Reversed-phase High-performance Liquid Chromatography Research Articles

Abstract 1381: Characterization of the 12-lipoxygenase expression in the megakaryoblastic cell line, Dami

Abstract Megakaryocytes are myeloid cells produced primarily in the bone marrow and are best known for releasing platelets in the blood stream. In the platelet production process, megakaryocytes transfer their bioactive content, including the 12-lipoxygenase (12-LO) enzyme, into newly formed platelets. The 12-LO has been shown to be implicated in platelet activation and is overexpressed in several chronic inflammatory conditions, including several types of cancers. The 12-LO is responsible for the conversion of the arachidonic acid into the 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a key lipid mediator implicated in several steps of the metastatic cascade. While 12-LO is expressed in mature human megakaryocytes, we recently identified two megakaryoblast cell lines isolated from leukemia patients lacking 12-LO expression. However, only one of the two cell lines expresses functional 12-LO protein upon megakaryocyte maturation. The goal of this study is to characterize the 12-LO expression during the megakaryocyte maturation process. We hypothesize that 12-LO expression is increased during cell maturation and that inhibition of the 12-LO expression will result in dysfunctional platelets. Both the Dami cell and MEG-01 cells were induced with the co-incubation of both phorbol myristate acetate and eltrombopag, a thrombopoietin receptor agonist. The maturation and viability of the cells were confirmed by flow cytometry. Expression of the 12-LO was assessed by RT-qPCR and immunoblot. Quantification of the 12(S)-HETE was performed by reversed-phase high performance liquid chromatography. Platelet release was quantified by flow cytometry using CD41 fluorescent labelled antibodies. We observed a 3-fold increase in 12-LO protein expression following cell maturation in the Dami cell line. Interestingly, mRNA expression between the undifferentiated Dami cells and mature Dami cells remained unchanged. Furthermore, we did not detect any 12-LO protein expression in the MEG-01 after the cell’s maturation. Finally, we identified a specific 12-LO antisens non-coding RNA that is implicated in the regulation of the protein expression in megakaryocytes. Our study reports the first mechanistic regulation of 12-LO expression implicated in the megakaryocyte maturation process. Since both platelets and platelet-specific 12-LO play an important role in cancer biology, it is important to better understand how megakaryocytes package the enzyme inside these small cells. It remains elusive whether 12-LO enrichment in platelet correlates with the disease severity. Citation Format: Luc H. Boudreau, Vanessa L. Gauvin, Jaël Richard, Marie-France N. Soucy, Mathieu P.A. Hébert, Eric P. Allain. Characterization of the 12-lipoxygenase expression in the megakaryoblastic cell line, Dami [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1381.

Determination of Enantiomer in Tofacitinib Citrate Using Reversed-Phase Chiral High-Performance Liquid Chromatography

Tofacitinib citrate (RR-isomer) is a janus kinase (JAK) inhibitor approved for the treatment of rheumatoid arthritis (RA) in adults who have had an inadequate response or intolerance to methotrexate. The presence of the enantiomer of tofacitinib citrate (SS-isomer) is monitored for quality control as a possible impurity in the final product. In this study, a reversed-phase high-performance liquid chromatography (RP-HPLC) method based on a chiral recognition mechanism for the separation of tofacitinib citrate and its enantiomer was established based on the principles of green analytical chemistry. A CHIRALPAK IH column was used with a mobile phase of ammonium acetate buffer (pH 8.0) and acetonitrile in a gradient elution at a detection wavelength of 285 nm. The calibration curve exhibited excellent linearity over the range of 0.1002–20.04 μg/mL (r = 0.9999). The average recovery of the enantiomer was 98.6% with a relative standard deviation (RSD) of 0.7%. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.04 and 0.1 μg/mL, respectively. This RP-HPLC method was suitable for detecting the enantiomers of tofacitinib citrate in tablets. Furthermore, the method proved to be environmentally friendly based on the evaluation by Analytical Eco-Scale, Analytical GREEnness (AGREE) and Green Analytical Procedure Index (GAPI).

Less is more: Validating a single method for comprehensive rh-insulin analysis

The objective of this current study is to establish a single method for potency and related proteins analysis of human insulin formulations using reverse-phase high performance liquid (RP-HPLC) chromatography technique which was validated and verified for the potency analysis in insulin formulations. Chromatographic separation was achieved using an octadecylsilane (C-18) stationary phase and a mobile phase composed of 55% (v/v) buffer (0.2 M sodium sulfate in water, {pH 2.3}) and 45% (v/v) acetonitrile. Detection was performed by UV detector at 214 nm with a flow rate of 1 ml/min and an injection volume of 20 µL, at 40°C. Currently there are separate methods available in Indian Pharmacopoeia for analysis of Potency and Related proteins in human insulin. We have validated a single method where quantitation of potency and related proteins can be performed in the same run. The method validation exhibited linearity over the concentration range of 0.08–4.5 mg/ml (r2=0.999) with limit of detection of 0.094 mg/ml The accuracy of the method was 99–102.8%. Thus, it is proposed that both potency and related proteins in insulin formulations can be precisely evaluated using a single run thus saving the time and cost for quality analysis of insulin preparations both at manufacturing and regulatory laboratories which in turn will increase the market availability of such standard quality insulin preparations for public health use.

Variations of Carotenoids, Total Bioactive Contents, and Antioxidant Activity in Leaves of Medicago polymorpha

Medicago polymorpha L. (MP) is a herbaceous plant commonly known as “bur clover.” It is aboriginal to Central and Western Asia and extensively distributed around the world. This study determines the changes in carotenoids and antioxidant potential of different MP of different origins. The sample leaves were analyzed for identification and quantification of carotenoids using a reversed-phase HPLC system. The result showed nine carotenoids and pigments in each sample. The major carotenoid compounds were phytoene, phytofluene, neoxanthin and its isomer (9-Z-neoxanthin), violaxanthin, lutein along with their isomers (9-Z-lutein, 9′-Z-lutein), and all-E-β-carotene. The major pigments were 15-hydroxy-lactone chlorophyll a, pheophytin a, pheophytin a′, pyropheophytin a, divinyl chlorophyll a, chlorophyll b′, chlorophyll b, 13′-hydroxy-lactone chlorophyll b, and hydroxy pheophytin a′. The carotenoids were detected in considerable amounts in the samples from Lower Dir (213 μg/g), Swat (171 μg/g), and Buner (157 μg/g). Chlorophylls were higher in Lower Dir (203.4 μg/g), Swat (184.0 μg/g), and Buner (152.2 μg/g) and significantly lower amounts in Malakand samples (141.7 μg/g). The total carotenoids in Lower Dir (51.2%) were higher than in Swat (48.2%), Buner (50.8%), and Malakand samples (44.6%). The amounts of violaxanthin and lutein were significantly higher in Lower Dir (73.6%) samples, followed by Malakand (51.7%), whereas Buner and Swat samples showed the lowest levels. In conclusion, MP leaves are a good source of important carotenoids having potential antioxidant properties, which are highly correlated to the violaxanthin and lutein contents.

Dietary exposure to aflatoxin B1 and ethanol from homemade and industrial fermented beverages in South Kivu, Democratic Republic of the Congo

BACKGROUND: The prevalence of AFB1 contamination in agricultural products used to produce fermented alcoholic beverages is increasing, raising concerns for human health. AIM: The aim of this study was to investigate dietary exposure to AFB1 and ethanol through homemade and industrial fermented beverages commonly consumed in South Kivu, DR Congo. METHOD: AFB1 and ethanol were measured using reverse-phase HPLC with a fluorescence detector and a refractive index detector, respectively. Data on fermented beverage consumption were collected from 847 adults using a food frequency questionnaire (FFQ). RESULTS: The findings revealed that industrial sample Man 8 had the highest exposure to AFB1 (77.8 ± 45.0 ng/kg b.w/day) and Man4 had the highest ethanol exposure (4.83 ± 2.40 mg/kg b.w/day); while among homemade samples, Kasiksi had the highest exposure to both AFB1 (8.8 ± 6.6 ng/kg b.w/day) and ethanol (2.46 ± 1.85 mg/kg b.w/day). The margin of exposure (MOE) for AFB1 was 1011.7 or less, and for ethanol, it was 818.2 or less. Men are more likely to be exposed. CONCLUSION: Increased consumption of homemade and industrial fermented beverages raises the risk of developing hepatocellular cancer (HCC) because the levels of AFB1 and ethanol MOE drop below the safe limit of 10000. Further research is needed to investigate the connection between AFB1, ethanol, and HCC, especially in regions where alcohol misuse is common.

Cross-column density functional theory-based quantitative structure-retention relationship model development powered by machine learning.

Quantitative structure-retention relationship (QSRR) modeling has emerged as an efficient alternative to predict analyte retention times using molecular descriptors. However, most reported QSRR models are column-specific, requiring separate models for each high-performance liquid chromatography (HPLC) system. This study evaluates the potential of machine learning (ML) algorithms and quantum mechanical (QM) descriptors to develop QSRR models that can predict retention times across three different reversed-phase HPLC columns under varying conditions. Four machine learning methods-partial least squares (PLS) regression, ridge regression (RR), random forest (RF), and gradient boosting (GB)-were compared on a dataset of 360 retention times for 15 aromatic analytes. Molecular descriptors were calculated using density functional theory (DFT). Column characteristics like particle size and pore size and experimental conditions like temperature and gradient time were additionally used as descriptors. Results showed that the GB-QSRR model demonstrated the best predictive performance, with Q2 of 0.989 and root mean square error of prediction (RMSEP) of 0.749min on the test set. Feature analysis revealed that solvation energy (SE), HOMO-LUMO energy gap (∆E HOMO-LUMO), total dipole moment (Mtot), and global hardness (η) are among the most influential predictors for retention time prediction, indicating the significance of electrostatic interactions and hydrophobicity. Our findings underscore the efficiency of ensemble methods, GB and RF models employing non-linear learners, in capturing local variations in retention times across diverse experimental setups. This study emphasizes the potential of cross-column QSRR modeling and highlights the utility of ML models in optimizing chromatographic analysis.

LC-HRMS and NMR studies for the characterization of degradation impurities of ubrogepant along with the in silico approaches for the prediction of degradation and toxicity

Ubrogepant is the first oral calcitonin gene-related peptide (CGRP) receptor antagonist which is used for the acute treatment of migraine in adults. The present study employs liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR) techniques for the identification and characterization of degradation impurities of ubrogepant. The forced degradation study of ubrogepant was performed as per the International Council for Harmonisation (ICH) Q1A and Q1B guidelines. The in silico degradation profile of ubrogepant was predicted by Zeneth. It was observed that ubrogepant was labile to acidic hydrolysis, basic hydrolysis, and oxidative degradation conditions (H2O2), although it was stable in neutral hydrolysis and photolytic (UV light and visible light) conditions. Eight degradation impurities were formed, which were separated on reversed-phase HPLC with a gradient program on an InertSustain C8 column (4.6 × 250 mm, 5 µm) using 10 mM ammonium formate (pH unadjusted) and acetonitrile as the mobile phase. The structures of all the degradation impurities were characterized using the exact masses obtained from the HRMS/MS. Further, NMR studies were conducted on two major degradation impurities (UB-4 and UB-7). A plausible mechanism was proposed to support the structures of all the degradation impurities of UBR. In silico toxicity and mutagenicity assessment were done by DEREK Nexus, SARAH Nexus, and ProTox-II.

Immunochemical Recognition of Bothrops rhombeatus Venom by Two Polyvalent Antivenoms.

The protein profile of Bothrops rhombeatus venom was compared to Bothrops asper and Bothrops atrox, and the effectiveness of antivenoms from the National Institute of Health of Colombia (INS) and Antivipmyn-Tri (AVP-T) of Mexico were analyzed. Protein profiles were studied with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase high-performance liquid chromatography (RP-HPLC). The neutralizing potency and the level of immunochemical recognition of the antivenoms to the venoms were determined using Western blot, affinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Bands of phospholipase A2 (PLA2), metalloproteinases (svMPs) I, II, and III as well as serine proteinases (SPs) in the venom of B. rhombeatus were recognized by SDS-PAGE. With Western blot, both antivenoms showed immunochemical recognition towards PLA2 and svMP. INS showed 94% binding to B. rhombeatus venom and 92% to B. asper while AVP-T showed 90.4% binding to B. rhombeatus venom and 96.6% to B. asper. Both antivenoms showed binding to PLA2 and svMP, with greater specificity of AVP-T towards B. rhombeatus. Antivenom neutralizing capacity was calculated by species and mL of antivenom, finding the following for INS: B. asper 6.6 mgV/mL, B. atrox 5.5 mgV/mL, and B. rhombeatus 1.3 mgV/mL. Meanwhile, for AVP-T, the following neutralizing capacities were found: B. asper 2.7 mgV/mL, B. atrox 2.1 mgV/mL, and B. rhombeatus 1.4 mgV/mL. These results show that both antivenoms presented similarity between calculated neutralizing capacities in our trial, reported in a product summary for the public for the B. asper species; however, this does not apply to the other species tested in this trial.

Development and Validation of a New Reversed Phase HPLC Method for the Quantitation of Azithromycin and Rifampicin in a Capsule Formulation.

This research aimed to develop a new method for simultaneously estimating the presence of azithromycin (AZT) and rifampicin (RIF) in a capsule formulation using reverse-phase high-performance liquid chromatography. The developed method utilized a Gemini column with a 60:40% v/v acetonitrile and potassium dihydrogen phosphate mobile phase, a flow rate of 1mL/min, and an injection volume of 20μL. The detection wavelengths of 210 and 254nm for AZT and RIF, respectively, were used. Validation ensures specificity with a peak purity index > 0.99999 for AZT and >0.99995 for RIF, affirming unambiguous analyte detection. The system suitability test, within acceptable limits, validates method reliability. Linearity calibration curves (R2 = 0.998) cover a 25-150% target concentration range. Accuracy studies employing the standard addition method yield recovery values between 96.6 and 103.9% for both drugs, confirming method accuracy. Precision studies reveal % relative standard deviation values consistently below 2%, highlighting reproducibility. Robustness testing supports method reliability under varying conditions. Application to a pharmaceutical capsule formulation demonstrates the method's practicality, accurately quantifying AZT (98.30%) and RIF (99.37%). This study provides a validated analytical approach for simultaneous quantification in commercial pharmaceutical products containing both drugs, enhancing pharmaceutical quality control for critical antibiotics in complex formulations.

Correlation of solvent strength parameter with two molecular descriptors in reversed-phase liquid chromatography

A new hydrophobicity index has been previously developed and reported (Rev. Roum. Chim., 2023, submitted). The new index is based only on the solvent strength parameter (S) that describes the linear dependences (log k = log kw - S) between the logarithm of the retention factor (k) and volume fraction of the organic modifier in mobile phase () in an HPLC separation. The correlations between the new hydrophobicity index and octanol-water partition constant (log Kow) were evaluated and reported in present study for a set of 20 solutes by using seven C18 HPLC columns and two organic modifiers, typically utilized in reversed-phase HPLC applications (acetonitrile and methanol). All the correlations were good, showing values above 0.85. Correlations between the extrapolated values of retention factor to zero percent of the organic modifier in mobile phase, log kw, and log Kow showed also good determination coefficients R2, unlike an older hydrophobicity index based on the ratio between log kw and S, which was generally characterized by lower R2 than 0.8 for acetonitrile and 0.7 for methanol.

A Novel Stability-Indicating RP-HPLC Method for the Simultaneous Estimation and In Vitro and In vivo Evaluation: Curcumin and Naringin Co-amorphous System

Curcumin (CUR) is a phytochemical widely used in food industries, cosmetics, and in the treatment of various ailments. It is a polyphenol derived from turmeric and is often considered the golden spice. CUR has a low solubility of less than 1 µg/ml and poor oral bioavailability which can be improved by co-amorphization with naringin (NRG). Analytical method to simultaneously quantify CUR and NRG is not reported in literature. This study aimed to develop a stability-indicating reverse phase HPLC method in gradient mode to simultaneously quantify CUR and NRG in co-amorphous system. The co-amorphous system of CUR and NRG in molar ratios 1:1 and 1:2 was prepared by quench cooling technique. The separation was attained on a Genesis C18, (4.6 mm × 150 mm, 4 µm) column with the mobile phase comprising of methanol and a 0.1% acetate buffer pH 3.8 at a single wavelength, 289 nm. CUR and NRG eluted at 5.1 and 11.1 min, respectively. For both the molecules, the linearity range was 0.125–16 µg/ml with LOD and LOQ of 0.063 and 0.125 µg/ml. The method developed was validated as per International Conference on Harmonization (ICH) guidelines for linearity, accuracy, precision, and robustness. The method was used to estimate CUR and NRG content in co-amorphous mixture and for in vitro evaluation.Graphical

Implementing analytical quality by design in reversed phase-high performance liquid chromatography for simultaneous estimation of teriflunomide and quercetin: Applicability in dual drug loaded topical microemulsion

The current study reports the development of a novel, robust, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of teriflunomide (TFD) and quercetin (QCN) in dual drug-loaded microemulsion. TFD and QCN have been reported to exhibit anti-inflammatory effects by targeting various inflammatory mediators involved in the pathogenesis of rheumatoid arthritis (RA). This analytical method employs a risk-based approach and follows the principles of analytical quality by design (AQbD). The study involved preliminary screening trials and systematic risk analysis to identify critical method attributes, that significantly impact the critical quality attributes (CQAs). For the optimization of method,a face centered central composite design was utilized, and 16 experimental runs were performed using design expert software version 7.0.0. Chromatographic conditions were carefully optimized using AQbD, falling within the design space and consisting of ammonium acetate buffer (pH 3.5) and acetonitrile (60:40, % v/v) with a flow rate of 1.0 mL/min. An analytical Hibar Lichospher 100 RP-18e column (250 × 4.6 mm, 5 µm) was employed, with detection wavelength of 280 nm and 367 nm for TFD and QCN, respectively. The optimized method underwent validation according to ICH guidelines and demonstrating applicability for evaluating topical microemulsion.

Ecofriendly bioanalytical validated RP-HPLC method for simultaneous determination of COVID-19 co-prescribed drugs employing quality by design and green chemistry

Molnupiravir, dexamethasone, ertapenem, and remdesivir are among the most frequently co-prescribed drugs used for COVID-19 virus treatment. For the first time, a simple isocratic reversed phase-high performance liquid chromatographic (RP-HPLC) method employing a photodiode array detector has been developed and validated for their simultaneous determination in spiked human plasma using rivaroxaban as an internal standard by the aid of quality by design and in accordance with the criteria of green chemistry. The separation was carried out utilizing a BDS Hypersil ® C18 column (300 × 4.6 mm), 5 μm particle size as the stationary phase using mobile phase consisted of methanol: acidified water; adjusted at pH 4.0 with 0.1 M orthophosphoric acid; with ratio (70:30, v:v) delivered at flow rate 0.6 mL/min, temperature adjusted at 20 °C, photodiode array detector maintained at 240 nm. The method was validated according to bioanalytical FDA guidelines with respect to linearity, accuracy, precision, selectivity, recovery, and stability. The method was found to be linear in the range of 4.00–120.00 μg/mL for molnupiravir, 10.00–140.00 μg/mL for dexamethasone, 10.00–160.00 μg/mL for ertapenem and 4.00–120.00 μg/mL for remdesivir. It is worth mentioning that the previously mentioned ranges covered the Cmax of the drugs. Finally, the green analytical score has been evaluated, and it was found to be an excellent green analytical method based on analytical eco-scale system penalty points. Moreover, it was assessed by analytical GREEnness metric approach and green analytical procedure index tool.

Unveiling Novel Chaotropic Chromatography Method for Determination of Pralidoxime in Nerve Agent Antidote Autoinjectors

Pralidoxime chloride, a highly hydrophilic antidote, cannot be effectively separated by reverse-phase high-performance liquid chromatography (RP-HPLC), unless the mobile-phase composition is varied. However, the use of ion-pairing reagents for pralidoxime separation is hindered by the persistent contamination of the stationary phase or chromatography system inside the HPLC system. Thus, this study aimed to develop a simple, rapid, and robust method based on RP-HPLC to determine pralidoxime chloride in antidote autoinjectors using a chaotropic salt as the mobile-phase additive. The use of UV detection at 270 nm allowed for the simultaneous detection of pralidoxime chloride and the internal standard, pyridine-2-aldoxime. The addition of chaotropic salts (NaPF6, NaBF4, and NaClO4) and an ionic liquid ([EMIM]PF6) increased the retention time of pralidoxime chloride. Among them, NaPF6 exhibited the highest capacity factor in the reverse-phase C18 column. Increasing the salt concentration increased the capacity factor and the number of theoretical plates. Analytical method validation was performed to assess the linearity, accuracy, precision, recovery, and repeatability, according to the Ministry of Food and Drug Safety guidelines. Additionally, this newly developed method exhibits an adequate separation capability, making it a potential substitute for the current method employed in the United States/Korean Pharmacopoeia, and it ensures the necessary durability to maintain the robustness and reliability of the analytical system.

Genome-Based Identification and Characterization of Bacteriocins Selectively Inhibiting Staphylococcus aureus in Fermented Sausages.

The bacteriocin-producing Lactiplantibacillus plantarum SL47 was isolated from conventional fermented sausages, and the bacteriocin SL47 was purified using ethyl acetate, Sephadex G-25 gel chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Bacteriocin SL47 was identified by HPLC-MS/MS combined with whole-genome sequencing, and the results showed it consisted of plantaricin A, J, K, and N. Further characterization analysis showed that the bacteriocin SL47 was highly thermostable (30min, 121°C), pH stable (2-10), sensitive to protease and exhibited broad-spectrum antibacterial ability against Gram-positive and Gram-negative bacteria. The mechanism of action showed that the bacteriocin SL47 increased cell membrane permeability, and 2 × minimum inhibitory concentration (MIC) treatment for 40min caused apoptosis of Staphylococcus aureus F2. The count of S. aureus in the sausage that was inoculated with L. plantarum SL47 and bacteriocin SL47 decreased by about 64% and 53% of that in the initial stage, respectively. These results indicated the potential of L. plantarum SL47 and bacteriocin SL47 as a bio-preservative in meat products.

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION FOR THE QUANTITATIVE ESTIMATION OF SODIUM BUTYRATE IN TABLETS

Objective: To develop a simple, accurate, precise, and linear reverse-phase high-performance liquid chromatographic (RP-HPLC) method and verify the quantitative estimation (assay) of sodium butyrate in tablets. Methods: The optimized RP-HPLC method uses a reverse phase stationary phase which is YMC Triart C18 column, having specifications of 250×4.6 mm; 5 μm, a mobile phase composition of pH 8.0 sodium dihydrogen phosphate buffer and acetonitrile in the proportion of 92:8 v/v, flow rate of 0.5 ml/min, injection volume of 10 μL, and detection wavelength of 210 nm using a UV/PDA detector. Results: The developed method gave sodium butyrate eluting at about 6 min. Sodium butyrate exhibited linearity in the range of 119.56–1195.6 μg/ ml. The precision was exemplified by a relative standard deviation of 0.30%. The percentage of individual recovery was found to be in the range of 97.0 and 103.0 during accuracy studies. Conclusion: A simple, specific, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (assay) of sodium butyrate in tablets and hence this method can be explored for the analysis of sodium butyrate in tablets in various pharmaceutical industries.

Phytochemical Diversity and Related Bioactivity of Ethyl Acetate Fraction from Different Organs and Provenances of the Edible Halophyte Crithmum Maritimum L.

Sea fennel (Crithmum maritimum L.) is a facultative halophyte from the Apiaceae family. The variation of phenolic compounds, the antioxidant and antibacterial activities of different extracts of different organs of sea fennel were investigated. The roots, stems, leaves and seeds were collected from three bioclimatic stages of Tunisia. Solvent fractionation method (using aqueous acetone, petroleum ether and ethyl acetate) was adopted to separate the extract ingredient and to concentrate phenolics. Total phenolics, total flavonoids, and condensed tannins significantly varied between organs and localities. In leaves, the highest level of phenols was detected in the upper and sub-humid zones. However, in stems the high amounts of phenols were observed in semiarid climate. The antioxidant activity was evaluated through four different methods and the antimicrobial activity was assessed against five human pathogenic bacteria and a fungal strain. Multivariate analysis showed significant effect of organ and locality on the antioxidant properties. Results of the antibacterial activities showed significant differences among extracts and was reflected by a large spectrum of activity ranging from totally not active (Zones of inhibition (zi) < 1 mm) to very high activity (zi >16 mm). Combined statistical results allowed to select the more potent fraction to be analyzed using reversed-phase HPLC (RP-HPLC). The flavanol epigallocatechin was identified as a major phenolic compound which contributed greatly to the antioxidant capacity. The results established in this study highlighted the importance of this halophyte cash crop in the human diet as natural source of active components.

THE DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR EVALUATING GALLIC ACID IN ETHYL ACETATE FRACTION (EAF) OF SNEDDS FORMULATION: QUANTITATIVE ANALYSIS WITH IN VITRO ASSAY

Objective: This study aimed to develop a simple, accurate, precise, sensitive, robust, and stable analytical method for the evaluation of gallic acid in Self-Nanoemulsifying Drug Delivery System (SNEDDS) incorporating ethyl acetate fraction (EAF) of Melastoma malabathricum leaves in combination with Gentamicin. Methods: Validation process followed ICH guidelines and applied a reverse phase HPLC method with a mobile phase of acetonitrile-phosphoric buffer at pH 3.03 (20:80 v/v). The stationary phase consisted of a VP-ODS shim-pack C-18 column (250x4.6 mm) with a flow rate of 0.2 ml/min and detection at 263 nm using an Ultraviolet detector. Additionally, antioxidant activity was assessed through the DPPH and FRAP methods, and SPF value was determined with a UV/Vis spectrophotometer in the 290-390 nm wavelength range. Results: The results showed that the retention time of quercetin was 16.648 min with a tailing factor of 1.623. The regression equation (y=224689x-989000) had a concentration range of 10-55 µg/ml and a correlation value of 0.9920. Limit of Detection (LOD) and Limit of Quantification (LOQ) were found to be 2.394±0.086 and 7.254±0.260 µg/ml, respectively. Method accuracy, determined by recovery values at concentrations of 50%, 100%, and 150%, ranged from 91.18% to 109.49%. Repeatability inter-day variations were expressed as %RSD values of 1.027-1.963% for AUC and 0.150-0.145 for RT. Moreover, the applied method showed stability within a temperature range of 14 °C–35 °C. Analysis showed gallic acid content of 1.773±0.049 mg/g in SNEDDS EAF formulation. Antioxidant activity measured through the DPPH and FRAP methods yielded IC50 values of 4.167±0.552 µg/ml and 20.253±0.619 µg/ml, respectively, while SPF value at SNEDDS concentration of 150 µg/ml was 36.993±0.183. Conclusion: This study successfully developed a precise, accurate, specific, and stable method for quantifying gallic acid levels in SNEDDS EAF of Melastoma malabathricum leaves in combination with Gentamicin. Therefore, SNEDDS EAF formulation exhibited an effective wound-healing potential, supported by a robust quality control process.

Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

Purification technology design, biochemical and immunological characteristics of the recombinant chimeric antigen for evaluation of T cell immunity against coronavirus infection

Diagnosis of specific T cell immunity to the antigenic determinants of SARS-CoV-2 in patients seems to be an increasingly important task due to accumulation of data about the role of T cell immune response in course of coronavirus infection and clearance of SARS-CoV-2 in case of secondary infection. Previously, we designed the recombinant CorD_PS antigen for evaluation of T cell antiviral immunity, containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus. E. coli CorD_PS producing strain with stable expression of the recombinant CorD_PS antigen was obtained. Aim of the present work is to design a laboratory technology for the production of recombinant antigen CorD_PS, to control the quality of the obtained chimeric protein and to study its immunological activity. Development of the cultivation conditions for E. coli CorD_PS cells was carried out in conical flasks in LB-M_Km medium at 37 °C, then scaled in a fermenter with a volume of 30 liters. Expression was induced by the addition of IPTG. Expression was controlled in culture lysates in 12% SDS-PAGE. The resulting biomass was lysed using an ultrasonic disintegrator with followed by centrifugation. The compositions of lysing and solubilizing buffers were selected, as well as conditions for refolding of the recombinant protein. Cation exchange (SP-sepharose), hydrophobic (Butylsepharose) and exclusive (Sephacryl S-200 HR) chromatography were used sequentially to purify the dissolved protein. Protein impurities in the preparation were determined by reverse-phase HPLC and electrophoresis in 12% SDS-PAGE, residual lipopolysaccharides were determined using a gel-thrombin variant of the LAL test, residual proteins of the producer strain were determined by enzyme immunoassay, residual DNA of the producer strain was determined by binding with PicoGreen dye. Specificity was controlled by indirect enzyme immunoassay. The evaluation of cytokine production by CD4+T lymphocytes in response to their stimulation by recombinant antigen ex vivo was performed on a flow cytofluorimeter. The biomass yield during cultivation of E. coli CorD_PS in a 30L fermenter was up to 20 g/L for 4 hours of 0.1 mM IPTG induction. Sequential washing of inclusion bodies from bacterial cellular components and their subsequent solubilization in a buffer containing 8 M urea allowed to obtain a solution of denatured antigen with a concentration of 10 mg/mL. The efficiency of refolding by dilution was 75%. After three stages of chromatographic purification, protein samples with a concentration of 1.2-1.4 mg/mL, HPLC purity of 98.43%, corresponding to key quality parameters according to the OFS.1.7.1.0007.15, were obtained. The recombinant antigen showed specific binding to a sample of the First WHO International Standard and sample Company Reference Standard No. 3 of anti- SARS-CoV-2 human immunoglobulins in a specified concentration range. CD4+T lymphocytes effectively responded to recombinant antigen treatment by increasing IFNγ production. The optimal concentration of recombinant coronavirus antigen was 5 μg/mL. The developed technological process makes it possible to obtain 5-7 grams of coronavirus recombinant CorD_PS antigen in one cultivation cycle in 30 liters of fermentation medium with key quality parameters according to the OFS.1.7.1.0007.15. As a result of specific immunological activity studies of the recombinant coronavirus antigen CorD_PS, the concept of its possible use as a diagnostic tool for determining the formation of a T cell immune response was confirmed.