Design of experiment-oriented development and validation of novel bioanalytical reverse phase high performance liquid chromatography method of palbociclib in rat plasma and tissues, and its application in pharmacokinetic studies.

The current research involved the development and validation of an easy, cost-effective, and sensitive bioanalytical reverse-phase high-performance liquid chromatography method for the assessment of palbociclib (PAL) in rat plasma and kidney, liver, spleen and heart. A response surface methodology-based Box-Behnken design was used to optimize critical chromatographic conditions such as buffer pH, organic phase concentration and flow rate to attain good sensitivity, tailing factor and retention time. The conditions were: pH of buffer, 4.5; organic phase concentration, 40%; Shimpac column with 1.0 ml/min flow rate. The responses were: tailing factor, 1.29 ± 0.03, sensitivity, 366,593 ± 8,592; and retention time, 4.5 ± 0.05 min. The samples were extracted by a protein precipitation method, and absolute recoveries were in the range of 88.99-95.08%. The linearity of the developed method was validated over the range 100-2,000 ng/ml (r2 ≥ 0.994) in all tested matrices. The developed bioanalytical method showed greater accuracy (0.98 and 4.01%) and precision (<4.88%). The method was optimized for the sensitive analysis of the PAL in rat plasma, and the kidney, liver, spleen and heart were effectively applied to pharmacokinetic studies.

Analytical quality by design based on knowledge organization: A case study of developing an ultrahigh-performance liquid chromatography method for the detection of phenolic compounds.

INTRODUCTION: Despite numerous successful cases, there are still some challenges in using analytical quality by design (AQbD) for the development of analytical methods. Knowledge organization helps to enhance the objectivity of risk assessment, reduce the number of preliminary exploratory experiments, identify potential critical method parameters (CMPs) and their scope. OBJECTIVE: In the present study, we aimed to develop a simple, rapid, and robust analytical method for detecting phenolic compounds in Xiaochaihu capsule intermediates utilizing knowledge organization. METHODS: Knowledge organization and AQbD were combined to obtain the initial analytical conditions through knowledge collection, extraction, reorganization, and analysis. The quantitative relationship between critical method attributes (CMAs) and CMPs was then established by a definitive screening design. The method operable design region was calculated using an exhaustive Monte Carlo approach based on the probability of reaching the standard. Robustness investigation and methodological validation were finally performed. RESULTS: Analytical target profiles, CMAs, potential CMPs, and initial analytical conditions were initially identified, and the optimized ranges of operating parameters were obtained. A UHPLC method was successfully established for the analysis of phenolic compounds in ginger-ginger pinellia percolate, and the method validation outcomes were also satisfactory. CONCLUSION: The developed method can be a reliable means to detect the phenolic compounds of Xiaochaihu capsule intermediates. Knowledge organization provides a new approach for making better use of prior knowledge, significantly enhancing the efficiency of analytical method development. The approach is versatile and can be similarly applied to the development of other methods.

Increasing Analytical Quality by Designing a Thin-Layer Chromatography Scanner Method for the Determination of the Radiochemical Purity of Radiopharmaceutical Sodium Iodide 131I Oral Solution.

The goal of this study was to apply the principles of analytical quality by design (AQbD) to the analytical method for determining the radiochemical purity (PQR) of the radiopharmaceutical sodium iodide 131I oral solution, utilizing thin-layer chromatography (TLC) with a radio-TLC scanner, which also enables the evaluation of product quality. For AQbD, the analytical target profile (ATP), critical quality attributes (CQA), risk management, and the method operable design region (MODR) were defined through response surface methodology to optimize the method using MINITAB® 19 software. This study encompassed the establishment of a control strategy and the validation of the method, including the assessment of selectivity, linearity, precision, robustness, detection limit, quantification limit, range, and the stability of the sample solution. Under the experimental conditions, the method parameters of the TLC scanner were experimentally demonstrated and optimized with an injection volume of 3 µL, a radioactive concentration of 10 mCi/mL, and a carrier volume of 40 µL. Statistical analysis confirmed the method's selectivity for the 131I iodide band Rf of 0.8, a radiochemical impurity IO3- Rf of 0.6, a linearity from 6.0 to 22.0 mCi/mL, and an intermediate precision with a global relative standard deviation (RSD) of 0.624%. The method also exhibited robustness, with a global RSD of 0.101%, a detection limit of 0.09 mCi/mL, and a quantification limit of 0.53 Ci/mL, meeting the prescribed range and displaying stability over time (at 0, 2, and 20 h) with a global RSD of 0.362%, resulting in consistent outcomes. The development of a method based on AQbD facilitated the creation of a design space and an operational space, with comprehensive knowledge of the method's characteristics and limitations. Additionally, throughout all operations, compliance with the acceptance criteria was verified. The method's validity was confirmed under the established conditions, making it suitable for use in the manufacturing process of sodium iodide 131I and application in nuclear medicine services.

The Applicability of Chromatographic Retention Modeling on Chiral Stationary Phases in Reverse-Phase Mode: A Case Study for Ezetimibe and Its Impurities.

Mechanistic modeling is useful for predicting and modulating selectivity even in early chromatographic method development. This approach is also in accordance with current analytical quality using design principles and is highly welcomed by the authorities. The aim of this study was to investigate the separation behavior of two different types of chiral stationary phases (CSPs) for the separation of ezetimibe and its related substances using the mechanistic retention modeling approach offered by the Drylab software (version 4.5) package. Based on the obtained results, both CSPs presented with chemoselectivity towards the impurities of ezetimibe. The cyclodextrin-based CSP displayed a higher separation capacity and was able to separate seven related substances from the active pharmaceutical ingredient, while the cellulose-based column enabled the baseline resolution of six impurities from ezetimibe. Generally, the accuracy of predicted retention times was lower for the polysaccharide CSP, which could indicate the presence of additional secondary interactions between the analytes and the CSP. It was also demonstrated that the combination of mechanistic modeling and an experimental design approach can be applied to method development on CSPs in reverse-phase mode. The applicability of the methods was tested on spiked artificial placebo samples, while intraday and long-term (2 years) method repeatability was also challenged through comparing the obtained retention times and resolution values. The results indicated the excellent robustness of the selected setpoints. Overall, our findings indicate that the chiral columns could offer orthogonal selectivity to traditional reverse-phase columns for the separation of structurally similar compounds.

Quality by Design (QbD) Based Method for Estimation of Xanthohumol in Bulk and Solid Lipid Nanoparticles and Validation.

The analytical quality by design (AQbD) approach is utilized for developing and validating the simple, sensitive, cost-effective reverse-phase high performance liquid chromatographic method for the estimation of xanthohumol (XH) in bulk and nanoformulations. The Box-Behnken design (BBD) is applied for method optimization. The mobile phase ratio, pH and flow rate were selected as independent variables, whereas retention time, peak area, peak height, tailing factor, and theoretical plates were selected as dependent variables. The chromatogram of XH obtained under optimized conditions has given optimum conditions such as retention time (5.392 min), peak area (1,226,737 mAU), peak height (90,121 AU), tailing factor (0.991) and theoretical plates (4446.667), which are contoured in the predicted values shown by BBD. Validation of the method has been performed according to ICH Q2(R1) recommendations, using optimized conditions for linearity, limit of detection (LOD) and limit of quantification (LOQ), accuracy, precision, robustness and system suitability. All the values of validation parameters lie within the acceptable limits prescribed by ICH. Therefore, the developed and validated method of XH by the AQbD approach can be applied for the estimation of XH in bulk and various nanoformulations.

Exploring an experimental combination of analytical quality by design and green analytical chemistry approaches for development of HPTLC densitometric protocol for the analysis of barnidipine hydrochloride.

An experimental combination of analytical quality by design and green analytical chemistry approaches is introduced to develop an high-performance thin-layer chromatography (HPTLC) approach to quantify barnidipine hydrochloride in the pharmaceutical matrix. The analytical quality by design approach was introduced to green analytical chemistry to enhance protocol knowledge while ensuring efficiency and reducing environmental impacts, energy consumption and analyst visibility. This analytical approach was systematically addressed by exploring failure mode effect analysis, risk assessment and optimization design. Subsequently, a screening of primary variables was performed to select the aptest proportion of solvents in the mobile phase resulting from the principles of green analytical chemistry. Failure mode effect analysis and a risk assessment study were attempted to estimate the critical method parameters (CMPs). The influence of the CMPs on critical analytical attributes, i.e. retention factor and peak area, was assessed through a screening design. A response surface methodology was then executed for the critical analytical attributes as a concern of the determined CMPs, and the conditions for excellent resolution were determined using a desirability procedure. The established protocol was validated in compliance with the International Conference on Harmonization guideline Q2(R1) and showed excellent specificity and sensitivity.

DoE-assisted development and validation of a stability-indicating HPLC-DAD method for simultaneous determination of five cannabinoids in Cannabis sativa L. based on analytical quality by design (AQbD) concept.

INTRODUCTION: Medical uses of Cannabis sativa L. have gained interest in recent decades, which highlights the need for defining appropriate quality specifications for Cannabis-based products. However, the complexity of plant matrices and structural similarity between cannabinoids make analytical development a challenging task. Thus, the application of analytical quality by design (AQbD)-driven approaches can favour the development of fit-for-purpose methods. OBJECTIVES: To develop a high-performance liquid chromatography diode array detector (HPLC-DAD) method for simultaneous quantification of cannabidiol, Δ9 -tetrahydrocannabinol, cannabidiolic acid, tetrahydrocannabinolic acid, and cannabinol in C. sativa by applying an AQbD-driven approach. MATERIALS AND METHODS: Critical method attributes (CMA) were established following the analytical target profile. Critical method variables (CMV) were categorised based on risk assessment and literature review. Selected CMV regarding sample preparation and chromatographic conditions were optimised using response surface methodology (RSM). The working point was estimated by multiple response optimisation using Deringer's desirability function. The validity of the optimal conditions was confirmed experimentally. Method validation was performed according to ANVISA and ICH guidelines. Relative response factors (RRFs) were also determined. RESULTS AND DISCUSSION: Baseline resolution of 12 major cannabinoids was achieved in a 35 min chromatographic analysis. All experimental responses obtained during confirmatory analyses were within the prediction intervals (PI95% ). Method's selectivity, linearity (10-100 µg/mL), precision, bias, extraction recovery, and ruggedness were satisfactorily demonstrated. CONCLUSIONS: The application of an AQbD-driven approach allowed for a better understanding of the effects of the ensemble of CMV on the analyte's behaviour, enabling the definition of appropriate conditions to ensure consistent achievement of the intended method's performance.

Implementation of QbD Approach to the Development of Chromatographic Methods for the Determination of Complete Impurity Profile of Substance on the Preclinical and Clinical Step of Drug Discovery Studies.

The purpose of this work was to demonstrate the use of the AQbD with the DOE approach to the methodical step-by-step development of a UHPLC method for the quantitative determination of the impurity profile of new CPL409116 substance (JAK/ROCK inhibitor) on the preclinical and clinical step of drug discovery studies. The critical method parameters (CMPs) have been tested extensively: the kind of stationary phase (8 different columns), pH of the aqueous mobile phase (2.6, 3.2, 4.0, 6.8), and start (20-25%) and stop (85-90%) percentage of organic mobile phase (ACN). The critical method attributes (CMAs) are the resolution between the peaks (≥2.0) and peak symmetry of analytes (≥0.8 and ≤1.8). In the screening step, the effects of different levels of CMPs on the CMAs were evaluated based on a full fractional design 22. The robustness tests were established from the knowledge space of the screening step and performed by application fractional factorial design 2(4-1). Method operable design region (MODR) was generated. The probability of meeting the specifications for the CMAs was calculated by Monte-Carlo simulations. In relation to literature such a complete AQbD approach including screening, optimization, and validation steps for the development of a new method for the quantitative determination of the full profile of nine impurities of an innovative pharmaceutical substance with the structure-based pre-development pointed out the novelty of our work. The final working conditions were as follows: column Zorbax Eclipse Plus C18, aqueous mobile phase 10 mM ± 1 mM aqueous solution of HCOOH, pH 2.6, 20% ± 1% of ACN at the start and 85% ± 1% of ACN at the end of the gradient, and column temperature 30 °C ± 2 °C. The method was validated in compliance with ICH guideline Q2(R1). The optimized method is specified, linear, precise, and robust. LOQ is on the reporting threshold level of 0.05% and LOD at 0.02% for all impurities.

Development and Validation of a Stability-Indicating UPLC Method for the Determination of Hexoprenaline in Injectable Dosage Form Using AQbD Principles.

A novel and efficient stability-indicating, reverse phase ultra-performance liquid chromatographic (UPLC®) analytical method was developed and validated for the determination of hexoprenaline in an injectable dosage form. The development of the method was performed using analytical quality by design (AQbD) principles, which are aligned with the future requirements from the regulatory agencies using AQbD principles. The method was developed by assessing the impact of ion pairing, the chromatographic column, pH and gradient elution. The development was achieved with a Waters Acquity HSS T3 (50 × 2.1 mm i.d., 1.8 µm) column at ambient temperature, using sodium dihydrogen phosphate 5 mM + octane-1-sulphonic acid sodium salt 10 mM buffer pH 3.0 (Solution A) and acetonitrile (Solution B) as mobile phases in gradient elution (t = 0 min, 5% B; t = 1 min, 5% B; t = 5 min, 50% B; t = 7 min, 5% B; t = 10 min, 5% B) at a flow rate of 0.5 mL/min and UV detection of 280 nm. The linearity was proven for hexoprenaline over a concentration range of 3.50-6.50 µg/mL (R2 = 0.9998). Forced degradation studies were performed by subjecting the samples to hydrolytic (acid and base), oxidative, and thermal stress conditions. Standard solution stability was also performed. The proposed validated method was successfully used for the quantitative analysis of bulk, stability and injectable dosage form samples of the desired drug product. Using the AQbD principles, it is possible to generate methodologies with enhanced knowledge, which can eventually lead to a reduced regulatory risk, high quality data and lower operational costs.

Analytical Method Lifecycle Management in Pharmaceutical Industry: a Review.

The adoption of Quality by Design (QbD) and Analytical Method Lifecycle Management (AMLM) concepts to ensure the quality of pharmaceutical products has been applied and proposed over the last few years. These concepts are based on knowledge gained from the application of scientific and quality risk management approaches, throughout method lifecycle to assure continuous improvement and high reliability of analytical results. The overall AMLM starts with the definition of the method's intended use through the Analytical Target Profile definition, including three stages: (1) Method Design, taking advantage of the well-known concept of QbD; (2) Method Performance Qualification; (3) Continued Method Performance Verification. This is intended to holistically align method variability with product requirements, increasing confidence in the data generated, a regulatory requirement that the pharmaceutical industry must follow. This approach views all method-related activities, such as development, validation, transfer, and routine use as a continuum and interrelated process, where knowledge and risk management are the key enablers. An increase in method robustness, cost reduction, and decreased risk failures are some of the intrinsic benefits from this lifecycle management. This approach is clearly acknowledged both by regulators and industry. The roadmap of the regulatory and industry events that mark the evolution of these concepts helps to capture the current and future expectation of the pharmaceutical framework.

Developing an Improved UHPLC Method for Efficient Determination of European Pharmacopeia Process-Related Impurities in Ropinirole Hydrochloride Using Analytical Quality by Design Principles.

This article presents the development of a reversed-phase ultra-high-performance liquid chromatographic method for determining process-related impurities in ropinirole hydrochloride drug substance applying the analytical quality by design approach. The current pharmacopeial method suffers from selectivity issues due to two coelutions of two pairs of impurities. The development of a new method began with preliminary experiments, based on which the Acquity UPLC BEH C8 was selected as the most appropriate column. The effects of six different critical method parameters (CMPs) were then investigated using a fractional factorial screening design. Column temperature, the ratio of methanol in mobile phase B, and gradient slope turned out to be highly significant CMPs in achieving critical resolutions, and they were further evaluated using a central composite face-centered response-surface design. Mathematical models were created by applying a multiple linear regression method. Based on the elution order of an unknown degradation impurity and impurity C, two design spaces were established, and for each design space an optimal combination of CMPs was determined. The method developed was validated for precision, accuracy, linearity, and sensitivity, and it was proven suitable for determining nine process-related impurities of ropinirole.

Development of a Unified Reversed-Phase HPLC Method for Efficient Determination of EP and USP Process-Related Impurities in Celecoxib Using Analytical Quality by Design Principles.

This article presents the development of a reversed-phase (RP) high-performance liquid chromatographic (HPLC) method for determination of process-related impurities in a celecoxib drug substance following Analytical Quality by Design (AQbD) principles. The method from European Pharmacopeia (EP) for celecoxib drug substance does not sufficiently separate celecoxib from its EP impurity B because the system suitability criterion is not achieved (resolution NLT 1.8). The same issue was observed with the proposed method from United States Pharmacopeia (USP) for celecoxib capsules, where EP impurity A elutes under the main peak. A new HPLC method was developed that eliminates the disadvantages of the two pharmacopeial methods and is capable of efficiently separating and determining all seven impurities listed in EP and the proposed USP monographs. The development of a new HPLC method started with method scouting, in which various C18 and phenyl stationary phases were tested. Improved selectivity was obtained only with a chiral stationary phase. An immobilized Chiralpak IA-3 column used in RP mode turned out to be the most appropriate for method optimization. The ratio of acetonitrile in the mobile phase, flow rate, and column temperature were recognized as critical method parameters (CMPs) and were further investigated using a central composite face response-surface design. A multiple linear regression (MLR) method was applied to fit the mathematical models on the experimental data to determine factor-response relationships. The models created show adequate fit and good prediction abilities. The Monte Carlo simulation method was used to establish the design space. The method developed was verified in terms of precision, sensitivity, accuracy, and linearity, and the results showed that the new method is suitable for determination of seven process-related impurities of celecoxib.

AQbD TLC-densitometric method approach along with green fingerprint and whiteness assessment for quantifying two combined antihypertensive agents and their impurities.

Preserving the environment, reducing the amount of waste resulting from chemical trials, and reducing the amount of energy consumed have currently become a pivotal global trend. An analytical quality by design (AQbD) based eco-friendly TLC-densitometric method was implemented for quantifying two antihypertensive agents, captopril (CPL) and hydrochlorothiazide (HCZ), along with their impurities; captopril disulphide (CDS), chlorothiazide (CTZ) and salamide (SMD). The analytical target profile (ATP) was first identified, followed by selecting the critical analytical attributes (CAAs), such as retardation factors and resolution between the separated peaks. Critical method parameters (CMPs) that may have a crucial influence on CAAs were identified and emanated through the quality risk assessment phase. A literature survey-based preliminary studies were performed, followed by optimization of the selected CMPs through a custom experimental design to attain the highest resolution with optimum retardation factors. Moreover, method robustness was also tested by testing the design space. Complete separation of the drugs and their impurities was achieved using ethyl acetate: glacial acetic acid (6: 0.6, v/v) as a developing system applied to a 12 cm length TLC plate at room temperature with UV scanning at 215 nm. Calibration graphs were found to be linear in the ranges of (0.70-6.00), (0.10-2.00), (0.20-1.00), (0.07-1.50) and (0.05-1.00) µg/band corresponding to CPL, HCZ, CDS, CTZ, and SMD, respectively. Four different green metric tools were used to evaluate the greenness profile of the proposed method, and results showed that it is greener than the reported HPLC method. Method whiteness assessment was also conducted. Moreover, the method performance was evaluated following the ICH guidelines, and the outcomes fell within the acceptable limits. The developed method could be approved for routine assay of the cited components in their pharmaceutical formulations and bulk powder without interference from the reported impurities. The issue of concern is saving money, especially in developing countries.

Analytical Quality by Design Driven Development and Validation of UV-Visible Spectrophotometric Method for Quantification of Xanthohumol in Bulk and Solid Lipid Nanoparticles.

Objectives: Xanthohumol (XH) is a prenylated chalcone available naturally and has diverse pharmacological activities. It has some limitations in the physiological environment such as biotransformation and less gastrointestinal tract absorption. To overcome the limitations, we prepared nanoformulations [solid lipid nanoparticles (SLNs)] of XH. Therefore, an analytical method is required for the estimation of XH in the bulk nanoformulations, so we developed and validated a quality by design (QbD)-based ultraviolet (UV)-spectrophotometric method as per the International Conference of Harmonization (ICH) Q2 (R1) guidelines. Materials and Methods: The new analytical Qbd based UV-visible spectrophotometric technique is developed and validated for estimation of XH in bulk and SLNs as per ICH guidelines Q2 (R1). Critical method variables are selected on the basis of risk assessment studies. Optimization of method variables was performed using the a central composite design (CCD) model. Results: Multiregression ANOVA analysis showed an R2 value of 0.8698, which is nearer to 1, indicating that the model was best fitted. The optimized method by CCD was validated for its linearity, precision, accuracy, repeatability, limit of detection (LOD), limit of quantification (LOQ), and specificity. All validated parameters were found to be within the acceptable limits [% relative standard deviation (RSD) <2]. The method was linear between 2-12 g/mL concentration with R2 value 0.9981. Method was accurate with percent recovery 99.3-100.1%. LOD and LOQ were found to be 0.77 and 2.36 µg/mL, respectively. The precision investigation confirmed that the method was precise with %RSD <2. Conclusion: The developed and validated method was applied to estimate XH in bulk and SLNs. The developed method was specific to XH, which was confined by the specificity study.

Peptidomics: LC-MS operational parameters do matter.

The sensitive and specific detection of peptides at low levels in biofluids is critical to increase the lab-to-human translation of peptidomic research. An interesting group of peptides with increasing evidence for involvement in human diseases are quorum sensing peptides. To obtain more reliable conclusions on peptide measurands in biofluids, a selection of often neglected parts of the analytical process using LC-MS were investigated, with novel approaches recommended for each part. Quorum sensing peptides were used as the main model-peptides. The peptidomic parts investigated and discussed here are: Our work addresses aQbD-approached solutions to these challenges, encompassing sample stabilization measures, a suitable peptide anti-adsorption tool, judicious choice of injection solvent versus gradient system and optimal duty cycle parameters. Our recommendations will improve the peptidomics bio-analytics of not only quorum sensing peptides, but can also be of value for other measurands at low concentrations.

Application of the Analytical Procedure Lifecycle Concept to a Quantitative 1H NMR Method for Total Dammarane-Type Saponins.

Dammarane-type saponins (DTSs) exist in various medicinal plants, which are a class of active ingredients with effects on improving myocardial ischemia and immunomodulation. In this study, a quantitative 1H NMR method of total DTSs in herbal medicines was developed based on the analytical procedure lifecycle. In the first stage (analytical procedure design), the Ishikawa diagram and failure mode effects and criticality analysis were used to conduct risk identification and risk ranking. Plackett-Burman design and central composite design were used to screen and optimize critical analytical procedure parameter. Then, the method operable design region was obtained through modeling. In the second stage (analytical procedure performance qualification), the performance of methodological indexes was investigated based on analytical quality by design. As examples of continued procedure performance verification, the method was successfully applied to determine the total DTSs in herbal pharmaceutical preparations and botanical extracts. As a general analytical method to quantify total DTSs in medicinal plants or pharmaceutical preparations, the developed method provides a new quality control strategy for various products containing dammarane-type saponin.

Drilling into "Quality by Design" Approach for Analytical Methods.

The need for consistency in analytical method development reinforces the dependence of pharmaceutical product development and manufacturing on robust analytical data. The Analytical Quality by Design (AQbD), akin to the product Quality by Design (QbD) endows a high degree of confidence to the method quality developed. AQbD involves the definition of the analytical target profile as starting point, followed by the identification of critical method variables and critical analytical attributes, supported on risk assessment and design of experiment tools for the establishment of a method operable design region and control strategy of the method. This systematic approach moves away from reactive troubleshooting to proactive failure reduction. The objective of this review is to highlight the elements of the AQbD framework and provide an overview of their implementation status in various analytical methods used in the pharmaceutical field. These methodologies include but are not limited to, high-performance liquid chromatography, UV-Vis spectrophotometry, capillary electrophoresis, supercritical fluid chromatography, and high-performance thin-layer chromatography. Finally, a critical appraisal is provided to highlight how regulators have encouraged AQbD principles application to boost the prevention of method failures and a better understanding of the method operable design region (MODR) and control strategy, ultimately resulting in cost-effectiveness and regulatory flexibility.

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 optimization of a LC-MS/MS compatible method for quantification of losartan, hydrochlorothiazide and their impurities using AQbD approach and measurement uncertainty evaluation.

The concept of Analytical Quality by Design (AQbD) comes as a more robust, economical, and scientifically based alternative for analytical development, to the detriment of OFAT (one factor at a time). This new understanding applicable to analytical development is recommended since regulatory flexibility can be achieved and ensure more reliable results throughout the life of the product. This new approach was applied to develop an analytical procedure indicative of stability for a pharmaceutical product of association of Losartan Potassium and Hydrochlorothiazide, considered a potential first line for the treatment of hypertension. The first stage of the analytical development consisted of defining analytical target profile (ATP), follow by a bibliographic survey of the physicochemical properties of the molecules in question to define an initial method. After defining the initial analytical conditions, statistical tools for design of experiments (DoE) were used for the screening and optimization steps. In the screening stage, the Plackett-Burman design was chosen, using 11 factors and 2 levels, through which it was possible to evaluate numerous variables and determine their significance in relation to the responses. Next, optimization was carried out with the experimental design of a central composite with 4 factors and 5 levels, which allowed modeling a complex response surface and evaluating the phenomena of interactions between the factors. Thus, the optimized analytical conditions were defined, considering a 0.3% formic acid gradient as eluent A and a mixture of acetonitrile and methanol (80:20) as eluent B, X-Bridge C18 chromatographic column (150 mm × 4 .6 mm × 3.5 µm), column temperature of 40°C, flow rate of 1.3 mL/min, injection volume of 10 µL. Through this methodology, it was possible to identify an unknown degradation product of Hydrochlorothiazide, formed by the reaction with lactose (excipient present in the drug formulation), proving that the method can be applicable both to DAD detectors and to spectrometry and mass detectors. It was also proven through the forced degradation study that the method is indicative of stability, in addition to being validated and robust for its purpose.

Quality-by-Design Approach for Chromatographic Analysis of Metformin, Empagliflozin and Linagliptin.

New analytical quality by design-oriented HPLC method with multiple response optimization (Derringer's desirability function) was demonstrated for simultaneous analysis of three antidiabetic drugs (metformin hydrochloride/empagliflozin/linagliptin) in a fixed-dose combination. Central composite design was employed for systematic optimization of critical method parameters, namely, % organic phase (X1), aqueous phase pH (X2) and flow rate (X3) while resolution, capacity factor and theoretical plate number as critical analytical attributes. Effective chromatographic separation of title analytes was accomplished on Std. Discovery C18 column at 30°C with mobile phase comprising acetonitrile: phosphate buffer pH 5 (38:62% v/v), pumped at a flow rate of 1 mL/min by isocratic elution pattern and UV detection at 222 nm. The model is rectilinear in the range of 1.0-200, 0.2-40 and 0.1-20 µg/mL at retention times of 3.04, 3.93 and 5.99 min for metformin, empagliflozin and linagliptin, respectively. The method obeyed all validation parameters of ICH Q2(R1) guidelines. The proposed HPLC method was highly robust for method transfer, regulatory flexibility within design space and can be used for assay of pharmaceutical dosage forms comprising these analytes. The proposed method was applied for stability studies of drugs under various stress conditions.