HPLC Method Research Articles

A novel QbD HPLC approach for the concurrent analysis of amoxicillin, amprolium, and ethopabate in food samples and dosage forms

A simple, rapid, and sensitive reversed-phase HPLC method coupled with photodiode array detection (PDA) was developed for the simultaneous estimation of three co-administered antibacterial drugs, amprolium, ethopabate, and amoxicillin for the first time. An analytical quality-by-design (QbD) approach was adopted to optimize the developed approach. A two-level full factorial design (25 FFD) was implemented to optimize how variable factors affected chromatographic responses. On a Hypersil BDS C18 column, the chromatographic separation was carried out using isocratic elution at 40.1 °C. The mobile phase consisted of methanol: potassium phosphate buffer containing 5.76 mM heptane sulphonate (9.05 mM; pH 3.32, 44.8:55.2 % v/v) pumped at a flow rate of 0.8 mL/min and an injection volume of 20.0 μL. In less than 7 min, the suggested method was able to separate the ternary mixture with a challenging ratio of 1: 15.6: 1 for amoxicillin, amprolium, and ethopabate, respectively. The calibration curves showed excellent linearity over the concentration ranges of 0.50–30.0, 0.30–30.0, and 0.05–20.0 μg/mL, respectively. Recovery percentages ranged from 90.30 % to 111.13 % with %RSD less than 2 % were obtained upon spiking to chicken tissues, liver, and egg samples, indicating the suitability of the proposed method for determining of the studied drugs in complicated food matrices. Furthermore, the proposed method was successfully applied to determine the studied drugs in their commercial formulations. Full validation was carried out according to ICHQ2 (R1) guidelines. Finally, the method's greenness and ecofriendliness were evaluated using various metrics tools, including the Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE).

Harnessing the hidden environmental power of Bjerkandera adusta laccase: Sustainable production, green immobilization, and eco-friendly decolorization of mixed azo dyes

This research unveils the untapped potential of laccase, derived from the Bjerkandera genus, utilizing it in an immobilized system aimed at detoxifying harmful azo dye effluents from the textile industry, thus contributing to environmental protection. Marking a pioneering achievement, we recorded the highest laccase activity at 94.52 U/g cultivating B. adusta's mycelium on brewer's spent grain, enhanced with lignocellulosic waste, under meticulously optimized conditions −2.69 g of alkali-pretreated beech sawdust, 0.91 g of cypress cone, and 8-day incubation period. The harvested laccase was subjected to an immobilization process on alkali-pretreated beech sawdust, where, through optimization, we established the ideal conditions (30 mg of carrier, pH 7, and 3.5 h), achieving an immobilization efficiency of 72.24% with a residual activity of 57.64%. Remarkably, both free and immobilized forms of the B. adusta TMF1 laccase enzyme demonstrated formidable efficacy in decolorizing a mix of three distinct azo dyes (Orange G, Eriochrome Black T, and Congo Red), eliminating over 63% of the coloration within just 30 min. The immobilized laccase showed consistent performance across four decolorization cycles. Moreover, the breakdown products of the azo dye mix were analyzed using the HPLC method, complemented by evaluations of potential antimicrobial activity, phytotoxicity and cytotoxicity, revealing a non-toxic composition without cytotoxicity, highlighting the process's safety for environmental release. The significance of this research is reflected in the distinguished construction of a green biocatalyst acting as a stable and time-efficient in remediation of targeted azo dye pollution from the textile industry following the principles of circular economy.

Study on Fingerprint and Multiple Indicators for Quantitative Detection Method Development in Powdered Milk Thistle Extract

Introduction: Silymarins are the main active ingredient in milk thistle extract, reflecting the quality of extract products. The detection methods for silymarins in different pharmacopoeia standards may vary, which may lead to inconsistent detection results. Method: Due to the lack of research on related fingerprint spectra, its qualitative work is hard to carry out. Hence, based on existing standard methods, an HPLC method was established by optimizing the gradient program of the mobile phase. Results: The effectiveness of this method and the fingerprint was also studied. The similarity of 31 batches of samples ranged from 0.791 to 1.000, indicating the characteristic spectrum formed can be well used for qualitative analysis. Conclusion: Under optimal analytical conditions, the six silymarin compounds in the samples can achieve good baseline separation from the matrix with good resolution. At the same time, the LOD was 25 mg/kg, and the recovery rates at three levels ranged from 92% to 108%, with RSDs less than 4%. The fingerprint spectrum and content determination method established in this study had good repeatability and high accuracy, which can provide a new method for quality control of milk thistle extract.

Development and Application of HPLC and UFLC Methods for the Analysis of Cardiovascular Drugs

Separation and identification of amiloride, metoprolol, hydrochlorothiazide, Carvedilol and amlodipine, in human plasma using the presented SPE and HPLC procedures was selective, efficient, robust, economical, environmentally friendly, and repeatable. Because plasma samples showed no evidence of a secondary peak, it was determined that the claimed drug-drug interaction between cardiovascular medications did not occur. Furthermore, no new peak was generated, indicating no metabolic product. This is the first report on isolating and identifying these nine cardiovascular medicines. Thus, SPE and HPLC techniques may be used to investigate these 5 cardiovascular medications. Successful monitoring of these medications in human plasma was achieved using the established SPE and HPLC technologies. The results of this inquiry were contrasted with those that had already been released. In comparison to other works in the literature, it was determined that the current study had the best complete baseline separation and lowest detection limit. The retention, separation, and resolution factors were discovered to have ranges of 0.07-9.14, 1.44-4.21, and 2.15-18.66, respectively. The SPE and UFLC techniques created and verified for this purpose were used to examine human plasma samples for the presence of cardiovascular drugs. Peak retention, separation, resolution, and symmetry all matched the reference samples' values. The results for symmetry, separation, and resolution were all in agreement with the reference samples. The methods used to separate and identify amiloride, metoprolol, hydrochlorothiazide; Carvedilol and Amlodipine in human plasma were found to be selective, effective, hardy, affordable, environmentally friendly, and reproducible, according to the authors. The selectivity of the SPE technique was validated by the absence of any secondary signal in the plasma samples. Additionally, there was no evidence of these drugs degrading in the plasma sample. This is the initial report on the simultaneous isolation and identification of these eight medications. The well-known SPE and UFLC techniques were successfully used to monitor these drugs in human plasma. Therefore, these drugs can be detected in any plasma sample using SPE or UFLC methods

Development and Application of HPLC and UFLC Methods for the Analysis of Anti Histaminic Drugs

Research focused on medications used to treat allergic reactions. The results of this inquiry were contrasted with those that had already been released. In comparison to other works in the literature, it was determined that the current study had the best complete baseline separation and lowest detection limit. The retention, separation, and resolution factors were discovered to have ranges of 0.07-9.14, 1.44-4.21, and 2.15-18.66, respectively. The SPE and UFLC techniques created and verified for this purpose were used to examine human plasma samples for the presence of antihistaminic drugs. Peak retention, separation, resolution, and symmetry all matched the reference samples' values. The results for symmetry, separation, and resolution were all in agreement with the reference samples. The methods used to separate and identify Phenylephrine, cetirizine, loratadine, montelukast, and Ebastine in human plasma were found to be selective, effective, hardy, affordable, environmentally friendly, and reproducible, according to the authors. The selectivity of the SPE technique was validated by the absence of any secondary signal in the plasma samples. Additionally, there was no evidence of these drugs degrading in the plasma sample. This is the initial report on the simultaneous isolation and identification of these eight medications. The well-known SPE and UFLC techniques were successfully used to monitor these drugs in human plasma. Therefore, these drugs can be detected in any plasma sample using SPE or UFLC methods.

Development and validation of stability indicating HPLC method for favipiravir used in the treatment of the Covid-19 disease

Development and validation of stability indicating HPLC method for favipiravir used in the treatment of the Covid-19 disease

Exploring environmental sustainability through dimension reduction algorithms: A cost-effective UV spectroscopic method for simultaneous determination of veterinary binary mixtures of doxycycline hydrochloride and tylosin tartrate

This study presents a cost-effective and environmentally friendly approach for the simultaneous determination of a veterinary binary mixture comprising doxycycline hydrochloride (DOX) and tylosin tartrate (TYZ) utilizing UV spectroscopy alongside dimension reduction algorithms (DRAs). Seventeen DRAs were evaluated, and their performances were compared based on four metrics: mean squared error (MSE), mean absolute error (MAE), median absolute error (MedAE), and coefficient of determination (R2). Based on the performance indices, Isomap algorithm demonstrated the highest predictive capacity among all DRAs. MSE, MAE, MedAE and R2 values of (0.38, 0.28, 0.19 and 0.999) and (0.08, 0.26, 0.22 and 0.998) were obtained for calibration and test datasets, respectively across a concentration range 4.67–30 μg mL−1 for DOX. MSE, MAE, MedAE and R2 values of (0.54, 0.34, 0.19 and 0.994) and (0.07, 0.19, 0.07 and 0.998) were obtained for calibration and test datasets, respectively across a concentration range 3.51–24 μg mL−1 for TYZ. The developed method underwent validation utilizing the accuracy profile approach. An ecological impact assessment was carried out employing six greenness evaluation tools: The Green Solvent Selection Tool (GSST), National Environmental Methods Index (NEMI), the Assessment of Green Profile (AGP), carbon footprint analysis, Analytical Greenness Calculator (AGREE), and Complementary Green Analytical Procedure Index (Complex GAPI). Additionally, we applied blueness and whiteness assessments using Blue Applicability Grade Index (BAGI) and Red-Green-Blue 12 (RGB 12) algorithms, respectively. The proposed method demonstrated higher GSST scores, a more "green" profile in NEMI, a superior AGP profile, and better environmental sustainability in Complex GAPI. The calculated carbon footprint value was 0.0002 kg CO2 equivalent per sample, The AGREE score was 0.87, BAGI was assessed at 72.5, and the whiteness assessment by the RGB12 algorithm was 89.6. Statistical comparison of the proposed method with a previously reported HPLC method for dosage form analysis revealed no significant differences at a 95% confidence level. This study highlights the novelty of combining UV spectroscopy with dimension reduction algorithms, offering significant advancements over traditional UV and chemometric methods for the analysis of these drugs. This approach not only enhances the efficiency and accuracy of determining active ingredients in pharmaceutical dosage forms but also contributes to environmental sustainability.

Physicochemical Characterization of the Oral Biotherapeutic Drug IMUNOR®.

IMUNOR is an oral biotherapeutic drug that had been developed, registered, and approved in 1997 in the Czech Republic and Slovakia. IMUNOR is a dialyzable leukocyte extract (DLE) prepared from swine leukocytes. It is characterized as a mixture of small peptides with molecular weights smaller than 12 kDa and a specific portion of nucleotides. The medical uses of IMUNOR include therapeutic applications within its registered range of indications, primarily for the treatment of immunodeficiencies, allergies, and certain acute or relapsing bacterial infections in adults and children. Despite the long-term clinical application of DLE, with strong evidence of positive therapeutic effects and no serious side effects, a detailed physicochemical specification of this mixture was lacking. We developed several methods for more in-depth physicochemical characterization of IMUNOR, including a spectrophotometric method for quantification of the total protein concentration and total DNA concentration in a mixture, several chromatographic methods for identification of individual components present in significant concentrations in IMUNOR, such as HPLC methods and the Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis method, and characterization of amino acid composition of this mixture. For the investigation of the variability among different batches of IMUNOR, five to nine representative batches from a standard manufacturing process on an industrial scale were utilized. Using the analytical methods, we verified and confirmed the batch-to-batch reproducibility of the biological product IMUNOR.

Preliminary studies on changes in the amount of tryptophan metabolites in human glioma tissues

BackgroundWe have developed and validated methods for the determination of three major tryptophan metabolites metabolized by the kynurenine pathway, namely kynurenine (KYN), 3-hydroxykynurenine (3-HK), and 3-hydroxyanthranilic acid (3-HAA). KYN and 3-HK were determined using RP-HPLC-UV, and 3-HAA using RP-HPLC-FL. We then developed a comparative method based on CE-UV. The developed methods were validated and 36 samples of human brain glioma tissue homogenates were assayed in all 4 grades of malignancy, and the concentration levels of assayed metabolites were compared with available clinical data. ResultsEach of the methods is characterized by high precision, accuracy and repeatability, and the determined LOQ values indicate the possibility of performing quantitative analysis on the available samples of human glioma tumors (36 samples in grades G1–G4). The concentration values of selected metabolites obtained using HPLC methods were subjected to statistical analysis and preliminary clinical data processing. We found statistically significant differences in the concentrations of KYN, 3-HK and 3-HAA between the various grades of the disease, and characterized these differences more precisely by means of the Dunn–Bonferroni post hoc test. We did not find that the patient's environment or habits significantly affected the metabolites concentration of the study samples population. In addition, we showed a high positive correlation between KYN, 3-HK and 3-HAA, which appears to be a characteristic that describes metabolic changes of Trp in relation to KYN, 3-HK and 3-HAA, and indicates potential diagnostic value. SignificanceThe preliminary studies carried out contribute new knowledge on the molecular basis of human brain glioma. They also provide valuable information useful for the development of glioma diagnostics, differentiation of disease grades and assessment of the patient's condition. The obtained relationships between metabolite concentrations and the grade of malignancy of the disease and correlations between metabolite concentrations constitute the basis for further broader biochemical and clinical analysis.

A factorial-design/assisted HPLC method for simultaneous monitoring of phenytoin, trimethoprim, and sulphamethoxazole in plasma samples to optimize therapy and mitigate toxicity risks

Phenytoin, an anticonvulsant drug, has a narrow therapeutic index and exhibits significant affinity to plasma proteins, mainly albumin. It is mainly metabolized by cytochrome P450 enzymes 2C9, 2C8, and 2C19. The potential for increased phenytoin toxicity may arise when a drug with enzyme-blocking properties are concurrently administered. The co-administration of trimethoprim is reported to decrease phenytoin clearance by 30 %, therefore, monitoring its plasma concentrations is recommended to minimize the risk of its toxicity and optimize its therapeutic efficacy. This study involves development of a novel and simple HPLC method for concurrent measurement of trimethoprim, sulphamethoxazole and phenytoin in plasma samples. The proposed method was optimized using full factorial design to optimize the variable factors affecting chromatographic responses. The separation was performed via isocratic elution on HyperClone C8 column, using a mobile phase consisting of 30: 38.5: 31.5 (v/v/v) of acetonitrile: 3.3 mM heptane-1-sulphonic acid sodium salt: 0.05 M phosphate buffer containing 0.1 % triethylamine at pH 3.7. The mobile phase was delivered at a flow rate of 1 mL/min with UV detection at 210 nm. The method was validated to verify agreement to the criteria set by the International Council for Harmonization (ICH) and Bioanalytical Methods validation. The results showed excellent linearity over the concentration ranges of 0.5–20.0, 1.0–25.0 and 1.0–20.0 µg/mL with detection limits of 0.41, 0.82 and 0.92 µg/mL for trimethoprim, sulphamethoxazole and phenytoin, respectively.The proposed method was extended for the assessment and of the studied drugs in spiked human plasma samples with % recoveries ranging from 92.60 to 105.30 %, indicating bioanalytical applicability. The proposed method can be utilized to study drug-drug interactions for in-patients therapeutic drug monitoring.

Stability-Indicating HPLC Method for Quantifying Process-Related Impurities in Fedratinib and Identification of Its Forced Degradation Products Using LC-MS/MS

Background: Pharmaceutical industry is characterized by rigorous quality standards to ensure the safety and efficacy of drugs. Despite stringent manufacturing processes, the presence of impurities or generation of degradation products (DPs) in pharmaceutical products remains a concern. This necessitates a comprehensive and systematic approach to analysis impurities and DPs. Objectives: This study deals with the optimization of the stable HPLC method for quantification of fedratinib impurities and its DPs characterization through LC-MS/MS. Method: Method optimization studies were conducted by analyzing standard solutions in various method parameters. The results noticed in every varied method condition were tabulated for finalizing the appropriate conditions for analyzing fedratinib. The mass spectral response of DPs was interpreted carefully for structural conformation of DPs. Results: The method is optimized as HIQSIL C18 (250mm×4.6mm;5µ) column employing 1.0 mL/min flow of phosphate buffer (pH 5.2) and acetonitrile in 45:65 (v/v) and 257 nm. This method elutes 5.4, 2.6, 9.2 and 3.5 min for fedratinib, impurity 1, 2 and 3 respectively. Method sensitivity was verified to be very sensitive that can evaluate up to 0.003, 0.015 and 0.004 µg/mL for impurity 1, 2 and 3 respectively. Well correlated calibration curve achieved in 50-200 µg/mL for fedratinib and 0.05-0.20 µg/mL for impurities. Various stress studies produce four stress DPs and were identified using LC-MS/MS. The molecular mass (g/mol) and formula of DPs were identified as 426 and C21H25N5O3S, 312 and C17H22N5O, 354 and C17H14N4O3S, 215 and C11H11N4O respectively for DP 1 to 4. Conclusion: The method proposed can successfully be helpful for quantifying the pharmaceutical impurities and DPs of fedratinib in bulk batch samples and formulations.

Effect of Nixtamalization on Aflatoxins, Functional and Acceptability of Maize from Mulindi, Rwanda

Rwanda relies highly on maize for human food as well as for animal feed and industrial uses. However, high temperature and wet climate conditions in Rwanda favor the growth of fungi that may lead to mycotoxin contamination in maize and subsequent degradation of its quality. Therefore, this study aimed to assess the effect of nixtamalization on the aflatoxins, the functional and acceptability of maize grains from Mulindi, Rwanda. The maize samples were collected from the Mulindi market in Rwanda; representative samples were collected by the guidelines for sampling food for mycotoxin determination. Subsequently, the treated maize samples were dry-milled to produce fine flour, and the flour obtained was analyzed for swelling capacity, water absorption capacity, water solubility, acceptability, and aflatoxins, which were quantified by the HPLC method. The HPLC results showed that the highest total aflatoxin level in maize grains was 44.99 ppb. Cooking maize in water and calcium hydroxide of 1% resulted in a reduction of aflatoxin with degradation percentages of 25% and 88%, respectively. However, in all treatments, aflatoxins B1 and B2 were less reduced compared to aflatoxins G1 and G2. Cooking maize in pure water showed the lowest reduction of aflatoxin compared to the other treatments. However, cooking maize in water with 1% calcium hydroxide decreased aflatoxin to a higher percentage. Cooking maize in lime enhanced the swelling capacity, water absorption capacity, and water solubility of maize, as well as its acceptability. However, the nixtamalization process was observed to reduce aflatoxins levels in maize and improve the functional properties of maize, and therefore, its application could be encouraged for safe products in the market.

Rapid and simple dual extraction for the analysis of lipids and autoantigenic peptides within phosphatidylserine-liposomes

Autoimmune diseases are a major health concern in developed countries. Currently, only palliative treatments based on anti-inflammatories and immunosuppressors are available. A novel antigen-specific therapy that uses a physiological process of tolerance generation is being developed. This is plausible by using phosphatidylserine rich liposomes (PS-liposomes), which bio-mimic apoptotic cells, encapsulated with the autoantigen responsible of generating the autoimmunity. In this way, tolerance against the own cells or tissues that were considered hostile can be achieved. In addition, only by changing the encapsulated peptide, different autoimmune diseases can be treated. Efficacy of this approach was demonstrated in type I diabetes, rheumatoid arthritis, multiple sclerosis, and myasthenia gravis.In the regulatory pre-clinical phase, analytical methodologies to evaluate the quality of the product need to be developed. In this regard, identification and quantification of the encapsulated peptide and lipids are considered critical quality attributes. In this study, a rapid and simple liquid–liquid extraction procedure, based on Bligh-Dyer method, is described for dual extraction of peptides and lipids within PS-liposomes formulation. This single step allows the separation of lipids and the encapsulated peptide in two different phases. For the subsequent analysis, two different HPLC methods were developed. The organic phase, which contains the lipids was analysed by HPLC-ELSD, while the aqueous phase, containing the encapsulated peptide, was analysed by HPLC-UV. Both methods were also validated in terms of accuracy, precision, linearity, LOD and LOQ. The extraction procedure has demonstrated highly efficient separation of lipids and peptides, avoiding interferences between them in the quantification.

Ultrasonic-driven chemical reduction synthesis of alizarin complexone-modified gold nanoparticles for dual-signal colorimetric and fluorometric sensing of histamine in seafood products

Alizarin complexone-modified gold nanoparticles (Au0-NPsALz) were synthesized using a proposed ultrasonic irradiation-assisted chemical reduction method. Ultrasonic irradiation powers, reaction time and alizarin complexone concentration had been proven to be the main parameters for controlling the nucleation and growth of Au0-NPsALz. In the synthesized ultrasonic irradiation-assisted chemical reduction conditions, Au0-NPsALz had a spherical oriented morphology with a uniform size of 17.84 ± 1.37 nm and are shiny red with a surface plasmon resonance (SPR) of 535 nm. A rapid colorimetric and fluorometric dual-mode detection strategy for selective detection of histamine in seafood was developed based on the self-assembly of Au0-NPsALz-Ni (II) complexes. Ni (II) can capture the histamine molecules close to Au0-NPsALz surfaces, making changes in the colorimetric and fluorometric responses of the solution. The quantitative analysis of histamine was realized through the variation of dual-signal colorimetric and fluorometric responses. Such Au0-NPsALz sensor offered good detection sensitivity for histamine with a detection limit (LOD) of 59.32 μmol L−1 and 116.20 μmol L−1 and wide linear response within the range of 10–10000 μmol L−1 (R2 = 0.9952) and 100–5000 μmol L−1 (R2 = 0.9947) for colorimetric and fluorometric measurement, respectively. Recoveries ranging from 94.99 to 103.29 % and 97.67–106.88 % for colorimetric and fluorometric assay were obtained, showing low levels of matrix effects. Particularly, the results of the dual-mode sensor were also validated by comparing with the HPLC method for improving the assay accuracy and dependability. Ultimately, the developed Au0-NPsALz colorimetric and fluorometric probe performs excellently in practical applications, with promising results for detecting histamine in seafood products.

Inhaled Ivermectin-Loaded Lipid Polymer Hybrid Nanoparticles: Development and Characterization.

Ivermectin (IVM), a drug originally used for treating parasitic infections, is being explored for its potential applications in cancer therapy. Despite the promising anti-cancer effects of IVM, its low water solubility limits its bioavailability and, consequently, its biological efficacy as an oral formulation. To overcome this challenge, our research focused on developing IVM-loaded lipid polymer hybrid nanoparticles (LPHNPs) designed for potential pulmonary administration. IVM-loaded LPHNPs were developed using the emulsion solvent evaporation method and characterized in terms of particle size, morphology, entrapment efficiency, and release pattern. Solid phase characterization was investigated by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Using a Twin stage impinger (TSI) attached to a device, aerosolization properties of the developed LPHNPs were studied at a flow rate of 60 L/min, and IVM was determined by a validated HPLC method. IVM-loaded LPHNPs demonstrated spherical-shaped particles between 302 and 350 nm. Developed formulations showed an entrapment efficiency between 68 and 80% and a sustained 50 to 60% IVM release pattern within 96 h. Carr's index (CI), Hausner ratio (HR), and angle of repose (θ) indicated proper flowability of the fabricated LPHNPs. The in vitro aerosolization analysis revealed fine particle fractions (FPFs) ranging from 18.53% to 24.77%. This in vitro study demonstrates the potential of IVM-loaded LPHNPs as a delivery vehicle through the pulmonary route.

Lavandula angustifolia Herb from Ukraine: Comparative Chemical Profile and in vitro Antioxidant Activity.

Lavandula L. genus plants have always been relevant as medicines for various purposes in food, medicine, pharmaceuticals, cosmetology and aromology. Ukraine is a new territory in the mass plant cultivation and lavender essential oil production. Therefore, the issue of integrated use of herbal raw materials and their intended use is still relevant. For the first time, ten samples of Lavendula angustifolia herb from 5 growing regions of Ukraine were studied for the composition and content of polyphenols and terpenoids using HPLC and HPTLC methods, respectively, to assess the prospects and quality of herbal raw materials. The results obtained showed that L. angustifolia herb has pronounced antioxidant activity due to the high content of phenolic compounds, namely hyperoside (5.665-11.629 mg/g), vanillic acid (5.986-11.196 mg/g), rosmarinic acid (0.211 to 1.488 mg/g), caffeic acid (0.369-3.835 mg/g), chlorogenic acid (0.239-4.619 mg/g), genistein-7-O-glucoside, as well as due to the presence of linalool and linalyl acetate, which was confirmed by qualitative analysis. The total antioxidant activity was the highest in samples from Lviv Botanical Garden (0.293 Trolox mg/mL), Kyiv OLawander (0.288 Trolox mg/mL), Kharkiv Bohodukhiv (0.270 Trolox mg/mL) which is due to the qualitative composition of phenolic compounds. At the same time, the most intense zones of terpenoids in lavender herb were noted for images from Kharkiv region Lebiazhe and Kitchenkivka villiges. Cluster analysis showed priority in the selection of marker compounds (vanillic acid, hyperoside, chlorogenic acid, rosmarinic acid) for lavender herb based on their quantitative content in the samples. In the future, lavender herb from Ukraine can be considered as a promising raw material with neuroprotective properties as part of its complex use, as research continues.

Simultaneous spectrophotometric and chromatographic determination of Ketorolac and Dexamethasone: Overall assessment regarding environmental Impact, Practicality, and functionality principles

Determination of a drug combination that contains a minor component that is also a poorly UV absorbing analyte represents greet challenges in drug analysis. A fixed-dose ophthalmic combination of ketorolac tromethamine (KET) and dexamethasone sodium phosphate (DEX) (in the ratio of 5:1) is used to relieve post-surgical inflammation and seasonal allergic conjunctivitis due to their analgesic and anti-inflammatory activity. Five spectrophotometric methods and a reversed phase chromatographic separation method were developed and validated for simultaneous determination of both drugs in lab-prepared eye drops. Direct spectrophotometric determination of KET (the major, highly UV absorbing analyte) was achieved from the zero-order UV absorption spectra at 323 nm while DEX (which is the minor, poorly UV absorbing analyte) was quantified after applying several spectrophotometric methods. Either fundamental zero-order spectra or ratio spectra were used, and measurements were done at either single or dual wavelengths. The developed methods were: isosbestic point method, absorption factor method, dual wavelength method, ratio difference UV spectrophotometric method, and simultaneous equation method. The developed spectrophotometric methods determine KET in the linearity range of 2.0–12.0 μg/mL and DEX in the range of 5.0 – 35.0 μg/mL. For accurate spectrophotometric determination of DEX in the ratio of the dosage form, multiple standard addition method was applied. HPLC technique was also applied for separation and quantification of both drugs. Chromatographic separation was attained by using Hypersil ODS-C18 (150 x 4.6 mm, 5 μm) column. The mobile phase was composed of 50 % methanol: 50 % phosphate buffer (pH 2.5). The column compartment was maintained at 25 °C. Flow rate was adjusted at 1.2 mL/min and detection was performed by DAD at 254 nm. The linearity of HPLC method was 5.0–100.0 μg/mL, and 5.0–50.0 μg/mL for KET and DEX, respectively. Simultaneous determination of both drugs was applied to lab-prepared eye drops that contain also benzalkonium chloride as inactive ingredient. The developed methods determined the cited drugs with good and acceptable % recovery. The results of the developed methods and a reported HPLC method were compared using one-way ANOVA test, F-test and student’s t-test at confidence level 95 %, and they show no significant statistical difference. Assessment of greenness of the developed methods in comparison with reported HPLC method was performed using different tools, including analytical eco-scale, GAPI and AGREE. HPLC-EAT was also performed to ensure greenness of developed HPLC method. Moreover, assessment of practicality, applicability, and functionality of developed and reported methods was evaluated by BAGI metric tool and WAC calculator, respectively. The developed methods allow simple, rapid, and accurate analysis of quality control samples that contain KET and DEX.

Effects of geographical variation on the phytochemicals gallic acid, corilagin, and ellagic acid, as well as medicinal properties of Emblica officinalis Gaertn (Fruit)

Geographical variation can significantly influence the abundance and distribution of phytochemicals within the raw drug, which in turn may affect its medicinal efficacy and potential health benefits. Understanding these variations is crucial for optimizing cultivation practices and harnessing the full therapeutic potential of herbal raw drug. Owing to the lack of availability of the comprehensive study on the effects of geographical variation on phytochemicals & micronutrient content and antioxidant & antibacterial potential of Emblica officinalis (EO), in the current study, EO fruit samples from different terrestrial regions of India were analyzed to identify variations in certain phytochemicals and nutrients content. A broad study including the identification, substantiation and phytochemical analysis of EO has been performed via a validated HPLC method. The complete profiling of various volatile metabolites present in the sample has been done by GC-MS/MS technique. The observed range of phytochemicals present in different EO samples using validated HPLC-DAD method are 4.38 to 69.41 mg/g for Gallic acid, 0.50 to 5.54 mg/g for Corilagin and 3.92 to 15.34 mg/g for ellagic acid. The total phenolic content (TPC) and total flavonoid content (TFC) for different samples of EO ranged from 52.86–175.47 mg GAE/g of wt. and 1.28–13.88 mg QCE/g of wt. respectively. ICP-OES analysis depicts substantial amount of various essential micronutrients such as Fe, Cu, Mn and Zn. In antibacterial assay, methanolic extracts of EO obtained from Pune zone exhibited promising activity against S. aureus, S. typhi and MRSA strains with inhibition zones of 28, 23 and 28 mm, respectively. Further, the antioxidant, antidiabetic and anti-inflammatory activities of EO on the basis of analyzed phytochemicals have also been evaluated through molecular docking study.

Utility of melatonin on brain injury, synaptic transmission, and energy metabolism in rats with sepsis.

Melatonin is a powerful endogenous antioxidant hormone. Its healing effects on energy balance and neuronal damage associated with oxidative metabolism disorders have been reported in pathologic conditions. We aimed to determinate the utility of melatonin on neuronal damage, synaptic transmission, and energy balance in the brain tissue of rats with sepsis induced with LPS. Rats was divided into four groups such as control, LPS (20mg/kg i.p.), melatonin (10mg/kg i.p. × 3), and LPS + Melatonin (LPS + Mel). After 6h from the first injection, rats were decapitated, and also tissue and serum samples were taken. Lipid peroxidation and neuron-specific enolase (NSE) levels were determined from the serum in all group. High energy compounds, creatine, and creatine phosphate are measured by HPLC methods from the homogenized tissue. Counts of living neurons are marked with NeuN (neuronal nuclei), degenerated neurons are marked with S100-ß and synaptic vesicles transmission is analyzed with synaptophysin antibodies immunoreactivities. One-way ANOVA and post hoc Tukey tests were used to statistical analysis. In LPS group, AMP, ATP, creatine, and creatine phosphate levels were significantly decreased (p < 0.05), and also ADP levels were significantly increased compared with the other groups (p < 0.01). Living neurons counts were significantly decreased in LPS (p < 0.01), melatonin, and LPS + Melatonin (p < 0.05) groups compared with control. Degenerated neurons counts were increased in LPS group compared with control (p < 0.01) and also decreased in both of melatonin and LPS + Melatonin groups (p < 0.01). Synaptophysin immunoreactivity was decreased in LPS group compared with the other groups (p < 0.05). We observed that melatonin administration prevents neuronal damage, regulates energy metabolism, and protects synaptic vesicle proteins from sepsis-induced reduction.

Gentiana capitata Buch.-Ham. ex D.Don Cell Suspension Culture as a New Source of Isosaponarin and 3,7,8-Trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-β-D-ribopyranosyl-β-D-allopyranoside and Their Effect on PC-12 Cell Viability.

Some species of the Gentianaceae family are a valuable source of secondary metabolites. However, the phytochemical knowledge of some of these species remains insufficient. Therefore, this work focused on the isolation of the two main secondary metabolites in the methanolic extract from a Gentiana capitata cell suspension using preparative HPLC and the determination of their structure using UHPLC-DAD-IT-MS/MS and NMR methods. Their content in the methanolic extract was quantified using a previously validated HPLC method. The toxicity of the extract and two isolated compounds was also tested on the PC-12 cell line. The structures of the main secondary metabolites were identified as isosaponarin and 3,7,8-Trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-β-D-ribopyranosyl-β-D-allopyranoside by comparing the UHPLC-DAD-IT-MS/MS and NMR results with the literature data. The content of isosaponarin was determined to be 0.76 ± 0.04%, and the content of 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-β-D-ribopyranosyl-β-D-allopyranoside was found to be 0.31 ± 0.02% in the dry extract. Additionally, a two-fold increase in the viability of the PC-12 cell line was observed compared to the control after treatment with the methanolic extract at a concentration of 500 µg/mL. These results suggest the potential use of G. capitata cell suspension methanolic extract as a new source of isosaponarin and 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-β-D-ribopyranosyl-β-D-allopyranoside, highlighting their lack of toxicity to the PC-12 (rat pheochromocytoma) cell line.