An Experimental Design Approach to Quantitative Expression for Quality Control of a Multicomponent Antidiabetic Formulation by the HILIC Method

A rapid and reproducible hydrophilic liquid chromatography (HILIC) process was established for concomitant determination of remogliflozin etabonate (RE), vildagliptin (VD), and metformin (MF) in a formulation. A face-centered central composite experimental design was employed to optimize and predict the chromatographic condition by statistically studying the surface response model and design space with desirability close to one. A HILIC column with a simple mobile phase of acetonitrile (65% v/v) and 20 mM phosphate buffer (35% v/v, pH 6, controlled with orthophosphoric acid) was used to separate RE, VD, and MF. RE, VD, and MF were separated in 3.6 min using an isocratic mode mobile phase flow at a flow rate of 1.4 mL at room temperature, and the analytes were examined by recording the absorption at 210 nm. The developed HILIC method was thoroughly validated for all parameters recommended by ICH, and linearity was observed in the ranges 20–150 µg/mL, 10–75 µg/mL, and 50–750 µg/mL for RE, VD, and MF, respectively, along with excellent regression coefficients (r² > 0.999). The calculated percentage relative deviation and relative error ascertained the precision and accuracy of the method. The selectivity and accuracy were further confirmed by the high percentage recovery of added standard drugs to the formulation using the standard addition technique. The robustness of the HILIC processes was confirmed by developing a half-normal probability plot and Pareto chart, as the slight variation of a single factor had no significant influence on the assay outcomes. Utilization of the optimized HILIC procedure for concurrent quantification of RE, VD, and MF in solid dosage forms showed accurate and reproducible results. Hence, the fast HILIC method can be regularly employed for the quality assurance of pharmaceutical preparations comprising RE, VD, and MF.     

Development and Validation of a Novel HPLC Method to Analyse Metabolic Reaction Products Catalysed by the CYP3A2 Isoform: In Vitro Inhibition of CYP3A2 Enzyme Activity by Aspirin (Drugs Often Used Together in COVID-19 Treatment)

Aspirin (also known as acetylsalicylic acid) is a drug intended to treat fever, pain, or inflammation. Treatment of moderate to severe cases of COVID-19 using aspirin along with dexamethasone has gained major attention globally in recent times. Thus, the purpose of this study was to use High-Performance Liquid Chromatography (HPLC) to evaluate the in vitro inhibition of CYP3A2 enzyme activity using aspirin in rat liver microsomes (RLMs). In this study, an efficient and sensitive HPLC method was developed using a reversed phase C18 column (X Bridge 4.6 mm × 150 mm, 3.5 µm) at 243 nm using acetonitrile and water (70:30 v/v). The linearity (r² > 0.999), precision (<15%), accuracy and recovery (80–120%), limit of detection (5.60 µM and 0.06 µM), limit of quantification (16.98 µM and 0.19 µM), and stability of the newly developed method were validated for dexamethasone and 6β-hydroxydexamethasone, respectively, following International Conference on Harmonization (ICH) guidelines. This method was applied in vitro to measure CYP3A2 activity. The results showed that aspirin competitively inhibits 6β-hydroxylation (CYP3A2 activity) with an inhibition constant (Ki) = 95.46 µM and the concentration of the inhibitor causing 50% inhibition of original enzyme activity (IC₅₀) = 190.92 µM. This indicated that there is a minimal risk of toxicity when dexamethasone and aspirin are co-administrated and a very low risk of toxicity and drug interaction with drugs that are a substrate for CYP3A2 in healthcare settings.     

Development of Improved High-Performance Liquid Chromatography Method for the Determination of Residual Caprylic Acid in Formulations of Human Immunoglobulins

Quality control of human immunoglobulin formulations produced by caprylic acid precipitation necessitates a simple, rapid, and accurate method for determination of residual caprylic acid. A high-performance liquid chromatography method for that purpose was developed and validated. The method involves depletion of immunoglobulins, the major interfering components that produce high background noise, by precipitation with acetonitrile (1:1, v/v). Chromatographic analysis of caprylic acid, preserved in supernatant with no loss, was performed using a reverse-phase C18 column (2.1 × 150 mm, 3 μm) as a stationary phase and water with 0.05% TFA–acetonitrile (50:50, v/v) as a mobile phase at a flow rate of 0.2 mL/min and run time of 10 min. The developed method was successfully validated according to the ICH guidelines. The validation parameters confirmed that method was linear, accurate, precise, specific, and able to provide excellent separation of peaks corresponding to caprylic acid and the fraction of remaining immunoglobulins. Furthermore, a 2⁴⁻¹ fractional factorial design was applied in order to test the robustness of developed method. As such, the method is highly suitable for the quantification of residual caprylic acid in formulations of human immunoglobulins for therapeutic use, as demonstrated on samples produced by fractionation of convalescent anti-SARS-CoV-2 human plasma at a laboratory scale. The obtained results confirmed that the method is convenient for routine quality control.     

Development and Validation of the UPLC-DAD Methodology for the Detection of Triterpenoids and Phytosterols in Fruit Samples of Vaccinium macrocarpon Aiton and Vaccinium oxycoccos L.

Cranberries are used in the production of medicinal preparations and food supplements, which highlights the importance of triterpene compounds determination in cranberry fruit raw material. The aim of our study was to develop and validate for routine testing suitable UPLC-DAD methodology for the evaluation of triterpene acids, neutral triterpenoids, phytosterols, and squalene content in cranberry samples. The developed and optimized UPLC-DAD methodology was validated according to the guidelines of the International Council for Harmonization (ICH), evaluating the following parameters: range, specificity, linearity (R² > 0.999), precision, LOD (0.27–1.86 µg/mL), LOQ (0.90–6.18 µg/mL), and recovery (80–110%). The developed and validated technique was used for the evaluation of triterpenic compounds in samples of Vaccinium macrocarpon and Vaccinium oxycoccos fruits, and their peels, pulp and seeds. The studied chromatogram profiles of Vaccinium macrocarpon and Vaccinium oxycoccos were identical but differed in the areas of the analytical peaks. Ursolic acid was the dominant compound in fruit samples of Vaccinium macrocarpon and Vaccinium oxycoccos. The highest amounts of triterpenic compounds were detected in the cranberry peels samples. The developed method for the detection of triterpene compounds can be applied in further studies for routine testing on the qualitative and quantitative composition of fruit samples of Vaccinium macrocarpon and Vaccinium oxycoccos species and cultivars.     

Development and Validation of Stability-Indicating HPLC Methods for the Estimation of Lomefloxacin and Balofloxacin Oxidation Process under ACVA,H2O2, or KMnO4 Treatment. Kinetic Evaluation and Identification of Degradation Products by Mass Spectrometry

The oxidation of lomefloxacin (LOM) and balofloxacin (BAL) under the influence of azo initiator of radical reactions of 4,4′-azobis(4-cyanopentanoic acid) (ACVA) and H₂O₂ was examined. Oxidation using H₂O₂ was performed at room temperature while using ACVA at temperatures: 40, 50, 60 °C. Additionally, the oxidation process of BAL under the influence of KMnO₄ in an acidic medium was investigated. New stability-indicating HPLC methods were developed in order to evaluate the oxidation process. Chromatographic analysis was carried out using the Kinetex 5u XB—C18 100A column, Phenomenex (Torrance, CA, USA) (250 × 4.6 mm, 5 μm particle size, core shell type). The chromatographic separation was achieved while using isocratic elution and a mobile phase with the composition of 0.05 M phosphate buffer (pH = 3.20 adjusted with o-phosphoric acid) and acetonitrile (87:13 v/v for LOM; 80:20 v/v for BAL). The column was maintained at 30 °C. The methods were validated according to the ICH guidelines, and it was found that they met the acceptance criteria. An oxidation process followed kinetics of the second order reaction. The most probable structures of LOM and BAL degradation products formed were assigned by the UHPLC/MS/MS method.     

Quantification of Zonisamide in Dried Blood Spots and Dried Plasma Spots by UPLC–MS/MS: Application to Clinical Practice

In this research, a UHPLC–MS/MS method was developed and validated for the determination of zonisamide in dried plasma spots (DPS) and dried blood spots (DBS). Detection of zonisamide and internal standard, 1-(2,3-dichlorphenyl)piperazine, was carried out in ESI+ mode by monitoring two MRM transitions per analyte. Total run time, less than 2.5 min, was achieved using Acquity UPLC BEH Amide (2.1 × 100 mm, 1.7 µm particle size) column with mobile phase comprising acetonitrile–water (85:15%, v/v) with 0.075% formic acid. The flow rate was 0.225 mL/min, the column temperature was 30 °C and the injection volume was 3 µL. Desolvation temperature, desolvation gas flow rate, ion source temperature and cone gas flow rate were set by the IntelliStart software tool in combination with tuning. All of the Guthrie cards were scanned, and DPS/DBS areas were determined by the image processing tool. The influence of hematocrit values (20–60%) on accuracy and precision was evaluated to determine the range within which method for DBSs is free from Hct or dependency is within acceptable limits. The validated method was applied to the determination of zonisamide levels in DPS and DBS samples obtained from patients confirming its suitability for clinical application. Finally, the distribution of zonisamide into the red blood cells was estimated by correlating its DPS and DBS levels.     

A Box–Behnken Extraction Design and Hepatoprotective Effect of Isolated Eupalitin-3-O-β-D-Galactopyranoside from Boerhavia diffusa Linn.

The objectives of this study were to optimize and quantify the maximum percentage yield of eupalitin-3-O-β-D-galactopyranosidefrom Boerhavia diffusa leaves using response surface methodology (RSM), as well as to demonstrate the hepatoprotective benefits of the bioactive compound. The Box–Behnken experimental design was utilized to optimize the eupalitin-3-O-β-D-galactopyranoside extraction procedure, which also looked at the extraction duration, temperature, and solvent concentration as independent variables. Boerhaviadiffusa leaves were extracted, and n-hexane, chloroform, ethyl acetate, and water were used to fractionate the dried extracts. The dried ethyl acetate fraction was thoroughly mixed in hot methanol and stored overnight in the refrigerator. The cold methanol was filtered, the solid was separated, and hot methanol was used many times to re-crystallize the solid to obtain pure eupalitin-3-O-β-D-galactopyranoside (0.1578% w/w). The proposed HPTLC method for the validation and quantification of eupalitin-3-O-β-D-galactopyranosidewassuccessfully validated and developed. The linearity (R₂ = 0.994), detection limit (30 ng), and quantification limit (100 ng) of the method, as well as its range (100–5000 ng), inter and intraday precision (0.67% and 0.991% RSD), specificity, and accuracy (99.78% RSD), were all validated as satisfactory. The separation of the eupalitin-3-O-β-D-galactopyranoside band was achieved on an HPTLC plate using toluene:acetone:water (5:15:1 v/v) as a developing system. The Box–Behnken statistical design was used to determine the best optimization method, which was found to be extraction time (90 min), temperature (45 °C), and solvent ratio (80% methanol in water v/v) for eupalitin-3-O-β-D-galactopyranoside. Standard silymarin ranged from 80.2% at 100 µg/mL to 86.94% at 500 µg/mL in terms of significant high hepatoprotection (cell induced with carbon tetrachloride 0.1%), whereas isolated eupalitin-3-O-β-D-galactopyranoside ranged from 62.62% at 500 µg/mL to 70.23% at 1000 µg/mL. More recently, it is a source of structurally unique flavonoid compounds that may offer opportunities for developing novel semi-synthetic molecules.     

Novel Fast Chromatography-Tandem Mass Spectrometric Quantitative Approach for the Determination of Plant-Extracted Phytosterols and Tocopherols

Phytosterols and tocopherols are commonly used in food and pharmaceutical industries for their health benefits. Current analysis methods rely on conventional liquid chromatography, using an analytical column, which can be tedious and time consuming. However, simple, and fast analytical methods can facilitate their qualitative and quantitative analysis. In this study, a fast chromatography-tandem mass spectrometric (FC-MS/MS) method was developed and validated for the quantitative analysis of phytosterols and tocopherols. Omitting chromatography by employing flow injection analysis—mass spectrometry (FIA-MS) failed in the quantification of target analytes due to analyte-to-analyte interferences from phytosterols. These interferences arise from their ambiguous MS fingerprints that would lead to false identification and inaccurate quantification. Therefore, a C18 guard column with a 1.9 µm particle size was employed for FC-MS/MS under isocratic elution using acetonitrile/methanol (99:1 v/v) at a flow rate of 600 µL/min. Analyte-to-analyte interferences were identified and eliminated. The false peaks could then be easily identified due to chromatographic separation. In addition, two internal standards were evaluated, namely cholestanol and deuterated cholesterol. Both internal standards contributed to the observed analyte-to-analyte interferences; however, adequate shift in the retention time for deuterated cholesterol eliminated its interferences and allowed for an accurate quantification. The method is fast (1.3 min) compared to published methods and can distinguish false peaks observed in FIA-MS. Seven analytes were quantified simultaneously, namely brassicasterol, campesterol, stigmasterol, β-sitosterol, α-tocopherol, δ-tocopherol, and γ-tocopherol. The method was successfully applied in the quantitative analysis of phytosterols and tocopherols present in the unsaponifiable matter of canola oil deodorizer distillate (CODD). β-sitosterol and γ-tocopherol were the most abundant phytosterols and tocopherols, respectively.     

The [DPPH●/DPPH-H]-HPLC-DAD Method on Tracking the Antioxidant Activity of Pure Antioxidants and Goutweed (Aegopodium podagraria L.) Hydroalcoholic Extracts

The 2,2-diphenyl-1-picrylhydrazyl (DPPH)-reverse phase (RP)-HPLC-diode array detector (DAD) method was tested on standard antioxidants (AOs), i.e., reduced glutathione (GSH), ascorbic acid (vitamin C), and alcoholic extracts of A. podagraria L. An elaborated HPLC procedure enabled the simultaneous measurement of the redox couple DPPH-R (2,2-diphenyl-1-picrylhydrazyl radical)/DPPH-H (2,2-diphenyl-1-picrylhydrazine). Both forms were fully separated (R_s = 2.30, α = 1.65) on a Zorbax Eclipse XDB-C18 column eluted with methanol–water (80:20, v/v) and detected at different wavelengths in the range of 200–600 nm. The absorbance increases of the DPPH-H as well as the DPPH-R peak inhibition were measured at different wavelengths in visible and UV ranges. The chromatographic method was optimized, according to reaction time (slow, fast kinetics), the linearity range of DPPH radical depending on the detection conditions as well as the kind of the investigated antioxidants (reference chemicals and the ground elder prepared from fresh and dry plants). The scavenging capacity was expressed by the use of percentage of peak inhibition and the IC₅₀ parameters. The evaluated extracts displayed antioxidant activity, higher than 20% inhibition against 350 µM DPPH free radical. The results show that extract prepared from dry plants in the ultrasonic bath exhibits the highest antioxidant potential (IC₅₀ = 64.74 ± 0.22 µL/mL).     

Development and Validation of a LC Method for the Determination of Pramipexole Using an Experimental Design

A rapid and sensitive RP-HPLC method with UV detection for routine control of pramipexole in tablets was developed. Chromatography was performed with mobile phase containing a mixture of acetonitrile/phosphate buffer (60/40; v/v) with a flow rate of 0.8 mL min⁻¹. Quantitation was accomplished with the internal standard method; the procedure was validated by linearity (correlation coefficient = 0.99892), accuracy, robustness and intermediate precision. Limit of quantitation and limit of detection were found to be 4.5 μg and 1.4 μg respectively, which indicates the method is highly sensitive. Experimental design was used during validation to calculate method robustness and intermediate precision, for robustness test three factors were considered; percentage v/v of acetonitrile, flow rate and pH; an increase in the flow rate results in a decrease of concentration found of the drug, while the percentage of organic modifier and temperature have no important effect on the response. For intermediate precision measure the considered variables were: analyst, equipment, days and obtained RSD value (0.56%, n=24) which indicated a good precision of the analytical method. The method was found to be applicable for determination of the drug in tablet formulations and the results of the developed method were compared with those of the UV spectrophotometric method to access the active pramipexole content. Revised: 13 March and 25 April 2006     

Thermodynamic and Computational (DFT) Study of Non-Covalent Interaction Mechanisms of Charge Transfer Complex of Linagliptin with 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and Chloranilic acid (CHA)

This study describes the non-covalent interactions of the charge transfer complex (CT), which was responsible for the synthesis of Linagliptin (LNG) with 2,3-Dichloro-5,6-Dicyano-1,4-benzoquinone (DDQ), or with Chloranilic acid (CHA) complexes in acetonitrile (MeCN) at temperatures of (25 ± 2 °C). Then, a UV–Vis spectrophotometer was utilized to identify Linagliptin (LNG) from these complexes. For the quantitative measurement of Linagliptin in bulk form, UV–Vis techniques have been developed and validated in accordance with ICH criteria for several aspects, including selectivity, linearity, accuracy, precision, LOD, LOQ, and robustness. The optimization of the complex synthesis was based on solvent polarization; the ratio of molecules in complexes; the association constant; and Gibbs energy (ΔG°). The experimental work is supported by the computational investigation of the complexes utilizing density functional theory as well as (QTAIM); (NCI) index; and (RDG). According to the optimized conditions, Beer’s law was observed between 2.5–100 and 5–100 µM with correlation coefficients of 1.9997 and 1.9998 for LGN-DDQ and LGN-CHA complexes, respectively. For LGN-DDQ and LGN-CHA complexes, the LOD and LOQ were (1.0844 and 1.4406 μM) and (3.2861 and 4.3655 μM), respectively. The approach was successfully used to measure LGN in its bulk form with high precision and accuracy.     

Quality by Design approach in the development of a solvent-modified micellar electrokinetic chromatography method: Finding the design space for the determination of amitriptyline and its impurities

A solvent-modified micellar electrokinetic chromatography method was set up for the simultaneous determination of the tricyclic antidepressant amitriptyline (AMI) and its main impurities. The method was developed following Quality by Design (QbD) principles according to ICH Guideline Q8(R2). QbD approach made it possible to find the design space (DS), where quality was assured. After a scouting phase, aimed at selecting a suitable capillary electrophoresis pseudostationary phase, risk assessment tools were employed to define the critical process parameters (CPPs) to be considered in a screening phase (applied voltage, concentration and pH of the background electrolyte, concentration of the surfactant sodium dodecyl sulphate, of the cosurfactant n-butanol and of the organic modifiers acetonitrile and urea). The effects of the seven selected CPPs on critical quality attributes (CQAs), namely resolution values between critical peak pairs and analysis time, were investigated throughout the knowledge space by means of a symmetric screening matrix. Response surface study was then carried out on four selected CPPs by applying a Doehlert Design. Monte-Carlo simulations were performed in order to estimate the probability of meeting the desired specifications on CQAs, and thus to define the DS by means of a risk of failure map. Additional points at the edges of the DS were tested in order to verify the requirements for CQAs to be fulfilled. A control strategy was implemented by defining system suitability tests. The developed method was validated following ICH Guideline Q2(R1), including robustness assessment by Plackett–Burman design, and was applied to the analysis of real samples of amitriptyline coated tablets.     

Determination of Chiral Impurity of Naproxen in Different Pharmaceutical Formulations Using Polysaccharide-Based Stationary Phases in Reversed-Phased Mode

A novel, validated, reversed-phase (RP), chiral high performance liquid chromatography (HPLC) method was developed for the enantiopurity control analysis of naproxen, a frequently used non-steroidal anti-inflammatory agent using polysaccharide-type chiral stationary phase (CSP). In the screening phase of method development, seven columns were tested in polar organic (PO) mode using mobile phases consisting of 0.1% acetic acid in methanol, ethanol, 2-propanol, and acetonitrile. Enantiorecognition was observed only in five cases. The best enantioseparation was observed on a Lux Amylose-1 column with 0.1% (v/v) acetic acid in ethanol with a resolution (R_s) of 1.24. The enantiomer elution order was unfavorable, as the distomer eluted after the eutomer. When the ethanolic mobile phase was supplemented with water, enantiomer elution order reversal was observed, indicating a difference in the enantiorecognition mechanism upon switching from PO to RP mode. Furthermore, by changing ethanol to methanol, not only lower backpressure, but also higher resolution was obtained. Subsequent method optimization was performed using a face-centered central composite design (FCCD) to achieve higher chiral resolution in a shorter analysis time. Optimized parameters offering baseline separation were as follows: Lux Amylose-1 stationary phase, thermostated at 40 °C, and a mobile phase consisting of methanol:water:acetic acid 85:15:0.1 (v/v/v), delivered with 0.65 mL/min flow rate. Using these optimized parameters, a R_s = 3.21 ± 0.03 was achieved within seven minutes. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of different pharmaceutical preparations, such as film-coated tablets and gel, as well as fixed-dose combination tablets, containing both naproxen and esomeprazole.     

Development and Validation for Quantification of 7-Nitroso Impurity in Sitagliptin by Ultraperformance Liquid Chromatography with Triple Quadrupole Mass Spectrometry

The purpose of this research study was to develop an analytical method for the quantification of 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4] triazolo [4,3-a] pyrazine (7-nitroso impurity), which is a potential genotoxic impurity. Since sitagliptin is an anti-diabetic medication used to treat type 2 diabetes and the duration of the treatment is long-term, the content of nitroso impurity must be controlled by using suitable techniques. To quantify this impurity, a highly sensitive and reproducible ultraperformance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-MS/MS) method was developed. The analysis was performed on a Kromasil-100, with a C18 column (100 mm × 4.6 mm with a particle size of 3.5 µm) at an oven temperature of approximately 40 °C. The mobile phase was composed of 0.12% formic acid in water, with methanol as mobile phases A and B, and the flow rate was set to 0.6 mL/min. The method was validated according to the current International Council for Harmonisation (ICH) guidelines with respect to acceptable limits, specificity, reproducibility, accuracy, linearity, precision, ruggedness and robustness. This method is useful for the detection of the impurity at the lowest limit of detection (LOD), which was 0.002 ppm, and the lowest limit of quantification (LOQ), which was 0.005 ppm. This method was linear in the range of 0.005 to 0.06 ppm and the square of the correlation coefficient (R²) was determined to be > 0.99. This method could help to determine the impurity in the regular analysis of sitagliptin drug substances and drug products.     

Quality-by-Design-Based Development of n-Propyl-Gallate-Loaded Hyaluronic-Acid-Coated Liposomes for Intranasal Administration

The present study aimed to develop n-propyl gallate (PG)-encapsulated liposomes through a novel direct pouring method using the quality-by-design (QbD) approach. A further aim was to coat liposomes with hyaluronic acid (HA) to improve the stability of the formulation in nasal mucosa. The QbD method was used for the determination of critical quality attributes in the formulation of PG-loaded liposomes coated with HA. The optimized formulation was determined by applying the Box–Behnken design to investigate the effect of composition and process variables on particle size, polydispersity index (PDI), and zeta potential. Physiochemical characterization, in vitro release, and permeability tests, as well as accelerated stability studies, were performed with the optimized liposomal formulation. The optimized formulation resulted in 90 ± 3.6% encapsulation efficiency, 167.9 ± 3.5 nm average hydrodynamic diameter, 0.129 ± 0.002 PDI, and −33.9 ± 4.5 zeta potential. Coated liposomes showed significantly improved properties in 24 h in an in vitro release test (>60%), in vitro permeability measurement (420 μg/cm²) within 60 min, and also in accelerated stability studies compared to uncoated liposomes. A hydrogen-peroxide-scavenging assay showed improved stability of PG-containing liposomes. It can be concluded that the optimization of PG-encapsulated liposomes coated with HA has great potential for targeting several brain diseases.     

Structural Elucidation of Alkali Degradation Impurities of Favipiravir from the Oral Suspension: UPLC-TQ-ESI-MS/MS and NMR

A novel stability-indicating, reversed-phase, high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination of favipiravir in an oral suspension. The effective separation of favipiravir and its degradation products was achieved on a Zorbax Eclipse Plus C18 column (5 μm particle size, 150 mm length × 4.6 mm diameter). The mobile phase was prepared by mixing 5 mM of phosphate buffer (pH 3.5) and methanol in a 75:25 v/v ratio delivered at a 1.0 mL/min flow rate. The eluents were monitored using a photodiode array detector at a wavelength of 322 nm. The stability-indicating nature of this method was evaluated by performing force degradation studies under various stress conditions, such as acidic, alkali, oxidative, thermal, and photolytic degradation. Significant degradation was observed during the alkali stress degradation condition. The degradation products generated during various stress conditions were well separated from the favipiravir peak. In addition, the major degradation product formed under alkali stress conditions was identified using UPLC-ESI-TQ-MS/MS and NMR. Method validation was performed according to the ICH Q2 (R1) guideline requirements. The developed method is simple, accurate, robust, and reliable for routine quality control analysis of favipiravir oral suspensions.     

Chemical Composition and Antioxidant,Antimicrobial, and Anti-Inflammatory Properties of Origanum compactum Benth Essential Oils from Two Regions: In Vitro and In Vivo Evidence and In Silico Molecular Investigations

The purposes of this investigatory study were to determine the chemical composition of the essential oils (EOs) of Origanum compactum from two Moroccan regions (Boulemane and Taounate), as well as the evaluation of their biological effects. Determining EOs’ chemical composition was performed by a gas chromatography–mass spectrophotometer (GC-MS). The antioxidant activity of EOs was evaluated using free radical scavenging ability (DPPH method), fluorescence recovery after photobleaching (FRAP), and lipid peroxidation inhibition assays. The anti-inflammatory effect was assessed in vitro using the 5-lipoxygenase (5-LOX) inhibition test and in vivo using the carrageenan-induced paw edema model. Finally, the antibacterial effect was evaluated against several strains using the disk-diffusion assay and the micro-dilution method. The chemical constituent of O. compactum EO (OCEO) from the Boulemane zone is dominated by carvacrol (45.80%), thymol (18.86%), and α-pinene (13.43%). However, OCEO from the Taounate zone is rich in 3-carene (19.56%), thymol (12.98%), and o-cymene (11.16%). OCEO from Taounate showed higher antioxidant activity than EO from Boulemane. Nevertheless, EO from Boulemane considerably inhibited 5-LOX (IC₅₀ = 0.68 ± 0.02 µg/mL) compared to EO from Taounate (IC₅₀ = 1.33 ± 0.01 µg/mL). A similar result was obtained for tyrosinase inhibition with Boulemane EO and Taounate EO, which gave IC_50s of 27.51 ± 0.03 μg/mL and 41.83 ± 0.01 μg/mL, respectively. The in vivo anti-inflammatory test showed promising effects; both EOs inhibit and reduce inflammation in mice. For antibacterial activity, both EOs were found to be significantly active against all strains tested in the disk-diffusion test, but O. compactum EO from the Boulemane region showed the highest activity. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for O. compactum EO from the Boulemane region ranged from 0.06 to 0.25% (v/v) and from 0.15 to 0.21% (v/v) for O. compactum from the Taounate region. The MBC/MIC index revealed that both EOs exhibited remarkable bactericidal effects.     

Phytochemical Analysis and Anti-Cancer Properties of Extracts of Centaurea castriferrei Borbás & Waisb Genus of Centaurea L

The Centaurea L. (Asteraceae) genus includes many plant species with therapeutic properties. Centaurea castriferrei Borbás & Waisb is one of the least known and least described plants of this genus. The aim of the study was the phytochemical analysis of water and methanol–water extracts (7:3 v/v) obtained from the aerial parts of the plant as well as evaluation of their anticancer activity. Quantitative determinations of phenolic compounds and flavonoids were performed, and the antioxidant potential was measured using the CUPRAC method. The RP-HPLC/DAD analysis and HPLC-ESI-QTOF-MS mass spectroscopy were performed, to determine the extracts’ composition. The antiproliferative activity of the obtained extracts was tested in thirteen cancer cell lines and normal skin fibroblasts using MTT test. Regardless of the extraction method and the extractant used, similar cytotoxicity of the extracts on most cancer cell lines was observed. However, the methanol–water extracts (7:3 v/v) contained significantly more phenolic compounds and flavonoids as well as showing stronger antioxidant properties in comparison to water extracts. Centaurea castriferrei Borbás & Waisb is a rich source of apigenin and its derivatives. In all tested extracts, chlorogenic acid and centaurein were also identified. In vitro research revealed that this plant may be a potential source of compounds with anticancer activity.     

Development and Validation of High-Throughput Bioanalytical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Newly Synthesized Antitumor Carbonic Anhydrase Inhibitors in Human Plasma

In the present study, a sensitive and fully validated bioanalytical high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitative determination of three newly synthesized carbonic anhydrases inhibitors (CAIs) with potential antitumor activity in human plasma. The analytes and the internal standard (IS) were extracted using 1.5 mL acetonitrile from only 450 µL aliquots of human plasma to achieve the desired protein precipitation. Chromatographic separations were achieved on Phenomenex Kinetex^® C₁₈ column (100 × 4.6 mm, 2.6 µm) using a binary gradient elution mode with a run time of less than 6 min. The mobile phase consisted of solvent (A): 0.1% formic acid in 50% methanol and solvent B: 0.1% formic acid in acetonitrile (30:70, v/v), pumped at a flow rate of 0.8 mL/min. Detection was employed using triple quadrupole tandem mass spectrometer (API 3500) equipped with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was selected for quantitation through monitoring the precursor-to-parent ion transition at m/z 291.9 → 173.0, m/z 396.9 → 225.1, m/z 388.9 → 217.0, and m/z 146.9 → 91.0 for AW-9a, WES-1, WES-2, and Coumarin (IS), respectively. Linearity was computed using the weighted least-squares linear regression method (1/x²) over a concentration range of 1–1000, 2.5–800, and 5–500 ng/mL for AW-9a, WES-1, and WES-2; respectively. The bioanalytical LC-MS/MS method was fully validated as per U.S. Food and Drug Administration (FDA) guidelines with all respect to linearity, accuracy, precision, carry-over, selectivity, dilution integrity, and stability. The proposed LC-MS/MS method was applied successfully for the determination of all investigated drugs in spiked human plasma with no significant matrix effect, which is a crucial cornerstone in further therapeutic drug monitoring of newly developed therapeutic agents.     

Characterization and Evaluation of Antimicrobial Potential of Trigonella incise (Linn) Mediated Biosynthesized Silver Nanoparticles

The goal of the research was to explore a new green method used to synthesize silver nanoparticles (Ag NPs) from an aqueous extract of Trigonella incise, which serves as a reducing and stabilizing agent. The obtained results showed an 85% yield of nanoparticles by using 2:5 (v/v) of 5% plant extract with a 0.5 M solution of AgNO₃. Different techniques were used to characterize the synthesized Ag NPs, including X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and UV–visible spectroscopy. The UV–visible spectra of green synthesized silver nanoparticles showed maximum absorption at a wavelength of 440 nm. The FT-IR studies revealed the stretching oscillation frequency of synthesized silver nanoparticles in the absorption band near 860 cm⁻¹. Similarly, the bending and stretching oscillation frequencies of the NH function group were assigned to the band in the 3226 cm⁻¹ and 1647 cm⁻¹ regions. The bending vibration of C-O at 1159 cm⁻¹ confirmed the carbonyl functional group that was also assigned to the small intensity band in the range of 2361 cm⁻¹. The X-ray diffraction analysis of Ag NPs revealed four distinct diffraction peaks at 2θ of 38°, 45°, 65° and 78°, corresponds to (111), (200), (220) and (311) of the face-centered cubic shape. The round shape morphology of Ag NPs with a mean diameter in the range 20–80 nm was analyzed via SEM images. Furthermore, the nanoparticles showed more significant antimicrobial activity against Salmonella typhi (S. typhi) and Staphylococcus aureus (S. aureus) with an inhibition zone of 21.5 mm and 20.5 mm at 6 μg/mL concentrations, respectively, once compared to the standard reference. At concentrations of 2 µg/mL and 4 µg/mL, all of the bacterial strains showed moderate activity, with inhibition zones ranging from 11 mm to 18.5 mm. Even at high concentrations of AgNPs, S. typhi showed maximum resistance. The best antifungal activity was observed by synthesized Ag NPs against Candida albicans (C. albicans) with 21 mm zone of inhibition, as compared to a standard drug which gives 22 mm of inhibition. Therefore, we conclude that the antibacterial and antifungal activities showed satisfactory results from the synthesized Ag NPs.