Reversed-phase High-performance Liquid Research Articles

Novel post-functionalized polymer microspheres containing capsaicin as reversed-phase chromatography stationary phase separation for hydrophilic compounds

Here, we created for the first time that highly stable polymer microspheres copolymerized with capsaicin derivative, divinyl benzene, and N-(Hydroxymethyl)acrylamide could be efficient for reversed-phase high-performance liquid chromatography (RP-HPLC) by using a simple “one-pot cooking” method, which had the potential to be produced in large quantities. Then, the spheres were post-functionalized with two different monomers, N-vinyl formamide and 4-acrylomorpholine by double coating, which had ideal mechanical strength and efficiency as stationary phase. In order to reach best separation condition, the effects of buffer concentration, pH, column temperature, and mobile phase polarity on the retention rate were studied. Specifically, the interaction mechanism showed that the stationary phase had hydrophobic and electrostatic interaction with the water-soluble vitamins. It is of significant interest that all columns had excellent precision and accuracy (RSD < 4 %). Taken together, this study played a specific role in promoting the development of a new reverse-phase mode for separating hydrophilic compounds.

Comparison between Conventional Ageing Process in Barrels and a New Rapid Aging Process Based on RSLDE: Analysis of Bioactive Compounds in Spirit Drinks

“Aging” is a practice that allows alcoholic beverages to mature and gives them particular flavors and colors. In this context, oak or durmast wooden barrels are used in this process, thus providing different types of aging. This conventional process produces a slow enrichment of organic compounds in the spirit inside the barrels. Organic substances present in the internal part of the barrels slowly undergo the phenomenon of extraction by the liquid phase (solid–liquid extraction). In this work, a new procedure based on rapid solid–liquid dynamic extraction (RSLDE) was used to evaluate the potential of obtaining the effects of aging in spirits in shorter times than conventional methods. For this purpose, a comparison between two solid–liquid extraction techniques, RSLDE and conventional maceration, was made. Four water/ethanol 60:40 (v/v) model solutions were prepared and put in contact with medium-toasted chips using the two extraction procedures (conventional and non-conventional) and determining dry residue and total polyphenol content. Reversed phase high-performance liquid chromatography (RP-HPLC) analyses allowed the identification and quantification of furfural, ellagic acid and phenolic aldehydes (vanillin, syringaldehyde, coniferaldehyde and sinapaldehyde). The aging procedure with medium-toasted chips was tested on a young commercial grappa using maceration and RLSDE.

Chronic exposures to cholinesterase-inhibiting pesticides adversely affects the health of agricultural workers in India

Biomonitoring of pesticide exposure has become a public concern because of its potential health effects. The present study investigated the acetylcholinesterase (AChE) inhibitory levels and their associated health effects in agricultural areas in Telangana, India. This cross-sectional included 341 exposed participants and 152 control participants from agricultural areas. A structured questionnaire was completed and blood and urine samples were collected to measure pesticides, dialkyle phosphate (DAP) metabolites, and AChE activity using liquid chromatography-tandem mass spectrometry and reversed-phase high-performance liquid chromatography. twenty-eight pesticides were detected in blood samples at concentrations ranging 0.42-45.77 ng/mL. Six DAP metabolites were also measured in urine, and all DAP metabolites were significantly higher in the exposed group. AChE activity is significantly reduced in individuals exposed for >10 years, raising concerns regarding possible neurological disorders. These results emphasise the urgent need to investigate the health effects of pesticides exposure, especially in agriculture.

Analytical Method Development and Validation of Brivaracetam in API and Marketed Formulation by RP-HPLC

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of brivaracetam in its pure form as well as in tablet dosage form. Chromatography was carried out on a Symmetry ODS RP C18,5mm, 15mm x 4.6mm column using a mixture of phosphate buffer: methanol: acetonitrile (30:35:35% v/v) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 285 nm. The retention time of the brivaracetam was 2.183. The method produces linear responses in the concentration range of 60-140μg/ml of brivaracetam. The method precision for the determination of assay was below 2.0%RSD. The method was found to be sensitive, accurate and precise useful in the quality control of bulk and pharmaceutical formulations.

Bioanalytical method development and validation for the quantification of raloxifene hydrochloride from mice plasma by RP-HPLC

Raloxifene hydrochloride (RLX) belongs to the Selective estrogen receptor modulator (SERM) class of drugs, exhibiting diverse affinity to estrogen receptors. The drug is known for its anti-cancer and anti-osteoporosis activity. The current study employs a Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method that is sensitive and time-efficient for quantitative evaluation of RLX from mice plasma, using tamoxifen citrate as an internal standard (IS). The protein precipitation technique was used to extract the RLX and IS. RLX was separated using a C8 column with an isocratic elution of a mobile phase consisting of acetonitrile and Milli-Q water (pH 3) at a 1 mL/min flow rate. The ultraviolet (UV) detection of the analyte and IS were carried out at 287 nm. The linearity plot was constructed in the range of 100–3200 ng/mL with a correlation coefficient of r2 ≥ 0.9996. The inter-day and intra-day accuracy and precision were found to be within acceptable limits. Stability studies revealed that the analyte remained stable. The validated RP-HPLC method was successfully applied to quantify RLX after oral administration in mice, and the pharmacokinetic (PK) parameters were established. The present RP-HPLC method could be a beneficial tool for the fast and reliable estimation of future PK interactions, PK analysis of formulations, and therapeutic drug monitoring.

Updated component analysis method for naturally occurring sophorolipids from Starmerella bombicola

Sophorolipids (SLs) are promising glycolipid biosurfactants as they are easily produced and functional. SLs from microorganisms are comprised of mixtures of multiple derivatives that have different structures and properties, including well-known acidic and lactonic SL (ASLs and LSLs, respectively). In this study, we established a method for analyzing all SL derivatives in the products of Starmerella bombicola, a typical SL-producing yeast. Detailed component analyses of S. bombicola products were carried out using reversed-phase high-performance liquid chromatography and mass spectrometry. Methanol was used as the eluent as it is a good solvent for all SL derivatives. With this approach, it was possible to not only quantify the ratio of the main components of ASL, LSL, and SL glycerides but also confirm trace components such as SL mono-glyceride and bola-form SL (sophorose at both ends); notably, this is the first time these components have been isolated and identified successfully in naturally occurring SLs. In addition, our results revealed a novel SL derivative in which a fatty acid is bonded in series to the ASL, which had not been reported previously. Using the present analysis method, it was possible to easily track compositional changes in the SL components during culture. Our results showed that LSL and ASL are produced initially and that SL glycerides accumulate from the middle stage during the fermentation process.Key points• An easy and detailed component analysis method for sophorolipids (SLs) is introduced.• Multiple SL derivatives were identified different from known SLs.• A novel hydrophobic acidic SL was isolated and characterized.Graphical

Exploring the phytochemical and biological properties of Salsola foetida: a promising wild plant from Southeastern Algeria

ABSTRACT This study presents an initial exploration of the phytochemical composition and biological properties of Salsola foetida, a wild plant native to southeastern Algeria. The investigation encompassed an analysis of the biochemical content, phytochemical profile, and bioactivity of the plant’s aerial parts. The biomass of S. foetida was found to comprise 51.2 ± 0.4% ash, with elemental analysis revealing the presence of carbon, oxygen, calcium, sodium, chlorine, magnesium, potassium, and sulfur. Carbohydrates were identified as the predominant constituents, surpassing the levels of proteins and lipids. Phytochemical screening, coupled with Fourier Transform Infrared (FT-IR) analysis, confirmed the presence of various secondary metabolites, including alkaloids and phenols. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis further delineated a spectrum of phenolic compounds, with rutin emerging as the predominant compound at 1.59 µg/ml. Quantification revealed a noteworthy phenolic content of 67.1 ± 0.2 µg GE/mg ED. The extract exhibited robust radical scavenging effects, with the radical 50% inhibitory concentration (IC50) being 61.8 ± 0.2 μg/ml, along with protective effects against H2O2-induced hemolysis and the ability to reduce ferric ions. Additionally, the extract demonstrated anti-inflammatory, antibacterial, and antidiabetic activities. Notably, it exhibited considerable sunscreen properties, boasting a sun protected factor (SPF) value of 21 ± 1. Overall, S. foetida presents promising antioxidant activity and a spectrum of potential biological activities, underscoring its significance as a novel antioxidant candidate for applications in animal nutrition, medicine, and natural product research.

Structural and Biochemical Characterization of a New Phage-Encoded Muramidase, KTN6 Gp46.

Endolysins are phage-encoded lytic enzymes that degrade bacterial peptidoglycan at the end of phage lytic cycles to release new phage particles. These enzymes are being explored as an alternative to small-molecule antibiotics. The crystal structure of KTN6 Gp46 was determined and compared with a ColabFold model. Cleavage specificity was examined using a peptidoglycan digest and reversed-phase high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS). The structure of KTN6 Gp46 could be determined at 1.4 Å resolution, and key differences in loops of the putative peptidoglycan binding domain were identified in comparison with its closest known homologue, the endolysin of phage SPN1S. Reversed-phase HPLC/MS analysis of the reaction products following peptidoglycan digestion confirmed the muramidase activity of Gp46, consistent with structural predictions. These insights into the structure and function of endolysins further expand the toolbox for endolysin engineering and explore their potential in enzyme-based antibacterial design strategies.

Therapeutic drug monitoring of six contraindicated/co-administered drugs by simple and green RP-HPLC-PDA; application to spiked human plasma

Therapeutic drug monitoring is an important clinical testing of the drugs to monitor their concentrations in plasma in order to guarantee their optimal impact, and to avoid any side effects resulting from drug-drug interactions. A green reversed-phase high-performance liquid chromatographic method using a photodiode array detector (RP-HPLC-PDA) was developed for the simultaneous determination of three carbapenem antibiotics (Imipenem, ertapenem, and meropenem) with the co-formulated drug (cilastatin) and contraindicated drugs (probenecid and warfarin) in spiked human plasma. The separation was achieved at 25 °C using a gradient elution of a mixture of mobile phase A: methanol and mobile phase B: phosphate buffer (pH 3.0). The photodiode array detector was adjusted at 220 nm. Bioanalytical method validation was carried out as per the FDA guidelines, and the method showed good linearity ranges for the six drugs that included their Cmax levels along with low limits of quantification. Based on the results, the method was found to be accurate and precise; with high % recovery and good % RSD, respectively. The method was successfully applied to spiked human plasma, signifying a good potential to be implemented in future TDM studies of these drugs when co-administered together.

A novel, high throughput, and low cost method for the detection of 40 amino acids relevant to inborn errors of metabolism in under 60 minutes using reverse phase high performance liquid chromatography

A novel, high throughput, and low cost method for the detection of 40 amino acids relevant to inborn errors of metabolism in under 60 minutes using reverse phase high performance liquid chromatography

Quantitative comparison and evaluation between aerial and underground parts of Gentiana straminea through simultaneous determination of five major compounds by RP-HPLC

Herbal Gentiana straminea Maxim. (Family Gentianaceae), “Ma Hua Jiao” in Chinese, is a commonly used Chinese medicine. Secoiridoids and flavonoids have been identified as the major active components of herbal medicines used in the treatment of hepatitis, rheumatism and many other diseases. It is the overharvesting of the roots of this plant for medicinal purposes that has led to a drastic decline in its population. In the present study, the above and below ground parts of Gentian Bitter Glycine were quantitatively compared and evaluated for the determination of the major active constituents. Five major compounds, loganic acid, swertiamarin, gentiopicroside, sweorside and isoorientin, were extracted by solvent extraction technique and analyzed by Reversed-phase High Performance Liquid Chromatography (RP-HPLC). By analysing the principal components and calculating the composite scores, the results show that the aboveground component in different areas ranked higher compared to the underground component, with the former being able to substitute to some extent for the latter's underground component. Finally, based on hierarchical cluster analysis, we identified the ideal natural growing region for aerial parts of G. straminea distributed on the Qinghai-Tibetan Plateau. The significance of this work is that we can balance the demand for herbs with environmental preservation by selectively picking the aerial parts, which can regrow next year, instead of removing the whole plant. It protects the fragile ecological environment of the Tibetan Plateau and is important for sustainable development.

Developed and Validated for the Estimation of Bupropion and Dextromethorphan in a Fixed Dose Combination of the Tablet.

The aim of this study was to develop a simple, accurate, and precise method for the estimation of bupropion and dextromethorphan in a fixed-dose combination of tablets and robust high-performance liquid chromatography for assay analysis of such a fixed combination. Chromatographic analysis was performed and separations were achieved on a Denali C18 150 × 4.6 mm, 5 micron using a mobile phase composition of ortho-phosphoric acid and acetonitrile in the ratio of 600:400 (v/v), flow rate of 1.0 mL/min, injection volume is 10 µL and run time of 6 min in isocratic elution. Ultraviolet (UV) detection was performed at a wavelength of 221 nm. The temperature was maintained at 30 °C. Well-resolved peaks were observed with a high number of theoretical plates, lower tailing factor, and reproducible relative retention time. The method was validated, and all validation parameters were found to be within the acceptance limits. A simple, accurate, and precise method has been developed for estimating bupropion and dextromethorphan in a fixed dose combination of tablets. The optimized method included the following parameters: column temperature of 30 °C, 40% acetonitrile as the mobile phase, and flow rate of 1.0 mL/min. Retention times were 2.25 min and 3.12 min for bupropion and dextromethorphan, respectively. The method was found to be linear in the range of 17.5-105 µg/mL [for R2 < 0.999) and 7.5-45 µg/mL (for R2 > 0.999] for bupropion and dextromethorphan, respectively. Both active pharmaceutical ingredients dissolved more than 90% within 5 min. The current study describes a new, simple, reliable, and economical elution reversed-phase high performance liquid chromatography method for estimating bupropion and dextromethorphan in a fixed combination tablet dosage form. The forced degradation studies were conducted using several degradation conditions such as acidic, alkali, oxidation, thermal, UV, and neutral conditions; the proposed method was effectively employed from the resolution of sample peaks. To the best of our knowledge, no such detailed and stability-indicating method has been reported for a fixed tablet dosage form.

Validation of HPLC-DAD Based Stability Indicating Assay Method for Ornidazole in Periodontal Polymeric Hydrogel: Robustness Study using Quality by Design (QbD) Approach

In the present study, a gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method has been designed and validated to quantify Ornidazole (OZ) in the marketed formulation (oral gel) with the application of QbD. The proposed HPLC method utilizes an RP C18 column (4.6 x 250 mm, 5 μm). The mobile phase contained HPLC grade Solvent A (water) and Solvent B (acetonitrile) and ran at a flow rate of 1 mL/min in a gradient mode. The linearity was plotted in an OZ range of 1–12 μg/mL and the correlation coefficient was found to be 0.9998. The limit of quantification, as well as the limit of detection, has been recorded as 0.70 μg/mL and 0.23 μg/mL. Inter- and intra-day precision (RSD) percentages were reported respectively within a range of 0.262–0.589 and 0.179–0.879. At 80, 100, and 120 percent different OZ levels, the average accuracy was 99.55, 99.58, and 99.92, respectively. The result of the study indicates that the OZ gel estimate HPLC method has good sensitivity, reproducibility, accuracy, and precision. This method may be used for the estimation of OZ in a gel formulation in quality control laboratories.

The Quality by design Approach for Analytical Method Development and Validation of the RP-HPLC Method for Estimation of Quercetin in Pure, Marketed, and Cream Formulation

ABSTRACT: A simple, precise reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the flavonoid quercetin, isolated from Tridax procumbens L. The method was validated by using Phenomenex C18 (250 x 4.6mm i.d) Column. A simple, cost effective mobile phase consisting of (ACN and 10 m/moL Phosphate buffer as mobile phase in proportion of 50:50 v/v ) pH 3, 1.0 ml/min Flow rate at 370nm by using UV Visible detector. The retention time of Quercetin was found to be 3.392 minutes. The Quercetin linearity range was found to be 05 to 25 μg/mL. The accuracy and precision of commercially available preparations and in-house cream formulations were investigated using a one-way ANNOVA test. The Percentage recovery of both formulations was found to be 99.83%, 99.88%, 99.82% and 98.92%, 98.18%, 98.86%. Robustness of analytical method was studied by using 2/3 full factorial design by using Design expert software. The Percentage assay and % RSD of marketed capsule and in house cream was found to be 98.38 %, 97.40% and 0.05273, 0.02053 respectively. The Limit of Quantitation and Limit of detection were found to be 0.9053μg/mL and 2.5435μg/mL respectively. The development method of quercetin is simple, accurate, precise, sensitive, and robust

Separation of inorganic anions on reversed-phase C18 columns with a phosphomolybdate mobile phase

Reversed-phase high performance liquid chromatography (RP-HPLC) is the most widely used chromatographic method. In addition to hydrophobic interactions, additional interactions such as electrostatic interactions may participate in the retention behaviour of an analyte. This makes it possible to use RP-HPLC for many types of analyte. We describe a simple method for separating inorganic anions on a C18 column, in which retention of inorganic anions is almost entirely due to electrostatic interactions. This leads to rapid separations as well as higher theoretical plate numbers. We used 2 mM phosphoric acid containing a low concentration of disodium molybdate as the mobile phase, which allows UV detection of non-UV-absorbing anions. With this method, we determined eight inorganic anions including several non-UV-absorbing anions photometrically at 220 nm. The detection limits of the examined eight inorganic anions calculated at a signal-to-noise ratio of 3 were between 0.3 and 10 μM. The detector response was linear over three orders of magnitude of inorganic anion concentration. The proposed RP-HPLC/UV method was successfully applied to determine inorganic anions in some water samples.

Optimization of a Validated Liquid Chromatographic Method with Chemometric Approach for Estimation of Eugenol from Seed Powder Extract and Perform Anti-oxidant Activity

Background:: Developed and validated a new reverse phase high-performance liquid chromatographic (RP-HPLC) method, and it is prompt, precise, sensitive and robust for the esti-mation of eugenol in seed powder extract of Myristica fragrans. Objective:: The chemometric approach was utilized to obtain a rugged and definitive chromato-graphic method for the purpose. Methods:: Method variables such as acetonitrile (%) and flow rate were investigated for robust-ness and optimization by using a face-centered cubic design (FCCD). The Design Expert 12.0.1.0 software has been employed for this optimization. Further, the effects of factors were monitored on the concentrations of eugenol recovered from seed powder extract. Chromatograms have been developed by using an optimized mobile phase mixture containing methanol-water-acetonitrile (10:40:50, v/v/v) and Symmetry® C18 column (5 μm, 3.9 ×150 mm). The mobile phase was de-rived at a flow rate of 1 mL/min, and estimation of eugenol was performed at ʎmax 272 nm. Results:: Validation of the method has been carried out to reveal its selectivity, linearity, preci-sion, accuracy, LOD, and LOQ. Linear calibration plot for eugenol was held over the concentra-tion across 6.25 and 100 μg/mL (R2 = 0.999). The coefficient of variation was less than 1%, and accurate recovery of eugenol was observed between 96.80 and 99.56 %. The LOD and LOQ have been established to be 1.97 and 6.25 μg/mL, respectively. Intraday and Inter-day coefficients of variation have 1.81-1.91 and 1.92-1.57, respectively. Antioxidant activity (AA) by DPPH assay of seed powder extract in five different solvents was performed, and % AA activity was calculat-ed against ascorbic acid. Conclusion:: The validated method has been found to be highly robust and will be applied for the analysis of eugenol in formulation. The highest % AA has been reported in hexane solvent.

Reverse Phase HPLC Methodology for the Determination of Bay K8644.

Following ICH guidelines for analytical validation, we report a common C18 column stability indicating isocratic reverse phase high performance liquid chromatography method for the determination of the ion channel modulator Bay K8644. Two main forced degradation products and a minor impurity were also tentatively identified by Mass Spectrometry. The mobile phase consisted of a 50/50 acetonitrile/buffer mixture at a flow rate of 2mL/min. Mean retention time for Bay K8644 was 3.030minutes. Excellent linearity (r = 0.9998) was achieved in the range 0.10-1.40μg/mL at 274nm wavelength. Analytical limits were 16.56 ± 1.04ng/mL for detection and 55.21 ± 3.48ng/mL for quantitation respectively. Accuracy and precision studies showed good results (95-105%). Robustness was assessed by varying ±3%, both temperature and flow rate. Five different stress conditions were applied to assess Bay K8644's stability. Only basic and photolytic treatments yielded degradation products, both correctly resolved in a total runtime of 4minutes. In conclusion, we developed a fast, simple, sensitive, accurate, precise, reliable and stability indicating method for detecting/quantifying Bay K8644, and tentatively characterized its main impurities/forced degradation products.

Ecological niche modelling guided chemotypic analysis of Ageratum conyzoides L. from varied geography of India

Ageratum conyzoides L., recognized globally as a noxious weed, holds significant value in traditional medicine, particularly in treating cuts, wounds, and tick infestations. This study focuses on identifying specific germplasms of this plant, rich in therapeutically valuable metabolites, across various phytogeographical zones in India. Using the Reverse Phase High-Performance Liquid Chromatography with Photodiode Array Detection (RP-HPLC-PDA) method, we analyzed 110 samples to quantify five key bioactive markers: Precocene-I (PRC-I), Coumarin (CM), Caryophyllene (CAR), Caryophyllene Oxide (CAO), and Humulene (HUM). Our findings revealed distinct geographical variations in these compounds. The highest concentrations of PRC-I (7.86±1.36 %) was found in sample NAC-12, CM (1.50±0.46 %) and HUM (0.0099±0.0004 %) in NAC-104, while CAR (6.33±0.87 %) and CAO (6.23±0.18 %) were predominant in NAC-01 and NAC-02, respectively. Ecological Niche Modelling (ENM) was used to predict the climatic suitability of regions for high yield of these metabolites. The ENM results showed that annual precipitation (Bio 12), precipitation of the coldest quarter (Bio 19) and minimum temperature of coldest month (Bio 6) were the principal climatic factors influencing metabolite content. Geographically, the Indo-Gangetic plain, Indus plain, and Central India were identified as suitable for higher content of PRC-I, CAO, CAR, whereas the North Eastern region favoured high content of CM and HUM. This study underscores the importance of bioactive marker-based assessment in understanding chemotypic diversity and demonstrates the effective use of ENM in predicting optimal habitats for harvesting specific chemotypes. These insights pave the way for location-specific cultivation strategies and targeted procurement of metabolite-rich germplasms, contributing to sustainable sourcing and conservation efforts, as well as to the advancement of herbal product standardization and efficacy.

Quantification of serine residue stereoinversion in a short peptide by reversed-phase high-performance liquid chromatography: analysis of mechanisms promoting serine stereoinversion.

Stereoinversion of Ser residues within proteins, which has been identified in long-lived proteins, influences protein function. To quantify the stereoinversion of Ser residues, we investigated the potential adaptation of our direct peptide analytical method originally established for analyzing the isomerization of asparaginyl/aspartyl residues. Peptide pairs containing L-Ser or D-Ser residues with lengths of four or five residues were synthesized. Separation conditions for these peptide pairs were systematically examined by precisely adjusting the pH of the elution solvent using reverse-phase high-performance liquid chromatography (HPLC). Optimal separation conditions were successfully developed for all peptide pairs, enabling the direct quantification of Ser residue stereoinversion through a single HPLC run. Subsequently, the degree of Ser stereoinversion within the model peptide, Gly-Ser-Gly-Tyr, was determined using the method established in this study. Surprisingly, the stereoinversion of Ser residues occurred only when the absolute configurations of Ser and Tyr residues of the peptide differed from each other, whereas no stereoinversion was observed when their absolute configurations were identical. The experiments using peptides similar to the model peptide reveal that both the N-terminal amino group and the hydroxyl group of the C-terminal Tyr residue are involved in the stereoinversion of the Ser residue. By applying a simple method to quantify the stereoinversion of Ser residues, valuable insights into the mechanisms governing these stereoinversions were obtained.

New Stability Indicating HPLC Method for Related Substances in Dapagliflozin

The requirements established by the International Council for Harmonization (ICH) for the measurement of dapagliflozin from tablets were satisfied by the development and successful implementation of an reversed-phase high performance liquid chromatography (RP-HPLC) technique that is characterized by its speed, precision, and accuracy. In order to separate the samples, an isocratic mode was used on a Princeton C18 column that was operating at a flow rate of 1-mL per minute. The phase was composed of acetonitrile and 0.1% triethylamine in a volume-to-volume ratio of 50:50, with a pH of 5.0 and a detection wavelength of 224 nm. 5.163 minutes was the retention time that was determined to be best under the circumstances of the chromatographic analysis. Based on the correlation value of 0.999, it can be concluded that the procedure adhered to Beer-Lambert’s law within the measurement range of 10 to 70 μg/mL. The findings not only verified the claim that was made on the label, but they also validated the mean percent drug concentration that was specified for the commercial tablet formulation, which was 100.57%. Following the completion of an investigation, it was discovered that the approaches that were proposed have sufficient proportions of precision measurements, robustness, accuracy, range, and linearity. As part of the technique for stress testing, a number of criteria were evaluated and evaluated overall. The elements that were considered to be among these were light, humidity, temperature, oxidation, and pH (acid/base).