Development Of Chromatographic Method Research Articles

Ultra‐high‐performance liquid chromatography method development for the quantification of Molnupiravir and its process‐related impurities using Box‐Behnken experimental design

AbstractMolnupiravir, a promising antiviral agent, has gained significant attention for its potential in treating viral infections, particularly in the context of the coronavirus disease 2019 pandemic. This study aimed to develop and validate an ultra‐high‐performance liquid chromatography (UHPLC) method for the quantification of Molnupiravir and its associated impurities (Imp‐I, Imp‐II, Imp‐III, and Imp‐IV). The method involved a systematic investigation of critical method parameters and their optimization using Quality by Design principles. The percentage of organic modifiers, column temperature, and flow rate were systematically optimized using the Box‐Behnken design. The developed method was assessed for specificity, system suitability, accuracy, precision, linearity, and detection limits. The results demonstrate strong specificity, with the analysis remaining accurate even in the presence of impurities. The developed method exhibits excellent precision, with repeatability and intermediate precision showing relative standard deviation values ranging from 0.78% to 1.14% for impurities and 0.82% to 1.91% for Molnupiravir. Furthermore, the method displays exceptional linearity, covering a wide range of concentrations. The linear regression analysis yields high coefficients of determination (r2, 9993–0.9997), confirming the linearity. The developed UHPLC method is well‐suited for the accurate and reliable analysis of Molnupiravir and its impurities, making it a valuable tool for quality control and pharmaceutical research applications.

Impurity Profiling and a Stability Indicating Advanced Liquid Chromatographic Method Development and Validation for the Estimation of Related Impurities of Anagrelide

A novel, streamlined and highly selective isocratic liquid chromatographic method has been developed for the quantification of impurities present in anagrelide. Exceptional resolution between anagrelide and its impurities was accomplished utilizing a Waters Nova Pack C18 column (250 mm × 4.6 mm, 4 µm), employing a phosphate buffer with a pH of 4.4 as mobile phase A and a blend of acetonitrile and methanol with buffer in a 30:40:30 v/v/v ratio as mobile phase B. The column oven was maintained at 35 ºC and the mobile phase flowed at a constant rate of 1.0 mL/min, while detection was set at 254 nm. Rigorous validation was conducted to assess accuracy, precision, specificity, linearity and sensitivity. Validation studies have unequivocally established the HPLC method’s precision, specificity, rapidity, reliability and reproducibility. Linearity was ascertained for both anagrelide and its impurities, exceeding an R2 value of 0.95. The limits of detection (LOD) and limit of quantitation (LOQ) were determined to be more than sufficient for precise estimation. This method was validated in accordance with the stringent ICH guidelines. The relative standard deviation (RSD) for intra- day and inter-day precision remained consistently below 5%. Percentage recovery demonstrated excellent agreement, affirming the simplicity, specificity, precision and enhanced accuracy of method for determining related substances in pharmaceutical substances and various anagrelide-containing dosage forms.

Development of chromatographic methods to determine multivitamins formulation depending on their solubility and polarity: comparative study using three greenness assessment tools

High performance liquid chromatography is one of the techniques of choice for the separation and quantitative determination of drugs in mixture form. Ipriflavone, ascorbic acid, pyridoxine, vitamin D3, and lysine are formulated together as an adjuvant combination in osteoporosis. In this work, we developed and validated two complementary high performance liquid chromatographic methods to determine the five compounds in their pharmaceutical dosage form. The first method (method A) was capable of determining ipriflavone, ascorbic acid, pyridoxine, and vitamin D3 in their bulk and combined pharmaceutical formulation. The method is based on Liquid Chromatographic separation with UV detection at 254 nm using Agilent Eclipse XDB-C18 column with a mobile phase consisting of 25 mM ammonium acetate buffer (pH 4.2): methanol in gradient mode. Due to the high polarity of lysine, it was difficult to achieve satisfactory retention on reversed phase columns. So, we separated it on a strong cation exchange column (Exsil 100 SCX) without derivatization with a mobile phase consisting of 10 mM sodium dihydrogen phosphate and 200 mM sodium chloride (pH 6) with UV detection at 210 nm (method B). Validation of the proposed methods was performed according to ICH guidelines Q2(R1). The proposed methods proved to be valid for selective analysis of the stated drugs in their bulk and combined pharmaceutical formulation. Greenness assessment of the developed methods was evaluated using three assessment tools: ESA, GAPI and the most recently developed tool AGREE, showing a satisfactory comprehensive guide of the greenness of the developed methods.

Simulation of gas chromatographic separations and estimation of distribution-centric retention parameters using linear solvation energy relationships

For method development in gas chromatography, suitable computer simulations can be very helpful during the optimization process. For such computer simulations retention parameters are needed, that describe the interaction of the analytes with the stationary phase during the separation process. There are different approaches to describe such an interaction, e.g. thermodynamic models like Blumberg's distribution-centric 3-parameter model (K-centric model) or models using chemical properties like the Linear Solvation Energy Relationships (LSER).In this work LSER models for a Rxi-17Sil MS and a Rxi-5Sil MS GC column are developed for different temperatures. The influences of the temperature to the LSER system coefficients are shown in a range between 40 and 200 °C and can be described with Clark and Glew's ABC model as fit function. A thermodynamic interpretation of the system constants is given and its contribution to enthalpy and entropy is calculated. An estimation method for the retention parameters of the K-centric model via LSER models were presented. The predicted retention parameters for a selection of 172 various compounds, such as FAMEs, PCBs and PAHs are compared to isothermal determined values. 40 measurements of temperature programmed GC separations are compared to computer simulations using the differently determined or estimated K-centric retention parameters. The mean difference (RSME) between the measured and predicted retention time is less than 8 s for both stationary phases using the isothermal retention parameters. With the LSER predicted parameters the difference is 20 s for the Rxi-5Sil MS and 38 s for the Rxi-17Sil MS. Therefore, the presented estimation method can be recommended for first method development in gas chromatography.

Quantification of residual organic bases in an active pharmaceutical ingredient using mixed-mode chromatography and UV detection

The use of organic bases is ubiquitous in chemical synthesis, yet quantifying these compounds with traditional HPLC methodologies is often hampered by poor peak shape, low retention, and limited UV absorption. When employed in the manufacture of an active pharmaceutical ingredient (API), these compounds must be controlled to levels that are safe for human consumption, requiring robust analytical methods with sufficiently low quantification limits. This work describes the development of an HPLC method for the quantification of imidazole and 1,8-Diazabicyclo[5.4.0]undec‑7-ene (DBU) in an API using mixed-mode chromatography. Through control of the pH and organic modifier gradients, the retention of the basic analytes and API can be tuned independently to achieve desirable retention and sensitivity for each compound. The resulting HPLC method exhibits good performance in linearity, accuracy, sensitivity, specificity, and solution stability. Notably, these conditions avoid more complex detection modalities, such as mass spectrometry, while maintaining a system pressure below 400 bar, making the method compatible with a broad range of instruments. This approach to mixed-mode chromatography method development could be extended to different organic bases in the presence of complex molecules to fit the needs of projects in an academic or industrial environment.

Characterization of antibody surface properties and selection of a diverse subset for development of a generic size-exclusion chromatography method

Aggregates are an important quality attribute for biotherapeutics because they can affect the safety and efficacy of the drug and they are therefore routinely monitored, mainly with size-exclusion chromatography (SEC). However, there is often a need to tailor a SEC method to a specific molecule. This study describes the development of a generic SEC method tested on 138 antibodies with the variable domains taken from clinical stage antibodies. We report on the discovery of a subset of 12 antibodies that represents the full range of physiochemical properties found in the 138 antibodies. This subset is shown to be an efficient and reliable test set when developing chromatographic methods for antibodies. An understanding of the nature of the analyte-stationary phase interactions was gained when using this set with its wide range of physiochemical properties. Highly hydrophobic antibodies interact strongly with some modern silica hybrid materials causing the elution time to increase significantly, while a hydrophilically modified hybrid surface showed highly reduced interactions for the hydrophobic antibodies. Highly hydrophilic antibodies, on the other hand, exhibited asymmetric peaks to a certain extent on all stationary phases, while the elution time was not affected. The developed SEC method was shown to have satisfactory performance in terms of linearity, repeatability, range, and accuracy and exhibit very narrow distributions of elution time and peak symmetry when testing the 138 antibodies indicating its generic performance.

Analytical quality by design aided stability indicating a robust ultra‐performance liquid chromatographic technique for the quantification of sonidegib and its organic impurities in bulk drug substance

AbstractA simple quality by design aided stability indicating method was developed for quantification of sonidegib (SONI) and its process related impurities using on ultra‐performance liquid chromatography in bulk drug substance. AutoChrom and Design‐Expert software were used to predict physicochemical properties, draw Ionization graphs, and generate analytical target profile. SONI was subjected to forced degradation conditions, such as oxidative, acid hydrolysis, base hydrolysis, hydrolytic, thermal, and photolytic hydrolysis. All degradation products and process contaminants were separated using an Acquity Ethylene Bridged Hybrid C18 column in gradient elution mode with a mobile phase containing 0.02 M ammonium acetate buffer and acetonitrile: methanol (80:20 v/v). The predicted physicochemical properties are accurate and they facilitated for selection of robust conditions in development of chromatographic method with minimal trials. The developed method can be used for quantification of drug and its process related impurities in bulk drugs.

Application of Quality by Design in Chromatographic Method Development for Drug Analysis in Pharmaceutical Formulations

Application of Quality by Design in Chromatographic Method Development for Drug Analysis in Pharmaceutical Formulations

Radiochemical isolation of 45Ti using ion chromatography

45Ti exhibits favorable decay properties for positron emission tomography (PET) imaging and can be easily produced by the bombardment of natural scandium (Sc) by protons using the 45Sc(p,n)45Ti nuclear reaction. However, separation of 45Ti from irradiated Sc targets is arduous due to the hydrolytic instability of Ti(IV) complexes, making it a significant bottleneck for routine application of this radionuclide. In the present work, we describe the development and optimization of an ion chromatographic separation method based on trapping of 45Ti on a hydroxamate-functionalized chelating resin and subsequent elution with oxalic acid at pH = 2.8. Under optimized conditions, this method enabled 45Ti-recovery of 61 ± 8% within 7 min. Sc contamination in scaled-up experiments was found to be only 3.0 ± 1.8 µg/mL. The resulting 45Ti-solution was directly used for complexation with CDTA as a model chelator, affording the corresponding [45Ti]Ti(cdta) complex with a radiochemical conversion of 73 ± 3%. Conclusively, this promising method could be transferred to automated synthesis modules and should enable the preparation of 45Ti-labeled compounds for PET imaging.

Development and Validation of the Ultra-Performance Liquid Chromatography Method for Quantification of Belzutifan in Pharmaceutical Dosage Form

Development and Validation of the Ultra-Performance Liquid Chromatography Method for Quantification of Belzutifan in Pharmaceutical Dosage Form

Development and validation of a novel high-performance liquid chromatography (HPLC) method for the detection of related substances of pralsetinib, a new anti-lung cancer drug.

Pralsetinib, a targeted inhibitor of the RET enzyme, plays a critical role in the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) characterized by RET gene fusion mutations following platinum-based chemotherapy. Nevertheless, impurities resulting from the manufacturing and degradation of pralsetinib have the potential to impact its therapeutic effectiveness and safety profile. To address this issue, a liquid chromatography method was developed and validated for the specific identification of pralsetinib and its related impurities. The separation of pralsetinib and its related impurities was achieved via a Waters X Bridge C18 column with dimensions of 4.6mm × 250mm and a particle size of 5μm. Mobile phase A was composed of 20mmol/L potassium dihydrogen phosphate (KH2PO4) and acetonitrile (ACN) at a volume ratio of 19:1, while mobile phase B consisted solely of ACN, utilizing a gradient elution technique. Detection was performed at a wavelength of 260nm, with an injection volume of 10μL and a flow rate of 1.0mL/min. The chromatographic method established in this study was validated according to the ICH Q2 (R1) guidelines. The method demonstrated excellent linearity over a specific concentration range (imp-A: 0.035-10.21μg/mL; imp-B: 0.09-10.16μg/mL; imp-C: 0.15-10.19μg/mL; pralsetinib: 0.04-10.32μg/mL). Additionally, the method possesses high sensitivity, with detection limits for impurities A, B, C, and pralsetinib of 0.01, 0.03, 0.015, and 0.013μg/mL, respectively, and quantification limits of 0.035, 0.09, 0.05, and 0.04μg/mL, respectively. In terms of specificity, stability, repeatability, accuracy, and robustness, the method met the validation acceptance criteria. Overall, the chromatographic technique established in this study can effectively separate pralsetinib and its impurities, providing reliable assurance for the accurate detection and quantification of impurities. The chromatographic method developed in this study can be utilized for the detection of pralsetinib and its impurities, offering a crucial reference for research on the quality of pralsetinib.

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

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

Quality by Design Based Stability Indicating Quantitative Reverse Phase High Performance Liquid Chromatography Method Development and Validation for Bilastine in Tablet Dosage Form

Quality by Design Based Stability Indicating Quantitative Reverse Phase High Performance Liquid Chromatography Method Development and Validation for Bilastine in Tablet Dosage Form

Development of High-performance Thin-layer Chromatography (HPTLC) Method for Quality Control of Actinidiae Fructus Vermicultus

Development of High-performance Thin-layer Chromatography (HPTLC) Method for Quality Control of Actinidiae Fructus Vermicultus

Response Surface Methodology Assisted RP-HPLC Method for the Determination of Meropenem in Human Plasma: Application to a Pharmacokinetic Study

A simple, accurate and precise development of an reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of meropenem in human plasma samples for therapeutic drug monitoring (TDM) was developed and validated after optimization of various chromatographic parameters. The best recovery of meropenem was achieved through sample preparation based on a simple extraction method that included deproteinization and extraction with acetonitrile. The developed method reported acceptable values for selectivity, linearity, accuracy and precision, carry-over, dilution integrity and stability. The developed method was efficiently shown to be applicable to a real-time monitoring and pharmacokinetic study of meropenem in critically ill patients.

Isolation, In-silico Studies, and Biological Evaluation of Higenamine from Annona squamosa L. against Breast Cancer

The efficacy of Annona squamosa L. in cancer treatment has been documented, inspiring the authors to investigate the plant further for potential novel anticancer compounds. Therefore, the current study aimed to isolate and characterize Higenamine from leaves extract of A. squamosa L. and studied for the in-vitro cancer cell line and molecular docking. The high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) method development of A. squamosa L. leaves extract furnished Higenamine of which characterization was established by HPLC, infrared radiation (IR), liquid chromatography-mass spectrometry (LC-MS) and its acute toxicity study and cytotoxic properties using MCF-7 cell lines by MTT assay were assessed. Molecular docking was done using Auto Dock Vina software to validate the anticancer activity. The extraction batches by ultrasonication and maceration methods showed good results using 80% methanol. Acute toxicity studies, after a single dose (2000 mg/kg) administration of methanolic extract don’t show clinical symptoms of toxicity and mortalities. From the extract, Higenamine was eluted and the dried fraction showed 66% of enrichment of Higenamine. The developed HPLC and TLC methods showed Higenamine (HGN) peak at 6.21 minutes band at Rf 0.61, respectively. The isolated compound was identified as Higenamine with FTIR spectroscopic analysis which revealed various characteristic band values with functional groups. HPLC-MS showed m/z at 272.1 (M+H+) corresponding to the molecular formula C16H17NO3. The IC50 value for Higenamine was 42.39 μg/mL were observed in MCF-7 breast cancer which was better than 5-Fluorouracil having 39.22 μg/mL. In-silico studies explored that higenamine showed good binding affinity to breast cancer PDBIDs and forms a stable complex. Furthermore, absorption, distribution, metabolism, and excretion (ADME) properties of isolated compound was calculated using the Swiss ADME online tool in which higenamine was found to have a good pharmacokinetic profile as compared with 5-fluorouracil and doxorubicin. Higenamine has been isolated and identified as a potential anticancer agent. Further research can explore its effectiveness through pharmaceutical formulation and in vitro cell line studies.

Stability Indicating Reverse Phase Ultra Performance Liquid Chromatography Method Development and Validation for Amiloride and Hydrochlorothiazide Tablet in the Presence of Degradation Products and Application to Green Analytical Principles

Amiloride (AML) and hydrochlorothiazide (HCTZ) combination is used alone or with other medicines to treat high blood pressure (hypertension). The present article provides the development and validation of an reverse phase ultra performance liquid chromatography (RP-UPLC) approach for the quantification of AML, HCTZ, and related impurities in pharmaceutical dosage forms. The analytical column used for the separation of impurities is the Waters X bridge C18 3.5 μm (50 mm X 4.6 mm). Used 0.1% formic acid and ethanol as MP in an isocratic mode with 0.3 mL/min as flow rate and 3 μL as injection volume, at 254 nm as detection in UV and 12 minutes as total run time. The samples were prepared specifically to undergo forced degradation, which involved subjecting them to hydrolysis, oxidation, thermal, and photolytic conditions. The technique underwent validation by the principles set forth by the ICH Q2 guidelines. The validation confirmed that the method possesses specificity, linearity, ruggedness, robustness and accuracy. The methodology employed exhibited a linear relationship extending from 10 to 150% for all impurities. The recovery analysis was conducted throughout a range of concentrations, starting from 10 to 150% concentration. The average recovery value was determined to be within acceptable limits. The evidence of degradation and the findings of the verified study suggest that the nature of the subject under investigation is stable. Hence, this approach might be employed within the domains of pharmaceutical research and development and quality control departments. Green analytical chemistry tools are used to assess the method’s greenness and calculated using GAPI, AGREE and ecological-scale and found excellent green of >75%.

A quality-by-design evaluated liquid chromatography method development and validation for the separation and quantification of nitroxoline and its impurities.

A novel, isocratic, sensitive, stability-indicating high-performance liquid chromatography method was developed for the separation and quantification of related substances in nitroxoline (NTL). The chromatographic separation has been achieved on Inertsil ODS-3V, (250 × 4.6mm, 5μm) at 240nm using ethylenediamine tetraacetic acid buffer and methanol in the ratio of 60:40 v/v as mobile phase. The performance of the method has been checked as per the International Conference on Harmonization guidelines for specificity, linearity, accuracy, precision, and robustness. Regression analysis showed a correlation coefficient value greater than 0.99 for NTL and its three impurities. The detection limit of impurities was in the range of 0.01% (0.05μg/mL)-0.22% (1.1μg/mL) indicating the sensitivity of the newly developed method. The accuracy of the method was established based on the recovery obtained between 94.7% and 104.1% for all the impurities. The percentage relative standard deviation obtained for the repeatability was less than 4.0% at the specification level for all impurities. Forced degradation was performed to establish the stability-indicating nature of the method and to know about the degradation products, the quality of a drug substance changes with time under the influence of stress conditions. Thus, the proposed method was validated and found to be specific, sensitive, linear, accurate, precise, reproducible, and beneficial for routine usage.

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 1.0ml/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 thin‐layer chromatography method development and validation for assay and content uniformity testing of the anti‐neoplastic drug Axitinib in tablet formulation

AbstractThe current study aims to develop and validate an analytical quality by design approach‐based high‐performance thin‐layer chromatographic (HPTLC) method for the analysis of Axitinib tablet samples. The chromatographic conditions in the TLC method were optimized using a three‐level full factorial design. The mobile phase composition and chamber saturation time served as the independent variables for optimization. The mobile phase used for TLC separation on pre‐coated aluminum plates with silica gel 60 F254 consisted of a mixture of ethyl acetate and isopropyl alcohol in a ratio of 9:1 (v/v). Axitinib was quantified at 330 nm using the densitometric method. Axitinib peak from tablet formulation was verified against the reference standard by comparing its single band at Rf 0.44 ± 0.02. Linearity was found to exist between 100 and 600 ng/band, with a correlation coefficient (r2) of 0.9978. The percentage recovery was obtained as 98.21%–99.05%. The system was validated by determining the parameters according to the guidelines of the International Council for Harmonization of Technical Conditions for Medical Products for Human Use. The proposed TLC method can be effectively applied to routine quality control of a pharmaceutical product.