Current Pharmaceutical Analysis - Volume 17, Issue 4, 2021

Background: Quality by design-based software’s in analytical research and development normally encompasses multiple objectives. For decades, this task has been attempted through trial and error, supplemented with the previous experience, knowledge, and wisdom of analytical researchers. Objective: The study analyzes the current QbD-assisted software’s, such as design-experts, minitab, fusion product development, etc., and its broad implementations in an analytical research and development. Methods: The traditional approach may fails to meet the intended purpose by trial and error procedure during analytical research and development. However, modern scientific technology is equipped with highly advanced features associated with the software of the QbD paradigm. The impact and interactions between the critical process variables and critical method attributes such as resolution, tailing, etc. can be well understood by the screening, optimization, and robustness studies based on the principles of experimental design. Results: The design of experiments assimilate statistical multi-variate analysis instead of one factor at a time approach. This also provides a prominent, most reliable quality output, which is also essential for getting highly robust method as well as to obtain homogenous product development. Conclusion: The present review, critically discussed about the various QbD based multivariate software and their applications in drug development and analytical research.

Background: Metabolomics is one of the main areas to understand cellular process at molecular level by analyzing metabolites. In recent years metabolomics has emerged as a key tool to understand molecular basis of diseases, to find diagnostic and prognostic biomarkers and develop new treatment opportunities and drug molecules. Objective: In this study, untargeted metabolite and lipid analysis were performed to identify potential biomarkers on premature ovarian insufficiency plasma samples. 43 POI subject plasma samples were compared with 32 healthy subject plasma samples. Methods: Plasma samples were pooled and extracted using chloroform:methanol:water (3:3:1 v/v/v) mixture. Agilent 6530 LC/MS Q-TOF instrument equipped with ESI source was used for analysis. A C18 column (Agilent Zorbax 1.8 μM, 50 x 2.1 mm) was used for separation of the metabolites and lipids. XCMS, an “R software” based freeware program, was used for peak picking, grouping and comparing the findings. Isotopologue Parameter Optimization (IPO) software was used to optimize XCMS parameters. The analytical methodology and data mining process were validated according to the literature. Results: 83 metabolite peaks and 213 lipid peaks were found to be in semi-quantitatively and statistically different (fold change >1.5, p <0.05) between the POI plasma samples and control subjects. Conclusion: According to the results, two groups were successfully separated through principal component analysis. Among the peaks, phenyl alanine, decanoyl-L-carnitine, 1-palmitoyl lysophosphatidylcholine and PC(O-16:0/2:0) were identified through auto MS/MS and matched with human metabolome database and proposed as plasma biomarker for POI and monitoring the patients in treatment period.

Background: Drugs based on natural products targeting the microtubule system remain an important component in cancer therapy. Compound 10, 4-((3-amino-4-methoxyphenyl) amino)-2Hcoumarin, derived from coumarin, showed excellent anti-proliferative activity through directly binding to the colchicine-binding site in β-tubulin, suggesting that it could be a perfect drug candidate for antitumor drug research and development. Identification and structural characterization of metabolites is a critical step of both drug discovery and development research. Objective: Compound 10, 4-((3-amino-4-methoxyphenyl) amino)-2H-coumarin, derived from coumarin. Method: In this study, an efficient and sensitive method using Ultra High-Performance Liquid Chromatography couple with Quadrupole Time of Flight tandem Mass Spectrometry (UHPLC/QTOF/ MS/MS) was successfully established and applied to identify the in vivo metabolites in plasma, urine and feces samples of rats after intravenous administration of Compound 10 with a single dose of 10 mg/kg. Result: A total of eight metabolites (including two phase I and six phase II metabolites) had been detected or tentatively identified in plasma, urine and feces, indicating the prominent metabolic pathways were glucuronidation, demethylation and hydroxylation. In addition, in order to understand the structure of metabolites more accurately, synthesis strategy was used to confirm the metabolite M3. Conclusion: The present study provides important information on the metabolism of Compound 10 in vivo for the first time, which would be helpful for understanding the potential metabolic processes of Compound 10 and paving the way for pharmacology and toxicology research.

Objective: The objective of the method was to develop a new, simple and reliable enantioselective Reverse Phase- Ultra-Fast Liquid Chromatography (RP-UFLC) method for the separation of Atenolol enantiomers. A comprehensive study was performed by extending the work to pharmacokinetic studies using rabbit plasma. Background: Many methods were reported for enantioseparation of Atenolol enantiomers but no attempts were made for chiral separation of Atenolol using rabbit plasma. Moreover, pharmacokinetic data to prove the efficiency of particular enantiomers in rabbit plasma was not studied. Methods: In the present examination, the binary RP-UFLC technique was developed on Phenomenex® Lux cellulose i5 segment (150×4.6 mm, 5μ) using di-sodium hydrogen phosphate buffer (pH 6.8): acetonitrile (35:65 v/v) as the mobile phase. Results: The elution of Atenolol was observed at 225 nm with a stream rate of 1 mL.min^-1. The described technique offered a linear relationship with a regression coefficient of r² = 0.997 and 0.996 for (R) and (S)-enantiomer respectively, between the concentration range of 2-10 ng.mL^-1. Atenolol enantiomers were detected at a retention time (tR) of 2.7 min and 3.10 min for R and S-enantiomer respectively. The rate of recovery of both Atenolol enantiomers was observed to be (R) 98.18% and (S) 100.45% individually. USFDA guidelines May 2018 were systematically followed for the development and validation of the bioanalytical method. Conclusion: The developed technique was applied for the separation of Atenolol enantiomers and for the pharmacokinetic determination of Atenolol enantiomers in rabbit plasma.

Background: Pyrogens are fever-inducing substances and pyrogen detection is mandatory in parenteral pharmaceuticals. Succinylated Gelatin Injection (SGI) is a biopharmaceutical product, containing multi-component, and it is administered parenterally. Objective: The study aimed to assess pyrogen in SGI and to evaluate the feasibility of the Monocyte Activation Test (MAT) for pyrogen detection in a multi-component pharmaceutical product. Method: In the present study, the Bacterial Endotoxin Test (BET) and the Monocyte Activation Test (MAT) were employed to assess pyrogen in SGI. The MAT method was developed on the basis of the HL-60/IL-6 assay. HL-60 cells were incubated with lipopolysaccharide (LPS) standards and sample solutions. The endotoxin produced by the incubation, interleukin-6 (IL-6), was measured by ELISA. The MAT method was validated and main parameters were investigated. Finally, the pyrogenicity of SGIs from two different enterprises was determined by the developed MAT method. Results: The BET failed in the test for interfering factors and the MAT was proved suitable for the pyrogen detection of SGI. All the products examined showed negative results in the pyrogen detection test. Conclusion: The MAT method is feasible in pyrogen detection of SGI. It can be applied in pyrogen detection for quality and safety control of multi-component biological products.

Background: Although chromatography and spectrometry-based methods have been used to analyse phenolic acids in Chinese traditional medicine Salvia miltiorrhiza Bunge (SMB), quantitative nuclear magnetic resonance (qNMR) has never previously been used to analyse fresh SMB root extracts. Objective: To establish a fast and simple method of quantitating danshensu, lithospermic acid, rosmarinic acid, and salvianolic acid B content in fresh SMB root using ¹H-NMR spectroscopy. Method: Fresh SMB root was extracted using a 70% methanol aqueous solution and quantitatively analysed for danshensu, lithospermic acid, rosmarinic acid, and salvianolic acid B using ¹H-NMR spectroscopy. Different internal standards were compared and the results were validated using highperformance liquid chromatography. Results: The established method was accurate and precise with good recovery. The LOD and LOQ indicated the excellent sensitivity of the method. The robustness was testified by the modification of four different parameters, and the differences among each parameter were all less than 2%. Conclusion: qNMR offers a fast, reliable, and accurate method of identifying and quantifying danshensu, lithospermic acid, rosmarinic acid, and salvianolic acid B in fresh SMB root extracts.

Introduction: Dryopteris marginalis L. is a local plant in Bangladesh which is widely used as folk medicine. Materials and Methods: In this study, the chemical composition of essential oil of D. marginalis L. as well as the antioxidant activity, antibacterial activity and total phenolic content of various organic extracts were analyzed. The gas chromatography mass spectrometry analysis was used to identify the chemical compositions. The antibacterial activity of different extracts of Dryopteris marginalis was examined against three gram-negative bacteria and two gram-positive bacteria. The antibacterial activity of different extracts was investigated using the disc diffusion method and minimum inhibitory concentration showing important antibacterial activity and was measured by using the broth dilution method. Results: The antioxidant activity was determined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and superoxide radical scavenging assay. The total phenolic portion of the extracts was ascertained by Folin-Ciocalteu in an alkaline medium. Twenty-two volatile compounds were identified from the essential oil. The methanol extract exhibited a significant inhibitory effect which was close to the value of standard antibiotic (Streptomycin). The zones of inhibition of different organic extracts against the tested bacteria were found in the range of (7-15) mm and the Minimum Inhibitory Concentrations (MIC) were recorded (15.62-1000) μg/ml. The antioxidant activity of D. marginalis was (82.13+1.2) % and (59.21+0.45) % highest activity with DPPH and superoxide radical scavenging assay, respectively. Conclusion: The methanol extract showed the highest (147.43+3.1) mg/g GAE of total phenolic content. The extracts and essential oil of D. marginalis showing strong antibacterial activity can be considered as a potential source of natural medicine and the ingredient of modern therapeutic agents.

Background: The indole-containing aroylhydrazone derivatives 3a-c with potent antimycobacterial activity against a referent strain M. tuberculosis H37Rv and low cytotoxicity were evaluated for their stability via the precise and accurate HPLC analytical method in aqueous media of different pH (2.0, 7.0, 9.0 and 12.0). Objective: The study describes the development and validation of a simple and reliable HPLC-UV procedure for the determination of aroylhydrazone derivatives and their hydrolytic stability. Additionally, to recognize if hydrolysis leads to generating undesired products, the degradation processes were identified. Method: The separation was achieved with a LiChrosorb®RP-18 (250 x 4.6 mm) column, at ambient temperature with isocratic mode with mobile phase containing mixture of component A (acetonitrile) and component B (0.001M NaH2PO4, with 5 mM 1-heptane sulfonic acid sodium salt, adjusted to pH 3.0) in a ratio 60:40 (v/v). The flow rate was 1.0 ml/min and the eluent was monitored at 297 nm. The proposed method was validated as per ICH guidelines. Result: The obtained results showed that the compounds were sensitive to hydrolytic decomposition in aqueous media, resulting in the splitting of the hydrazone bond. Rapid hydrolysis of substances was observed in the acid medium. The elevated temperature significantly accelerated the hydrolytic reaction. Relatively slow hydrolysis of 3a-c was observed in a neutral solution and aqueous solutions buffered to pH 9. The hydrolysis of 3a-c in neutral, alkaline and strong alkaline medium followed the pseudo- first-order reaction rate and showed a linear dependence of lnC versus time. Conclusion: A validated high-performance liquid chromatographic assay for the determination of the hydrolytic stability of a series of aroylhydrazones was developed and optimized for the first time. The methods devised are successfully applicable to the development of pharmaceutical formulations.

Background: Curcumin (CUR), a natural isolated compound from turmeric, helps in fighting many diseases, but the broad application of curcumin has been limited ascribed to low bioavailability. Objective: The aim of this study is to pursue the enhancement of curcumin bioavailability through coadministration of vitamin C. Methods: Such purpose was achieved through the analysis of curcumin pharmacokinetics by highperformance liquid chromatography coupled with electrospray ionization - tandem mass spectrometry (HPLC - ESI - MS/MS). The plasma was separated on a C18 reverse-phase column using acetonitrile and ammonium formate solution (pH 6.5; 2.0 mM) at 0.8 mL/min. MS/MS detection was carried out in negative mode using mass patterns of m/z 367.0 > 216.7 for curcumin and m/z 265.2 > 223.9 for internal standard (honokiol). Results: Successful application of the proposed method in the pharmacokinetic study presented clear changes in key pharmacokinetic parameters, including the growth of AUC (0-t) up to 2.4 times, a 2.2- fold increase of Cmax, 2.2-fold loss of CL, and 1.5-fold diminishment of t1/2. Conclusion: An HPLC-ESI-MS/MS method for the determination of curcumin in rat plasma and validated the improvement of bioavailability of curcumin through co-administration of vitamin C was determined. These changes were reasoned to the inhibition of lipid peroxidation induced by the use of vitamin C. Such a simple strategy is possible to become an alternative for enhancing curcumin efficiency in practice.

Introduction: In this study, a precise, rapid and accurate ultra-performance liquid chromatography- tandem mass spectrometry (UPLC-MS/MS) method for the quantification of zhebeirine in mouse blood was developed, and pharmacokinetics of zhebeirine was studied for the first time after intravenous and oral administration. Methods: The mobile phase consisted of acetonitrile-0.1% formic acid, with a flow rate at 0.4 mL/m during 4 min run time. MRM modes of m/z 414.5→81.0 for zhebeirine and m/z 430.2→412.2 for 3- dehydroverticine (internal standard) were utilized to perform quantitative analysis. Protein in mouse blood was directly precipitated with acetonitrile for sample preparation. Results: The linear range was 1-3000 ng/mL with r>0.995, and LLOQ was 1 ng/mL. The intra-and inter-day precision of zhebeirine in mouse blood was less than 13%. The accuracy ranged from 91.2% to 112.5%, while the matrix effects were between 84.8% and 106.4%. Conclusion: The UPLC-MS/MS was successfully applied to the pharmacokinetic study on zhebeirine after intravenous and oral administration, and the bioavailability was determined to be 22.8%.

Background: A simple quantification technique by liquid chromatography electrospray ionization- tandem mass spectrometry (LC-ESI-MS/MS) is required for regorafenib in biological matrices with bioavailability studies in healthy rabbits, when compared with reported techniques. Objective: The main aim of the research work was to develop a validated LC-ESI-MS/MS technique for the quantification of regorafenib and application to bioavailability studies in healthy rabbits. Methods: Chromatographic separation was achieved with hypersil-C18 analytical column (50mm×4.6 mm, 4μm) and mobile phase composition of acetonitrile and 5mM ammonium acetate in the proportion of 70:30. The mobile phase was infused into the column with high pressure to get a 0.7 ml/min flow rate. The total retention time of the analyte is promising when compared with the existed methods for regorafenib. Quantitation was processed by monitoring transitions of m/z -483.0/262.0 and 450.0/260.0 for regorafenib and internal standard respectively in multiple reaction monitoring. Results: The linearity equation and correlation coefficient (R2) findings were y =0.9948x+2.6624 and 0.998 respectively. The intra and inter-day precision of the developed technique was found between 1.00 – 8.50% for the QC-samples (2, 4, 240 and 480ng/ml). From bioavailability study, the drug was shown Tmax of 3.688 ± 0.754; average AUC0→α and AUC0→t were 6476.81 ± 259.59 and 6213.845 ± 257.892 respectively and Cmax was found to be 676.91 ± 22.045 in healthy rabbits. Conclusion: The developed technique was validated and successfully applied in the pharmacokinetic study of the drug (40 mg tablet) administered through the oral route in healthy rabbits.

Introduction: Impurity analysis is an important step in the quality control of pharmaceutical ingredients and the final product. Impurities can arise from drug synthesis or excipients and even at small concentrations may affect product efficacy and safety. In this work, two methods using highperformance liquid chromatography (HPLC) were developed and validated for the evaluation of besifloxacin and its impurity synthesis, with isocratic elution and another with gradient elution. Method: The analysis by HPLC in isocratic elution mode was performed using a cyano column maintained at 25 °C. The mobile phase was composed of 0.5% triethylamine (pH 3.0): acetonitrile (88:12 v/v) eluted at a flow rate of 1.0 mL/min with detection at 330 nm. The gradient elution method was carried out with the same column and mobile phase components only modifying the rate between organic and aqueous phase during analysis. The procedures have been validated according to internationally accepted guidelines, observing results within acceptable limits. Results: The methods presented were found to be linear in the 140 to 260 μg/ mL range for besifloxacin and 0.3 to 2.3 μg/ mL for an impurity named A. The limits of detection and quantification were, respectively, 0.07 and 0.3 μg/ mL for impurity A, with a 20 μL injection volume. The precision achieved for all analyses performed provided RSD inter-day equal to 6.47 and 6.36% for impurity A with isocratic elution and gradient, respectively. The accuracy was higher than 99% and robustness exhibited satisfactory results. In the isocratic method, an analysis time of 25 min and 15 min was obtained for the gradient. For impurity A, the number of theoretical plates in the isocratic mode was about 5000 while in the gradient mode it was about 45000, hence, it made the column more efficient by changing the mobile phase composition during elution. In besifloxacin raw material and in the pharmaceutical product used in this study, other related impurities were present but impurity A was searched for and not detected. Conclusion: The proposed methods can be applied for the quantitative determination of impurities in the analysis of the besifloxacin raw material, as well as in ophthalmic suspension of the drug, considering the quantitation limit.