DEVELOPMENT AND VALIDATION OF HIGH-PERFORMANCE THIN LAYERCHROMATOGRAPHIC METHOD FOR CIPROFLOXACIN BY QUALITY BY DESIGN APPROACH

Objective: The aim of this paper is to create a new, systematic high-performance thin-layer chromatography (HPTLC) method for ciprofloxacin that is based on quality by design (QbD). Methods: The mobile phase was chloroform: IPA: H2O: Formic Acid (2:7:0.5:0.5V/V), and the chromatographic separation was performed on aluminum-backed silica gel 60 F254 plates. Ciprofloxacin was detected using UV light at 278nm. In factor screening studies, a 3-factor 17-run standard 3-level factorial design was used, and a Box-Behnken design was used to optimize HPTLC experimental parameters for obtaining anticipated chromatographic conditions. The basic method parameters were tested to understand risk assessment. Three independent parameters, such as saturation time, band duration, and migration distance, were chosen and analyzed based on the risk assessment to see if these three parameters influenced the responses. For ciprofloxacin, the method produces a compact and well-resolved band at Rf = 0.40 0.02. In the linear regression analysis performed on ciprofloxacin, the regression coefficient was found to be r2 = 0.996. Results: According to the International Council on Harmonization (ICH) guidelines, it was validated for validation parameters such as accuracy, precision, robustness, the limit of detection, and the limit of quantification. The proposed method for ciprofloxacin determination was found to be straightforward, precise, reliable, stable, and sensitive. Conclusion: The QbD method produced a more robust method that can generate accurate, high-quality, and reliable data during the process, and it can be effectively used in the routine inspection of Ciprofloxacin in the tablets dosage form.

Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic Bacillus aryabhattai B8W22

Chitin deacetylase (CDA) (EC 3.5.1.41) is a hydrolytic enzyme that belongs to carbohydrate esterase family 4 as per the CAZY database. The CDA enzyme deacetylates chitin into chitosan. As the marine ecosystem is a rich source of chitin, it would also hold the unexplored extremophiles. In this study, an organism was isolated from 40 m sea sediment under halophilic condition and identified as Bacillus aryabhattai B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in E. coli Rosetta pLysS and purified using a Ni–NTA affinity chromatography. The enzyme was found active on both ethylene glycol chitin (EGC) and chitooligosaccharides (COS). The enzyme characterization study revealed, maximum enzyme velocity at one hour, optimum pH at 7 with 50 mM Tris–HCl buffer, optimum reaction temperature of 30 ºC in standard assay conditions. The co-factor screening affirmed enhancement in the enzyme activity by 142.43 ± 7.13% and 146.88 ± 4.09% with substrate EGC and COS, respectively, in the presence of 2 mM Mg2+. This activity was decreased with the inclusion of EDTA and acetate in the assay solutions. The enzyme was found to be halotolerant; the relative activity increased to 116.98 ± 3.87% and 118.70 ± 0.98% with EGC and COS as substrates in the presence of 1 M NaCl. The enzyme also demonstrated thermo-stability, retaining 87.27 ± 2.85% and 94.08 ± 0.92% activity with substrate EGC and COS, respectively, upon treatment at 50 ºC for 24 h. The kinetic parameters Km, Vmax, and Kcat were 3.06E−05 µg mL−1, 3.06E + 01 µM mg−1 min−1 and 3.27E + 04 s−1, respectively, with EGC as the substrate and 7.14E−07 µg mL−1, 7.14E + 01 µM mg−1 min−1 and 1.40E + 06 s−1, respectively, with COS as the substrate. The enzyme was found to be following Michaelis–Menten kinetics with both the polymeric and oligomeric substrates. In recent years, enzymatic conversion of chitosan is gaining importance due to its known pattern of deacetylation and reproducibility. Thus, this BaCDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application.

Novel Methotrexate-Loaded Zein Nanoparticles With Improved and Extended Biopharmaceutical Performance: QbD-Steered Development, and Extensive In Vitro and In Vivo Evaluation

The current work entails QbD-enabled preparation of methotrexate-loaded nanoparticles (NPs) using zein as the release-controlling natural polymer. Initially, quality risk estimationand factor screening studies using Taguchi design were undertaken to delineate “vital few” process and material attributes among “plausible so many”. Further, formulation optimization using central composite design and validation using correlation plots and percent predictive bias was carried out. Optimized NPs exhibited mean size of 159 nm, zeta potential of 14.85 mV and entrapment of 50.23%. In vitro dissolution kinetic modelling unearthed non-Fickian drug release extension mechanism from the proposed zein NPs. In vitro MTT and apoptosis assay using MCF-7 cells and cellular uptake studies using Caco-2 cells indicate remarkably superior anticancer potential of zein NPs over pure methotrexate, ascribable to their nanometric size and cationic nature. In vivo pharmacokinetic studies in rat construed significant enhancement by 2.15-fold in AUC48h (p<0.001), 1.30-fold in Cmax (p<0.05), 3.67-fold in Tmax (p<0.001), and 1.38-fold in T1/2 (p<0.01), along with notably reduced variability in biopharmaceutical performance. Establishment of significant point-to-point level A in vitro/in vivo correlations (IVIVC) and kinetic modeling construed the robustness and prognostic ability of drug release studies. Robustness of the nanoformulation was ratified under refrigerated storage through six months’stability studies. Overall, the studies unequivocally indicate development of a stable nanoparticulate formulation with significantly enhanced extent, extension and consistency of biopharmaceutical performance, along with improved anticancer potential of methotrexate.

QBD APPROACH TO ANALYTICAL METHOD DEVELOPMENT AND ITS VALIDATION FOR ESTIMATION OF LENVATINIB IN BULK AND PHARMACEUTICAL FORMULATION

Objective: The objective of this research was to develop a simple, very rapid, sensitive, accurate, precise reverse phase High-Performance Liquid Chromatography (RP-HPLC) technique for the estimation of Lenvatinib in bulk and its dosage form. Methods: To perform this study, we employed a central composite design (CCD) to make method robust and effective to create chromatographic database. The factor screening studies were performed using 2-factor 10-runs. The factors were selected as the mobile phase ratio and buffer pH. Results: The desirability value of the optimized model was found to be 0.869 and The optimized chromatographic condition was achieved on Enable C18 analytical column with 0.01M Ammonium acetate buffer pH 3.84: methanol (33.17:66.83 v/v) as the mobile phase and flow rate of 1 ml min-1 and detection wavelength was set to 240 nm. The retention time of Lenvatinib was found to be 5.122 min. Linearity was established for Lenvatinib in the range of 10-50 µg/ml with a correlation coefficient (r2=0.9995). The accuracy values were found to be in the range of 98–102%. Intraday precision and Interday precision were in prescribed (Less than 0.98% RSD). Robustness was found to be less than 1.22% RSD. Conclusion: The proposed method was useful for best analysis of Lenvatinib in Bulk pharmaceutical dosage forms. Central Composite Design was an effective tool for the proposed RP-HPLC method.

Analytical quality by design methodology for botanical raw material analysis: a case study of flavonoids in Genkwa Flos

The present study introduces a systematic approach using analytical quality by design (AQbD) methodology for the development of a qualified liquid chromatographic analytical method, which is a challenge in herbal medicinal products due to the intrinsic complex components of botanical sources. The ultra-high-performance liquid chromatography-photodiode array-mass spectrometry (UHPLC-PDA-MS) technique for 11 flavonoids in Genkwa Flos was utilized through the entire analytical processes, from the risk assessment study to the factor screening test, and finally in method optimization employing central composite design (CCD). In this approach, column temperature and mobile solvent slope were found to be critical method parameters (CMPs) and each of the eleven flavonoid peaks’ resolution values were used as critical method attributes (CMAs) through data mining conversion formulas. An optimum chromatographic method in the design space was calculated by mathematical and response surface methodology (RSM). The established chromatographic condition is as follows: acetonitrile and 0.1% formic acid gradient elution (0–13 min, 10–45%; 13–13.5 min, 45–100%; 13.5–14 min, 100–10%; 14–15 min, 10% acetonitrile), column temperature 28℃, detection wavelength 335 nm, and flow rate 0.35 mL/min using C18 (50 × 2.1 mm, 1.7 μm) column. A validation study was also performed successfully for apigenin 7-O-glucuronide, apigenin, and genkwanin. A few important validation results were as follows: linearity over 0.999 coefficient of correlation, detection limit of 2.87–22.41, quantitation limit of 8.70–67.92, relative standard deviation of precision less than 0.22%, and accuracy between 100.13 and 102.49% for apigenin, genkwanin, and apigenin 7-O-glucuronide. In conclusion, the present design-based approach provide a systematic platform that can be effectively applied to ensure pharmaceutically qualified analytical data from complex natural products based botanical drug.

DEVELOPMENT AND STATISTICAL OPTIMIZATION OF GASTRORETANTIVE FLOATING MICROSPHERES OF PREGABALIN PREPARED BY W/O/O MULTIPLE EMULSION METHOD

Objective: The aim of this study is to develop a gastroretentive microsphere of pregabalin using design of experiment (DoE) to decrease dosing frequency and increasing bioavailability. Methods: Pregabalin microsphere was prepared by W/O/O multiple emulsion method using a mixture of ethyl cellulose (EC) and polyvinyl pyrrolidone (PVP) as rate-controlling polymer. Mixed solvent system comprising of dichloromethane (DCM) and acetonitrile (ACN) and light liquid paraffin was chosen as primary and secondary oil phase respectively. Taguchi design was employed for factor screening and Box Behnken design was used for the optimisation of critical process parameters. Results: Taguchi design revealed that polymer: drug, DCM: ACN and PVP: EC is the critical factor for the preparation of microspheres. The optimized formulation was prepared using polymer: drug (4.95:1), DCM: ACN (53.76: 46.24) and PVP: EC (2:5) which showed mean particle size of 203.34±4.82 µm, practical yield of 87.52±2.91 %, encapsulation efficiency of 96.43±3.14 %, floating ability up to 90.42±1.64 % and T60% of 332.81±5.84. Drug release from microsphere followed Higuchi Kinetic model. Conclusion: In a nutshell, microspheres with excellent flowability and great encapsulation efficiency were successfully developed. These can be useful in improving patient compliance by reducing frequent dosing.