosphate buffer (25:75) at pH 6.3 as the mobile phase in an isocratic elution mode on a Luna ODS C18 column kept at 25 °C as the stationary phase. The method was linear from 0.25 to 20 µg/mL, for both the drugs with R2 values of 0.9993 and 0.9997 for amoxicillin and tinidazole, respectively. This validated RP-HPLC technique demonstrated selectivity in the presence of possible degradation products and excipients present in the mucoadhesive GRDDS beads. The method was used for the determination of entrapment efficiency and in vitro release profile for tinidazole and amoxicillin in the mucoadhesive GRDDS formulation. Graphical Abstract

AQbD-guided development and validation of an innovative extraction procedure and stability-indicating RP-HPLC method for quantification of posaconazole in tablet formulation

The objective of this work is to develop a stability-indicating HPLC method for the quantification of Posaconazole (PCZ) in tablet formulation using an Analytical Quality by Design (AQbD) approach. The development process involved the Design of Experiments (DOE) utilizing distinctive constraints mixture design for mobile phase ratio optimization and a 2-level factorial design for selection of extraction diluent compositions. Key responses measured included % assay and system suitability parameters. Method operable design regions (MODR) were determined, and final optimum conditions were selected. Forced degradation studies were conducted to assess method stability. The optimized HPLC method employed a Zorbax C18 column with a mobile phase consisting of pH 3.5 10mM phosphate buffer, acetonitrile, and methanol in a ratio of 30:53:17 % v/v/v. The method demonstrated stability-indicating capabilities, with PCZ degradation observed in acidic and oxidative environments, while remaining stable in alkali. Peak purity analysis from Empower software confirmed the absence of interaction with degradants. Validation according to ICH Q2 (R2) guidelines showed precision, linearity over the range of 0.25 µg/mL to 376 µg/mL, and accuracy demonstrated through recovery studies from 50 to 150%. The developed HPLC method utilizing AQbD approach is specific, robust, precise, and accurate for the quantification of PCZ in tablet formulations, thus suitable for routine analysis.

Determination of Mesalamine in Bulk and Suppository Dosage Forms through the Development and Validation of Stability- indicating RP-HPLC Method

Background: In the current study, a simple and cost-effective stability-indicating RP-HPLC method was developed and validated to estimate the mesalamine from both bulk and pharmaceutical dosage forms. Methods: An isocratic HPLC method using a reverse phase HiQSilC18 column (250 x 4.6 mm, 5μm) and a mobile phase methanol: ammonium acetate buffer (90:10 v/v) were employed as the mobile phase with a flow rate of 1 mL/min at 25°C. Detection was carried out at 305 nm, and the injection volume was 20μl. The developed method was validated as per ICH Q2 guidelines. Mesalamine has been subjected to various stress testing conditions, such as hydrolysis of acid and base, thermal degradation, oxidation, and photolysis. Also, methods have been validated with regard to linearity, accuracy, precision, and robustness. Results: The RT of mesalamine was determined to be 3.550 min ± 0.024 minutes, providing a reliable marker for its identification. The method was found to be linear between 5-30 μg/mL concentration with (R²) of 0.994. This demonstrated the method's ability to measure varying concentrations of mesalamine accurately. Additionally, the percentage recovery of mesalamine was approximately 100%, confirming the accuracy of the developed method. The parameters for system suitability have also been found to be within acceptable limits. Force degradation studies reinforced the method's selectivity and sensitivity in detecting mesalamine under various degradation scenarios. Notably, mesalamine significantly degraded in an acidic environment. Conclusion: In conclusion, our proposed RP-HPLC method provides a sensitive, accurate, and precise means of analyzing mesalamine in both bulk and pharmaceutical dosage forms.

Development and Validation of Green Stability-Indicating RP-HPLC Method for Estimation of Ertapenem: Degradation Kinetics and Structural Mass Elucidation Using MS

Development and Validation of Green Stability-Indicating RP-HPLC Method for Estimation of Ertapenem: Degradation Kinetics and Structural Mass Elucidation Using MS

A Validated Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Aloe Emodin, Aloin, and Emodin in a Marketed Formulation of Aloe Vera

A Validated Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Aloe Emodin, Aloin, and Emodin in a Marketed Formulation of Aloe Vera

A Novel Stability-Indicating RP-HPLC Method for the Simultaneous Estimation and In Vitro and In vivo Evaluation: Curcumin and Naringin Co-amorphous System

Curcumin (CUR) is a phytochemical widely used in food industries, cosmetics, and in the treatment of various ailments. It is a polyphenol derived from turmeric and is often considered the golden spice. CUR has a low solubility of less than 1 µg/ml and poor oral bioavailability which can be improved by co-amorphization with naringin (NRG). Analytical method to simultaneously quantify CUR and NRG is not reported in literature. This study aimed to develop a stability-indicating reverse phase HPLC method in gradient mode to simultaneously quantify CUR and NRG in co-amorphous system. The co-amorphous system of CUR and NRG in molar ratios 1:1 and 1:2 was prepared by quench cooling technique. The separation was attained on a Genesis C18, (4.6 mm × 150 mm, 4 µm) column with the mobile phase comprising of methanol and a 0.1% acetate buffer pH 3.8 at a single wavelength, 289 nm. CUR and NRG eluted at 5.1 and 11.1 min, respectively. For both the molecules, the linearity range was 0.125–16 µg/ml with LOD and LOQ of 0.063 and 0.125 µg/ml. The method developed was validated as per International Conference on Harmonization (ICH) guidelines for linearity, accuracy, precision, and robustness. The method was used to estimate CUR and NRG content in co-amorphous mixture and for in vitro evaluation.Graphical

Analysis of Tolfenamic Acid using a Simple, Rapid, and Stability-indicating Validated HPLC Method.

Tolfenamic acid (TA) belongs to the fenamates class of nonsteroidal anti-inflammatory drugs. Insufficient information is available regarding the availability of a reliable and validated stability-indicating method for the assay of TA. A relatively simple, rapid, accurate, precise, economical, robust, and stabilityindicating RP-HPLC method has been developed to determine TA in pure and tablet dosage forms. The method was validated according to the ICH guideline, and parameters like linearity, range, selectivity, accuracy, precision, robustness, specificity, and solution stability were determined. TLC and FTIR spectrometry were used to ascertain the purity of TA. The specificity was determined with known impurities and after performing forced degradation, while the robustness was established by Plackett-Burman's experimental design. The mobile phase used for the analysis was acetonitrile and water (90:10, v/v) at pH 2.5. The detection of the active drug was made at 280 nm using a C18 column (tR = 4.3 min.). The method's applicability was also checked for the yellow polymorphic form of TA. The results indicated that the method is highly accurate (99.39-100.80%), precise (<1.5% RSD), robust (<2% RSD), and statistically comparable to the British Pharmacopoeia method with better sensitivity and specificity. It was observed that the stress degradation studies do not affect the method's accuracy and specificity. Hence the proposed method can be used to assay TA and its tablet dosage form.

QbD-Based Stability-Indicating RP-HPLC Method Development and Validation for the Estimation of Favipiravir-An Eco-Friendly Approach.

Analytical quality by design (AQbD) affords a systematic scaffolding to triumph a continuously validated, robust assay as well as life cycle management. The resuscitative repurposed drug favipiravir, an oral drug approved for reemerging pandemic influenza in Japan in 2014, is used for the treatment of life-threatening pathogens such as Ebola, Lassa virus, and currently COVID-19. Favipiravir is gaining a great deal of medical importance due to its pharmaceutical applications. To develop and validate a risk-based stability-indicating RP-HPLC method employing an AQbD approach using Central Composite Design (Design Expert Software 13.0) for the estimation of favipiravir. The Quality Target Product Profile optimized were flow rate and mobile phase composition, thus assessing the critical analytical attributes (retention time, tailing factor, and number of theoretical plates) as the constraints of method robustness. The proposed technique was optimized with a C18 (150 × 4.6 mm, 5 µm) column and 0.1% orthophosphoric acid buffer-acetonitrile (50:50, v/v) as the mobile phase at a flow rate of 1 mL/min using diode-array detector (230 nm) eluted favipiravir at 2.3 min. The optimized method validated as per ICH guideline Q2 (R1) was found to be eco-friendly, simple, precise (RSD 0.0051-1.2%), accurate (99.86-100.22%), linear (25-150 µg/mL), rugged (RSD 0.70%), and robust (RSD 0.6-1.6%) with a limit of detection and limit of quantitation of 1.140 µg/mL and 4.424 µg/mL, respectively. Forced degradation studies (acidic, alkaline, thermal, photolytic, and oxidative conditions) revealed the suitability of the AQbD method for the analysis of favipiravir in tablet formulation.The developed and validated AQbD method is less time consuming and can be used in the industry for routine quality control/analysis of bulk drug and marketed Favipiravir products. A robust Design of Experiment enhanced stability-indicating analytical method was developed and validated for the estimation of favipiravir. Furthermore, the contemporary method would aid in extending the analysis of favipiravir in other formulations.

Characterization of Forced Degradation Products of Netarsudil: Optimization and Validation of a Stability-Indicating RP-HPLC Method for Simultaneous Quantification of Process-Related Impurities.

The aim of this study is to examine resolution, identification, and characterization of forced degradation products of netarsudil by liquid chromatography-tandem mass spectrometry by validating a simple and sensitive high-performance liquid chromatography method for the resolution, identification, and quantification of two process-related impurities in netarsudil. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB C18 (250 x 4.6 mm; 5 µ id) column at room temperature as the stationary phase and 257 nm as the detector wavelength with the mobile phase consisting of acetonitrile, methanol, and pH 4.6 phosphate buffer in 45:35:20 (v/v) at 1.0 mL/min flow rate in isocratic elution. The method reported very sensitive detection limits of 0.008 µg/mL for impurity 1 and 0.003 µg/mL for impurity 1. The method produces a calibration curve linear in the concentration level of 25-200 for netarsudil and 0.025-0.2 µg/mL for impurities. The proposed method gives acceptable results for other validation parameters such as accuracy, precision, ruggedness, and robustness. The drug was subjected to various stress conditions such as acid, base, peroxide, and thermal and ultraviolet light to investigate the stability-indicating ability of the method. Considerable degradation was observed in stress studies, and the degradation products were well resolved from process-related impurities. The characterization of degradation products was performed on the basis of collision-induced dissociation mass spectral data, and the possible structures of the six degradation compounds of netarsudil were proposed. The outcomes of other validation studies were likewise satisfactory and proven adequate for the regular analysis of netarsudil and its process-related impurities in bulk drug and pharmaceutical dosage forms and can also be applied for the evaluation of the stress degradation mechanism of netarsudil.

Development and Validation of a Stability-Indicating RP-HPLC Method for Estimation of Glibenclamidei Bulk and Pharmaceutical Formulation

Glibenclamide is the choice of medicine for diabetes type II. There are numerous methods for estimation; however the methods are either not suitable or expensive. A simple, cheap, precise, and accurate stability-indicating HPLC method was developed and further validated. The wavelength of 229 nm with a constant flow rate of (Methanol: Mixed Phosphate Buffer, 70:30% v/v (pH 7.3)) 1 mL/min was used during the experimentation. The run time was (run time). The internal standard used was (Internal standard). The sample eluted and showed a sharp peak at 2.1 minutes. It was discovered that calibration curve experiments had a 5–50 ppm linearity. The precision of the developed method was lower than 2% RSD. Recovery studies showed that the method’s accuracy and precision ranged from 99.25 to 101.81%. Stress testing was used to determine specificity. The developed method was capable of separating the drug peak from any probable degradation products. There was no evidence of recipient or impurity interference. The method can be used to anticipate how long glibenclamide will be stable. The method was feasible and appropriate for analyzing the presence of glibenclamide in a drug substance as well as a drug product.

Simultaneous determination of paracetamol and caffeine by RP-HPLC in soft gelatin capsules

A rapid and stability-indicating RP-HPLC method was developed for simultaneous estimation of paracetamol and caffeine in soft gelatin capsules form especially to get some more advantages over other methods already developed for this combination. The method was validated according to ICH guideline with respect to accuracy, precision, specificity, linearity, solution stability, robustness, sensitivity, forced degradation and system suitability. For this, an isocratic condition of mobile phase comprising acetonitrile and phosphate buffer (pH 3.5) in a ratio of 15:85, v/v over YMC C18 column (250 × 4.6 mm, 5 μm) at a flow rate of 1 mL/minute at ambient temperature was maintained. The method showed excellent linear response with correlation coefficient (R2) values of 0.999 and 1.0 for paracetamol and caffeine respectively, which were within the limit of correlation coefficient (R2 &gt; 0.995). The percent recoveries for two drugs were found within the acceptance limit of (98.0–102.0%). % RSD values for repeatability, reproducibility and Intermediate precisions were below 2.0. Forced degradation of the drug product was carried to establishing the stability-indicating property of this method and providing useful information about the degradation pathways, degradation products, and how the quality of a drug substance and drug product changes with time under the influence of various stressing conditions.

Analytical Method Development by HPLC to Evaluate the Stress Degradation Stability Profile of Ertugliflozin

Diabetes is a chronic disease in which the body does not make or properly utilize the insulin, a hormone that is required to convert blood sugar, starches, and other food into energy by circulate the glucose from blood into the body cells”. People are going on making new drugs and new formulations of the existing drugs within very short period of time. To get the regulatory permission for marketing, company has to submit required data including the analysis reports as to prove that their drug product is of required quality for its intended use. T2DM, the predominant type of diabetes accounting for &gt;90% of all diabetes cases, is a progressive disease involving parallel defects of glucose metabolism in multiple tissues. Common risk factors for T2DM include increasing age, smoking, being overweight or obese, physical inactivity and poor nutrition. Ertugliflozin is an oral, selective inhibitor of sodium glucose co-transporter-2 (SGLT2) which inhibits renal glucose reabsorption and results in urinary glucose excretion (UGE) and reductions in plasma glucose and haemoglobin A1c (A1C) in patients with type 2 diabetes mellitus (T2DM).The present developed analytical method was developed using 0.1% Formic acid in water: 0.1% Formic acid in ACN as the mobile phase. The developed method was found to be sensitive and time and cost effective with the reduced the use of organic chemicals in the mobile phase. Improved stability-indicating RP-HPLC method was developed for the estimation of ertugliflozin. Retention times compared to the best method reported. The developed method could be suitable for routine analysis of the drugs in bulk and tablet formulation after complete validation as per ICH guideline.

Development and validation of a stability-indicating RP-HPLC method for estimation of Lefamulin in pure and pharmaceutical drug products

BackgroundCommonly occurring serious lung parenchymal infection is community-acquired bacterial pneumonia (CABP). Lefamulin acetate drug products are useful for the treatment of community-acquired bacterial pneumonia (CABP). The main objective of the study was the development and validation of a quick reverse-phase high performance liquid chromatographic analytical testing method for Lefamulin in pure and pharmaceutical dosage forms. The C18 column was the stationary phase for reverse-phase high performance liquid chromatographic separation, and the mobile phase consisted of 0.05 M phosphate buffer with a pH adjustment of 2.5 mixed with an equal volume of acetonitrile. A flow rate of 1.0 mL/min, a column temperature of 30 °C, an injection volume of 10 µL, and a detection wavelength of 210 nm were used as optimum conditions.ResultsThe developed method reported that Lefamulin peak’s retention time was 3.1 min, the average theoretical plate count 5000, and the average peak asymmetry 1.12. In both pure and pharmaceutical dosage forms, the method was found to be accurate, precise, specific, linear calibration curve, and robust. Quantitative assay results from laboratory-made formulations showed similarity to existing commercial products of Lefamulin. Lefamulin is stable in acidic and photolytic environmental circumstances, while it degraded in oxidative, basic, and thermal humidity environment, according to the results of stress studies.ConclusionsThe developed analytical method is fast, economic and stability indicating. It is useful for routine pharmaceutical analysis where the Lefamulin is synthesized in pure form, pharmaceutical tablet formulation, and parenteral dosage form, for their quality and safety.

A New Stability-Indicating RP-HPLC Method for Simultaneous Quantification of Diacerein and Aceclofenac in Novel Nanoemulgel Formulation and Commercial Tablet Dosage Form in the Presence of their Major Degradation Products Using DoE Approach.

The present study aimed to develop a simple, robust, sensitive and effective stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous quantification of diacerein (DCN) and aceclofenac (ACE) in novel nanoemulgel formulation and commercial tablets in the presence of their main degradation product: rhein (RH) and diclofenac sodium (DLS), respectively. A fractional factorial design was used to screen the crucial independent factors, whereas a central composite design was used for the optimization of the chromatographic conditions. The separation was carried out on Phenomenex C18 column (5μm, 250 × 4.6mm), using a mobile phase consisting of phosphate buffer pH3 (0.1% v/v orthophosphoric acid) and acetonitrile (40:60v/v) at a flow rate of 1mL/min with detection at 264nm. The analytes were exposed to a variety of stress conditions, including heat, alkali, acid, oxidation, photochemical, humidity and hydrolysis. DCN, ACE, RH and DLS were found to have retention times of 4.32 ± 0.15, 5.77 ± 0.07, 8.28 ± 0.20 and 9.10 ± 0.18min, respectively. The percent recovery for all four analytes was found to be between 98 and 102, and the procedure was discovered to be linear in the range of 0.1-64μg/mL with R2 value > 0.999. The established method was validated as per ICH guidelines and successfully used to assay DCN and ACE in their combined marketed tablet dosage form and developed nanoemulgel formulation.

Development and Validation of Robust, Highly Sensitive and Stability-Indicating RP-HPLC Method for Estimation of Deferasirox and its Degradation Products.

A rapid, simple and highly sensitive stability-indicating reverse-phase high-performance liquid chromatographic technique, coupled with a photodiode array detector, was developed and validated for the estimation of Deferasirox (DFS). The chromatographic separation was achieved using a C-18 (250 × 4.6 mm, 5μm) stationary phase and a mobile phase composed of 0.1% orthophosphoric acid and acetonitrile at a flow rate of 1mL/min. The detection was carried out at a wavelength of 245nm with a constant injection volume of 10μL throughout the analysis. With an R2 value of 0.9996, the calibration curve was determined to be linear over an appropriate concentration range of 50-500ng/mL. According to the International Conference on Harmonization (ICH) Q1 (R2) guideline, DFS was evaluated under stress conditions that included hydrolytic (acid, alkali and neutral), oxidative and thermal degradation. The findings demonstrated that significant degradation was observed in acidic degradation conditions, whereas drug substance was found to be stable when exposed to neutral, basic, oxidative and thermal degradation. The developed method was validated as per ICH guidelines. The developed method was employed successfully to estimate the amount of DFS in bulk and pharmaceutical formulation.

Determination of Olmesartan in Bulk and Pharmaceutical Dosage Forms through the Development and Validation of Stability-indicating RP-HPLC Method.

Angiotensin II type 1 (AT 1) receptor antagonist (angiotensin receptor blocker [ARB]) called Olmesartan medoxomil (OLM) prevents angiotensin II from acting on the renin-angiotensin-aldosterone pathway, which is a crucial factor in the development of hypertension. OLM is reported to rapidly hydrolyze into its active metabolite, Olmesartan, in plasma after oral treatment. The objective of the ongoing study was to develop an easy-to-use, precise, and reliable RP-HPLC method for the determination of Olmesartan in bulk as well as pharmaceutical dosage forms. The stability indicating HPLC method for assay includes the use of Kromasil 100-5-C8 (100 mm × 4.6 mm) column, UV detector 265 nm, and mobile phase composition was a mixture of Acetonitrile: water (70:30) and flow rate of 1.0 mL/min. ICH guidelines were followed in the method's validation. To assess the method's specificity and stability in showing characteristics, stress degradation studies were carried out. The working standard solution of Olmesartan was exposed to 0.1 N HCl at room temperature, 0.1 N NaOH at room temperature, 30 percent hydrogen peroxide by volume, and UV radiation in order to conduct a degradation study. The retention periods of the drug were found to be 1.36 and 1.47 min for standard and sample solutions, respectively. The degradation behaviour of drug under different conditions was studied. The drug was found susceptible to acidic, alkaline and oxidative conditions while it was found stable in photolytic condition. The developed stability-indicating RP-HPLC method for assay was validated as per ICH Q2 guidelines and the validation parameters such as accuracy, precision and specificity were obtained within the accepted criteria. It may be concluded that this method is stability-indicating and specific and can successfully be applied to analyze tablet dosage form containing Olmesartan.

Stability-indicating RP-HPLC method applied to the quantification of anti-histaminic drug ebastine in its oral suspension dosage form

For the quantification of ebastine in pharmaceutical suspension, a simple, quick, accurate, and exact stability-indicating HPLC approach was developed and validated. The drug was determined using a phase reverse system and the separation was performed in an analytical C18 column (250 mm x 4.6 mm, 5 μm). The mobile phase consists of 0.1% orthophosphoric acid and methanol in a 25:75, v/v ratio. Using a concentration range of 10–90 μg mL-1, the technique demonstrated a strong linear response (r=0.999). Effluents were measured at 262 nm while the flow rate was kept at 1.0 mL min-1. There was a retention time of 3.506 min. The method was statistically validated to determine its accuracy, precision, linearity, ruggedness, robustness, solution stability, selectivity, and forced degradation assessments. The stresses that were used were acid, alkali hydrolysis, water stress, oxidation, photolysis, and heat. Since the degradation products did not affect the capacity to identify ebastine, this technique may be taken as a stability indication. This methodology may be utilized for the analysis of Ebastine in pharmaceutical suspension, since the findings obtained were within the limits set by ICH standards.

Development and Validation of Stability-Indicating RPHplc Method for Simultaneous Determination of Canagliflozin and Metformin in Fixed-Dose Combination

Development and Validation of Stability-Indicating RPHplc Method for Simultaneous Determination of Canagliflozin and Metformin in Fixed-Dose Combination