ICH Q2 Research Articles

Box-Behnken Design-Aided Validation and Optimization of a Stability-Indicating Reverse Phase-HPLC Method for the Estimation of Tamoxifen Citrate in Lipidic Nano-Vesicles.

Stability indicating a reverse-phase HPLC analytical method for the quantification of tamoxifen citrate (TMX) in the bulk and lipidic nano-vesicles (LNVs) was developed. The optimized method was validated according to the ICH Q2 (R1) guidelines by following a three-factor interaction Box-Behnken design using Design-Expert® software. The responses measured at 236nm were retention time (Rt), peak area, tailing factor (TF) and the number of theoretical plates. TMX was eluted best using the Luna® C18 LC Column along with a mobile phase of methanol (MeOH) and ammonium acetate buffer (AAB pH4.5) 80:20v/v mixture at 25 ± 2°C temperature. The currently developed method was linear in 100-5,000ng/mL range with a detection limit of 4.55ng/mL and a quantification limit of 13.78ng/mL. The optimized method was utilized to evaluate the stability of TMX in different stress conditions by performing forced degradation studies. The results from the degradation study stipulated that on exposure to various stressors namely acid, alkali, oxidative, thermal and UV light, the TMX did not show considerable degradation except for UV light exposure. Further, the method was successfully used for the quantification of TMX in LNVs.

HYDROLYTIC DEGRADATION STUDY OF ROXADUSTAT BY RP-HPLC AND HPTLC

Objective: Simple, rapid RP-HPLC and HPTLC methods have been developed in order to study the degradation of Roxadustat under various stress conditions. The Kinetics of hydrolytic degradation is studied. Methods: Optimum separation of Roxadustat and its degradation products was achieved using the following conditions in HPLC, Agilent eclipse XDB-C8 (150×4.6 mm) column, the mobile phase was composed of methanol: phosphate buffer (pH 5, 0.05 M) (70:30 v/v) with UV detection at 262 nm. The flow rate was at 1.0 ml/min. The RT was 4.6±0.02 min. HPTLC work for Roxadustat was performed on Aluminium plates precoated with silica gel 60 F254, (10 cm × 10 cm with 250 μm layer thickness). The mobile phase was composed of Toulene: Ethyl Acetate: Glacial acetic acid (5:5:0.5 v/v/v) and then scanned. The system was found to give a compact spot for Roxadustat (Rf value of 0.58±0.02). Results: In HPLC the calibration curves plotted were found to be linear over the concentration range of 2.5-25μg/ml, with a correlation coefficient of R2=0.9994. In HPTLC the calibration curves plotted were found to be linear over the concentration range of 500-2500 ng/band, with a regression coefficient of R2=0.9957. The analytical performance of the proposed methods was validated as per ICH Q2 (R1) guidelines. The degradant peaks were well resolved from the Roxadustat peak. Significant degradation was observed in acid hydrolysis, alkali hydrolysis, and oxidative degradation. The drug is relatively stable towards photolysis, neutral hydrolysis, and thermal conditions. Conclusion: In the current work, simple RP-HPLC and HPTLC analytical methods for the determination of Roxadustat in the presence of its degradation products have been developed. The information presented herein could be very useful while developing formulation procedures to prevent hydrolytic degradation. It can be used as a routine quality control test.

Interlaboratory Co-validation of a UPLC-ToF MS MAM Method for Truncations of a Fc Fusion Protein.

Peptide-Fc fusion proteins are inherently heterogeneous and complex molecules. Protein post-translational modifications (PTMs) or truncation can arise during manufacturing or product storage. Some of these product attributes could potentially impact the efficacy or safety of the bio-molecule and are thus classified as critical quality attributes (CQAs). These CQAs should be controlled in order to ensure manufacturing and quality consistency. A subunit UPLC-ToF MS based MAM method was developed for identity test and quantitatively monitored two critical quality attributes (CQAs) resulting from two truncations of that fusion protein (fragment 1 and 2). Three independent laboratories are involved in the method validation according to ICH Q2(R1), ICH Q6B, FDA and NMPA guidance. This developed method fully meets the pre-defined analytical target profile (ATP), including specificity, accuracy, precision, quantitation limit, linearity, range and robustness. Three independent labs co-validate a UPLC-ToF MS based MAM method for protein drug QC release and stability testing. The experimental design of method validation can be a reference for LC-HRMS-based subunit MAM methods that have been widely used in the characterization of antibodies, ADCs and other protein-based biologics. This work paves the way for implementing MAM in QC with more targeted control of product quality.

Development of a New, Fully Validated LC-MS/MS Method for the Analysis of Flibanserin in Pharmaceutical Preparations and Comparison of the Chromatographic Performance with Six Stationary Phase Types

Background: Initially synthesized as an antidepressant and potentially rapid onset of action, flibanserin (FLB) was approved by the Food and Drug Administration (FDA) in August 2015 with a warning to dispense the drug through a dedicated risk management program, despite the removal of HSDD from the DSM-5TM. The drug is the first noteworthy FDA-approved drug for treating premenopausal women with acquired, generalized HSDD. Objective: In literature, studies are plasma analyses or metabolite determinations to meet pharmacokinetic analyses and some analytical targets. For this reason, in this thesis, a new method has been developed for analysing FLB from pharmaceutical preparations, which is our target, providing all optimization conditions and method validity parameters. Methods: The chromatographic separation was also investigated using Chromolith® and Ascentis® Express models with a total of six stationary phases. The mobile phase mixture was acetonitrile:ammonium formate (0.020 M, pH 6.0) and was used at the ratio (60:40, v/v). The optimum column temperature was chosen as 40.0±0.1°C, and the autosampler thermostat temperature was chosen as 15±0.1°C. The sample injection volume is optimized to be 1 μL. Results: The developed method is linear in the range of 2.63–105.0 ng/mL, and the regression coefficient is 0.999 intraday and 0.986 interday. In the method, LOD and LOQ were obtained as 128 pg/mL and 384 pg/mL, respectively. In addition, the ANOVA P values were calculated as 0.586 and 0.914, respectively, in the validation studies conducted intraday and interday. Conclusion: FLB chromatographic behaviors were studied and compared in detail with six different stationary phases. The developed method was fully validated according to the ICH Q2 (R1) guideline, and its pseudo-pharmaceutical formulation was analyzed.

QUANTITATIVE ESTIMATION OF 6-GINGEROL, E-GUGGULSTERONE AND Z-GUGGULSTERONE IN A FIXED DOSE COMBINATION NANOEMULGEL BY RP-HPLC

Polyherbal formulations have proved to be efficacious for the therapeutic treatment of various diseases, However, the development of validated robust analytical methods for quantification is a major challenge. The aim of this project was to develop a simple analytical method for the quantification of 6-gingerol (6-GIN), E-guggulsterone (E-GGS) and Z-guggulsterone (Z-GGS) in nanoemulsion based gel using reverse phase high performance liquid chromatography (RP-HPLC). 6-GIN, E-GGS and Z-GGS were quantified using acetonitrile: water: methanol (70:20:10 V/V/V) as the mobile phase at 1.0 mL min-1 flow rate with photodiode array detection. The developed method was validated for linearity, accuracy, precision, specificity and robustness as per ICH Q2 (R1) guidelines. The drug content of the three actives in the developed nanoemulgel was found to be between 90% to 110% w/w. The developed analytical method is simple and can be used for quantification of 6-GIN, E-GGS and Z-GGS in fixed dose product containing these actives.

A Novel RP-Chiral Separation Technique for the Quantification of Brivaracetam and its process Related Isomeric Impurities using High Performance Liquid Chromatography

Reverse‐phase high‐performance liquid chromatography method has been developed for the determination of brivaracetam (BRV) with its stereoisomeric impurities (BRV-SS), (BRV-RR) and (BRV- RS). Brivaracetam with its impurities has been eluted with good resolution using the chiral column, chiralpakIG (250 × 4.6mm, 5µ) column at the detection wavelength of 210nm with the flow rate of 0.50ml/minute. The separation was achieved with the mobile phase consisted of methanol: acetonitrile and trifluoroacetic acid in the volume ratio of 90:10:0.1. The column temperature was maintained at 30°C and detection wavelength was maintained at 210 nm. Brivaracetum (BRV), and stereoisomeric impurities, BRV-SS, BRV-RR and BRV-RS, were eluted at 13.829, 11.810, 10.448 and 9.449 min, respectively. The method showed adequate specificity, sensitivity, linearity, accuracy, precision, and robustness inline to ICH tripartite guidelines. The limit of detections were 0.2 μg/mL for BRV-SS and BRV-RR, for BRV-RS it was found 0.1μg/mL.The limit of quantification limits were 0.5μg/mL for BRV-SS, BRV-RR and for BRV-RS as well.The developed method was found to be linear over the concentration range of 0.5-20µg/mL for BRV-RS, for BRV-RR and BRV-RS it was 0.5-3µg/mLfor stereoisomeric impurities with a correlation coefficient of 0.999. The developed method was found precise (%RSD = 0.5%, 0.6% and 0.5% for BRV-SS, BRV-RR and BRV-RS,) accurate (with 96%–107% recovery) and found robust. The validation parameters of the developed method were within the limits as per the regulation of ICH Q2(R1) guidelines. Therefore, in this present method a good separation was achieved specifically for the identification of brivaracetam and its processed related stereoisomeric impurities using high performance liquid chromatography and found suitable for the quantification of stereoisomeric impurities of brivaracetam in bulk scale as well.

Exploring fluorescence of metal nanoparticles for effective utility in drug sensing: A Promising ''on-off'' fluorescence probe for analysis of cephalosporins is fabricated

A promising fluorescent nano sensor was fabricated exploiting the unique optical and physicochemical properties of silver nanoparticles (AgNPs). AgNPs were prepared following a chemical reduction technique to get a highly water-soluble nano sensor, stable for at least 1 month without the need of organic stabilizers. Full characterization of AgNPs was done using different spectroscopic and microscopic techniques. They exhibit excellent water solubility, physicochemical and optical properties, enabling them to be successfully applied in chemical sensing of drugs. The prepared AgNPs could be conceived as a fluorescent probe for the fluorimetric determination of two commonly administered cephalosporins ceftriaxone (CTX) and cefepime (CFP) based on the quenching behavior of the fluorescence omitting the need for pre-derivatization or chromogenic reagents. The fluorescence intensity of AgNPs at 485 nm after excitation at 242 nm was quantitively quenched upon increasingly adding the studied drugs over the concentration ranges of 1–10 µg/mL and 0.9–9 µg/mL with detection limits of 0.178 µg/mL and 0.145 µg/mL for CTX and CFP, respectively. The quenching mechanisms were investigated and illustrated. The influence of different experimental parameters was studied and optimized. The suggested sensor provides an innovative, sensitive, and eco-friendly approach for the assay of the drugs in their pharmaceutical vials and quality control laboratories with excellent % recoveries of 99.88 ± 1.15, 99.95 ± 1.15 for CTX and CFP, respectively. The method was validated in accordance with ICH Q2 R1 recommendations. The greenness evaluation was performed through both Eco-Scale and GAPI revealing the green criteria of the developed method.

Plumbagin Extraction: A Green Study

A green study was undertaken for the extraction of plumbagin, an important natural naphthoquinone, from the roots of Plumbago pulchella Boiss. In the first stage, four solvents were comparatively evaluated, acetone, n-hexane, ethyl acetate, and methanol; attending Principle #5, only green solvents (ethyl acetate and acetone) were underscored. In a second step, considering Principle # 6, various green alternative activating modes were assessed: two electromagnetic methods, microwave (MW) and near-infrared (NIR), in addition to mechanical approaches, ultrasound (US), and tribochemistry-mechanochemistry (TB-MCh). These green modes were studied in comparison to the classical mantle heating (MH) and finally with maceration (M). MW and US-MH ensued the better modes, and EtOAc (green) and n-hexane (non-green) displayed the best yields (0.027% and 0.025%, respectively). Focusing on Principle #11, the monitoring and quantification of the extractions were performed by GC-MS employing 8-hydroxyquinoline as a reference. Some other involved Principles (#1, #3, #7, #12) will be commented on in the discussion. As an important complement, a statistical technique based on the analysis of variance (ANOVA) and response surface methodology was used to establish the degree of dependence of the amount of plumbagin extracted with the parameters time and amount of root, in addition to a GC-MS validation process carried out according to ICH Q2 R2 2022 guidelines. Finally, it is convenient to highlight that the target plant is native to Mexico, and according to a profound literature search, no chemical information for Plumbago pulchella Boiss is accessible.

A Novel Spectrophotometric Method Development for Quantification of Desidustat in Bulk and Pharmaceutical Dosage Form.

Desidustat (DES) belongs to a new category of drugs, i.e., Hypoxia-Inducible Factor (HIF) propyl hydroxylase inhibitor, and is used for the treatment of anemia in chronic kidney disease. However, no method has yet been reported in the literature for the estimation of drugs. The objective of the study is to develop a simple, precise, and accurate method for determining DES in bulk and pharmaceutical dose form. The physicochemical characterization of the drug was performed using methanol as a solvent to establish the identity. According to ICH Q2 criteria, validation characteristics, such as specificity, linearity, accuracy, precision, limits of detection and quantification, and robustness, were assessed. Maximum absorbance wavelength was observed at 229 nm. The sample solution remained stable for up to 12 hours. The linear response from 2 to 12 μg/ml of DES was y = 0.1087x + 0.0962 and r2 = 0.9963. The accuracy was between 100 to 101%. Precision was recorded under three criteria: repeatability, intraday and interday, for which results fell within the acceptable ranges (<2%). The limit of detection (LOD) and limit of quantification (LOQ) of the technique were 0.434 μg/ml and 1.316 μg/ml, respectively. The proposed method was found to be beneficial for drug monitoring and the ongoing analysis of DES in research and quality control laboratories. This approach is simple, precise, rapid, economical, and sensitive.

Analytical Method Development, Validation and Forced Degradation Study of Dapagliflozin by RP-HPLC.

Worldwide, it is projected that 285 million individuals have diabetes, and by 2030, this number is expected to climb to 438 million. About 90% of cases of diabetes mellitus are type 2 (T2DM). Insulin sensitizers, such as metformin and thiazolidinediones; insulin secretagogues, such as sulfonylureas and glinides; dipeptidyl peptidase 4 (DPP-4) inhibitors; glucosidase inhibitors, or oral combination therapy are currently available treatments for type 2 diabetes. Some of these drugs exhibit serious limitations; thus, it is crucial to design an innovative therapy that is efficient and depends on a new channel. In the current work, a stability-indicating reverse phase HPLC (RP-HPLC) technique was developed and subsequently validated for the detection of dapagliflozin in its API. The stability-indicating HPLC method for assay included the use of Kromasil 100-5-C8 (100 mm × 4.6 mm) column, UV detector 224 nm, mobile phase composition involving a mixture of acetonitrile:water (52:48), and a flow rate of 1.0 mL/min. ICH guidelines were followed for the method's validation. To assess the method's specificity and stability in showing characteristics, stress degradation studies were carried out. The working standard solution of dapagliflozin was exposed to 1 and 2 N HCl by refluxing 1 and 2 N NaOH with 30% hydrogen peroxide by volume and UV radiation in order to conduct a degradation study. All system suitability parameters were determined to be within the intended ranges, and the drug's retention duration was discovered to be 1.67 minutes. It was also investigated as to how the drug degraded under various circumstances. The drug was discovered to be stable under situations of photolytic, thermal, neutral, alkaline, and oxidative deterioration. The developed stabilityindicating HPLC technique was validated in accordance with ICH Q2 recommendations, and the validation parameters, such as linearity, precision, and robustness, were achieved within the approved standards. It may be concluded that this method is stability-indicating and specific, and it can be successfully applied to analyze tablet dosage forms containing dapagliflozin.

A stability indicating UPLC method development and validation for the simultaneous estimation of nateglinide and metformin hydrochloride in bulk and tablet dosage form

BackgroundNateglinide and metformin HCl are used in combination for the treatment of type 2 diabetes. A simple, sensitive and reliable UPLC method was developed for simultaneous estimation of nateglinide and metformin HCl using Phenomenox C18 (50*2.1 mm, 3.5 µm) column at ambient temperature as stationary phase in addition to mobile phase containing 75 volumes of ammonium formate buffer (pH = 3) along with 25 volumes of acetonitrile with a flow rate of 0.2 mL/min with UV detection at 260 nm and a run time of 3 min. The developed method was validated as per ICH Q2(R1) guidelines.ResultsThe separation of metformin HCl and nateglinide was done at retention times of 1.014 min and 1.435 min, respectively. The mean % recovery for nateglinide and metformin HCl in the accuracy study was observed to be 99.9% and 99.2%, respectively. LOD and LOQ values were determined considering the S/N ratio and were found to be 0.09 µg/mL and 0.3 µg/mL, respectively, for nateglinide and 0.75 µg/mL and 2.5 µg/mL, respectively, for metformin HCl. The method was found to be precise with % RSD values of 0.58 and 0.45, respectively, for repeatability and intermediate precision of nateglinide and 0.43 and 0.43, respectively, for repeatability and intermediate precision of metformin HCl which were within acceptance criteria. The method was found to be linear in the range of 7.5–45 µg/mL and 62.5–375 μg/mL for nateglinide and metformin HCl, respectively. The regression equations for nateglinide and metformin HCl were found to be y = 17377x + 6543.4 and y = 18439x + 43,537, respectively. The method was found to be robust by deliberate changes in the method parameters like flow rate and mobile phase composition. Forced degradation studies were performed as per ICH Q1A(R2) and Q1B guidelines, and peak purity was observed in all types of degradation studies for both the drugs.ConclusionThe developed method was found to be satisfactory as it is simple, sensitive, accurate, precise, robust, rapid, economical and yet stability indicating and can be applied successfully in the routine laboratory analysis for the simultaneous estimation of nateglinide and metformin HCl in the bulk and pharmaceutical dosage forms.

QbD Approach for Analysis of Tirzepatide in its Bulk and Marketed Formulation by Stability Indicating RP-HPLC

The core intentions of the stated work has been to create and validate a simple, sensitive, specific, precise and cost-effective RP-HPLC method with good performance for the investigation of tirzepatide in API powder and its marketed formulation. HPLC system (WATERS) equipped with DAD detection system was used to develop the current system. The procedure conditions of BDS C18 (150 x 4.6 mm,5 m), 0.01N KH2PO4: Acetonitrile in the ratio of 41:59 (% v/v), a flow of 0.9 mL/min, and a temperature of 31°C were successfully optimized by central composite design of QbD experiments. The optimized wavelength selected was 250 nm. RT of tirzepatide was observed to be 2.841 minutes with good system suitability. The ICH Q2(R1) standards functioned as a validation for the planned action strategy. Linearity was observed for 5 to 30 μg/mL concentration series of tirzepatide with R2 of 0.999. The %RSD results of both precisions were found in the range of 0.40 to 0.41.% recovery of Tirzepatide in spiked samples was assessed to be 99.89%. The LoD and LoQ of tirzepatide were calculated to be 0.05 and 0.14 μg/mL, respectively. The results assured that the established procedure was simple, sensitive, specific, accurate and costeffective. Exploration of tirzepatide under a diversity of FD conditions represents the stability representing the quality of the established HPLC procedure. Hence, the anticipated process has significant credit in the pharmaceutical segment.

Rheology of Complex Topical Formulations: An Analytical Quality by Design Approach to Method Optimization and Validation.

Analytical method validation ensures that a method provides trustworthy information about a particular sample when applied in accordance with the predefined protocol. According to regulatory standards, the rheological characteristics of topically applied semisolid formulations are one of the key elements involved in microstructure equivalence documentation. Therefore, for generic drug product manufacturers, it is a dire need to take a step forward in rheology method development and validation procedures. This paper aims to apply Analytical Quality by Design (AQbD) principles towards the development and validation of rheology methods for topical creams, as complex semisolid formulations. Risk assessment was carried out through an Ishikawa diagram and an estimate failure mode, effects, and criticality analysis (FMECA). Sample application, peltier temperature control, and sample rest time were identified as critical method variables (CMVs), and a 23 full factorial design was applied to understand their impact on rotational, creep recovery and, oscillatory measurements. The development of the method was carried out as per the ICH Q8-Q10, and Q14 guidelines and validated according to ICH Q2 (R2) guideline. The method demonstrated adequate precision (RSD < 15%), as well as selectivity. AQbD provided a comprehensive framework for developing a reliable and effective rheology method for this type of formulation.

Development and Validation of RP-HPLC Method for the Quantitative Analysis of Bempedoic acid in Bulk and Pharmaceutical Dosage form Using Surface Response Methodology

Objectives: A specific and novel RP-HPLC method has been optimized for the quantitative analysis of bempedoic acid using response surface methodology. Method: Response surface methodology (RSM) helps in studying the empirical relationship between one or more measured responses and many independent variables in the form of a polynomial equation. Mapping of those responses related to the experimental domain helps in generating an optimized method. In this present study, the empirical relationship between measured responses like % organic phase, column temperature, and flow rate and independent variables like retention time and theoretical plates are drawn in the form of a polynomial equation and an optimized method was developed from that using Box-Behnken Design (BBD). Results: The analytes chromatogram was run through SB C18 100x1.8mm, 2μm. The optimized data from Design Expert software consists of 0.01% OPA: Acetonitrile (56.05:43.95%, v/v) as mobile phase pumped through column with 1.02ml/min flow rate at 30.09°C gave highest desirable function of 1. The retention times of the drug were found to be 2.231 min. The optimized method was validated as per instructions given in ICH Q2 (R1) guidelines. Conclusion: Based on the results of the analysis of variance, the selected model for the responses like retention time and tailing factor were found to be significant with p=0.05. 2D contour plots were inured to visualize the effect of factors and their interactions on the response.

Quality-by-design driven analytical method (AQbD) development and validation of HPLC–UV technique to quantify rivastigmine hydrogen tartrate in lipidic nanocarriers: Forced degradation, and assessment of drug content and in vitro release studies

The main purpose of current investigation was to design and further validate a technique for the quantitative determination of rivastigmine hydrogen tartrate (RHT), a cholinesterase inhibitor in the nano formulations. With the employment of a reverse phase C18 column and a very simple mobile phase, an isocratic HPLC–UV analysis technique was developed, optimized, and validated following ICH Q2 (R1) requirements along with the inculcation ofAnalytical Quality-by-Design (AQbD) methodology. A two-level full factorial screening design was applied to filter out the variables influencing responses, followed by the application of central composite design (CCD) to further optimize the method using Design-Expert® software. Analyses were performed at room temperature with 0.9 mL. min−1 flow rate and detection wavelength of 215 nm. The optimized HPLC technique exhibited superior linearity, selectivity, and precision. The inherent stability of RHT was evaluated using the optimized method byconductingforced degradation studies. The RHT peak was clear and unaffected by matrix interference in the specimens from the forced degradation studies, drug content analysis, and in vitro release study. The assay had a limit of detection (LOD) of 14.32 ng. mL−1 and a limit of quantitation (LOQ) of 43.40 ng. mL−1. With acceptable intra- and interday precision, the methodologywas confirmedto berobust. The devised stability-indicating HPLC technique for analyzing RHT content, its stability in the nano formulations, and drug release in vitro proved to be selective, accurate, sensitive, and consistent.

QbD-Engineered Development and Validation of a RP-HPLC Method for Simultaneous Estimation of Rutin and Ciprofloxacin HCl in Bilosomal Nanoformulation.

In the given study, a new reverse-phase high-performance liquid chromatography (RP-HPLC) method has been reported for the simultaneous estimation of ciprofloxacin hydrochloride (CPX) and rutin (RUT) using quality by design (QbD) approach. The analysis was carried out by applying the Box-Behnken design having fewer design points and less experimental runs. It relates between factors and responses and gives statistically significant values, along with enhancing the quality of the analysis. CPX and RUT were separated on the Kromasil C18 column (4.6 × 150 mm, 5 μm) using an isocratic mobile phase combination of phosphoric acid buffer (pH 3.0) and acetonitrile with the ratio of 87:13% v/v at a flow rate of 1.0 mL/min. CPX and RUT were detected at their respective wavelengths of 278 and 368 nm using a photodiode array detector. The developed method was validated according to guideline ICH Q2 R (1). The validation parameters taken were linearity, system suitability, accuracy, precision, robustness, sensitivity, and solution stability which were in the acceptable range. The findings suggest that the developed RP-HPLC method can be successfully applied to analyze novel CPX-RUT-loaded bilosomal nanoformulation prepared by thin-film hydration technique.

RP-UFLC based Bioanalytical Method Development, Optimization, and Validation for the Estimation of Isradipine in Rabbit Serum

Introduction: The objective of this study is to provide a rapid, sensitive, consistent, and costeffective method for quantifying isradipine using ultra-fast liquid chromatography. Methods: Quality by Design principles will form the basis of this approach, grounded on response surface analysis. Shimadzu liquid chromatographic system equipped with a photodiode array detector and LC solution software was used to conduct the RP-UFLC method development and validation. An ODS C18 (250 x 4.6 mm; 5 μm) UFLC column was used to complete the analysis. The RSM methodology utilized a central composite design to perform the optimization studies. Results: The mobile phase ratio and flow rate were considered crucial method parameters, as well as the peak area, retention time, and USP plate were considered critical analytical attributes. The optimal conditions for chromatographic separation were followed using 80% acetonitrile and water (20% v/v) as mobile phase, a 1 mL/min flow rate, an injection volume of 20 μL, 40°C of column oven temperature, and maximum absorption at λmax 254 nm using graphical optimization technique. When examining concentrations between 5 and 150 ng/mL, linearity was observed with an R2 of 0.999. The method created was validated by employing stability testing per the recommendations provided by ICH Q2 (R1). The analysis of blood serum was modified so that it could be used to examine the pharmacokinetic parameters. Conclusion: As a result, high accuracy, precision, sensitivity, linearity, and robustness were established for predicting the amount of isradipine present in its freeze-dried nano-formulations.

A novel UPLC – PDA method for simultaneous determination of hydroquinone, oxybenzone and octinoxate: An in-vitro study

Melasma, a type of hyperpigmentation, is a difficult condition to treat. Several topical therapies have been proposed to meet the therapeutic goals of this condition, one of which is the combination of hydroquinone, oxybenzone, and octinoxate. The current study aims to develop a robust and reliable analytical method for determining hydroquinone, oxybenzone, and octinoxate in fixed dosage forms simultaneously. In pharmacopoeias, the scope for simultaneous drug determinations is always limited, so the current work is an attempt to develop a method for routine analysis of this combination in quality control. On Water's Acquity system with an X-Bridge C8 column (100 ​mm 3.0 ​mm, 3.5 ​μm), an isocratic mobile phase of 30:70 v/v acetonitrile and buffer were used to achieve chromatographic separation. The flow rate is 0.5 ​ml per minute, and the detection wavelength is 230 ​nm. By subjecting the APIs to various stressors, the method's ability to indicate stability was demonstrated. The method validation followed ICH guidelines. The results of the research for system acceptability, reproducibility, detection limits, quantitation limits, linearity, accuracy, precision, and specificity are all within acceptable bounds. The reliability and analytical performance of the proposed method for its linearity ranges, system suitability, accuracy, precision, robustness detection, and quantification limits were statistically validated and found to be in consistent with ICH Q2 (R1) recommendations.

Identification and characterization of the forced degradation products of lenvatinib mesylate by liquid chromatography-high resolution mass spectrometry

Lenvatinib mesylate (LEN) is a new generation anti-cancer medication included under the class of multiple kinase inhibitor and approved for the treatment of differentiated thyroid cancers that have not responded to the conventional radioactive iodine. It is marketed under the trade name Lenvima®. Forced degradation studies play an important part in the drug development process by providing crucial information about the inherent stability of the molecule under various conditions that are representative of the drug life cycle. The present study focused on exposing the active pharmaceutical ingredient, LEN to various stress conditions as per the ICH guidelines. LEN was found to degrade under hydrolytic stress whereas relatively stable in the thermal, oxidative and photolytic stress conditions. All the degradation products were separated on a Waters Acquity PFP (100 × 2.1 mm, 1.7 µm) column with mobile phase consisting of ammonium acetate in water and acetonitrile in gradient elution mode. LEN produced a total of nine degradation products (L1 to L9) which were structurally characterized with the help of LC-QTof-MS. The developed method was validated as per the ICH Q2 (R1) guidelines and was found to be specific, precise, accurate, linear and robust. The developed method can be effectively used for the routine batch analysis of LEN in bulk drug and various dosage forms in the presence of its degradation products /impurities.

Development and Validation of Stability Indicating RP-HPLC Method for the Estimation of Glycopyrrolate and Neostigmine in Bulk and Injection.

A stability indicating RP-HPLC method is suggested for determination of Glycopyrrolate-Neostigmine (GLY/NEO) in bulk drugs and injection formulation. GLY/NEO were eluted from a Chromolith High Resolution RP-18e (100 mm×4.6mm) with buffer solution (pH3.0) as mobile phase A and a mixture of HPLC grade acetonitrile and water mixture (90:10) as mobile phase B. The gradient was optimized with a flowrate of 0.5mL/min and wavelength of 222nm. A complete analytical method validation was effectively carried out as per ICH Q2 (R1) guidelines. Recovery studies were performed at 50-150% level of working concentrations, and results were in the range of 99-101%. The linearity was detected in the range of LOQ to 200% of the specification limits i.e., 0.5% each for NEO and GLY, 0.01% for NEO Impurity B and 1.0% for rest of the impurities with respect to the test concentration of the respective components. For stability study, various stress conditions such as acid, base, oxidation and thermal as per ICH guidelines were studied. The high recovery and low relative standard deviation confirm the suitability of proposed method that can be employed for the routine analysis in bulk and pharmaceutical formulation.