Reversed-phase High-performance Liquid Chromatography Research Articles

Methanolic Leaves Extract of Ziziphus spina-christi Inhibits Cell Proliferation and Migration of HER2-Positive Breast Cancer via p38 MAPK Signaling Pathway

Human epidermal growth factor receptor 2 (HER2) is a subtype of breast cancer that is associated with poor prognosis and low survival rates. The discovery of novel anti-cancer agents to manage this subtype of cancer is still needed. Ziziphus spina-christi (ZSC) is a plant species that is native to Qatar. It exerts various biological activities, including cytotoxicity as it contains different essential bioactive constituents, mainly rutin and quercetin. To examine the outcome of ZSC on HER2-positive breast cancer, we standardized the ZSC methanolic leaves extracted by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) analysis using the flavonoids rutin and quercetin as marker compounds. Here we used two HER2-positive breast cancer cell lines, ZR-75-1 and SK-BR-3, and the chorioallantoic membrane as an angiogenesis model. We found that ZSC extract significantly reduces viability, alters the normal morphological phenotype of HER2-positive breast cancer cells, and inhibits cell migration as well as colony formation; this is accompanied by deregulating different apoptotic markers such as Bax/Bcl-2 and NF-κB in both cell lines. Additionally, ZSC methanolic extract significantly represses the angiogenesis of the chorioallantoic membrane model. Moreover, the molecular pathway investigations pointed out that ZSC extract represses the activity of HER2 and p38 MAPK which could be the main pathways behind the effect of ZSC in HER2-positive cells. Collectively, our results support the potential role of ZSC in the management of HER2-positive breast cancer and form the basis for future investigations.

Amino acid composition, antioxidant activity and mineral content of Achatina fulica snail slimes and edible bird’s nest

This study was performed to investigate whether the snail slimes which also well-known for their anti-aging properties, can be served as an alternative for edible bird’s nest (EBN) in our daily lives. The amino acid composition of samples was evaluated using a reversed-phase High Performance Liquid Chromatography. The four major amino acids detected in snail slimes were aspartic acid (3.80 µmol/mL), glutamic acid (2.87 µmol/mL), alanine (2.23 µmol/mL) and serine (1.80 µmol/mL), while for EBN, the top four amino acids were proline (5.31 µmol/mL), serine (5.27 µmol/mL), aspartic acid (4.78 µmol/mL) and threonine (3.97 µmol/mL). In overall, EBN possessed significantly (p < 0.05) higher total amino acid content (42.63 µmol/mL) than the snail slimes (23.59 µmol/mL). Besides, the EBN was found to have significantly higher (p < 0.05) DPPH free radical scavenging activity (25.12 %) as compared to the snail slimes (16.02 %). The four major elements identified in snail slimes and EBN were sodium, calcium, potassium and magnesium. Snail slimes contained higher levels of minerals compared to EBN. In conclusion, the snail slimes can potentially be served as an alternative of EBN to those people suffered from mineral deficiencies.

Development and validation of a RP-HPLC method for simultaneous determination of cimetidine, metoprolol tartrate and phenol red for intestinal perfusion studies.

A new reversed phase high-performance liquid chromatography (RP-HPLC) method, with a short analysis time and easy to apply, was developed for the simultaneous detection of cimetidine (CIM), metoprolol tartrate (MT) and phenol red (PR) for use in intestinal perfusion studies. The analysis was performed with phosphate buffer (pH 5.0, 12.5mM)-acetonitrile mixture as mobile phase and C18 column (Inertsil ODS-3; 5µm, 4.6×250 mm) as stationary phase. Gradient analysis conditions were used and the acetonitrile ratio in the mobile phase varied from 10 to 50% in 10min. Total run time for analysis was 10min and the injection volume was 20µL. Detection of compounds was performed at 207nm. Under optimum HPLC conditions, retention times were 4.03min for CIM, 6.99min for MT and 8.49min for PR. The method was validated according to ICH Q2 (R1) guideline for specificity, linearity, sensitivity, precision, accuracy, stability and robustness. Developed method was linear and determination coefficients of the calibration curves were 0.9993, 0.9991 and 1.0 for CIM, MT and PR, respectively. The limits of quantification were 6.20, 2.78 and 0.45μg/mL for CIM, MT and PR, respectively. The precision and accuracy values of the developed analytical method met the ICH Q2 (R1) limits. The applicability of the method was demonstrated by preliminary in-situ intestinal perfusion studies. In conclusion, samples obtained from in-situ intestinal perfusion studies performed to examine the absorption/permeability of CIM, MT, and PR can be analyzed with the developed HPLC method.

Undescribed and known phenolic acid derivatives with significant antioxidant activity from the Scorzonera parviflora Jacq. roots.

The antioxidant activity of Scorzonera parviflora Jacq. roots were assessed by measuring their ability to scavenge ABTS and DPPH radicals. Bioactivity-guided fractionation was utilized to identify the compound(s) responsible for this activity. The most active phase, ethyl acetate, was isolated using column chromatography. The resulting fractions were then purified using preparative TLC on reverse phase and semi-preparative HPLC. The structures of the pure compounds were elucidated by spectral analysis (MS and 1H, 13C, 2D-NMR). Three undescribed phenolic acid derivatives, namely parvifloric acid A (1), B (2), and C (3), and one new sesquiterpene lactone, parviflorin (4) together with seven known compounds were isolated and identified as scopolin (5), scopoletin (6), caffeic acid (7), protocatechuic acid (8), 4,5-O-dicaffeoylquinic acid (9) 3,5-O-dicaffeoylquinic acid (10), and 3,5-O-dicaffeoylquinic acid methyl ester (11). Finally, the pure compounds obtained were tested to evaluate their antioxidant capacities, using ABTS and DPPH radical scavenging potencies. The highest activity was observed with 3,5-O-dicaffeoylquinic acid (10), followed by its methyl ester.

Method Validation of Crisaborole using Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and Quantitation of Crisaborole in Nanoemulsion Formulation for its Application in Ex Vivo Transdermal Permeation Study

Crisaborole is a boron containing phosphodiesterase 4 (PDE4) inhibitor which in this study was formulated in nanoemulsion form to treat atopic dermatitis. The study aims to develop and validate a selective analytical method for determining crisaborole in nanoemulsion formulations, facilitating its application in ex vivo transdermal permeation studies for the treatment of atopic dermatitis. Crisaborole was eluted using gradient elution method in reverse phase (C18) chromatography. The mobile phases were comprised of phosphate buffer (pH3) and acetonitrile, which increasing acetonitrile volume from 30 to 95 percent within 15 min with constant flow rate of 0.65 ml/min. Column temperature was set at 40 ºC and detection wavelength was at 252 nm. The method produced linear responses (R2=0.99) in concentrations ranges from 1.56-100 μg/ml (n=7). The developed method was sensitive with limit of detection (LOD) and limit of quantitation (LOQ) values of 1.84 μg/ml and 5.58 μg/ml; respectively. Crisaborole was stable in various working conditions which were within the acceptance value provided by guidelines (±15%). In ex vivo studies, the concentration of crisaborole was plotted in cumulative permeated graph and this quantitative method was proven to provide a selective and sensitive measurement for detection of crisaborole.

PROSPECTIVE STUDY ON GENETIC DETERMINANTS OF CARBAMAZEPINE RESPONSE: THE CYP3A5-RS15524 GENE POLYMORPHISM IN PASHTUN PATIENTS WITH EPILEPSY

Objective: To evaluate the impact of CYP3A5-rs15524 polymorphism on the pharmacokinetics and clinical response to carbamazepine (CBZ) monotherapy in Pashtun epilepsy patients. Materials and Methods: A longitudinal study was carried out among Pashtun patients with epilepsy in Khyber Pakhtunkhwa from October 2020 to April 2022. Participants were recruited from Lady Reading Hospital, while laboratory work was performed at the Pharmacology Department of Khyber Medical University, Peshawar. Patients were genotyped for the CYP3A5-rs15524 polymorphism using Sanger sequencing, with variants analyzed via Finch TV. The study correlated each genotype with clinical response and serum CBZ levels, which were measured using reversed-phase HPLC during each follow-up visit. Results: A sample of 223 patients was analyzed, including 63.2% males and 36.8% females. Generalized tonic-clonic (GTC) seizures comprised 82.5% of cases, while partial seizures accounted for 17.5%. Genotype analyses showed a significant improvement in CBZ response in subsequent follow-up. At 3rd month, the non-responders / responders were recorded to be 18.3% / 31.3% for AA, 44.6% / 16.9% for AG, and 63.3% / 18.2% for GG genotype carriers. At 6th month it was recorded as 9.5% / 53% for AA, 32.3% / 32.3% for AG, and 27.3% / 54% among GG carriers, demonstrating a highly significant differences in response (p-value=0.001). Highest and lowest plasma levels across both follow-ups were observed in AA & AG genotypes, respectively with a p-value less than 0.001. Conclusion: Our findings suggest that CYP3A5-rs15524 polymorphism may impact the pharmacokinetics and clinical response to carbamazepine in Pashtun patients with epilepsy. Clinical pharmacogenetic research has the potential to greatly enhance personalized treatment within our study population.

Stability-Indicating Liquid Chromatographic Method Development for the Simultaneous Determination of Amitriptyline Hydrochloride and Propranolol Hydrochloride in Tablet Dosage Form.

The combination of the tricyclic antidepressant amitriptyline hydrochloride (AMH) and the non-selective beta-adrenergic blocker propranolol hydrochloride (PPH) is used for migraine prophylaxis. Higher doses of AMH trigger cardiac arrhythmias, anxiety, tachycardia, convulsions, hyperglycemia and anticholinergic side effects. The combined dosage formulation of AMH and PPH leads to drug-drug interactions; causes sedation, xerostomia, dysuria, insomnia and bradycardia; and results in patient non-compliance. The quantification of AMH and PPN becomes essential, especially for combination formulations, in addition to regular quality control to avoid clinical issues. Considering these facts into account, the reverse-phase -high-performance liquid chromatography (RP-HPLC) method was developed in accordance with International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Q2(R1) guidelines for the simultaneous determination of AMH and PPH. The HPLC separation was performed on an HPLC system (Shimadzu, Japan, Prominence I series 2030C) using a Shimadzu Shim-Pack GIST C18 column (100mm × 4.6mm, 5μ), which was equipped with an ultraviolet detector at the isosbestic point 238nm. The mixture of acetonitrile and orthophosphoric acid (pH3.5) in a ratio of 35:65v/v with a flow rate of 0.75mL/min was used as the mobile phase. The regression coefficients of AMH (r2 > 0.998) and PPH (r2 > 0.999) show good linearity between peak areas and drug concentration ranges. The limits of detection (AMH = 0.67μg/mL, PPH = 0.67μg/mL) and limits of quantification (AMH = 2.04μg/mL, PPH = 2.05μg/mL) demonstrated the higher detection sensitivity of the proposed method.

Isocratic liquid chromatography technique for the analysis of cyanocobalamin from beef liver and heart muscle extracts

Reverse-phase high-performance liquid chromatography (RP-HPLC) is among the most widely recommended techniques for analyzing water-soluble vitamins such as vitamins C and B-complexes. The research study was conducted to detect and quantify cyanocobalamin (vitamin B12) from the beef liver and heart muscle extracts using a validated isocratic RP-HPLC procedure. The analytical column was Luna® Phenomenex 5 µm C18 (2) 100 A LC-column (150 × 4.6 mm). The mobile phase consisted of water/ethanol in a ratio of 60:40 (v/v). An enzymatic digestion with 1% potassium cyanide was used for samples (beef liver and heart muscle) extraction. The validated method showed to be linear, R = 0.9977; fast, with a retention time of less than 6.00 min; precise, %RSD of 1.16 and 1.74%; and sensitive, with limits of detection and quantification of 0.00033 and 0.00100 µg/mL. The detected values of cyanocobalamin from the beef liver (BLV) and heart muscle (HRM) extracts were 52.04 ± 0.13 and 42.04 ± 0.29 µg/mL, respectively. BLV extract indicated a higher level of cyanocobalamin. Hence, the validated isocratic RP-HPLC technique can be recommended to analyze cyanocobalamin and related compounds in research laboratories such as diagnostics, foods, and pharmaceuticals.

Leucinostatins target Plasmodium mitochondria to block malaria transmission

BackgroundMalaria remains a critical disease. Leucinostatins from the fungus Purpureocillium lilacinum inhibited the transmission of Plasmodium falciparum to mosquitoes via contact.MethodsHere, we modified the leucinostatin B (LB) C-terminus to make derivatives and examined their inhibition against malaria transmission to mosquitoes. Fluorescence-labeled leucinostatins were incubated with intact gametocytes and were examined under microscopy to detect the targets of leucinostatins. We also analyzed leucinostatins’ general cytotoxicity and hemolysis.ResultsThe results showed that the derivatives with –H, –CH3, –Atto495, and –Biotin at C-terminus had EC50 of 1.5 nM, 0.2 nM, 4.2 nM, and 42 nM, respectively. Atto495 and biotin are similar in size and much bigger than -CH3 and -H. Based on reverse-phase HPLC elution time, we found that LB-Biotin had much higher hydrophobicity than the others, consistent with its lowest malaria transmission-blocking activity. Fluorescence microscopy showed that LB-Atto495 colocalized with mitochondria inside intact P. falciparum gametocytes. We found that leucinostatin A significantly inhibited the proliferation of human nucleated cells with IC50 around 47 nM and it did not lyse erythrocytes at 100 μM.ConclusionsWe conclude that the leucinostatins pass through the cytoplasmic membrane without lysing cells and interact with molecules specifically in mitochondria. Therefore, leucinostatins should be ideal inhibitors against mobile parasites, such as ookinetes and sporozoites, during malaria transmission.Graphical

Non-affinity platform for processing knob-into-hole bispecific antibody

Bispecific antibodies hold significant potential as next-generation biotherapeutics owing to their ability to simultaneously bind to two targets. However, the development of bispecific antibodies as biotherapeutics has been hindered by the high levels of byproducts produced, including both high molecular weight and low molecular weight variants. In addition, the inevitable expression of homodimers in host cells presents further obstacles to the commercial development of bispecific antibodies as therapeutics. These byproducts, which share similar physicochemical properties with the target, pose several challenges for downstream purification processes. In this study, we present a non-protein A purification platform that employ a one-step polishing chromatography to purify bispecific antibodies. Mixed-mode Capto adhere resin was used to capture the target protein at pH 7.90 ± 0.10, followed by anion exchange chromatography as a polishing step. Overall, the results of this two-step chromatography purification method demonstrated at final product purity of 98% as assessed by size-exclusion high-performance liquid chromatography (SEC-HPLC) and 98% by reversed-phase-high-performance liquid chromatography (RP-HPLC), with residual host cell proteins controlled at 10 ppm and an excellent recovery rate of approximately 60%. This study presents a non-protein A capture platform, offering a simplified, streamlined, and competitive alternative to conventional affinity chromatography.Graphical

Characterization of Siderophores Produced by Bacillus velezensis YL2021 and Its Application in Controlling Rice Sheath Blight and Rice Blast.

Bacillus velezensis YL2021 has extensive antimicrobial activities against phytopathogens, and its genome harbors a catechol-type siderophore biosynthesis gene cluster. Here, we describe the characterization of siderophores produced by strain YL2021 and its antimicrobial activity in vitro and in vivo. A few types of siderophores were detected by chrome azurol S plates coupled with Arnow's test, purified, and identified by reversed-phase high-performance liquid chromatography. We found that strain YL2021 can produce different antimicrobial compounds under low-iron M9 medium or iron-sufficient Luria-Bertani medium, although antimicrobial activities can be easily observed on the two media as described above in vitro. Strain YL2021 can produce at least three catechol-type siderophores in low-iron M9 medium, whereas no siderophores were produced in Luria-Bertani medium. Among them, the main antimicrobial siderophore produced by strain YL2021 was bacillibactin, with m/z 882, based on the liquid chromatography-tandem mass spectrometry analysis, which has broad-spectrum antimicrobial activities against gram-positive and gram-negative bacteria, the oomycete Phytophthora capsici, and phytopathogenic fungi. Moreover, the antifungal activity of siderophores, including bacillibactin, observed in vitro was correlated with control efficacies against rice sheath blight disease caused by Rhizoctonia solani and rice blast disease caused by Magnaporthe oryzae in vivo. Collectively, the results demonstrate that siderophores, including bacillibactin, produced by B. velezensis YL2021 are promising biocontrol agents for application in rice disease control.

Phytochemical characterization and anti-arthritic potential of green-synthesized CuO nanoparticles derived from the Bistorta amplexicaulis root extract.

Rheumatoid arthritis is an autoimmune disease that mainly causes joint damage. The patient experiences loss of appetite, pain, fever, and fatigue. The present study was designed to phytochemically characterize and evaluate the anti-arthritic activity of green-synthesized copper oxide (CuO) nanoparticles (NPs) using the hydroalcoholic extract of Bistorta amplexicaulis roots in an adjuvant-induced arthritic rat model. For this purpose, crude powdered plant material was used for proximate analysis, and the plant extract was assessed for qualitative phytochemical analysis, mineral contents, and flavonoid and phenolic contents, as well as quantitative phytochemical analysis through reversed-phase high-performance liquid chromatography (RP-HPLC) and Fourier-transform infrared (FTIR) spectroscopy. The in vitro antioxidant activity of both extracts was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The biosynthesized CuO NPs from the Bistorta amplexicaulis extract showed anti-arthritic activity due to the presence of flavonoids and phenols, which showed a pain reliever effect by blocking the cyclo-oxygenase enzyme and has immune suppressant activity, thus securing the joint from destruction. The nanoparticles were characterized by zeta size, zeta potential, scanning electron microscopy (SEM), and FTIR spectroscopy. Forty-eight albino rats were divided randomly into six treatment groups. The zeta size and zeta potential of the nanoparticles were 186.8nm and -9.23mV, respectively. Joint stiffness, spleen weight, thymus weight, and paw thickness showed a significant decrease after treatment with NPs. The hematological parameters such as red blood cells (RBCs) and hemoglobin showed a significant increase, while platelets and white blood cells (WBCs) showed a significant decrease in NP-treated groups. C-reactive protein (CRP), rheumatoid factor (RF), liver and kidney function biomarkers, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels showed a significant decrease at both low and high doses of green-synthesized CuO nanoparticles from the Bistorta amplexicaulis root extract. The final data were analyzed by one way and two-way analysis of variance (ANOVA) and Tukey's multi-comparison test. So, from this study, it was concluded that both the plant root extract and green-synthesized CuO nanoparticles have anti-arthritic potential, but CuO NPs showed remarkable results.

Development and validation of RP-HPLC method for simultaneous estimation of lidocaine and nifedipine in pure and combined dosage form

The simultaneous estimation of Lidocaine and Nifedipine using reversed-phase high-performance liquid chromatography (RP-HPLC) is essential for analysing both the bulk drug and its topical dosage form. The objective of this study was to develop a novel HPLC method for the simultaneous estimation of Lidocaine and Nifedipine. The method aimed to provide accurate and precise results for both the bulk drug and topical dosage form. The HPLC method employed a C18 column measuring (150 mm × 4.6 mm × 5 µm). A mobile phase consisting of a mixture of methanol and water in a ratio of 80:20 v/v was used. The flow rate was set at 1.0 ml/min, and the detection wavelength was set at λ-max 236 nm. The retention time for Lidocaine was determined to be 2.560 min, while for Nifedipine it was found to be 5.470 min. The developed HPLC method demonstrated good linearity for both Lidocaine and Nifedipine, as evidenced by the obtained correlation coefficients (R2) of 0.9890 and 0.9986, respectively. The accuracy studies yielded results within the acceptable range of 98.11% - 105%. Additionally, the precision of the method was established with % RSD values of less than 2% for routine analysis of lidocaine and nifedipine. This method provides a reliable and effective approach for the simultaneous estimation of lidocaine and nifedipine, contributing to the quality control and safety assessment of pharmaceutical formulations containing these compounds.

An Underlying Cause and Solution to the Poor Size Exclusion Chromatography Performance of Antibody-Drug Conjugates.

Antibody-drug conjugate (ADC) size variants are frequently assessed by size exclusion chromatography (SEC). However, poor chromatography performance is often observed during SEC analysis. Existing studies have primarily focused on qualitatively describing non-specific interactions between ADCs and the column matrix. The purposes of the current study are to introduce an underlying cause from a novel perspective on the protein-protein interaction (PPI) mechanism, characterized by quantifying diffusion interaction parameter (kD) values, and to provide several strategies to reduce PPI and improve column performance during SEC analysis. Two kinds of ADCs with varying hydrophobicity properties and their corresponding monoclonal antibodies are used as models. The hydrophobicity of these products was verified using the relative calculated logarithm of the partition coefficient of a substance in n-octanol (oil) and water (rCLogP) and reversed-phase high performance liquid chromatography (RP-HPLC), and the size variants were analyzed using SEC. Finally, the PPI was characterized by kD values of these four products. The results of rCLogP and RP-HPLC indicated that ADC-1 is relatively hydrophobic, whereas ADC-2 is relatively hydrophilic. In the SEC analysis of the ADC-1, substituting sodium chloride with L-arginine hydrochloride or adding a specific concentration of acetonitrile as an organic solvent to the mobile phase resulted in reduced PPI and enhanced column performance. Conversely, the impact on ADC-2 was negligible. This study provides insights into improving the performance of SEC analysis for ADCs through strategies involving alterations in mobile phase composition. The changes in column performance can be quantitatively explained by the PPI mechanism.

Development and Validation of RP-HPLC Method for Roflumilast Nano Particles in Rat Plasma for Pharmacokinetic Study Analysis

Aim: The aim of this study is to develop a sensitive, specific, rapid, and precise reverse- phase high-performance liquid chromatography (RP-HPLC) method and validate it to determine the Roflumilast in rat plasma. Background: This study was aimed at developing a simple HPLC method for the detection of Roflumilast in rat plasma after ingestion of nanoparticles into the rat. A bioanalytical method was developed and validated. Methodology: The drug sample (Roflumilast) was eluted isocratically using a mobile phase of Ammonium acetate buffer (pH = 7.0), acetonitrile, and methanol (20:40:40, v/v/v) made up the mobile phase on a Phenomenex C-18 (150mm x 4.68mm pore size 5-micron analytical column. The drug was quantitatively determined at a UV wavelength of 254 nm using a standard calibration curve spanning the range of 1.5, 3.0, 4.5, 6.0, 7.5μg/mL-1 Results: The results showed that the limits of quantitation (LOQ) and detection (LOD) were 0.18 μg/ml and 0.54μg/ml, respectively. For both the intra-day and inter-day analysis, relative standard deviation (% RSD) was less than 2%, and the drug’s percentage accuracy was between 96% and 98%. Conclusion: As per the established protocols, the developed method was quantitatively assessed for its intended use in terms of specificity, linearity, accuracy, precision, robustness, and sensitivity analysis. Roflumilast-loaded nanoparticles were effectively used to determine the drug entrapment efficiency using the currently validated RP-HPLC method. Besides, highly efficient drug extraction from plasma was attained, and mobile phase composition was suitable to the pharmacokinetic study of Roflumilast-loaded nanoparticles in rats. other: NA

Forced Degradation Study of an Anti-Diabetic Drug Imeglimin: Impurity Profiling and Structure Elucidation Using LC-Q-ToF-MS/MS and NMR.

The present study aims to establish structures of the degradation products of an anti-diabetic drug, Imeglimin (IMG) approved for the treatment of type 2 diabetes mellitus in the year 2021. Degradation pathways are proposed along with in silico toxicity assessments of the observed degradation products (DPs) of the drug. A reversed-phase high-performance liquid chromatography (RP-HPLC), equipped with a photodiode array detector, was used to separate the observed DPs with a Phenomenex Luna PFP (250 × 4.6 mm, 5 μm) column, using 10 mM ammonium formate (pH 4.5) and methanol as mobile phase. Liquid chromatography quadrupole time of flight mass spectrometry (LC-Q-ToF-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed for structural elucidation. Zeneth and Derek suites were used for in silico assessments. A total of four degradation products were observed, which were successfully separated on an RP-HPLC. The structural characterization of three of the four DPs was achieved using LC-Q-TOF-MS/MS by employing electro spray ionization as well as atmospheric pressure chemical ionization. Additionally, DP-3 was isolated using a preparative HPLC and was characterized by NMR. Computationally predicted structures were compared with the experimental observations. An HPLC method, capable of separating the Imeglimin and its four DPs, was developed and validated as per the ICH Q2(R1) guideline. Structure elucidation reveals a variety of products with metformin as one of the identified DPs along with a metabolite. The toxicity potential of DPs was assessed through docking studies.

Developed and validated for simultaneous estimation of telmisartan, and cilnidipine, metoprolol in tablet form, a method using reversed-phase high-performance liquid chromatography (RP-HPLC)

Developed and validated for simultaneous estimation of telmisartan, and cilnidipine, metoprolol in tablet form, a method using reversed-phase high-performance liquid chromatography (RP-HPLC)

Sorption of flavonoids and aromatic acids on hypercrosslinked polystyrene in systems with addition of imidazolium ionic liquids

In this work the effect of the addition of five imidazolium ionic liquids (ILs) to the water-acetonitrile eluents on the sorption of aromatic acids and flavonoids on hypercrosslinked polystyrene (HPS) was evaluated by reversed-phase HPLC (RP HPLC). It was found that both the nature of the cation and the nature of the anion of the used IL influence the retention of the studied compounds. The retention of aromatic acids and flavonoids in the systems without and with the addition of trifluoroacetic acid (TFA) to the eluent was investigated. It was shown that the addition of TFA to the water-acetonitrile eluent changes the mechanism of action of imidazolium ILs on the retention of sorbates on HPS. In particular, the sequence of elution of ferulic acid, salicylic acid and caffeic acid in the systems with addition of ILs to the mobile phase on HPS varies depending on the pH of the medium (depend of the addition of TFA). The Snyder-Soczewiński and Soczewiński–Wachtmeister models were used to evaluate the influence of water-acetonitrile eluent composition on the retention of studied compounds. The angular coefficients of the corresponding models were calculated.

Development And Validation of a RP-HPLC Method For Simultaneous Estimation of Risperidone and Trihexyphenidyl Hydrochloride in Pharmaceutical Formulations

A rapid and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous determination of Risperidone and Trihexyphenidyl hydrochloride in pharmaceutical formulations. The chromatographic separation was achieved using a Thermosil C18 column (4.0×125mm, 5μm) with a mobile phase consisting of methanol and sodium acetate buffer (pH 3.0) in the ratio of 70:30 v/v. The flow rate was maintained at 1.0 mL/min with UV detection at 252 nm. The retention times for Risperidone and Trihexyphenidyl hydrochloride were 2.566 and 3.417 minutes, respectively. The method demonstrated excellent linearity in the concentration ranges of 5-25 μg/mL for Risperidone and 50-250 μg/mL for Trihexyphenidyl hydrochloride, with correlation coefficients of 0.999 for both compounds. The method validation parameters including accuracy, precision, specificity, and robustness met ICH guidelines. The mean recoveries were 99.56% and 99.48% for Risperidone and Trihexyphenidyl hydrochloride, respectively. The limits of detection were 3.17 μg/mL and 0.0172 μg/mL, while the limits of quantification were 5.80 μg/mL and 0.212 μg/mL for Risperidone and Trihexyphenidyl hydrochloride, respectively. The validated method proved suitable for routine quality control analysis of both drugs in pharmaceutical formulations.

Preparation and in-vitro characterization of triamcinolone acetonide-loaded lipid liquid crystal nanocarriers for ocular delivery

Purpose: This study aimed to develop sustained-release Triamcinolone acetonide (TA) formulations using lipid liquid crystals (LLC) for ocular drug delivery and to characterize the designed formulations. Method: Eighteen dispersed LLC formulations were prepared through a top-down approach, incorporating varying concentrations of TA and different proportions of glyceryl monooleate, deionized water, and Pluronic F127. An additional formulation comprising TA: HPβCD complex was also developed to investigate the influence of hydroxypropyl beta-cyclodextrin (HPβCD) on the properties of the formulations. The formulations were evaluated for their rheological properties in room temperature using a rheometer, syringeability by passing them through a 27G needle, size measurements via dynamic light scattering, and morphology through polarized light microscopy (PLM). Furthermore, the prepared formulations were injected into a dialysis tube and placed in a phosphate buffer at pH 7.4 and 37°C for in vitro release evaluation. Samples were taken at predetermined intervals and stored in a refrigerator until HPLC analysis. The percentage of Encapsulation efficacy and drug loading were evaluated using an indirect method. A reversed-phase HPLC method was employed to quantify the drug concentrations in the samples. Results: All selected formulations demonstrated acceptable parameters, including particle size (less than 200 nm), polydispersity index ranging from 0.202 to 0.355, and zeta potential values between -14.3 and -32.8 mV. Additionally, the formulations showed good syringeability and achieved 100% drug release within 48 hours (except for the formulation containing HPβCD). PLM analysis revealed the presence of hexosomes and cubosomes, indicating that an increase in hexosomes contributed to a more uniform drug release from the formulations. Conclusion: Overall, the study findings suggest that liquid crystalline carriers can be a promising formulation for sustained ocular drug delivery of TA.