Reversed-phase High-performance Liquid Chromatographic Method Research Articles

A simple and quick run time RP-HPLC method for simultaneously analysis of pyrazosulfuron ethyl and pretilachlor content on GR formulation

Aim of the study was to develop and validate simple and quick run time reverse phase HPLC method for the simultaneous determination of pyrazosulfuron ethyl and pretilachlor in the pesticide formulation of GR. The method was executed toward using the Agilent Eclipse XDB C-18 column (150 x 4.6 mm; 3.5 μm), alongside the mobile phase comprising of 0.1 % (ortho phosphoric acid) with water: acetonitrile (30:70) at a column rate of flow 1.2 mL per minute and determination has been carried out in 220 nm. The retention time for Pyrazosulfuron Ethyl as well as Pretilachlor have been determined at 1.99 as well as at 4.58 minute, correspondingly with runtime 7 min. The method's effectiveness has been validated using SANCO recommendations for LOD, robustness, accuracy, precision, linearity, selectivity and specificity. There are several analytical methods reported for individual content estimation, but single method is not available to determine simultaneous content. The developed method has been utilized to estimate pyrazosulfuron ethyl and pretilachlor content in industries.

A sensitive, expandable AQC-based LC-MS/MS method to measure amino metabolites and sphingolipids in cell and serum samples

Sphingolipids are a major lipid species found in all eukaryotes. Among structurally complex and diversified lipids, sphingoid bases have been heavily linked to various metabolic diseases. However, most current LC-MS-based methods lack the sensitivity to detect low-abundant sphingoid bases. The 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization reagent, which efficiently forms covalent bonds with amino groups, has been widely used for amino acid detection. Nevertheless, the commonly used reverse-phase HPLC method for amino acid analysis is not suitable for amphipathic sphingolipids. To address this issue, we report a robust reverse-phase HPLC-MS/MS method capable of separating and detecting hydrophilic amino acids and sphingoid bases in a single run with high sensitivity. This method is also inclusive of other amino metabolites with an expandable target list. We tested this method under various conditions and samples, demonstrating its high reproducibility and sensitivity. Using this approach, we systematically analyzed human serum samples from healthy individuals, dyslipidemia, and type II diabetes mellitus (T2DM) patients, respectively. Two sphingolipids and five amino acids were identified with significant differences between the control and T2DM groups, highlighting the potential of this method in clinical studies.

LC–MS/MS-based phospholipid profiling of plant-pathogenic bacteria with tailored separation of methyl-branched species

Plant-pathogenic bacteria are one of the major constraints on agricultural yield. In order to selectively treat these bacteria, it is essential to understand the molecular structure of their cell membrane. Previous studies have focused on analyzing hydrolyzed fatty acids (FA) due to the complexity of bacterial membrane lipids. These studies have highlighted the occurrence of branched-chain fatty acids (BCFA) alongside normal-chain fatty acids (NCFA) in many bacteria. As several FA are bound in the intact phospholipids of the bacterial membrane, the presence of isomeric FA complicates lipid analysis. Furthermore, commercially available reference standards do not fully cover potential lipid isomers. To address this issue, we have developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method with tandem mass spectrometry (MS/MS) to analyze the phospholipids of various plant-pathogenic bacteria with a focus on BCFA containing phospholipids. The study revealed the separation of three isomeric phosphatidylethanolamines (PE) depending on the number of bound BCFA to NCFA. The validation of the retention order was based on available reference standards in combination with the analysis of hydrolyzed fatty acids through gas chromatography with mass spectrometry (GC/MS) after fractionation. Additionally, the transferability of the retention order to other major lipid classes, such as phosphatidylglycerols (PG) and cardiolipins (CL), was thoroughly examined. Using the information regarding the retention behavior, the phospholipid profile of six plant-pathogenic bacteria was structurally elucidated. Furthermore, the developed LC–MS/MS method was used to classify the plant-pathogenic bacteria based on the number of bound BCFA in the phospholipidome.Graphical

Estimation of CDK inhibitors by RP-HPLC: application for pharmacokinetic interactions studies with PPIs.

Background: The study investigated pharmacokinetic interactions between palbociclib and ribociclib with proton pump inhibitors (PPIs) using the reverse-phase high-performance liquid chromatography (RP-HPLC) method.Methods: Developed RP-HPLC method quantified palbociclib and ribociclib in biological matrices. In vitro metabolic stability assays and in vivo studies in rats evaluated effect of omeprazole and esomeprazole on pharmacokinetics of palbociclib and ribociclib.Results: The RP-HPLC method was sensitive, accurate and linear. Esomeprazole and omeprazole decreased metabolic clearance of palbociclib and ribociclib by several folds. In vivo, esomeprazole elevated Cmax of palbociclib and ribociclib by 90.1% and 86.4%, whereas omeprazole reduced it by 32.0% and 16.8%, respectively.Conclusion: The RP-HPLC method was used to analyze in vitro and in vivo samples. Long-term treatment with PPIs affects pharmacokinetics of palbociclib and ribociclib, necessitating optimal chemotherapy regimen.

Development of simple stability indicating RP-HPLC method to estimate ferulic acid in niosomes and marketed products using quality by design approach

Ferulic acid found in Feru-guard which is obtained from rice bran. This compound occurs in a wide range of plant sources including pineapple, various grasses, rice, leaves, beans, coffee beans, oats, grains, vegetables, flowers, fruits, peanuts and nuts. Particularly, it received a lot of attention due to its potential therapeutic uses in different pathologies, including metabolic, viral infections, cancer, cardiovascular, skin disorders, diabetes, hepatotoxicity and neurodegeneration. The article describes the methodical development of a simple, fast, highly sensitive, durable, efficient and economically viable reversed-phase high-performance liquid chromatography (RP-HPLC) technology for measuring ferulic acid. The analysis was Performed using the Central composite design (CCD), which offers the advantage of employing a reduced number of design points and experimental runs. Employing the response surface methodology via central composite design (CCD), the study thoroughly assessed the influence of these factors. The proposed analytical method underwent comprehensive validation, ensuring its reliability as per the guidelines outlined in ICH Q2A and ICH Q2B.The preferred method employed a mobile phase consisting of Methanol and OPA buffer (50:50 % v/v, pH maintain to 4.2 with 0.1 % orthophosphoric acid), operating at a flow rate of 1 mL min⁻1,with a Chemsil ODS column, the wavelength of detection was set at 320 nm. According to the novel analytical method, Ferulic Acid exhibited a peak retention time of approximately 6.13 min. The method demonstrated linearity range of 0.5–16 μg/ml. The developed approach proved to be robust, precise and accurate, with a recovery rate of 98 %. It achieved % relative standard deviation (RSD) of below 2 %,The current stable RP-HPLC method was effectively employed for the characterization of niosomes loaded with Ferulic Acid. Furthermore, degradation studies were conducted under various conditions to elucidate the degradation pathways and resultant products.

Optimization and Evaluation of Column Efficiency in RP-HPLC Method for Pharmaceutical and Forensic Drug Analysis

Reverse Phase High Performance Liquid Chromatography (RP-HPLC) is an efficient and reliable technique employed in pharmaceutical and forensic analysis. The assay determination in pharmaceutical and certain molecular detection is performed using this technique. Performance of chromatographic columns play a central role in development of more efficient separation methods. It is one of the main component of system suitability testing in a chromatographic system. The column efficiency can be assessed by using various traditional methods and reproducible results can be evaluated using liquid chromatography We studied the column efficiency using RP-HPLC technique for pharmaceutical drug product Aceclofenac in a validated method. Two mobile phases combinations Acetonitrile: Methanol (80:20 v/v) and Acetonitrile: Methanol: Ammonia (225:50:1 v/v) were compared on C-8 Lichrospher RP-HPLC column for Aceclofenac and three tablet samples (30 ppm). The height equivalent to theoretical plate (HETP) referred as direct measure of column efficiency produced efficient results (530-545 and 190-230) for respective mobile phases. The lower HEPT is an indication of efficient elution in second mobile phase. The change in polarity as per pKa of the analyte may enhance elution and resolution. helpful in method development for pharmaceutical and forensic drug analysis using liquid chromatography.

A Novel Reversed Phase HPLC Assay Method for Simultaneous Estimation of Glucose, Sodium Citrate and Chlorides in Pharmaceutical Formulations and Drug Solution for Oral Rehydration

A rapid, straightforward, sensitive, efficient, and cost-effective reverse-phase high-performance liquid chromatographic method was employed to simultaneously determine Glucose, Sodium Citrate, and chloride in a drug solution for oral solution. Glucose, Sodium Citrate, and Chlorides are all listed in the USP monograph. While various assay methods are available, titrimetric methods are ineffective for trace-level quantification. Although IC, AAS, and ICP-MS offer high accuracy, they are expensive and often inaccessible to many testing facilities. When selecting methods, it’s crucial to consider quality control requirements and user-friendly techniques. A simple proprietary HPLC method has been developed to simultaneously quantify Chlorides, Glucose, and Sodium Citrate, with a shorter run time. The separation was carried out using a Shim-pack SCR-102(H) ion exclusion analytical column (7.9 mm x 300 mm, 7 µm) with a flow rate of 0.6 mL/min. The column compartment was kept at 40°C, and the injection volume was 10 µL, with detection at 200 nm. All measurements were performed in a 0.1% phosphoric acid solution. The analytical curves showed excellent linearity (r > 0.9999) within the concentration ranges of 0.74 to 1.74 mg/mL for Sodium Citrate (SC), 1.0 to 2.5 mg/mL for Sodium Chloride (SC), and 3.0 to 7.3 mg/mL for Glucose. The method was validated according to the guidelines of the International Conference on Harmonization (ICH Q2B) and USP<1225>. The method demonstrated precision, robustness, accuracy, and selectivity. During accelerated stability testing over six months, no significant variations were observed in organoleptic analysis and pH. Consequently, the developed method is considered suitable for routine quality control analyses, enabling the simultaneous determination of Sodium Citrate, Chloride, and Glucose in pharmaceutical formulations and solutions for oral rehydration.

An Innovative RP-HPLC Method for Simultaneous Estimation of Montelukast and Ebastine in Tablet Formulations: Development, Validation, and Stability Assessment

This study aimed to develop and validate an innovative reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of montelukast sodium (MNK) and ebastine (EBA) in tablet formulations. Utilizing a Shimadzu LC2010CHT system with a Phenomenex Luna C18 column, the mobile phase consisted of ammonium acetate buffer, acetonitrile, and methanol in a 12:55:33 ratio, with a flow rate of 1.0 mL/min and detection at 250 nm. Validation followed ICH guidelines, assessing linearity, accuracy, precision, robustness, and stability under various stress conditions. Both MNK and EBA showed excellent linearity within 16 to 24 μg/mL with correlation coefficients of 0.999. Accuracy was confirmed through recovery studies showing 98 to 102% mean recoveries. Precision was demonstrated with %RSD values below 2%. The method remained robust under slight variations in conditions, and stability studies indicated minimal degradation of both drugs. The developed RP-HPLC method proved rapid, precise, and robust for the simultaneous quantification of montelukast and ebastine in tablet formulations, demonstrating practicality and reliability for pharmaceutical quality control.

RP-HPLC Method for Estimation of Caspofungin in Bulk and in Parenteral Dosage Form

In order to more precisely and accurately estimate the antifungal medicine caspofungin and its parenteral dosage form, a new, straightforward, and cost-effective reverse-phase high-performance liquid chromatography (RP-HPLC) technique has been created. The accuracy, precision, linearity, specificity, and reproducibility of the method for the measurement of caspofungin in used equipment were determined during the present study’s validation according to the International Council of Harmonization (ICH) criteria. Elution was accomplished using a CHEMSIL ODS-C18 (5μm) column in isocratic mode with a mobile phase consisting of acetonitrile:ammonium acetate buffer at pH 4.8 (60:40%v/v). The mobile phase was pumped into the column at a flow rate of 1.0 mL/min, and the eluent was detected using a variable wavelength UV detector set at 210 nm. The column dimensions are 250 × 4.6 mm. The chromatogram for the optimized RP-HPLC method of caspofungin estimation had 28,99 theoretical plates (N) and a strong peak at a resolution time of 16,105 minutes. The thorough validation process led to the conclusion that the approach is stable and could be applied for the duration of the drug’s shelf life.

Simultaneous RP-HPLC Estimation, Validation and Stability Indicating Assay of Two-Component Tablet Formulation Containing Grazoprevir and Pibrentasvir

The determination of a two-component tablet formulation containing grazoprevir and pibrentasvir for method development and validation has been done using the reverse-phase high-performance liquid chromatography (RP-HPLC) method as per International Council of Harmonization (ICH) guidelines. Analytical grade acetonitrile and 0.01 N of potassium dihydrogen phosphate buffer were used as mobile phase while Kromasil C18 column was opted for separation. In order to elute the analyte a flow rate of 1-mL/min having 260 nm λ has been maintained. An RT of 2.909 and 2.358 minutes while linearity concentration range from 25 to 150 μg/mL and 10 to 60 μg/mL was obtained for grazoprevir and pibrentasvir, respectively, with 4.4 resolution. Both had a high correlation coefficient of 0.999. The limit of quantification was 0.24 and 0.09 μg/mL, while the detection limit at 0.72 and 0.27 μg/mL was noticed for grazoprevir and pibrentasvir, respectively. For grazoprevir and pibrentasvir, the regression equation was determined to be y = 16332x + 5313.1 and y = 17261x + 601.95, correspondingly

Development and Validation of a Novel Stability-Indicating Reverse Phase High-Performance Liquid Chromatography Method for the Quantification of Capivasertib in Bulk Drug and Pharmaceutical Dosage Form

The quantification of capivasertib has been achieved using a Waters Symmetry column with UV detection at 260 nm through a novel stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method that has been established and verified. The analysis can be completed in just 5 minutes. The separation and collection of capivasertib occurred at a retention time of 3.3475 minutes. The concentration range of capivasertib was shown to be linear, ranging from 50 to 300 μg/mL. The regression equations for capivasertib were determined as y = 13485.85x + 1723.04. The detection limit for capivasertib was determined to be 0.6 μg/mL, while the quantification limits were established at 2.00 and 0.5000 μg/mL, respectively.

Estimation of Glyoxylic Acid in Emtricitabine by a New Validated RP-HPLC Method

To establish a simple, sensitive, as well as specified method for the separation and quantitative determination of glyoxylic acid in emtricitabine by using reversed-phase high-performance liquid chromatography (RP-HPLC). A superior separation of glyoxylic acid in emtricitabine was done by using a C8 column and a mobile system of (0.1% OPA and ACN: H2O) with 1-mL/min flow rate in gradient mode and a wavelength of 210 nm. The glyoxylic acid was eluted at 5.37 minutes with good efficiency and system suitability. The method has shown good linearity ranges from a limit of detection (LoQ) to 150% level of glyoxylic acid standard solution (0.1% or 0.001 mg/mL). ICH principles validated the suggested approach, and every parameter met the requirements for the International Council of Harmonization (ICH) Q2 recognition. %RSD of System, method precision, and precision at LoQ were in the range of 0.46 to 1.91. The developed method was sensitive, specific, and precise in estimating the trace levels of glyoxylic acid in emtricitabine.

Comparative study of UV spectroscopy, RP-HPLC and HPTLC methods for quantification of antiviral drug lamivudine in tablet formulation

BackgroundIn the current study, estimation of lamivudine (LMU) by UV spectroscopy, reverse-phase HPLC (RP-HPLC) and HPTLC methods in tablet formulation was developed, and comparative studies between the methods were investigated by analytical results and statistical test analysis of variance (ANOVA) to find out best method. In the UV spectral method, LMU was quantified at 271 nm absorption maxima using methanol as the solvent. In the RP-HPLC method, the Shimadzu C18 column (250 mm × 4.6 mm i.d., 5 µm particle size) was employed for chromatographic separation. The mobile phase used consists of methanol: water (70:30 v/v) in an isocratic mode with a 1.0 mL/min flow rate. In the HPTLC method, the chromatogram was developed on a pre-coated plate of silica gel 60 F254 with a mobile phase composition of chloroform: methanol (8:2 v/v). The quantification was performed at an absorbance mode of 271 nm by densitometry. The methods were validated according to the International Conference on Harmonization (ICH) guideline Q2 (R1). The degradation conditions were employed as per ICH guidelines Q1A(R2) and Q1B which include acid, alkaline, neutral, thermal and photostability to determine the intrinsic stability of the drug in varied environmental conditions.ResultsLMU absorption maxima was found to be 271 nm. The retention time of LMU was 3.125 min, and the total analysis time was 5 min. The Rf value of LMU was 0.49–0.62. The methods were linear within 2–12 μg/mL range. The correlation coefficient (r2) for UV, HPLC and HPTLC was 0.9980, 0.9993 and 0.9988, and percent recoveries were calculated as 98.40–100.52%, 99.27–101.18% and 98.01–100.30%, respectively, with percentage relative standard deviation (RSD) less than 2% showing that methods were precise and accurate.ConclusionDeveloped UV, RP-HPLC and HPTLC methods are free from intervention caused by excipients present in tablets and thus can be used for regular quantitative analysis of LMU in tablet formulation. Based on analytical results and statistical tests, ANOVA, it is inferred that the HPLC method is best for LMU quantification tablet formulation due to its high reproducibility, good retention time and sensitivity; it has a higher percent recovery and has less analysis time, i.e., 5 min. The degradation peaks were well separated from the LMU peak indicating stability of the HPLC method.

Development of a Robust and Reliable RP-HPLC Method for the Estimation of Finerenone in Tablet Dosage Form

An advanced analytical method was explored, specifically tailored for estimating finerenone, a vital drug found in tablet formulations intended to reduce the risk of chronic kidney disease in patients with type-2 diabetes. This sophisticated technique employs an Inertsil ODS 3V C18 column (5 μm particle size x 250 x 4.6 mm) for precise separation of analyte. The mobile phase consists of an ammonium dihydrogen phosphate buffer (pH 4.5) and acetonitrile (60:40), which is a carefully balanced mixture that flows consistently at 1.0 mL/min. UV detection is achieved at a wavelength of 238 nm. Finerenone’s characteristics are unveiled with a brief retention time of 4.06 minutes, highlighting the efficiency of the method. The method demonstrates linearity within the concentration range of 2.5 to 15 μg/mL, offering a comprehensive scope for accurate analysis. This robust reversed-phase high-performance liquid chromatography (RP-HPLC) method undergoes thorough validation following International Council for Harmonisation (ICH) guidelines, ensuring its reliability. Notably, the accuracy is impressive, with a mean recovery falling comfortably within the acceptable range of 99.76 to 101.09%. In summary, our analytical approach stands out for its simplicity, precision, sensitivity, rapidity, and robustness in estimating finerenone in tablet formulations.

To Synthesize and Characterize Metabolite Enalapril: PKPD and Toxicity Predictions by Computational Approaches

Enalapril is a prodrug that facilitates the transformation of angiotensin I to angiotensin II, an ingredient that brings constriction of blood vessels by action of the enzyme angiotensin-converting enzyme (ACE). Reversed-phase high-performance liquid chromatography (RP-HPLC) method is the focus of this work, which also includes its characterization, optimization, and synthesis. The objective is to correctly, precisely, and sensitively determine both enalapril and its synthesized metabolite, enalaprilat, when present as an impurity. The paper also forecasts the ADME and toxicity characteristics of ENLP by utilizing several ADME databases, including SWISS ADME and molesoft. The laboratory synthesized enalaprilat, the metabolite derived from enalapril, and characterized. The Kinetex C18 stationary phase was employed in the analytical approach. Flow rate was 1.0 mL/min, and the injection volume was 20 μL. With a wavelength of 244 nm, the run lasted for 10 minutes. The mobile phase used was an 80% acetonitrile: 20% pH 3 phosphate buffer. The HPLC technique validation, which involved the identification of the enalapril metabolite, followed the parameters outlined in ICH Q2B (R1). The analysis exhibited a range of accuracy and precision between 98.75 and 102.5% for all substances being tested. This approach is appropriate for identifying and measuring the metabolite in enalapril bulk or formulations. The method for assessing enalaprilat in biological fluids has the potential to be further refined and applied in clinical and bioequivalence research. The study furthermore presents an evaluation of toxicity utilizing computational methods such as the Swiss tool, with the objective of aiming to mitigate potential risks.

Stability Indicating Force Degradation Study of Nintedanib in Bulk and Pharmaceutical Dosage Form

Assay of the nintedanib drug substance can be determined using a newly designed and verified reverse-phase high-performance liquid chromatography (RP-HPLC) method, even when degradation products from forced degradation experiments are present. By using gradient elution, the mobile phase was a mixture of 70% acetonitrile and 30% water by volume. Acid, base, peroxide, thermal, and photolytic degradation were among the stress conditions that the product was subjected to. During the thermal and photolytic breakdown processes, no extra contaminants were detected. Using validation criteria such as specificity, linearity, limit of quantitation (LoQ), accuracy, precision, robustness, and ruggedness, the developed technique was validated in accordance with International Council for Harmonization (ICH) recommendations. At a concentration of 12.4 ng/mL, the LoQ value was attained. The results showed good linearity (r2 > 1.00) across doses ranging from 2 to 10 μg/mL. Verification of recovery was done by adding concentrated solutions of 5, 10, and 15 μg/mL. So, newly developed RP-HPLC technology can separate Nintedanib from its main degradation products, and it can also estimate the drug substance’s concentration.

Study on the impurity profiles of cloxacillin and flucloxacillin based on liquid chromatography tandem ion trap/time-of-flight mass spectrometry.

Cloxacillin and flucloxacillin are prone to degradation and polymerization in humid and hot environments, and their polymers have long been recognized to trigger allergic manifestations. A series of the degradation and polymerized impurities in cloxacillin and flucloxacillin were separated and characterized to ensure safe use of these drugs by the public. By studying the chromatographic behavior of the degradation impurities and polymerized impurities in reversed-phase high-performance liquid chromatography (RP-HPLC) gradient elution, the impurities in cloxacillin and flucloxacillin were effectively separated and eluted. RP-HPLC tandem ion trap/time-of-flight mass spectrometry (MS) was applied to characterize the structures of unknown impurities eluted from the RP-HPLC methods for cloxacillin and flucloxacillin. The mechanisms of formation of the impurities in cloxacillin and flucloxacillin were also investigated. The structures of 10 unknown impurities in cloxacillin and 8 unknown impurities in flucloxacillin were elucidated based on the high-resolution MSn data at positive and negative modes, respectively. Six polymerized impurities were found and characterized, of which three were from the polymerization of cloxacillin and three were from the polymerization of flucloxacillin. The study on the impurity profiles of cloxacillin and flucloxacillin provided a scientific basis for improving their production processes and quality control.

A stability indicating method development and validation of a rapid and sensitive RP-HPLC method for Nintedanib and its application in quantification of nanostructured lipid carriers.

Nintedanib (NTB) is a multiple tyrosine kinase inhibitor, been investigated for many disease conditions like idiopathic pulmonary fibrosis (IPF), systemic sclerosis interstitial lung disease (SSc-ILD) and non-small cell lung cancer (NSCLC). NTB is available as oral capsule formulation, but its ability to detect degradants formed through oxidative, photolytic and hydrolytic processes makes it difficult to quantify. In the current work, a novel reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated. The developed method is simple, precise, reproducible, stable and accurate. The inherent stability of NTB was evaluated using the proposed analytical method approach and force degradation studies were carried out. NTB was separated chromatographically on the Shimadzu C 18 column as stationary phase (250 ×4.6 mm, 5 µm) using an isocratic elution method with 0.1% v/v triethyl amine (TEA) in HPLC grade water and acetonitrile (ACN) in the ratio 35:65% v/v. The mobile phase was pumped at a constant flow rate of 1.0 ml/min, and the eluent was detected at 390 nm wavelength. NTB was eluted at 6.77±0.00 min of retention time (t R) with a correlation coefficient of 0.999, the developed method was linear in the concentration range of 0.5 µg/ml to 4.5 µg/ml. The recovery rate was found to be in the range of 99.391±0.468% for 1.5 µg/ml concentration. Six replicate standards were determined to have an % RSD of 0.04. The formulation excipients didn't interfere with the determination of NTB, demonstrating the specificity of the developed method. The proposed approach of the analytical method developed can be used to quantify the amount of NTB present in bulk drugs and pharmaceutical formulations.

Stability-indicating Reverse Phase-HPLC Method Development and Method Validation for Quantitative Determination of Degradation Products in Favipiravir API and Drug Product

Introduction: Favipiravir is an antiviral medication shown to be broad spectrum activity against RNA viruses, and potentially treating the COVID-19. Methodology: In this study, the HPLC method for the quantification of degradation impurities (Favipiravir Acid Impurities) were developed and validated for Favipiravir in Tablet dosage form. The specificity of the method was achieved in analytical column Agilent HPLC-C18, 5µm, (4.6 x250) mm. using a suitable mobile phase was 10 mM Phosphate buffer (pH 3.5 with orthophosphoric acid) and Acetonitrile in the Isocratic more of 70:30 v/v. The flow rate is 1.0 mL/min. the injection volume is 10 µL, detection at 320 nm in UV and total run time is 8.0 minutes. The samples were made for forced degradation under hydrolysis, oxidation, photolytic and thermal conditions. The method was validated for specific, selective, linear, robust and accurate as per the ICH guidelines. Results and Conclusion: The linearity of the method for Impurities and the analytes was found from 25 to 150 % concentration level with the correlation coefficient (r2) > 0.999. The accuracy for impurity and the analytes was performed from 50 to 150% level concentration, and mean recovery was found from 98-102%. The analytical degradation and validated study results indicate its unstable nature in acidic, basic and thermal conditions. Therefore, this method is simple, selective and sensitive, this method can be used in pharmaceutical research and development and quality control departments.

Development and validation of stability-indicating assay method and identification of force degradation products of glucagon-like peptide-1 synthetic analog Exenatide using liquid chromatography coupled with Orbitrap mass spectrometer.

Exenatide is a synthetic glucagon-like peptide 1 analog, widely used in the management of type 2 diabetes mellitus. The stability of pharmaceutical products is significantly impacted by various environmental stress conditions. The present study reports the development of a validated reverse-phase high-performance liquid chromatography (RP-HPLC) stability-indicating method for the identification of force degradation products (DPs) of synthetic glucagon-like peptide-1 analog Exenatide using UHPLC-Orbitrap fusionTM mass spectrometer. Force degradation studies were performed by subjecting Exenatide to various stress conditions, such as hydrolytic, oxidative, photolytic and thermal to investigate the stability indicating ability of the method. Significant degradation was observed during acidic, oxidative, photolytic and thermal stress conditions. Exenatide and its major DPs identification and characterization were demonstrated by employing LC-HRMS and MS/MS method. In total, five major stress DPs were characterized, and their fragmentation pathway was proposed using MS/MS studies. Finally, the proposed RP-HPLC method was validated as per ICH guidance.