Reversed-phase High-performance Liquid Chromatography Research Articles

Polydopamine coated surfactin micelles for brain delivery of ibudilast in multiple sclerosis: Design, optimization, in vitro and in vivo evaluation

Ibudilast inhibits TNF-α and IL-12 release from the astrocytes and microglial cells, reducing neuronal degeneration and may be a potential therapy in multiple sclerosis with neuroprotective effects. Our study aimed to prepare polydopamine coated micelles of surfactin, a biosurfactant, loaded with ibudilast to control the release and its targeted delivery through nose to the brain by targeting D2-like dopamine receptors limited in olfactory nerve of the bulb. Micelles were prepared by solvent evaporation and film hydration method and optimized to quantify the effects of excipients on particle size and entrapment efficiency. Polydopamine coating on the surface of surfactin micelles was characterized by studying the physicochemical properties of the micelles. Drug concentration in brain after 14 days of administration of the oral, and nasal free drug and also 3 doses of intranasal PDA coated micelles of ibudilast were determined by reverse-phase HPLC method. FTIR results confirmed successful coating of PDA on surfactin micelles and DSC and XRD results indicated solubilization of ibudilast in the core of micelles in an amorphous state. The morphology of the micelles by TEM showed spherical structures with coating of PDA on the shell of the micelles. Particle size of the micelles was 131.3 ± 12.6 nm, zeta potential 27.3 ± 1.7 mV, ibudilast loading percent was 90.2 ± 1.6% and drug release efficiency percent was 72.3 ± 6.5% in the best formulation for surfactin micelles. However, after PDA coating the particle size changed to 174.6 ± 15.7 nm, zeta potential varied to −41.0 ± 2.7 mV, PDA content was 70.3 ± 5.2%, and drug release efficiency percent was 52.9 ± 2.9%. Ibudilast brain concentration was significantly (p < 0.05) higher in PDA coated micelles than oral or nasal free drug.

Polycyclic aromatic hydrocarbons in sewage-irrigated vegetables from industrial cities in Haryana, India.

The majority of Indians consume a lot of vegetables because of their health advantages. High concentrations of polycyclic aromatic hydrocarbons (PAHs) in vegetables may be seriously harmful to consumers' health. The method for identifying and measuring 16 USEPA polycyclic aromatic hydrocarbons (PAHs) in samples of sewage-irrigated vegetables from three industrial cities in Haryana, India, is described in this research. Ultrasonication, liquid-liquid extraction using n-hexane as a solvent, clean-up using a Florisil column, and reversed-phase high-performance liquid chromatography with a UV detector were all included in the process. The PAHs were successfully linearized (R2 > 0.99) at various doses. Results for PAH recovery ranged from 90 to about 100%. The limit of quantification was 0.002-0.580, and the limit of detection was 0.0006-0.174 µgkg-1. Data indicated that the highest mean concentrations of ∑16 PAHs were detected in Spinach (123.36 µgkg-1), in carrot (105.09 µgkg-1), and in cucumber (63.40 µgkg-1) among leafy, underground, and fruity vegetables, respectively.

Rapid Development and Validation of Atoltivimab, Maftivimab and Odesivimab in Pharmaceutical Dosage form by using the RP-HPLC Method

Background: The reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the quantitative measurement of monoclonal antibodies (Maftivimab, Atoltivimab, and Odesivimab) in the pharmaceutical dosage form. The Food and Drug Administration (FDA) has approved these monoclonal antibodies for the treatment of Zaire ebolavirus infection in adults Methods: Maftivimab, Atoltivimab, and Odesivimab were separated chromatographically on the Waters Alliance-e2695 platform using the Luna Phenyl Hexyl (250 x 4.6 mm, 5 μm) column and a mobile phase made up of Acetonitrile (ACN) and ortho-phosphoric acid (OPA) buffer in a ratio of 70:30 (v/v). Results: The flow rate was 1.0 ml/min, and a photodiode array (PDA) detector operating at room temperature was used to measure absorption at 282 nm. For Maftivimab, Atoltivimab, and Odesivimab, the theoretical plates were not less than 2000, and the tailing factor shouldn't be greater than 2, accordingly. All measurements have a constant relative standard deviation of peak areas that is less than 2.0. Conclusion: The suggested procedure was approved following the International Conference on Harmonisation (ICH) recommendations. When used for the quantitative analysis of Maftivimab, Atoltivimab, and Odesivimab, the approach was found to be straightforward, affordable, appropriate, exact, accurate, and robust.

Droplet digital polymerase chain reaction-based quantitation of therapeutic lentiviral vector copies in transduced hematopoietic stem cells

Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). After HSPCs were transduced with an LV encoding the therapeutic β-globin (βA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10-3 ng and 5 × 10-6 ng of target copy per reaction. We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of βA-T87Q protein detected by reverse-phase high-performance liquid chromatography. Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.

Development And Validation Of Rp-Hplc Method For The Estimation Of Tenofovir And Emtricitabine In Bulk And Pharmaceutical Dosage Form.

A novel, simple, efficient, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the estimation of Tenofovir and Emtricitabine in the bulk and pharmaceutical dosage form. The currently developed method was subsequently validated according to ICH guidelines in terms of linearity, accuracy, precision, the limit of detection, the limit of quantification, robustness, etc. Methods: The separation of the selected drugs was optimized after several trials including change of mobile phase and its composition, stationary phase, flow rate, column temperature, etc. The separation was performed by using an Inertsil ODS C18 column (250 mm x 4.6 mm, 5μ) and UV absorption was measured at 231 nm. Methanol: Acetonitrile: Water was selected as the mobile phase in the ratio of 50:20:30 (V/V/V) at a flow rate of 1 mL/min. As per International Conference on Harmonization (ICH) Q2 R1 guidelines, several validation parameters were evaluated which include specificity, linearity, precision, accuracy, the limit of detection (LOD), and the limit of quantitation (LOQ). The acceptable degree of linearity range was found to be 40-100 µg/mL. The standard solution exhibited retention times of 3.06 minutes and 5.07 minutes for Tenofovir and Emtricitabine respectively. The LOD and LOQ obtained were 0.05µg/ml and 0.02µg/mL, 15µg/mL, and 0.08µg/mL for Tenofovir and Emtricitabine respectively. The percent recovery was found to be 98 to 102%. Hence, the proposed method is simple, selective, and specifically meets the requirements of ICH guidelines for the validation of the analytical method.

Synthesis of a Hydrophobic Phenanthrene-Containing Universal Support for Solid-Phase Oligonucleotide Synthesis.

Universal solid supports are widely used in solid-phase oligonucleotide (ON) synthesis based on phosphoramidite chemistry. Herein, we describe the synthesis of hydrophobic universal linkers, namely phenanthrene ring-fused 7-oxabicyclo[2.2.1]heptane-2,3-diol derivatives (PT linkers), their coupling to solid supports [e.g., controlled pore glass (CPG) and polystyrene (PS)], and the use of the resulting PT-linker-modified solid supports in ON synthesis. PT linkers were synthesized in four steps from commercial materials and subsequently attached to CPG and PS resins through succinyl and diethylene glycol-containing spacers, respectively. Cleavage of the desired ON from the resins was accomplished under standard basic conditions, indicating that the reactivity of the PT linkers was comparable to that of conventional universal linkers. Furthermore, owing to their high hydrophobicity, the desired ON could be readily separated from impurities originating from the PT linker by reversed phase HPLC. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phenanthrene ring-fused 7-oxabicyclo[2.2.1]heptane-2,3-diol (PT linker) derivatives Basic Protocol 2: Preparation of PT-linker-modified CPG and PS resins Basic Protocol 3: Solid-phase ON synthesis using PT-linker-modified solid supports and cleavage of ONs from resins.

RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR QUANTITATIVE ESTIMATION OF TEMOZOLOMIDE AND (S) - PERILLYL ALCOHOL IN NANOPARTICULATE DOSAGE FORM

A quick reverse-phase high-performance liquid chromatography (RP-HPLC) approach for the quantitative measurement of temozolomide (TMZ) and (s) - perillyl alcohol [(S)-POH] in a nanoparticulate system was developed and validated in the current work. The RP-HPLC method for the simultaneous estimation of TMZ and (S)-POH was developed using Agilent (Infinity 1260) HPLC system and ZorbaxC18 (4.6 x 150 mm i.d., 5µ; Agilent) as stationary phase. The optimized mobile phase comprised of ACN: water: MeOH (42:12:46 V/V/V; 42:08:50 V/V/V and 20:30:50 V/V/V) pumped at a flow rate of 0.8 mL min-1, 0.8 mL min-1 and 1 mL min-1, respectively. Drug separation was accomplished in an isocratic mode, and a PDA detector operating at 210 nm was used to track elution. The procedure was validated in accordance with ICH-Q2R1 standards. The responses of TMZ and (S)- POH were found to be linear at 50-175 μg mL-1 (ACN: water: MeOH 42:12:46 V/V/V and 42:08:50 V/V/V) and 50-175 μg mL-1 (ACN: water: MeOH 20:30:50 V/V/V) respectively. The percent recovery was determined to be between 97% and 103%, demonstrating that the method’s accuracy was adequate. The precision study’s percent relative standard deviation (% RSD) was less than 2, indicating the accuracy of the suggested procedure. It was discovered that the established method for the quantitative determination of TMZ and (S)- POH in bulk and in hollow gold nanoparticles was accurate, precise, and specific. The developed technique can be applied to TMZ and (S)- POH routine testing and quality control in bulk and nanoparticulate systems.

Unique heterocyclic phenolic compounds from shrimp (Pandalus borealis) and beyond

Shrimps are the most consumed species among crustaceans and are well appreciated for their flavor and high nutritional value. This work investigated the antioxidative compounds of Northern shrimp (Pandalus borealis) and its processing discards (shells and heads) using various solvents and chromatographic techniques as well as mass spectrometry. Ethanol served as the best extraction medium, and the extracts obtained demonstrated strong antioxidant activity in a β-carotene-linoleate emulsion system. The antioxidative compounds of the extract were isolated using a silica gel column chromatography, followed by thin layer chromatography. This led to the identification of two highly polar and two low polarity compounds. These compounds, for the first time, were isolated and purified by using reversed-phase HPLC. Their structures were tentatively elucidated using electrospray mass spectrometry. These unique compounds were heterocyclic phenolic compounds and identified as 7-(3-butenyl)-2-hydroxy-6-(1,2,5,6-tetrahydropyridin-2-yl) quinoline and 7-(3-butenyl)-2-hydroxy-6-(1,2,5,6-tertahydropyridin-2-yl)-7,8-dihydroquinoline and their isomers. The proposed novel compounds in shrimp provide a reason for their oxidative stability and potential benefits, as well as possibly serving as substrates for the discoloration of shrimp.Graphical

New Method for 5'-Nucleotidase Preparation and Evaluation of Its Catalytic Activity.

In this study, we established a new methodology for preparing 5'-nucleotidase (5'-NT) with the aim of enhancing our understanding of its enzyme activity and laying a basis for regulating the content of umami-enhancing nucleotides in pork. 5'-NT was prepared with Sephadex gel filtration and reverse-phase high-performance liquid chromatography, and its enzymatic properties and catalytic activity were evaluated. The results show that the molecular weight of the prepared 5'-NT was 57 kDa, the optimal catalytic temperature was 40 °C, and the optimal pH was 8. Zn2+, and sucrose showed inhibitory effects on the activity of 5'-NT, while K+, Na+, Ca2+, Mg2+, glucose, fructose, and trehalose promoted the activity of the studied compound. The prepared 5'-NT exhibited higher catalytic activity and selectivity against IMP compared with its commercial counterpart, while its catalytic activity against XMP was not significant (p > 0.05). In brief, we established a new methodology for preparing 5'-NT, enhancing our understanding of its enzyme activity and providing a solid basis for regulating the content of umami-enhancing nucleotides in pork through the control of endogenous 5'-NT activity.

Immune priming modulates Galleria mellonella and Pseudomonas entomophila interaction. Antimicrobial properties of Kazal peptide Pr13a.

Galleria mellonella larvae repeatedly infected with Pseudomonas entomophila bacteria re-induced their immune response. Its parameters, i.e. the defence activities of cell-free hemolymph, the presence and activity of antimicrobial peptides, and the expression of immune-relevant genes were modulated after the re-challenge in comparison to non-primed infected larvae, resulting in better protection. No enhanced resistance was observed when the larvae were initially infected with other microorganisms, and larvae pre-infected with P. entomophila were not more resistant to further infection with other pathogens. Then, the peptide profiles of hemolymph from primed- and non-primed larvae infected with P. entomophila were compared by quantitative RP-HPLC (Reverse Phase - High Performance Liquid Chromatography). The level of carbonic anhydrase, anionic peptide-1, proline peptide-2, and finally, unknown so far, putative Kazal peptide Pr13a was higher in the primed infected animals than in the larvae infected with P. entomophila for the first time. The expression of the Pr13a gene increased two-fold after the infection, but only in the primed animals. To check whether the enhanced level of Pr13a could have physiological significance, the peptide was purified to homogeneity and checked for its defence properties. In fact, it had antibacterial activity: at the concentration of 15 µM and 7.5 µM it reduced the number of P. entomophila and Bacillus thuringiensis CFU, respectively, to about 40%. The antibacterial activity of Pr13a was correlated with changes observed on the surface of the peptide-treated bacteria, e.g. surface roughness and adhesion force. The presented results bring us closer to finding hemolymph constituents responsible for the effect of priming on the immune response in re-infected insects.

Fixed combination of rivaroxaban and Aspirin: Tablet formulation and analytical method validation

Rivaroxaban and aspirin are commonly used antithrombotic agents in combination to prevent coronary artery disease (CAD) and atherothrombotic events in adult patients after acute coronary syndrome (ACS) with elevated cardiac biomarkers, or with coronary artery disease (CAD) or symptomatic peripheral artery disease (PAD) in high-risk patients. The recommended dosage is 2.5 mg rivaroxaban twice daily in combination with 75–100 mg aspirin once daily. This study aimed to develop a fixed-dose combination tablet of rivaroxaban (2.5 mg) and aspirin (50 mg) to enhance medication adherence and decrease pill burden.Compatibility studies were conducted on the active ingredients to develop the product formula. The formula and the manufacturing procedure were chosen based on the risk assessment for each active substance, wet granulation with both actives intragranular was found to have faster dissolution than direct mix formulae. Furthermore, a validated reverse-phase HPLC stability indicator method was developed to detect APIs and their possible degradants.

Features and Advantages of the Recurrent Approximation of Retention Times in Reversed-Phase High-Performance Liquid Chromatography

Background: Recurrent approximation of retention parameters in reversed-phase HPLC is effective for revealing anomalies that are otherwise difficult to detect, namely, the reversible hydration of analytes. This was demonstrated previously for restricted sets of analytes with acetonitrile–water eluents. Expanding the number of analytes and eluents seems to be a topical problem. Two kinds of derivatives of aromatic carbonyl compounds were characterized: unsubstituted hydrazones and oximes. Methods: If analyte demonstrates no anomalies in dependences of retention times vs. concentration of organic modifier, the recurrent approximations of these dependences are linear. To explain the features of recurrent approximations, the numerical experiments were proposed and considered. The artificial shifting of one, two, or more points allows for the modeling the different kinds of deviations of approximations from linearity. Results: It was shown that hydrazones are the class of analytes having no anomalies of retention parameters. On the contrary, several anomalies were detected for oximes. Downward deviations of points in the plots of recurrent approximations of retention times are the signs of reversible hydration. This effect for methanol–water eluents was detected for the first time. Some of oximes underwent hydrolysis. Conclusions: Recurrent approximation of retention times allows detecting chemical transformations of analytes during RP HPLC analysis.

The Oxidative Status and Na+/K+-ATPase Activity in Obsessive-Compulsive Disorder: A Case Control Study.

Oxidative stress is involved in pathogenesis of some psychiatric disorders. To examine the role of oxidative stress in the etiopathogenesis of obsessive-compulsive disorder (OCD), we aimed to determine oxidative stress indices, including malondialdehyde (MDA) levels in serum and red blood cells (RBC) membrane, total antioxidant capacity (TAC), serum glutathione (GSH) levels, serum antioxidant vitamins (A and E), and Na+/K+-ATPase activity, in patients with the mentioned disorder vs. healthy controls. 39 OCD patients diagnosed based on Diagnostic and Statistical Manual of Mental Disorders (DSM-V) and 39 volunteer healthy subjects were included in this study. MDA levels in serum and RBC membrane were measured using fluorometric method. Serum TAC level, serum GSH level, and Na+/K+-ATPase activity were also measured using spectrophotometric methods. Serum levels of vitamins were calculated by reversed-phase high-performance liquid chromatography (RP-HPLC). There was a significantly higher MDA level in serum (p < 0.0001) and RBC membrane (p = 0.002) of OCD patients compared with those in controls. A significant reduction in vitamin A (p = 0.001) and vitamin E (p = 0.024) levels was found in OCD patients vs. controls. There was significantly lower activity of erythrocyte membrane Na+/K+-ATPase in RBC membrane of OCD patients vs. controls (p < 0.0001). Our findings indicate significantly higher levels MDA in both serum and RBC membrane, lower levels of serum vitamins A and E, and lower activity of membrane Na+/K+-ATPase in OCD patients compared to controls. These suggest an imbalance between oxidant and antioxidant factors in OCD patients that might play a fundamental role in the etiopathogenesis of OCD.

Isolation and antioxidant activity of peptides from Chinese hairy tofu.

Hairy tofu is a famous Chinese snack that is made from soybeans and rich in various nutrients. In order to further explore the antioxidant peptides of hairy tofu hydrolysates, seven proteases were used to hydrolyze hairy tofu. The results of in vitro radical scavenging activity showed that hairy tofu hydrolysates obtained by pancreatin exhibited the highest antioxidant activity. After Sephadex G-25 gel filtration and reversed-phase high-performance liquid chromatography (RP-HPLC), 97 peptides were identified in the most antioxidant fraction using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Among them, nine peptides were synthesized and their antioxidant activities were assessed using a H2O2-induced oxidative 293T cell model. Finally, four peptides (QCESHK, LAWNEGR, NLQGENEWDQK, and FTEMWR) at concentrations of < 50 μg/ml significantly decreased the malondialdehyde content compared with the model group, displaying in vivo antioxidant activity and low cytotoxicity. Overall, this research provided the choice of using hairy tofu peptides as antioxidant products in the pharmaceutical and food industries.

A novel Six Sigma approach and eco-friendly RP-HPLC technique for determination of pimavanserin and its degraded products: Application of Box–Behnken design

Abstract A green analytical chemistry study attempts to generate environmentally friendly alternatives to dangerous compounds while reducing waste output. The study thoroughly analyzed eight green tools to determine their greenness. Given its importance in treating hallucinations and delusions produced by Parkinson’s disease psychosis, it is critical to have a reliable and precise method for identifying pimavanserin (PVS) in both pure form and pharmaceutical formulations. The current study used a straightforward approach to forced degradation experiments to establish a novel reversed-phase high-performance liquid chromatography method for evaluating stability. The method was executed on the Agilent Zorbax Eclipse Plus C18 column (100 × 4.6 mm, 3.5 μm particle size) with ultraviolet detection at 226 nm. The Box–Behnken design is the ultimate solution for identifying optimal chromatographic conditions in a timely and efficient manner, with minimal trials required. The study investigated the impact of three factors: acetonitrile ratio, column oven temperature, and flow rate on various responses, namely, retention time, tailing factor, and theoretical plates. Desirability and overlay plots were utilized to forecast the best mobile phase containing a buffer solution: acetonitrile: tetrahydrofuran in a ratio of (65: 20: 15, v/v/v), which proved highly effective in the experiments. Linearity was conducted for PVS in the 3–50 µg·mL−1 range with an R 2 coefficient of determination of 0.9997. PVS had detection and quantification limits of 1.1 and 3.5 µg·mL−1, respectively, indicating a highly significant correlation between the variables studied. PVS’s recovery percentage was determined to be 101.30%. We also used the Six Sigma lean technique to ensure precision and productivity. PVS was tested for acid, base, oxidative hydrolysis, photodegradation, and heat, as per International Council for Harmonisation guidelines. The highest degradation was obtained from oxidative hydrolysis and thermal degradation.

Simultaneous Identification and Quantification of Phenylalanine, Glycine and Lysine amino acids and conjugated heterocyclic amines in Maillard chemical model system using RP-HPLC-DAD

In this study, we developed a simple reversed-phase high-performance liquid chromatography (RP-HPLC) methodology for simultaneous identification and quantification of three types of mix: (1) phenylalanine and PHIP; (2) glycine and MeIQx; (3) lysine and 4,8 DiMeIQx in Maillard chemical model systems. In addition, we investigated the effects of different conditions on decreasing amino acids and heterocyclic amines (HCA) formation in chemical model systems. The results show that the developed methods used to separate the proposed mixes gave a relative standard deviation (RSD)% of less than 2% of peak area and retention time, thus proving the robustness and high reproducibility of the proposed method. The relationship between concentration and peak areas is linear with high statistical significance (p&lt;0.001). The residual and slope of linear regression give a limit of detection LoD) and (LoQ) of phenylalanine and PHIP were (2.92- 0.029 ppm),(9.73- 0.098 ppm),respectively, which indicate the high sensitivity of the developed method. Moreover, the findings illustrate that a water ratio of 20% leads to a decrease in phenylalanine versus the largest amount of PHIP, and phenylalanine is the most active amino acid with a decreasing percentage up to 99%.

Simultaneous Analysis of Anti-Hypertensive Drugs by RP-HPLC: An Overview

High performance liquid chromatography (HPLC) is an important qualitative and quantitative technique, generally used for the estimation of pharmaceutical and biological samples. In now a day pharmaceutical market, many newer antihypertensive combination drugs are available to control hypertension as well as to keep society healthy and stress free. All presently available old drugs have frequent dosing produces various side effects. So, there is need to analyse such antihypertensive drugs. The aim of this review is to analysed such commonly used antihypertensive combination drugs by using reverse phase high performance liquid chromatography (RP-HPLC). Reversed-phase high-performance liquid chromatography (RP-HPLC) involves the separation of molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase. The drugs like Amlodipine besylate and Valsartan, Irbesartan and Hydrochlorothiazide, Atenolol and Chlorthalidone, Hydrochlorothiazide and Candesartan, Ramipril and Amlodipine, Hydrochlorothiazide and Enalapril, Quinapril and Hydrochlorothiazide etc. About fifteen combination drugs are analysed by using RP-HPLC. This review assists in appropriate selection of column, mobile phase, pH, flow rate, detector and form of such combination drugs.

Purification and Identification of Peptides from Hydrilla verticillata (Linn. f.) Royle with Cytoprotective and Antioxidative Effect against H2O2-Treated HepG2 Cells.

Antioxidant peptides were purified from Hydrilla verticillata (Linn. f.) Royle (HVR) protein hydrolysate by ultrafiltration, gel filtration chromatography, and semipreparative reversed-phase HPLC and identified by UPLC-ESI-MS/MS. Therein, TCLGPK and TCLGER were selected to be synthesized, and they displayed desirable radical-scavenging activity to ABTS (99.20 ± 0.56-99.20 ± 0.43%), DPPH (97.32 ± 0.59-97.56 ± 0.97%), hydroxyl radical (54.32 ± 1.27-70.42 ± 2.01%), and superoxide anion (42.93 ± 1.46-52.62 ± 1.11%) at a concentration of 0.96 μmol/mL. They possessed a cytoprotective effect against H2O2-induced oxidative stress in HepG2 cells in a dose-dependent manner. 1.6 μmol/mL of the two peptides could perfectly protect HepG2 cells from H2O2-induced injury. The TCLGPK exhibited higher antioxidant activity and cytoprotective effect than TCLGER. Western blot and molecular docking results indicated that the two peptides achieved antioxidant ability and cytoprotective effect by combining with Kelch-like ECH-associated protein 1 (Keap1) to activate the Keap1-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response elements signaling pathway, leading to the activity and expression of the related antioxidases in the pathway significantly up-regulating and the intracellular reactive oxygen species level, lipid peroxidation, and cell apoptosis rate significantly down-regulating.

Quantification of Riboflavin and Thiamine in GI (Geographical Indication) Branded Yogurts Collected from Bogura, Bangladesh Using HPLC Equipped with a Fluorescence Detector

Thiamine (vitamin B1) and riboflavin (vitamin B2) are both essential vitamins for human health. Thiamine (vitamin B1) is essential for energy metabolism, nervous system function, carbohydrate metabolism, cardiovascular health, digestion, muscle function, etc. On the other hand, Riboflavin (vitamin B2), which acts as an antioxidant, is essential for energy production, healthy skin, hair and nails, eye health, red blood cell production, nervous system function, etc. Due to its specialty, Bogura's famous yogurt was recognized as a Geographical Indication (GI) on June 26, 2023 by the Directorate of Patents, Designs, and Trademarks (DPDT) under the Ministry of Industry, the Government of the People’s Republic of Bangladesh (Prothom Alo, 2023). The purpose of the study was the quantitative determination of two water-soluble vitamins, thiamine and riboflavin, in the GI branded yogurt and whey (Ghol), collected from Bogura, Bangladesh to ensure health benefits. The methods employed for the quantitative analysis of these vitamins were reversed-phase high performance liquid chromatography (RP-HPLC) equipped with a fluorescence detector. The extraction process involved acid hydrolysis followed by enzymatic hydrolysis with takadiastase enzyme. Chromatographic separation was achieved with a Shimadzu Prominence HPLC system using isocratic elution mode on a C18 column (4.6 mm × 250 mm, 5 μm). The correlation coefficients for thiamine and riboflavin were 0.9975 and 0.9974, respectively. The recovery rates for thiamine and riboflavin were found as 97.70% and 105.40%, respectively. Twenty-two samples were analyzed. Thiamine and riboflavin were found in the range of 0.0647 to 0.1396 mg/100 g and 0.0227 to 0.3749 mg/100 g, respectively. It can be concluded that these yogurts were rich in thiamine and riboflavin. Therefore, these yogurts are beneficial for health. Bangladesh Pharmaceutical Journal 27(1): 59-66, 2024 (January)

A comparative novel green reversed-phase high-performance liquid chromatography method for simultaneous determination of ivabradine and carvedilol in tablets and human plasma using UV and fluorescence detectors

A rapid, green, and sensitive dual detection technique method was applied using both UV and fluorimetric detection to determine ivabradine and carvedilol in their pure form, combined tablet formulation, and in the spiked human plasma. The method was achieved by using HyperClone™ MOS C8 column (150 × 4.6 mm, with particle size 5 μm), and a mixture composed of (70:10:20%v/v) ethanol: methanol: 0.02 M potassium phosphate buffer adjusted to pH = 3 b y using o-phosphoric acid. The dual detection technique was performed at 210 nm in the case of UV detection, and at 337 nm after excitation at 282 nm in the case of fluorimetric detection with a flow rate of 1 mL/min. The retention time in the proposed method was found to be 2.13 and 4.72 min for carvedilol and ivabradine respectively (UV detection) and 2.16 and 4.5 min for carvedilol and ivabradine respectively (fluorescence detection). It was found to be a rectilinear method (r > 0.999), precise (RSD <2 %), and accurate (recoveries 99.92 and 100.52). The method was simple, specific, sensitive, and robust. Calibration graphs were found to be rectilinear from 0.25 to 11.25 μg/mL and 0.50–16 μg/mL for carvedilol and ivabradine for UV detection, while from 30 to 210 ng/mL and 48–400 ng/mL for carvedilol and ivabradine for fluorimetric detection. A correlation coefficient (r) was 0.9999 for the two drugs in UV and fluorimetric detection approaches. Fixed–dose combination tablets containing both medications could be examined routinely by the validated method without interference from excipients. Moreover, the fluorimetric detection HPLC technique can be applied to determine the studied drugs in the human plasma with high accuracy and precision. Greenness of the method was assessed by using Eco-scale and AGREE metrics, and the method was found to be green.