Практичне застосування високоефективної рідинної хроматографії та високоефективної тонкошарової хроматографії у фармацевтичному аналізі.

Objective: Paclitaxel (PTX) is one of the oldest chemotherapeutic agents for cancer treatment. However, PTX is a class IV drug under the Biopharmaceutical Classification System (BCS), and its oral administration is restricted due to its low bioavailability. Complexing PTX with Beta-Cyclodextrin (β-CD) is an option to overcome the low solubility and bioavailability. This study aims to optimize and develop an RP-HPLC analytical method for quantifying PTX from the fabricated β-CD complex. Methods: The HPLC settings were optimized using Design-of-Experiments (DOE) software. The independent variables for the optimization process were buffer ratio, buffer pH, flow rate, and injection volume. The responses were Retention Time (RT), peak area, Tailing Factor (TF), and number of Theoretical Plates (TP) of PTX. The validated method was then used to measure the % entrapment from the PTX-β-CD complex. Results: The developed and optimized RP-HPLC method was validated as per International Council for Harmonisation (ICH) Q2 (R1) guidelines. The developed method showed linearity R2 = 0.999 with a 0.5-20 µg/ml range. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were 95 and 125 ng/ml, respectively. The accuracy and precision for the developed method came under the acceptance criteria. The developed method was used to evaluate the enhancement of solubility of the prepared PTX-β-CD complex. The method was also used in the evaluation of % drug loading, % drug release and stability of the PTX-β-CD complex. The study clearly showed that the solubility of PTX increased from 0 to 1.14±0.53 µg/ml at pH 1.2 and 0 to 3.18±0.61 µg/ml at pH 6.8, respectively. The PTX-β-CD complex showed 73±3.75% drug release in 120 min at pH 1.2 and 87±3.51% at pH 6.8. The developed RP-HPLC method was found to be eco-friendly as per the Analytical Greenness (AGREE) metric approach and software analysis. Conclusion: An eco-friendly RP-HPLC analytical method was successfully developed and optimized for the quantification of PTX from the PTX-β-CD complex.

The present studies describe the systematic development and validation of a simple, rapid, sensitive and cost-effective reversed-phase high-performance liquid chromatographic bioanalytical method for the estimation of valsartan in rat plasma employing analytical quality by design (AQbD) principles quality risk management was applied for identifying the critical method parameters (CMPs) and subsequently method optimization was performed employing Box-Behnken design by selecting mobile phase pH, flow rate and % organic modifier as the CMPs and evaluated for critical analytical attributes (CAAs) such as peak area, retention time, peak tailing and number of theoretical plates. The developed method was then transferred to bioanalysis, where liquid-liquid extraction process was used for separating the drug from rat plasma. The optimization of extraction process was performed with the help of face-centered cubic design by selecting centrifugation speed and centrifugation time as the CMPs for maximizing % recovery, signal-to-noise ratio and purity threshold of the drug peak after extraction as the CAAs. Optimum chromatographic solution was chosen by mathematical and graphical search techniques, and design space was demarcated. Validation studies performed for the developed method indicated linearity ranging between 5 and 100ng.mL-1, whereas accuracy and precision study showed good percent recovery (99-102%) along with % relative standard deviation within ±2%. Sensitivity evaluation revealed limit of detection and limit of quantification were found to be 0.76ng.mL-1 and 2.29ng.mL-1, respectively. In a nutshell, the present work demonstrates significant merits of AQbD approach for holistic process understanding and analytical method development and validation with enhanced robustness and performance.

Quality-by-Design-based development of an eco-friendly HPLC method for the estimation of nisoldipine in nanoformulations: Forced degradation studies and in-vitro release studies

Full factorial design for optimization, development and validation of HPLC method to determine valsartan in nanoparticles

A new, normal phase high performance liquid chromatography (NP-HPLC) method was developed for separation of Bortezomib (BZB) enantiomers and quantitative determination of (1S,2R)-enantiomer of BZB in active pharmaceutical ingredient (API) samples. The developed method was validated based on International Conference on Harmonisation (ICH) guidelines and it was proved to be accurate, precise and robust. The obtained resolution (RS) between the enantiomers was more than 2. The calibration curve for (1S,2R)-enantiomer was found to be linear in the concentration range of 0.24-5.36 mg/L with regression coefficient (R2) of 0.9998. Additionally, the limit of detection (LOD) and limit of quantification (LOQ) were 0.052 and 0.16 mg/L, respectively. Also, in this study, a precise, sensitive and robust gradient reversed-phase HPLC (RP-HPLC) method was developed and validated for determination of BZB in API samples. The detector response was linear over the concentration range of 0.26-1110.5 mg/L. The values of R2, LOD and LOQ were 0.9999, 0.084 and 0.25 mg/L, respectively. For both NP-HPLC and RP-HPLC methods, all of the RSD (%) values obtained in the precision study were <1.0%. System suitability parameters in terms of tailing factor (TF), number of theoretical plates (N) and RS wereTF < 2.0, N > 2,000 and RS > 2.0. The performance of two common integration methods of valley to valley and drop perpendicular for drawing the baseline between two adjacent peaks were investigated for the determination of diastereomeric impurity (Imp-D) in the BZB-API samples. The results showed that the valley to valley method outperform the drop perpendicular method for calculation of Imp-D peak areas. Therefore, valley to valley method was chosen for peak integration.

To solve the complexity problems that the batch distillation column design must be referred to the continuous distillation column design theories in many cases or to overcome the uncertainty shortcomings that the batch distillation column design have to be originated from an empirical method or from a semi-empirical method, a new shortcut design approach is proposed to determine the number of theoretical and actual plates and the height of the batch distillation column in this paper. Effects of equipment parameters of batch distillation column on the yield proportion are discussed and analyzed, relations between maximal yield proportion and column equipment parameters are correlated. Under the assistance of the separation difficulty defined, the minimum number of the theoretical plates is determined by the limit loss proportion method given, and further the actual number of the theoretical plates and the height are calculated by using the redundancy coefficient found to complete the whole design as shown in the computational sample. Research show that the actual number of the theoretical plates and the height of batch distillation column with the column diameter 0.6 m are 17 and 5.1 m in an alcohol mixture separation system of the example provided. Moreover, the approach can be extended to the design of batch distillation column with a separation system of multi-component liquid mixture after those adjacent components are treated as numerous binary component systems. Finally, recent trends with regarding to extension and application of separation difficulty in investigation areas of separation engineering are expounded based on the concept given, and potential hot spots such as the relations between the entropy value and the separation difficulty for a hybrid system are predicted.

Design of experiments-based RP – HPLC bioanalytical method development for estimation of Rufinamide in rat plasma and brain and its application in pharmacokinetic study

A benchmark UPLC-based method for concurrent analysis of seventeen Stevia rebaudiana metabolites (steviol, isosteviol, and fifteen steviol glycosides) in a single analytical run and its implementation in diverse products.

Core shell stationary phases for a novel separation of triglycerides in plant oils by high performance liquid chromatography with electrospray-quadrupole-time of flight mass spectrometer

Purpose: The main purpose of the study is to develop and test an image classification algorithm inspired by the mechanisms of gas chromatography. The research aims to transfer theoretical concepts from analytical chemistry—such as retention time and the number of theoretical plates—into the field of image processing. The motivation stems from the need for effective extraction and differentiation of visual features, particularly in domains where internal structural differences are subtle or where shape deformation occurs. Design/methodology/approach: The proposed method treats input images as complex mixtures of visual components (analogous to chemical substances) and decomposes them into subimages that are transformed into chromatograms. The algorithm performs feature extraction using simulated chromatographic separation, followed by background subtraction and peak comparison. The classification is based on matching the extracted chromatographic peaks against reference classes. Two types of datasets were used to validate the approach: synthetic blood cell images (medical domain) and geometric shape figures (industrial domain), both with controlled distortions. The influence of subimage size and the number of theoretical plates on classification performance was systematically tested. Findings: The experiments confirm that the classifier’s effectiveness strongly depends on both the resolution of the chromatographic decomposition (i.e., the number of plates) and the subimage size. For blood cells, the best results (precision 0.78) were achieved with a subimage size of 4×4 and a high number of plates (100,000). In the case of shape classification, the highest precision (0.84) was obtained for 60×60 subimages and 50 plates. The study shows that excessive resolution can lead to overfitting, while too little generalization limits the algorithm’s sensitivity to subtle differences.Research limitations/implications: The approach is tested on synthetic datasets, which—while offering control over feature variance—may not reflect all the complexities of real-world data. Future research should include validation on real medical images and industrial visual inspection systems. Additionally, comparative benchmarks against conventional machine learning classifiers (e.g., CNNs) could strengthen the results. Practical implications: The proposed method is applicable in areas requiring robust and explainable classification of complex image data. In medicine, it can support blood diagnostics by highlighting morphologically relevant features of leukocytes. In industry, it can enhance automated inspection systems by enabling recognition of deformed parts without reliance on purely edge-based or shape-based detection. Originality/value: The article presents an original and interdisciplinary algorithm that combines principles of gas chromatography with image analysis. It introduces a novel metaphor for feature decomposition and demonstrates its applicability in both medical and industrial scenarios. This approach expands the set of tools available for interpretable and structure-aware image classification. Keywords: gas chromatography, image classification, feature extraction, synthetic dataset, biomedical imaging. Article classification: Research paper, Technical paper.

Validamycin A, a main component of the antibiotic validamycin complex, which is widely used to control sheath blight disease of rice plants, can be determined by capillary zone electrophoresis with indirect UV detection. The influence of various separation conditions including background electrolyte and modifier concentration, pH, and voltage was investigated. By using 10 mM aminopyrine-2 mM ethylenediaminetetraacetic acid at pH 5.2 as the carrier electrolyte, high efficiency separation of validamycin A was achieved with the number of theoretical plates up to 350000 plates/m. The linear range was across 3 orders of magnitude. The relative standard deviations for migration times and peak areas were less than 0.5 and 3.0%, respectively. The limit of detection for validamycin A was 1.0 microg/mL. The average recoveries ranged from 103.0 to 104.8%. This method has many advantages as compared with high-performance liquid chromatography and micellar electrokinetic capillary chromatography in the determination of commercial formulations.

UHPLC assisted simultaneous separation of apigenin and prednisolone and its application in the pharmacokinetics of apigenin

Determination of spore concentration in Bacillus thuringiensis through the analysis of dipicolinate by capillary zone electrophoresis

Background: The current investigation explains the validation of High Pressure Liquid Chromatography (HPLC) technique for quantification of Ruxolitinib in active ingredient and marketed tablet dosage form. For assessment of Ruxolitinib, a specific column chromatographic method has been developed using UV detection. Methods: The chromatographic elution was executed on ODS Phenomenex analytical column (250mm × 4.6mm, 5μm particle size). The solvent ratio utilized was methanol: water (pH 3.5 adjusted with OPA) (70:30 V/V). A constant ratio of eluting phase flow rate 1.0ml/min was used to detect RTB at 236nm. Results: The developed method was justified using various variables such as accuracy, limit of detection, precision, specificity, robustness, limit of quantification. The retention time for Ruxolitinib was obtained at 8.59 min. The designed method was detected linear within 20 to 120μg/ml range. Correlation coefficient (r2) of the regression equation was found to be 0.9999. Detection and quantitation limit achieved was 0.1496 and 0.4483 respectively. The accuracy of the method was determined using recovery study. The % recovery obtained was in the range of 98.68 – 99.80%. All system suitability factors like tailing factor, retention factor, number of theoretical plates were obtained within acceptance range. The obtained result demonstrates that the presented method is reliable, precise, and economical. Conclusion: The specified method was effectively authenticated as per guidelines of ICH in practice and can be claimed successful for the determination of Ruxolitinib in commercial formulation.

Comparison of piperine content, antimicrobial and antioxidant activity of Piper chaba root and stem