Validation Of High-performance Liquid Chromatography Method Research Articles

Validation of chromatographic methods, TLC and HPLC, to quantify known radiochemical impurities and the radiochemical purity the radiopharmaceutical [177Lu]Lu-PSMA I&T

The Energy and Nuclear Research Institute (IPEN) is studying the production of the radiopharmaceutical [177Lu]Lu-PSMA I&T, in accordance with the good manufacturing practices recommended by the National Health Surveillance Agency (ANVISA), to be used in the therapy of prostate cancer. This work aims to validate chromatographic methods, Thin-Layer Chromatographic (TLC) and high-performance liquid chromatography (HPLC), to determine the radiochemical purity (RCP) of the product. The entire validation process of this work was based on ANVISA's RDC 166, 2017 and the Guide 10, version 1, 2017, guided the statistical treatments adopted. With the selectivity study we can observe that the presence of impurities or excess excipients in the sample does not interfere with the quantification of the product. The proposed methods were linear with linear correlation coefficients (r) above 0.99. The precision and repeatability presented relative standard deviation values lower than specified (RSD < 5 %). The small controlled variations in the method suggested for the robustness test also did not affect the radiochemical purity of the product. In view of the results and in accordance with the criteria established by the National Health Surveillance Agency (ANVISA), the two chromatographic methods were validated in accordance with RDC 166, 2017, proving to be selective, precise, linear and robust. The validation of TLC and HPLC methods enables their application in the batch release routine of the new radiopharmaceutical at Radiopharmacy Center of IPEN.

Development and Validation of an HPLC Method for Niraparib Analysis: A Comprehensive Approach.

Background: Niraparib is a significant PARP inhibitor utilized in cancer therapy. This study aims to develop and validate a high-performance liquid chromatography (HPLC) method for the quantification of Niraparib in pharmaceutical formulations and biological samples. Results: A Phenomenex Kinetex XB-C8 column (150 x 4.6 mm, 5 µ) was employed with a mobile phase of 0.1% formic acid in acetonitrile (60:40, % v/v). The analysis was performed at an oven temperature of 30℃ with a flow rate of 0.5 mL/min, and detection was achieved at 240 nm. The method demonstrated excellent linearity (R² = 0.9998) across concentrations from 40 to 60 μg/mL. Validation studies confirmed high accuracy, with a mean recovery of 99.96%. Conclusion: The validated HPLC method is robust and reliable for quantifying Niraparib in various formulations, aiding in the stability assessment and ensuring the efficacy and safety of cancer treatment protocols. This method facilitates informed decisions in pharmaceutical development.

High-Performance Liquid Chromatographic Method Development and Validation for Analysis of Expired and Marketed Doxofylline Tablets.

The objectives of the study were to develop a simple, reliable, sensitive and accurate high-performance liquid chromatography (HPLC) method for doxofylline pure and pharmaceutical dosage forms. HPLC method was developed for the estimation of doxofylline in pure and marketed, recently expired, and 1-year expired tablet formulations. Chromatographic separation of the drug was achieved on a stainless steel column 25 cm × 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm), Cosmosil® C18, column using a mobile phase of Potassium dihydrogen orthophosphate buffer: Acetonitrile pH 5.1 (60:40 v/v) at a flow rate of 1-mL/min. The drug eluted was monitored at 274 nm and the retention time of doxofylline was found to be 3.575 minutes, respectively. The validation of developed methods was done according to ICH Q2 (R1) guidelines. The calibration curve was linear over the range of 5 to 50 µg/mL. The correlation coefficient was found to be 0.9943 for doxofylline. The average retention time was found to be 3.538 minutes. The results were reproducible, showing the effectiveness of the system. The plate number was found to be 4909, which is also within the limit, i.e. ≥ 2000 prescribed in I.P. The dissolution studies of all three marketed, recently expired and 1-year expired formulations were carried out by using 0.01 M HCl and the results showed almost a similar rate of release profile and all three formulations accomplished the drug release within the limit. The developed and validated HPLC method offers a precise, accurate, and efficient analytical tool for the determination of doxofylline in pharmaceutical formulations.

Unveiling Impurities: A Comprehensive RP-HPLC Method for Accurate Atorvastatin Impurity Analysis in Pharmaceuticals Using Accuracy Profile Approach.

Accurate determination of active pharmaceutical ingredients and impurities is essential for ensuring the safety and effectiveness of medications. This study focuses on the validation of a high-performance liquid chromatography (HPLC) method for quantifying Atorvastatin and its impurities, addressing a critical aspect of pharmaceutical analysis. The primary objective is to conduct a comprehensive validation study for the HPLC method, covering specificity assessment, response function establishment, and a detailed analysis of precision, trueness, and tolerance intervals. The emphasis is on demonstrating the method's precision, accuracy, and stability-indicating capabilities across various concentrations and compounds. The HPLC method is validated through rigorous assessments, including specificity, response function establishment, and analyses of precision, trueness, and tolerance intervals. Induced degradation experiments are conducted to explore Atorvastatin's behavior under extreme conditions. Insights into the compound's synthesis and degradation pathways are provided through a proposed mechanism for intramolecular esterification. The results affirm the precision, accuracy, and stability-indicating capabilities of the validated HPLC method. The method effectively differentiates between Atorvastatin and its impurities, showcasing its suitability for pharmaceutical quality control. The validated HPLC method emerges as a robust and reliable tool for Atorvastatin analysis, contributing significantly to pharmaceutical research and quality control. Its application ensures the safety and efficacy of medications, reinforcing its role in pharmaceutical analysis. This study not only validates a crucial HPLC method for Atorvastatin analysis but also provides insights into the compound's behavior under extreme conditions and its synthesis and degradation pathways. The validated method serves as a cornerstone in pharmaceutical research and quality control, ensuring medication safety and efficacy.

Determination of Water-Soluble Vitamins in Palmyrah Sweet Sap and Its Derived Products using RP-HPLC Method

Palmyrah sweet sap, obtained from the inflorescence of the Borassus flabellifer L. palm tree, is a popular natural drink in Sri Lanka, available seasonally from January to August. It contains a blend of water, sugars, and essential vitamins and minerals. The sweet sap is processed thermally to produce sugary products like crude sugar, jaggery, and treacle. It is crucial to analyze them to ensure their nutritional value, due to the possibility of vitamin decomposition during the production stage. This study aimed to quantify the existing vitamins in sweet sap and its derived products using validated high-performance liquid chromatography (HPLC). The HPLC method validation parameters, such as linearity, accuracy, precision, range, limit of detection (LOD), and limit of quantification (LOQ), were determined. The results showed that the derived products from palmyrah sap had high concentrations of vitamins, with thiamine ranging from 2.81 to 35.16 mg/100g, niacin from 4.35 to 45.03 mg/100g, pyridoxine from 4.44 to 87.16 mg/100g, and ascorbic acid from 5.42 to 52.52 mg/100g. The accuracy of the HPLC method for vitamin analysis was between 98.43 and 100.64%, with a limit of detection ranging from 0.357 to 1.152 ppm and a limit of quantification from 1.081 to 13.573 ppm. The study successfully quantified the existing vitamins in the sap and its derived products, with overall results indicating that the vitamins were retained even after thermal processing.

Time-efficient HPLC Validation Methodology for the Qualitative Analysis of 68Ga PSMA-11 in Routine Clinical Usage under Isocratic Method

Background: Prostate-specific membrane antigen (PSMA) has shown to be a promising agent for prostate cancer imaging under PET-CT. With the automation in radiolabeling with 68Ga, using iTG 68Ge/68Ga generator, it has helped introduce various new diagnostic agents and achieve good manufacturing practices (GMP) simultaneously. However, before any radiopharmaceutical is put into clinical usage, it should always be checked for its radiochemical purity and other quality parameters before injecting in the patient. Chromatography techniques such as Gas Chromatography (GC), High-Performance Liquid Chromatography (HPLC), and Thin-Layer Chromatography (TLC) are the most frequently utilized separation technique for purity analysis. A rapid quality control HPLC based methodology was required for radiopharmaceuticals. Aim &amp; Objective: In our current setting, we conducted quality control analysis and standardized and validated HPLC method for the routine quality check of 68Ga-PSMA-11. Materials and Methods: The QC of 68Ga PSMA-11 was performed under ITLC and HPLC. Results: Linearity, accuracy, precision and specificity were assessed and quantified in accordance with International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use (Q2 (R1) ICH) guidelines, which can be implemented in resource-limited settings to check the quality. Conclusion: The current HPLC based methodology is rapid, with a retention time of 2.24 min, rendering it a favorable analytical standard operating procedure for QC analysis of 68Ga-PSMA-11.

Quantitative Analysis of 5-hydroxyindoleacetic Acid in Rat Urine by High-performance Liquid Chromatography Method

ABSTRACT Background: 5-hydroxyindoleacetic acid (5-HIAA) is a metabolite of serotonin, a neurotransmitter primarily associated with regulating mood, appetite, and sleep. When serotonin is broken down in the body, it forms 5-HIAA, which is then excreted in urine. Quantification of 5-HIAA levels in urine is often used as a diagnostic tool for certain conditions, particularly carcinoid tumors, which can produce excess serotonin. Elevated levels of 5-HIAA in urine can indicate increased serotonin production, which may be indicative of carcinoid tumors or other conditions affecting serotonin metabolism. However, there is limited work on employing high-performance liquid chromatography (HPLC)–ultraviolet to estimate monoamines in biological samples. Aim and Objectives: The present study explores the detection of 5-HIAA in rat urine samples, developing an easy, precise validated HPLC method. Materials and Methods: Mobile phase combination acetonitrile and o-phosphoric acid in the ratio of 30:70% v/v and flow rate 0.5 mL/min was able to separate 5-HIAA distinctly. Results: The detection was observed at λmax 277 nm with better sensitivity. According to the parameters indicated in the international council for harmonisation of technical requirements for pharmaceuticals for human use guidelines (Q2A; Q2B), the procedure was verified. The linearity range was selected from 20 to 45 μg/mL, r 2 = 0.992, limit of detection (4.65 μg/mL), limit of quantification (15.52 μg/mL), retention time (6.3 min). Conclusion: The technique has demonstrated that it is repeatable and recoverable within the given range. Thus can be used for routine analysis of 5-HIAA in urine samples.

Validation of HPLC and TLC analytical methods to determine radiochemical purity of 99mTc-cAbVCAM1-5, a new experimental radiotracer

Cardiovascular diseases, including fatal myocardial infarctions from atheromatous plaques, are the primary global mortality cause. Detecting stenotic atheromatous plaques is possible through coronary angiography, but vulnerable plaques with eccentric remodeling are undetectable with current diagnostic methods. Addressing this challenge, our group developed a radiopharmaceutical drug targeting vascular cell adhesion molecule 1 (VCAM-1), radiolabeled with technetium-99m. Given the absence of a monograph in the European Pharmacopoeia, and in order to draft the investigational medicinal product documentation, analytical methods had to be validated by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) to determine the radiochemical purity (RCP) of 99mTc-cAbVCAM1–5. This study therefore presents the results of the validation of analytical methods obtained in this context. The method validation followed the European Association of Nuclear Medicine (EANM) recommendations adapted from ICH Q2(R1), ensuring conformity with specificity, accuracy, repeatability and intermediate precision, linearity, robustness, quantification limit (LoQ), and range criteria. Regarding the results of specificity, both HPLC and TLC methods demonstrated excellent separation of 99mTc-cAbVCAM1–5 from impurities 99mTcO4-. Accuracy results indicated recovery percentages within the range of 99.52–101.40% for the HPLC and 99.51–101.97% for TLC, ensuring reliable measurements for each concentration of 99mTcO4-. Precision of the methods was validated by assessing repeatability and intermediate precision. Linearity was determined over the usual concentrations range and the correlation coefficient was greater than 0.99 for both methods. The limit of quantification was measured by diluting the 99mTcO4- to obtain a signal-to-noise ratio of around 10:1. Under these conditions, we obtained an LOQ of 2.10 MBq/mL for HPLC and 2Mbq/mL for TLC. In conclusion, the analytical methods developed in this study comply with EANM recommendations. This therefore allows us to correctly assess the radiochemical purity of 99mTc-cAbVCAM1–5, a new radiotracer targeting inflammation in vulnerable plaques.

Advances in High-Performance Liquid Chromatography (HPLC) Method Development and Validation: A Comprehensive Review

High-Performance Liquid Chromatography (HPLC) stands as the cornerstone technique for the detection, separation, and quantification of drugs in various matrices. Method optimization necessitates a thorough investigation of multiple chromatographic parameters, encompassing sample pretreatment, mobile phase composition, column selection, and detector configuration. This article aims to comprehensively address the processes of method development, optimization, and validation within the realm of HPLC. The inherent advantages of HPLC, including its rapidity, specificity, accuracy, precision, and automation feasibility, render it an indispensable tool for analyzing a wide spectrum of drugs in complex multi-component dosage forms. Furthermore, HPLC method development and validation hold paramount importance across the spectrum of drug discovery, development, and manufacturing, as well as in diverse human and animal studies. The validation of analytical methods during drug development and manufacturing is imperative to ensure their fitness for the intended purpose. Adhering to Good Manufacturing Practice (GMP) requirements, pharmaceutical industries must establish comprehensive validation policies outlining the validation procedures. This article primarily delves into the optimization of HPLC conditions, emphasizing the critical role of method refinement in ensuring robust and reliable analytical methods.

A high-performance liquid chromatography method validation and a Box-Behnken experimental design for the extraction optimization of quercitrin from Nectandra reticulata.

The ethanolic extract of Nectandra reticulata contains a high amount of quercetin-3-O-rhamnoside (quercitrin) that has exhibited a significant activity toward Alzheimer's disease, specifically with LXR receptors. In this work, a methodology was validated following the specifications of the International Conference of Harmonization in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), repeatability (intra-assay), intermediate precision (intra-laboratory), reproducibility (inter-laboratory), robustness, and specificity. The effect of location (Oiba, Granada, and Chiquinquira) and the extraction method (percolation, maceration, and ultrasound-assisted extraction) towards the chromatographic profile and quercitrin recovery was studied. Furthermore, a Box-Behnken design was conducted to optimize quercitrin extraction and extraction yield by ultrasonic-assisted extraction. The chromatographic method was validated, with a linear range from 5 to 180 mg quercitrin per L, LOD 0.26 mg L-1, and LOQ 0.86 mg L-1. Accuracy [recovery of 93.8% (w/w)], repeatability (relative standard deviation, RSD, 3.3%), intermediate precision (RSD 5.4%), and reproducibility (RSD 1.4%) were within the acceptable values. The method was robust and specific, except for the variation in the formic acid concentration. The location had a greater influence than the extraction method towards both the chromatographic profile and quercitrin recovery. Quercitrin extraction was maximized at 60% (v/v) ethanol and 50 °C, independent of the solvent : material ratio used. The highest yield values were achieved at 60% (v/v) ethanol and 50 °C, with a solvent : material ratio of 40 mL g-1.

ISOLATION, DEVELOPMENT AND VALIDATION OF QUERCETIN FROM THE ALLIUM ASCALONICUM’S TUNIC BY HPLC METHOD

Allium ascalonicum, the tunic of which holds various bioactive compounds including including quercetin - a potent flavonoid, is renowned for its medicinal properties. This study presents the development and validation of a High-Performance Liquid Chromatography (HPLC) method for quantifying quercetin in Allium ascalonicum tunic. Validation of the method was conducted following International Conference on Harmonization (ICH) guidelines, evaluating parameters such as linearity, precision, and accuracy. This validated HPLC method offers an efficient and reliable means of quantifying quercetin in Allium ascalonicum tunic. Its application holds promise for routine analysis in pharmaceutical and nutraceutical industries, offering insights into the medicinal potential of Allium ascalonicum peels as a natural source of quercetin.

Stability assessment and content determination test of oral liquid forms of beclomethasone dipropionate prepared in hospital pharmacy

ObjectivesOral beclomethasone dipropionate (BDP) is known for its use as a therapeutic medicine for gastrointestinal graft-versus-host disease (GI-GVHD). Despite growing demand for oral BDP formulation, no commercial forms have yet...

A Review on Method Development of High Performance Liquid Chromatography (HPLC) and its Applications

The most common method for identifying, separating, and measuring the medication is HPLC.To improve the approach, a variety of chromatographic factors including sample pretreatment, mobile phase selection, column selection, and detector selection were examined. Reviewing the technique creation, optimization, and validation is the article's main goal. The chemistry of the molecules, the synthetic pathway, solubility, polarity, pH and pKa values, and the activity of the functional groups are all factors that affect the creation of an HPLC technique. Information on several stages and properties including accuracy, specificity, linearity, limit of detection, and limit of quantification are provided through the validation of HPLC methods in accordance with ICH Guidelines1.

Validation of a HPLC method for the determination of diclofenac diethylamine and three of its impurities in a gel pharmaceutic form.

Monitoring impurities in drug products is a principal requirement of pharmaceutical regulatory authorities all over the world to ensure drug safety. For this reason, there is a great need of analytical quality control of dugs products. In this study, a simple, efficient and direct high performance liquid chromatography (HPLC) method was developed for the determination of three impurities of diclofenac. The HPLC method was developed using the mobile phase which consisted of HPLC grade mixture (Acetonitrile and 0.01M phosphoric acid adjusted to pH = 2.3) in the ratio of (25:75 v/v) respectively. The separation was performed in 15 minutes. The calibrations curve of the three impurities were linear; the correlation coefficient was 0.999 at a concentration of (0.00015 - 0.003 µg/mL). The validation of this method shows that this method meets all validation criteria. This shows the reliability of the use of this method for the routine control of diclofenac impurities. The validation of a robust HPLC method for the determination of diclofenac impurities is of great importance for the pharmaceutical industry to control its products.

Advancing posaconazole quantification analysis with a new reverse-phase HPLC method in its bulk and marketed dosage form.

Introduction: Posaconazole is a widely used antifungal drug, and its accurate quantification is essential for quality control and assessment of its pharmaceutical products. This study aimed to develop and validate a reverse-phase high-performance liquid chromatography (HPLC) analytical method for quantifying Posaconazole in bulk and dosage form. Methods: The HPLC method was developed and validated based on International Conference on Harmonisation (ICH) guidelines. The developed method was then applied to quantify Posaconazole in a marketed tablet formulation. The method's specificity, linearity, precision, accuracy, robustness, and stability were evaluated. Results: The developed HPLC method showed good linearity over a 2-20 μg/mL concentration range. The percentage recovery of Posaconazole from the bulk and marketed formulations was found to be 99.01% and 99.05%, respectively. The intra-day and inter-day precisions were less than 1%, and the method was stable under different conditions. The HPLC method was successfully applied to quantify Posaconazole in the marketed formulation. Conclusion: The developed and validated HPLC method is reliable and efficient for analyzing Posaconazole in bulk and dosage forms. The method's accuracy, precision, specificity, linearity, robustness, and stability demonstrate its effectiveness. The method can be used for the quality control and assessment of Posaconazole-containing pharmaceutical products.

Discrimination of BaphicacanthisCusiae Rhizoma et radix and its adulterant species and establishment of an assay method for quality control

BackgroundBaphicacanthisCusiae Rhizoma et Radix, commonly known as Nan-Ban-Lan-Gen (NBLG), is an essential traditional Chinese medicine that possesses diverse bioactivities, particularly noteworthy for its antiviral properties. Although NBLG has been listed in the Chinese Pharmacopoeia as an independent Chinese medicine, the establishment of a comprehensive quality standard for NBLG remains elusive. The absence of assay for marker compound in its quality standards has led to the lack of corresponding quality control measures for NBLG-containing preparations, and its discrimination from adulterant species in the market which thereby can significantly impact the efficacy and safety of NBLG-containing products.MethodsUltra-high performance liquid chromatography (UHPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was employed for comprehensive profiling of the chemical constituents of NBLG, the stem of Baphicacanthus cusia (Nees) Bremek (NBLJ), and the roots of Isatis indigotica Fort. (Bei-Ban-Lan-Gen, BBLG). Additionally, multivariate analysis was conducted to compare the chemical components of NBLG with those of NBLJ and BBLG. Furthermore, we established an optimized and validated HPLC method to obtain the fingerprint of NBLG and quantify the content of 2-benzoxazolinone and acteoside in the samples.ResultsA total of 73 compounds belonging to six classes were assigned in NBLG, with alkaloids being the most abundant and diverse species. High compositional similarities with significant differences in content were observed between NBLG and NBLJ. Moreover, the chemical profile of BBLG markedly differed from that of NBLG. An informative high performance liquid chromatography (HPLC) fingerprint of NBLG comprising seven characteristic peaks that can be used for quality assessment was established. Notably, we propose a quality control standard for NBLG, stipulating that the limit of content in dry weight for both 2-benzoxazolinone and acteoside should not be less than 0.010%.ConclusionThis study provides the most comprehensive chemical information to date on NBLG, offering valuable insights into its authentication and quality control. Our findings highlight the importance of comprehensive chemical profiling to differentiate potential substitutions and adulterations of herbal medicines, particularly when the original source is scarce or unavailable. These results can aid in the development of quality control measures for NBLG-containing preparations, ensuring their safety and efficacy.

Cytotoxicity Activity of Graviola Fruit Extract with Carbamazepine and Valproic Acid Show Antagonistic and Indifferent Effects.

Graviola is a tropical fruit with medicinal properties, used for treating various diseases such as inflammation, diabetes, and cancer. Histone deacetylase inhibitors (HDACIs), including carbamazepine (CBZ) and valproic acid (VPA), have been proven strong inhibitors against cancer cell growth. This study investigated the effect of Graviola fruit extract (GFE) on CBZ in healthy rat plasma using high-performance liquid chromatography (HPLC). In addition, the effect of GFE in combination with CBZ and VPA on two human cancer cell lines (PC3 and MCF-7) was explored. The CBZ levels were analyzed using a simple validated HPLC method. The linearity was achieved at a 0.9998 coefficient of determination over a range of 75-5000 ng/mL CBZ. The MTT assay was used to quantify the percentage of viable cells. The maximum plasma concentration (Cmax) and area under the curve (AUC) for CBZ alone were 4,631 ng/mL and 49,225 ng. h/mL, respectively. However, in the presence of GFE, the values reduced significantly to 2,994 ng/mL and 26,587 ng. h/mL, while the p-value was <0.05. The 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay results for VPA showed a weak cytotoxicity activity on PC3 and MCF-7 cell lines. A simple and validated HPLC method was used to determine CBZ levels in rats' plasma. The plasma CBZ levels (Cmax) were significantly reduced in the presence of GFE, indicating the importance of drug-herb interactions. For in vitro studies, two human cancer cell lines, MCF-7 (breast cancer cells) and PC3 (prostate cancer cells), were used to screen the cytotoxicity activity of GFE, CBZ, and VPA. We observed an antagonism effect for GFE and CBZ combination in both cell lines with FIC values > 4. On the contrary, the combination of GFE and VPA showed an additive or indifferent effect.

Advancing posaconazole quantification analysis with a new reverse-phase HPLC method in its bulk and marketed dosage form

Introduction: Posaconazole is a widely used antifungal drug, and its accurate quantification is essential for quality control and assessment of its pharmaceutical products. This study aimed to develop and validate a reverse-phase high-performance liquid chromatography (HPLC) analytical method for quantifying Posaconazole in bulk and dosage form. Methods: The HPLC method was developed and validated based on International Conference on Harmonisation (ICH) guidelines. The developed method was then applied to quantify Posaconazole in a marketed tablet formulation. The method's specificity, linearity, precision, accuracy, robustness, and stability were evaluated. Results: The developed HPLC method showed good linearity over a 2-20 μg/mL concentration range. The percentage recovery of Posaconazole from the bulk and marketed formulations was found to be 99.01% and 99.05%, respectively. The intra-day and inter-day precisions were less than 1%, and the method was stable under different conditions. The HPLC method was successfully applied to quantify Posaconazole in the marketed formulation. Conclusion: The developed and validated HPLC method is reliable and efficient for analyzing Posaconazole in bulk and dosage forms. The method's accuracy, precision, specificity, linearity, robustness, and stability demonstrate its effectiveness. The method can be used for the quality control and assessment of Posaconazole-containing pharmaceutical products.

Stability indicating HPLC method development and validation of Fostemsavir in bulk and marketed formulations by implementing QbD approach

Background: Achieving a predictable degree of quality with intended and planned specifications is known as "quality by design." QbD (Quality-by-Design) is an alternative to conventional method development that places more attention on identifying and mitigating potential risks. Component of the Quality-by-Design methodology involves conducting a series of experiments to learn how various factors, including the dependant variables, affect the answers of interest. Here, we use a QbD (Quality-by-Design) loom to detail the creation and verification of a stability-indicating high-performance liquid chromatography (HPLC) method for Fostemsavir in both bulk and finished-goods forms.&#x0D; Results:&#x0D; In this work, we present a workable experimental design for optimising the RP-HPLC separation technique by identifying the optimum mobile phase concentration and flow rate. Below are the ideal chromatographic conditions as calculated by Design Expert version 13.0: Mobile phase: 80 parts acetonitrile to 20 parts formic acid (v/v), flow rate: 0.8 millilitres per minute, retention time: 3.24 minutes, column dimensions: GIST C18 (250 mm × 4.6 mm × 5.0 μm).&#x0D; Here we propose a practical experimental layout for determining the optimal mobile phase concentration and flow rate for the RP-HPLC separation technique. Using Design Expert version 13.0, the optimum chromatographic conditions were determined to be as follows: Shim-pack GIST C18 (250 mm 4.6 mm, 5.0 μ), mobile phase acetonitrile to 1% formic acid (80:20, v/v), flow rate 0.8 ml/min, and retention period 3.24 min.&#x0D; At a detection wavelength of 266 nm, it was discovered that the devised technique was linear over a concentration range of 50-90 μg/ml (r2 = 0.997). Test parameters for the system's appropriateness were determined to be 1.124 for the tailing factor and 9480 for the theoretical plates. Intraday RSD was found to range from 0.70 to 0.94, whereas interday RSD was found to range from 0.55 to 0.95 percent. Values for robustness were under 2%. The solution stability % RSD was calculated to be 0.83. The result of the assay was 100.05 percent. The created methodologies were used to studies of forced degradation, and the stressed materials were analysed. The parameters used to validate the procedure fell within the acceptable range recommended by ICH.&#x0D; Conclusion: Using Design Expert 13.0, we created a central composite design experiment that illustrates the relationships between mobile phase and flow rate across three levels, with retention duration, tailing factor, as well as theoretical plates as the responses of interest. By this work, we gain insight into the variables that affect chromatographic separation and strengthen our conviction that the HPLC method we've devised will serve our needs. Quantitative method development was applied to improve comprehension of multi-tiered method variables.

DEVELOPMENT AND VALIDATION OF HPLC METHOD ALONG WITH ANTI-AGING ACTIVITY FOR XIMENYNIC ACID IN SANTALUM ALBUM LINN.

A new analytical approach and validation study were developed to assess the quantity of ximenynic acid (XMA) in Santalum album Linn. extract. High-performance liquid chromatography (HPLC) in reverse phase is the technique that is used in this procedure. The anti-aging properties of the medicine were tested using enzymes that inhibit hyaluronidase and elastase, respectively, in order to gather information and findings. Methanol and water were used as the mobile phase for the analysis, which was carried out using a ZORBAX SB-C18 column with a size 5 µ (4.6 x 250 mm) column size. During the course of this HPLC method validation, a number of different tests that were pertinent to specificity, linearity, accuracy, precision and robustness were carried out. The technique was tested to see whether or not it would be able to fulfill the prerequisites set out by the International Conference on Harmonization (ICH). Research conducted on anti-aging found that XMA was much more efficient than catechin at decreasing the activities of elastase and hyaluronidase. Because of its use, speed and reliability, the HPLC method that was described has the potential to be effectively utilized in industry for the standardization of herbs and phytomedicines. Because XMA has the potential to be a breakthrough anti-aging treatment, the pharmaceutical and cosmetics industries may benefit from its development.