Reversed-phase High-performance Liquid Chromatographic Method Research Articles

Development and Validation of RP-HPLC method for the estimation of Plecanatide from Bulk and Pharmaceutical Dosage form

A novel, simple, efficient, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the estimation of Plecanatide in the bulk and pharmaceutical dosage form. The currently developed method was subsequently validated according to ICH guidelines in terms of linearity, accuracy, precision, the limit of detection, the limit of quantification, robustness, etc. The separation of the selected drugs was optimized after several trials including change of mobile phase and its composition, stationary phase, flow rate, column temperature, etc. The separation was performed by using an Develosil ODS HG-5 and UV absorption was measured at 246 nm. Ortho phosphoric acid, Triethylamine buffer : Methanol [520 : 480] was selected as the mobile phase at a flow rate of 1 mL/min. As per International Conference on Harmonization (ICH) Q2 R1 guidelines, several validation parameters were evaluated which include specificity, linearity, precision, accuracy, the limit of detection (LOD), and the limit of quantitation (LOQ). The acceptable degree of linearity range was found to be 10 to 60 µg/mL. The percent recovery was found to be 98.55 to 101.88%. Hence, the proposed method is simple, selective, and specifically meets the requirements of ICH guidelines for the validation of the analytical method.

Determination of Nifedipine by Validated RP-HPLC Method in Bulk and Pharmaceutical Dosage Form

The present paper deals with the development and validation of reverse phase HPLC method for the determination of Nifedipine on Nucleosil 100, 5 μm, C8, 250 x 4.0 mm column. A mobile phase consisting of 40 ml 2-propanol: 60 ml phosphoric acid 0.85% was employed in this study. The flow rate was kept at 0.8 ml/min and the injection volume was 10 µl. The separation was performed at 40°C. Eluents were monitored by UV detector set at 237 nm. The developed method was statistically validated for the linearity, precision, robustness, specificity and solution stability. The specificity of the method was ascertained by force degradation studies by acid and alkali hydrolysis, oxidation, heat and photo degradation. The degraded products were well resolved from the analyte peak with significant differences in their retention time values.

Development and Validation of an RP-HPLC Method for the Quantitative Analysis of Triclosan in Human Urine

Triclosan (TCS), a synthesized chlorinated phenolic compound, is commonly utilized in consumable products as an antimicrobial agent. TCS has sparked widespread awareness because of its toxicity and possible negative effect on public health in recent years. In this study, a highly sensitive, fast, and cost-effective isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method coupled with solid-phase extraction for analysis of triclosan in human urine samples was developed. The method utilized methanol and water in a ratio of 90 : 10 as the mobile phase on a Phenomenex Luna 3 µm C18(2) 100 Å, 150 × 4.60 mm stationary phase, with a runtime of 5 minutes. The method showed good resolution of triclosan in the presence of the sample matrix. Validation of the method was performed according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). Linearity was tested over a range of 0.00625 µg/mL to 6.4 µg/mL, as accuracy recorded a recovery of 89.25%, 91.0%, and 92.75%. Limits of detection (LOD) and quantification (LOQ) were obtained to be 0.0173 µg/mL and 0.0525 µg/mL, respectively. The method proved to be robust over a temperature range of 26°C, 30°C, and 35°C and a flow rate of 0.5 ml, 1.0 ml, and 1.5 ml. The developed method was employed to detect and quantify triclosan in 153 urine samples, comprising 60 samples from Ibadan, Nigeria, and 93 samples from Kumasi, Ghana. Triclosan was detected in a total of 52 samples with an average content of 0.054588 µg/ml. This method can therefore be used for the routine analysis of triclosan in urine samples.

Quercetin Bioavailability Evaluation on Standardized Herbal Medicine Containing Guava Leaf Extract with HPLC

Standardized herbal medicines are classified as one of Indonesia's natural medicine ingredients in addition to herbal medicine and phytopharmaceuticals. The safety and efficacy of standardized herbal medicines are scientifically proven through preclinical trials, and raw materials and products have been standardized. One includes standardized herbal medicines is diapet, psidii, lelap, fitolac, and glucogarp. To determine the bioavailability of standardized herbal products containing guava leaf extract (Psidium guajava L.). which uses single and combined natural materials. The method used is experimental with a crossover design. Blood samples are taken from the marginalis vein of the rabbit ear at 0.5 hours; 1; 2; 4; and 6. The level of quercetin in the blood is determined by the reverse-phase HPLC method. The mobile phase used is methanol:aquabidest (59:41,v/v), stationary phase octadecyl silica (C18), flow rate 1 mL/min, UV-Vis detector 370 nm, and injection volume 20 ?L. The value of bioavailability parameters obtained in the parameters Cpmax, Tmax, and AUC of product A is 1.486454 ?g / ml; 1.4 hours and 10.2615291 ?g/ml/h, product B is 1.29224019 ?g/ml; 1.5 hours and 11.30810501 ?g/ml/hour. Based on the results of this study, it can be concluded that the bioavailability profile of the two products is not much different, so it is expected that the effects caused are the same.

A Novel, Economical RP-HPLC Method for Quantification of Cefixime in Bulk and Pharmaceutical Solid Dosage form

A new, precise, economical, and linear gradient reverse phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for assay determination in Cefixime. The chromatographic separation was achieved with Hypersil C18(250*4.6)mmand 3 µm particle size column. The flow rate was 1.0 mL/min and eluents were detected at 220 nm using PDA detector. The retention time was found to be 7.3 min.The percentage recoveries for molecules were found to be in the range of 99–101%. The calibration curve demonstrated good linearity in the range of 20–100 gm/ml. for Cefixime. The approach has been validated in accordance with the International Conference on Harmonization's regulatory criteria. The evaluated parameters are precision, linearity, detection limit, quantification limit, specificity, accuracy, and robustness.The technique may be applied to stability investigations as well as routine analysis to identify and quantify Cefixime in pharmaceutical dosage form .
 Keywords: Cefixim. Reversed Phase High Performance Liquid Chromatography. Assay substances. Methanol

Identification and Structural Characterization of Forced Degradation Impurities of Sacubitril by HRMS – Development and Validation of a Stability indicating RP-HPLC method

The current work identifies and characterises the stress degradation products of the anti-hypertensive drug sacubitril. The stability profile of sacubitril was evaluated under several stress settings including hydrolytic, oxidative, thermal and photolytic conditions in accordance with ICH recommendations. The degradation products of sacubitril were separated using reverse phase liquid chromatography and structural characterization was carried out using HRMS. The chromatographic separation was carried out by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) using a Fortis C18 column (150 mm X 4.6 mm; 5μm particle size) with mobile phase A (consisting of 0.1% TFA in Milli-Q water) and mobile phase B (100% Acetonitrile) in gradient mode. With a sample injection volume of 10μL, the mobile phase flow rate was 0.7 mL/min. A quadrupole time-of-flight (Q-TOF) mass spectrometer was used for the LC-MS analysis. Sacubitril is relatively stable in oxidative, thermal and photolytic conditions; however, considerable degradation was observed in acid, base and neutral hydrolysis. Three degradation products were identified and the structural characterization of these impurities was performed using the HRMS data and mass fragmentation. The proposed RP-HPLC method was robust, precise and specific in determining the impurity profile of sacubitril under different stress conditions.

Method Development and Validation of Curcuminoids in Curcuma Angustifolia Roxb. By RP-HPLC and Its Antifungal Activity

Curcuma angustifolia Roxb., a rhizomatous herb, belongs to the Zingiberaceae family of plants. The species is rich innutrients and is used mostly as a source of starch for Indian dishes and medicines. The published research articles for themeasurable estimation of components in Curcuma angustifolia Roxb. rhizomes are very scarce. The present study aimed to developa quantitative analysis method for estimating the constituents in the methanolic extract of Curcuma angustifolia Roxb. rhizome andto investigate its biological activity. Within the context of this challenge, the current work is a unique effort that recommends ahigh-performance liquid chromatography (HPLC) method for estimating curcuminoids, namely curcumin, demethoxycurcumin,and bis-demethoxycurcumin, present in the methanolic extract of Curcuma angustifolia Roxb. rhizome. A reverse-phase HPLCmethod was developed to estimate curcuminoids in the extract. The method was validated and found to be precise, sensitive,specific, linear, accurate, and robust. The method validation study's findings demonstrated the method's suitability for routine use.The HPLC method was successfully applied to a marketed formulation containing rhizome powder from Curcuma angustifolia Roxb.as one of its constituents. This study investigates the antifungal activity of the methanolic extract in vitro using an agar well diffusionassay method. A minimum inhibitory concentration (MIC) was established, and the antifungal activity was evaluated against Candidaalbicans and Candida glabrata with fluconazole as the standard reference. The development and validation of an HPLC method fortesting curcuminoids in Curcuma angustifolia Roxb. rhizome and the evaluation of rhizome extract antifungal activity are excellentexamples of herbal medicine quality control in its entirety. The in vitro antifungal activity results reaffirm the usage of Curcumaangustifolia Roxb. as a source of antifungal agents. They can lead to the developing of new herbal medicines in the coming days.

Poly (lactic-co-glycolic acid) nanoparticles for drug delivery of rupatadine fumarate: development and evaluation

This study aimed to consolidate rupatadine fumarate (RF) into nanoparticles to control its release. Ten RF nanoparticles were developed by nanoprecipitation using poly (lactic-co-glycolic acid) (PLGA) , polyvinyl alcohol (PVA), and Poloxamer 407 (Kolliphor P 407) in different percentages. A valid reverse-phase HPLC method was developed to assess RF in the formulated nanoparticles. The RF nanoparticles were tested chemically and morphologically. RF nanoparticles containing PLGA and PVA and Kolliphor P 407 have zeta potentials ranging from -24.4 mV±0.24 to -26.7 mV±0.05, higher than other formulations, and their release profiles were optimised. The formula (RPX3) had the best zeta potential (-26.7 mV±0.05), released about 86% of RF after 8 h and extended for 24 h. In summary, the formulation (RPX3), including 2:10:3:1.5 ratios of the drug PLGA: PVA: Kolliphor P 407 was the optimised RF-loaded nanoparticles formulation.

VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF GLECAPREVIR AND PIBRENTASVIR BY USING ANALYTICAL QUALITY BY DESIGN (AQBD) METHOD

The quantitative measurement of Glecaprevir and Pibrentasvir has been created using a simple, quick, precise, sensitive, and reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method. It is more difficult to analyse varying amounts of pharmaceutical active medicinal ingredients in dosage forms without interferences. Therefore, the objective of the current work is to estimate Glecaprevir and Pibrentasvir simultaneously by adopting an Analytical Quality by Design (AQbD), a rotatable central composite-based technique using RP-HPLC-based method development and validation. Glecaprevir and Pibrentasvir were separated by chromatography using a Kinetex RP-18 Column (100x4.6mm, 2.6µ) column and a mobile phase made up of Acetonitrile: 0.1% tri fluoro acetic acid in a ratio of 26.464:73.536 v/v. The flow rate was 1.0 ml/min, and a photodiode array detector operating at room temperature was used to detect absorption at 236 nm. ICH criteria have been used to validate the offered techniques' linearity, accuracy, precision, and other attributes. The degradation study's findings showed that the medications deteriorated in high-stress situations. The chemical and pharmaceutical sectors might easily implement this unique AQbD-based analytical technique for routine analysis without any regulatory constraints.

Computational quality-by-design strategy to validate high-performance liquid chromatography method for the estimation of meloxicam in bulk dosage forms and milk samples.

Bovine clinical mastitis has significant repercussions for farmers across the globe. Meloxicam, a COX-2 inhibitor, attenuates mastitis symptoms and is also approved for veterinary use. An RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) method development and validation is essential in the pharmaceutical industry to assess API (Active Pharmaceutical Ingredient) quantity present in the pharmaceutical dosage forms. RP-HPLC method of meloxicam was developed and optimized with the aid of QbD (Quality by Design) using Box-Behnken design (BBD). The pH of the aqueous mobile phase, acetonitrile (ACN) percentage, and column temperature were chosen as influence variables, and retention time (RT) and tailing factor (Tf) were selected as response variables. The optimum experimental conditions were selected as pH ~ 3 of the aqueous mobile phase, 65% v/v ACN, and 30˚C as column temperature. The drug was eluted at 6.02min RT with 1.18 as Tf. The method was subjected to validation for accuracy, linearity, precision, range, sensitivity, and robustness and was found to comply with ICH Q2 (R1) guidelines. The in vitro bioequivalent studies were performed in hydrochloric acid, pH ~ 1.2; acetate buffer, pH ~ 4.5; and phosphate buffer, pH ~ 6.8 for two veterinary brands of meloxicam tablets, and their release profiles were compared by mathematical models. Both the brands, brand 1 and 2 exhibited significant (Unpaired t-test, P < 0.05) differences in dissolution efficiency (DE) and mean dissolution time (MDT) except DE at pH 1.2. However, brands 1 and 2 showed similarity (f2 > 50) in terms of release of meloxicam except at pH 6.8 (f2 = 47.01). The in vitro release of meloxicam followed Peppas kinetics except for brand 2 at pH 6.8, where it followed the Higuchi model. Moreover, the recovery of meloxicam extracted with ACN in the milk sample was estimated to be 99.67 ± 0.58% significantly (Unpaired t-test, P < 0.05) higher than 90.34 ± 6.77% extracted with methanol. In conclusion, the optimized and validated RP-HPLC method of meloxicam may further be used for the analysis of drug content in pharmaceutical dosage forms in addition to biological fluids.

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

Background: 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. Methods: 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 C18 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. Results: NTB was eluted at 6.77±0.00 min of retention time (tR) 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. Conclusion: 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.

Validation and development of RP-HPLC method for quantification of glibenclamide in rat plasma and its application to pharmacokinetic studies in wistar rats

Herein, we report a novel, simple, specific, accurate and cost-friendly validated reverse phase-high performance liquid chromatographic (RP-HPLC) method for the quantification of second generation sulphonylurea based antidiabetic drug, glibenclamide (GLB) in rat plasma and its application to calculate pharmacokinetic parameters in wistar rats. The internal standard used was flufenamic acid. The chromatographic separation was conducted on C18 column (250 mm × 4.6 mm x 5 μm, Agilent-Zorbax, SB) using isocratic elution with mobile phase containing Acetonitrile: Water (1:1; v/v) pH adjusted to 4.0 with 0.03 % glacial acetic acid and detected by photo-diode array as detector. Calibration curves made in the rat plasma were linear in the range of 50–1200 ng/ml with r2 = 0.998. The LLOQ was 40 ng/ml. This method was effectively applied for pharmacokinetic studies of Glibenclamide following administration through oral route as solid dispersion formulation to Wistar rats. Several methods are available in the literature which can be employed for the quantification of Glibenclamide but such methods are tedious, provide lower sensitivity, less simultaneous resolution and are time-consuming. Therefore the present methods suits best for the quantification of Glibenclamide from Wistar rats.

Reversed phase HPLC method development and validation for the analysis of amlodipine besylate in tablets dosage form and human plasma

Introduction: Several analytical methods for quantitative determination of amlodipine either in pharmaceuticals or biological fluids have suffered different setbacks. Therefore, we aimed at developing a cost effective, simple, robust, and sensitive reversed phase high performance liquid chromatography (RP-HPLC- UV) method for quantification of amlodipine in dosage form and in human plasma. Method: The separation was performed on an analytical column 150 mm x 4.6 mm i.d., Agilent Eclipse XDB C18 Column packed with 5 μm particle size and protected by a guard column (30 mm x 4.6 mm i.d.). The mobile phase composition contained acetonitrile: phosphate buffer (25 mM KH2PO4) adjusted to pH 3.1 with orthophosphoric acid in the ratio of 45:55 v/v. The flow rate was 1.2 mL/min and the UV detector was set at a wavelength of 240 nm. The column was maintained at ambient temperature. Results: The calibration curve gave good linearity in the concentration range studied. The column efficiency was highly evident by high theoretical plates and small height equivalent to theoretical plate (HETP). The percent relative standard deviation (% RSD) was less than 3% in the inter- and intra-day o assay. Amlodipine, 50 ng/mL in plasma stored frozen at -80 C was stable There was no statistical difference in the precision of amlodipine assayed after 12 months when compared to the initial assay (P&gt;0.05). The % recovery of amlodipine in human plasma was 97.79±0.17% to 99.52±0.63%. Conclusion: The new RP-HPLC method is sensitive, reproducible, precise, accurate and robust. We conclude that the method is suitable for estimation of amlodipine besylate in human plasma for pharmacokinetic studies and assay of pharmaceutical dosage form

RP-HPLC Method Development, Validation Including Stability Indicating and Forced Degradation Studies for the Estimation of Pemetrexed in API and Pharmaceutical Dosage Form

Stability indicating analytical RP-HPLC (Reverse Phase- High performance Liquid Chromatography) method was developed for the analysis of the drug in the presence of its degradation products and subsequent validation of Pemetrexed that was conducted in API and pharmaceutical dosage form. Pemetrexed was subjected to different stress conditions as per International Conference on Harmonization (ICH) guidelines, stress conditions applied were including the effect of acid, base, oxidative, hydrolysis, thermal and photolytic degradation conditions. Chromatographic separation of API (Active Pharmaceutical ingredient) was achieved on Water’s HPLC instrument and ODS C18, 250x4.6 mm, 5µm particle size column with gradient elution of 0.1% ortho phosphoric acid buffer: Acetonitrile 80:20 ratio was taken as the eluent (mobile phase). The buffer pH was maintained at 3, the flow rate was 1.0 ml/min and wavelength were detected at 225 nm. The Retention time (RT) of Pemetrexed was Found at 2.9 minutes, column temperature was kept ambient and the runtime was 6 min. System suitability parameters like theoretical plates, tailing factor, and Asymmetry values were showed at 3391, 1.41, and 1.101 respectively. The percentage drug purity was found to be 99.99%. Validation of the method was proved that linear relationship over the range of 25-150 µg/mL, with linear regression curve (correlation coefficient) r2 value was noticed at 0.999 and LOD (limit of detection), LOQ (limit of quantification) values was established at 0.33µg/mL, 0.99µg/mL respectively. Recovery studies carried out at the concentrations of 50%, 100% and 150% with percentage recovery values of 100.06, 99.8 and 99.87 respectively. The precision of intraday was 0.64% and inter-day was 0.95%. Robustness of flow rate and mobile phase was changed even though the method was Robust. Forced degradation studies were also conducted to check the stability and suitability of this method to resolve the degradation products. Method validation was proved to be accurate, precise, linear, repeatable and robust. Thus, the accepted method can be proceeded for the estimation of Pemetrexed in pharmaceutical dosage form in a daily basis, due to their simplicity, suitability, accuracy, robustness and reproducibility.

A QbD Based RP-HPLC Method for Stability Indicating Impurity Profiling of Pyridoxine: Method Development, Validation, and Application

Pyridoxine impurity profiling in bulk and formulations was devised and validated using a reversed-phase high-performance liquid chromatography (RP-HPLC) strategy. The technique was fine-tuned using an Analytical quality by design (QbD) approach, ensuring its dependability and sturdiness. Linearity, accuracy, and precision were carefully examined as key performance indicators. With a relative standard deviation (RSD) &lt; 2%, the approach showed exceptional precision, excellent recovery rates (100 and, 101.2%), and a strong correlation value (R2 = 0.9990). The technique has been used successfully for impurity profiling, and because it indicates stability, it can be used for long-term stability studies. The work contributes to the body of knowledge and has applications for pharmaceutical quality assurance.

Application of Quality by Design in RP-HPLC for Robust Impurity Profiling and Stability Assessment of Doxylamine

The primary purpose of this research was to create and validate a doxylamine detection method using reverse phase high performance liquid chromatography (RP-HPLC) for use in pharmaceutical formulations and bulk materials. Quality by design (QbD) served as the inspiration for this approach. The method was developed by running a C18 column at 1-mL/min through a mobile phase of 60 parts methanol to 40 parts 0.1% OPA water (pH 2.8). The detection was done with a UV detector set to 259 nm. All requirements for system applicability were satisfied by the suggested approach, including an acceptable asymmetry factor and an adequate number of theoretical plates. A correlation coefficient (R2) of 0.9990 was used to confirm linearity over a concentration of 10–50 g/mL. The approach showed good accuracy by a mean %recovery ranging from 99.59–101.45% and a %RSD between 0.11 and 0.81. Precision tests conducted both intraday and across days showed that the medication content stayed within allowable bounds. The approach was found to be robust, with only minor variations in flow rate, mobile phase composition, and detecting wavelength significantly affecting the accuracy and specificity. A 0.71 g/mL LoQ and a 0.23 g/mL LoD were determined to be analytical limits of detection and quantification, respectively. This study substantiates the development of a reliable, long-lasting, and cost-effective QbD-based RP-HPLC method suitable for the comprehensive analysis of doxylamine in tablet formulations and bulk dosage

Development of a Simple Accurate Method, Validation and It’s Degradation Studies of Nirmatrelvir, Ritonavir in Bulk and Marketed Formulation by RP-HPLC

An easy, accurate, and precise method used for simultaneous quantification of nirmatrelvir, ritonavir in bulk and marketed formulation by reverse phase high performance liquid chromatography (RP-HPLC) using a standard column Inertsil ODS (150 x 4.6 mm, 5 μm ) at a rate of flow 1-mL/min, acetonitrile and buffer containing hexane sulphonic acid, 50:50 v/v as a mobile phase was introduced through the HPLC column, detected at a wavelength 258 nm. Nirmatrelvir and ritonavir retention times were 2.481 and, 3.873 minutes, respectively. %Recovery rates for nirmatrelvir and ritonavir were 100 and 100.3%, respectively. LoD and LoQ values for nirmatrelvir and ritonavir were 1.5, 1 and 4.5, 3 g/mL, respectively. Nirmatrelvir regression equation is y = 32885.25x + 4223.04 while ritonavir is y = 39086.65x + 1680.21. Stability indicating studies done by acid, alkali, peroxide, Reduction, thermal, neutral and ultra violet light. The newly created reverse phase HPLC method, for bulk and marketed formulation, was rapid, stability indicating and accurate

Understanding the Stress Testing Characteristics of Apixaban, Structural Elucidation of a Novel Degradation Impurity, and Stability-Indicating Method Development for Quantification of Related Substances.

People who have non-valvular atrial fibrillation may benefit from taking a new oral anticoagulant called apixaban, which has recently been given the green light by the U.S. Food and Drug Administration. During stress testing, apixaban was found to have a high degree of degradability when subjected to both acidic and basic conditions, and one significant unknown impurity was observed in addition to the major known impurities. Our aim is the isolation and characterization of degradation product observed in stress/forced degradation studies, and also the development of a single HPLC method that is both reliable and accurate for quantifying all 10 related impurities of apixaban. Preparative HPLC was used to isolate the degradation product, and 1H NMR, 13C NMR, and MS were used to elucidate the structure of the product. Additionally, a single reverse-phase (RP) HPLC method was developed for quantification of all related impurities of apixaban. Based on the spectral characterization data, the identified unknown degradation impurity was found to be a pH-independent hydrolysis degradation impurity of apixaban. The developed method is specific, linear, accurate, robust, and rugged. The isolated and characterized impurities were the same as those found during stress testing. The developed method has been validated for its intended purpose in accordance with the regulatory requirements that were outlined. The unknown impurity is a new apixaban degradation impurity that helps us understand its toxicity. The scientific community will benefit from the developed analytical method information as it relates to understanding drug product impurity profiling.

DEVELOPMENT VALIDATION OF QUERCETIN COMPOUNDS USING RP-HPLC AND IN VITRO ACTIVITY STUDIES ON MELASTOMA MALABATHRICUM LEAF NANOCREAM FOUNDATION PREPARATIONS

Objective: A simple, accurate, precise, sensitive, and robust analytical method has been developed to analyze quercetin in the nano-cream foundation formula added to the ethyl acetate fraction of Melastoma malabathricum leaves. Methods: The validation is consistent with ICH guidelines using a reversed-phase HPLC method with mobile-phase acetonitrile-phosphoric buffer conditions at pH 5.51 (40:60 v/v). The stationary phase has a C-18 column, a 0.5 ml/min flow rate, and an Ultraviolet detector at wavelength 370 nm. Furthermore, antioxidant activity testing used the FRAP method, and the SPF value was measured using a Elisa reader with wavelength 615nm and UV/Vis spectrophotometer in the wavelength range of 290-390 nm. Results: The results showed that the retention time of quercetin was 11.462 min with a tailing factor of 1.045. Regression equation y=170220x-85497 had a concentration range of 3-18 µg/ml and a correlation value of 0.9998. The limit of Detection (LOD) and Limit of Quantification (LOQ) were 0.236 and 0.786 µg/ml, respectively. The accuracy method for the recovery value at concentration levels of 80%, 100%, and 120% was 95.233%-109.446%. The repeatability of intra-day and inter-day variations express the %RSD values of 1.258-1.378% and 1.012-1.258%. Meanwhile, the method's strength by changing the column temperature, maximum wavelength, mobile phase composition, and the flow rate was expressed by the %RSD value of 0.045-0.067%, 0.069-1.642%, 0.107-0.165%, and 0.141-0.318%, respectively. The analysis results of the quercetin content in the nano cream foundation formula were 0.021728±5.83x10-4 mg/g. Antioxidant activity with an IC50 value was 1444.157±15.538 µg/ml, and the SPF at a concentration of 8000 µg/ml was 11.370±1.083. Conclusion: The development method of quercetin is simple, accurate, precise, sensitive, and robust. Quantitative analysis of quercetin biomarkers in nano cream preparations containing the ethyl acetate fraction of Melastoma malabathricum leaves using the RP-HPLC method.

Development and Validation of a Method for Trace Level Zinc Quantification in Pharmaceutical Zinc Supplements Using a Carboxyl Functional Group Packed Column and Refractive Index Detector.

Zinc helps with cell division, growth, wound healing, and carbohydrate breakdown. Humanbeings have to obtain zinc from food or supplements because our bodies do not produce it naturally. In view of the greater advantages (such as low cost, time of analysis, and stability-indicating) compared to other quantification methods (titration, ion chromatography, Atomic absorption spectroscopy) proposed in the literature, a refractive index detector coupled with HPLC has been used in quantification of zinc. The goal of this research is to develop and validate a sensitive, low-cost, high-resolution, and stability- indicating method for detecting and quantifying zinc levels in zinc supplement pharmaceutical products (injectables, tablets, and capsules). A novel isocratic reverse-phase HPLC method with a refractive index detector (with sensitivity 64, detector temperature 35°C, and positive polarity) was developed using a carboxyl functional group packed column and 0.8% (v/v) formic acid as the mobile phase to detect and quantify zinc content. The reported method has a good optimal sensitivity (LOQ: 0.006 mg/mL and LOD: 0.002 mg/mL). The correlation coefficient (r) obtained from the zinc calibration plot was greater than 0.998, indicating that the method was linear and that there was a strong correlation between zinc concentration (0.006 mg/mL to 0.375 mg/mL) and peak response. The accuracy at LOQ level was found to be 95-105% and 97-103% at the remaining levels (50, 100, and 150%). The proposed method was successfully developed and validated as per International council for harmonisation (ICH) guidelines. Therefore, this method can be used for the quantitative testing of zinc in the QC laboratory. A novel method was developed for zinc levels determination in pharmaceutical products using HPLC with a refractive index detector. The present approach has a quick run time of 10 min and is inexpensive.