Reversed-phase High-performance Liquid Chromatography Research Articles

DETERMINATION OF CHAMAZULENE BY REVERSED PHASE HPLC

A method for the determination of hamazulene in essential oils using reverse-phase high-performance liquid chromatography with diode-matrix detection has been developed. The possibility of qualitative determination of hamazulene from the electronic absorption spectra recorded in the detector cell and quantitative determination of this substance during detection at several different wavelengths is shown. To obtain chromatograms, it is proposed to use C18 stationary phase with a mobile phase containing 20 vol. % water in acetonitrile. Samples for the study were prepared by dissolving essential oils in acetonitrile. The chromatographic determination of hamazulene is not hindered by the accompanying components of the studied essential oils of four plants: Matricaria chamomilla, Achillea millefolium, Tanacetum annuum and Artemisia absinthium; the concentration of hamazulene in them was 0.184, 0.311, 0.299 and 0.0025 mol/l, respectively. The wavelength of 603 nm provides the most selective detection of hamazulene along a complex electron-vibrational band for a solution in the mobile phase. When the detection wavelength changes from 603 to 350 and up to 280 nm, the sensitivity of hamazulene detection increases by 15.5 and 187 times. The chromatographic method has a linear response of the detector despite the known features of the electronic absorption spectra of azulene derivatives. It was found that when a blue-colored spot of hamazulene is isolated from TLC plates, other nonpolar components of oils that are detected when recording a chromatogram under HPLC conditions at a detector wavelength of 210 nm also enter the extract. It was also found that when recording chromatograms at 200 nm, differentiation of oils is possible, since most of the components of these oils have at least one isolated double C=C bond in the structure, which allows detecting compounds at 190-200 nm. For citation: Deineka V.I., Salasina Ya.Yu. Determination of chamazulene by reversed phase HPLC. ChemChemTech [Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol.]. 2024. V. 67. N 6. P. 6-20. DOI: 10.6060/ivkkt.20246706.6989.

RP-HPLC-CAD method for the rapid analysis of lipids used in lipid nanoparticles derived from dual centrifugation

The use of lipids as suitable excipients for drug carrier systems has been established for years. Liposomes or lipid nanoparticles (LNPs) in general have been shown capable of delivering both hydrophilic and hydrophobic drugs. The Covid-19 pandemic and the resulting vaccines have significantly increased interest in the potential for these lipid-based systems, which can carry different types of therapeutic RNAs. LNPs used for the transfection of RNA are usually a multi-component mixture of phospholipids and other lipids. Essential components are positively charged or ionizable lipids such as DOTAP or SM-102, but also uncharged helper lipids such as cholesterol, DOPE, DSPC, DMG-PEG2000 or DSPE-PEG2000. Due to the differences in charge, simultaneous detection is a challenge. Here, we present a reversed-phase high-performance liquid chromatography charged-aerosol-detector method (RP-HPLC-CAD method) using a C-18 column for the simultaneous determination of charged and uncharged lipids. Our method has been validated according to the ICH-Q2 (R2) guideline for accuracy, precision, specificity and working range, including the limit of detection (LOD) and quantification (LOQ), as well as the calibration range. We were able to show satisfactory results in both precision and accuracy. The working range also shows great potential with a calibration range from 9.375 to 1000 μg/ml, LODs <1.85 μg/ml and LOQs <6.16 μg/ml. This method represents a fast and reproducible procedure for quantifying the lipids mentioned. In combination with the novel approach for the production of LNPs using dual centrifugation (DC), it offers the possibility of extremely rapid production of RNA-loaded LNPs, and the immediate analysis for their lipid components.

Abstract PO5-13-03: Early Detection of Breast Cancer using Targeted Plasma Metabolomic Profiling

Abstract Background: Breast cancer (BC) is the second leading cause of cancer death among women. Accordingly, early diagnosis is key to the successful treatment, management, and care of BC. Recent studies confirmed that plasma metabolites could be reliable cancer biomarkers, allowing for the development of a minimally invasive routine blood test that can be used for screening, as well as for monitoring disease evolution in patients. The exact metabolic pathways involved in early BC development remain unclear. Metabolomic profiling of women with BC may help to identify new biomarkers to predict breast cancer long before symptoms appear. The purpose of this study was to validate a plasma metabolomic biomarker panel for an improved risk assessment for early detection BC in 241 patients, and to understand the potential role and the relationship between BC subtypes and hormone receptor status. Methods: Our study included a total of 185 plasma samples from women with biopsy-confirmed BC and 56 plasma samples from healthy controls. A targeted, quantitative mass spectrometry (MS)-based metabolomics approach was used to analyze 138 metabolites in plasma samples using a combination of direct injection (DI) MS and reverse-phase high performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). The sample set was split into a discovery set and validation set. Metabolite concentration data, clinical data, and hormones receptor status were used to determine optimal biomarker sets. The same biomarkers and regression models were used and assessed on the validation models. The area under the receiver operator characteristic curves (AUROC), sensitivities and specificities at selected cut off points were calculated for each subgroup. Results: A large proportion of BC patients were at an early stage, with 98 at stage I (53.0 %), 70 at stage II (37.8%), and 17 at stage III (9.2%). The BC patients were of all subtypes: 138 luminal A (76,2%), 23 luminal B (12.7%), 5 hormone receptor-negative/HER2-positive (2.8%), and 15 triples negative (8.3%). A feature selection was performed on the training set using Partial Least-Squares Discriminant Analysis (PLS-DA), and the top performing metabolites were identified as the most important in discriminating BC from healthy subjects. The impact of age was also investigated. Features with &amp;gt;80% missing values were removed. To predict BC, the best signature comprised 9 variables implicated in fatty acid metabolism, amino acid metabolism, polyamine biosynthesis and related signaling pathways We further developed and validated a logistic regression with AUROC &amp;gt; 0.9 using these metabolites and other clinical data for detecting different stages and subtypes of BC. Conclusion: This study identified and validated a simple, high-performing, metabolite-based test for the early detection of BC. After confirmation in other independent cohort studies, our findings could provide the foundations for the development of a blood-based routine test for women at the highest risk for BC that is cost-effective, accurate and reliable. In addition, this approach could be used to complement other modalities especially for BC patients with dense breasts. Citation Format: Jean-Francois Haince, Lun Zhang, Rashid Ahmed Bux, Paramjit S. Tappia, Bram Ramjiawan, David Wishart, Andrew Maksymiuk. Early Detection of Breast Cancer using Targeted Plasma Metabolomic Profiling [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-03.

Optimising the recovery of phenolic compounds and antioxidant activity from orange peels through solid-state fermentation

It is widely recognised that orange peels contain a considerable quantity of phenolics, primarily in the form of glycosides. The process of fermentation holds potential as a viable method for extracting phenolic compounds and facilitating their biotransformation into novel metabolites. The aim of this study was to assess the enhanced release of phenolic compounds through the process of solid-state fermentation of orange peels using microorganisms. Following a 6-day incubation period, the methanol extract obtained from the sample fermented with starter Banh men exhibited the highest concentration of total phenolic compounds (17.57 ± 0.34 mg GAE/g DW) and demonstrated the most significant DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity (55.03%). The Reverse Phase High Performance Liquid Chromatography (RP-HPLC) analysis revealed that the predominant phenolic compounds in all fermented samples were flavonoid aglycones, specifically naringenin, hesperetin, and nobiletin. Conversely, in the unfermented orange peels, the major compound observed was the glycoside derivative hesperidin.

Different Detection Strategies of Pediocin-Like Produced by Pediococcus pentosaceus.

In this study, Pediococcus pentosaceus C-2-1 and C23221 contained genes encoding penocin and pediocin PA-1, mined by antiSMASH. The penocin structural gene pedA from Pediococcus pentosaceus C-2-1 was successfully expressed in Escherichia coli BL21. The presence of a 6.5kDa recombinant penocin was confirmed by Tricine-SDS-PAGE, and the specific activity increased by 1.54-fold. The bacteriocins produced by Pediococcus pentosaceus C23221 were purified using acetic ether extraction, Sepharose Fast Flow, Sephadex G-25 gel chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC); the amino acid sequence of this bacteriocin was identical to pediocin PA-1 by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), which confirmed the expression of pediocin PA-1 gene; and the specific activity increased by 24.39-fold. The heterologous expression and purification of bacteriocins have proved the expression of pediocin-like produced by Pediococcus pentosaceus. This provides a theoretical basis for the subsequent development and application of pediocin-like.

Retention mechanisms of amitriptyline and its impurities on amide, amino, diol, and silica columns in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) has been widely applied to challenging analysis in biomedical and pharmaceutical fields, bridging the gap between normal-phase high-performance liquid chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). This paper comprehensively explores the retention mechanisms of amitriptyline and its impurities A, B, C, D, F, and G on amide, amino, diol, and silica columns. Dual HILIC/RP-HPLC retention mechanisms were developed, and transitional points between HILIC and RP-HPLC mechanisms were calculated on amide, diol, and silica columns. Adsorption and partition contributions to overall retention mechanisms were evaluated using Python software in HILIC and RP-HPLC regions. The cation exchange mechanism dominates overall retention for ionized analytes in the silica column (R2>0.995), whereas the retention of ionized analytes increases with pH. Impacts of acetonitrile content, buffer ionic strength, and pH, along with their interactions on the retention of ionized analytes in the silica column, were determined using the chemometric approach. Acetonitrile content showed the most significant impact on the retention mechanisms. These findings highlight that a detailed investigation into retention mechanisms provides notable insights into factors influencing analyte retention and separation, promising valuable guidance for future analysis.

The enzymatic synthesis of theaflavin-3-gallate oxidation product and its determination

In this work, the oxidation of theaflavin-3-gallate (TF-3-G) was investigated using (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG) as substrates in a one-pot reaction. The resulting TF-3-G oxidation product was acquired by employing acetonitrile/water and ethanol/water as eluents, respectively, which was identified as theanaphthoquinone-3′-gallate (TNQ-3′-G). Surprisingly, we discovered that TNQ-3′-G could react with certain protic solvents to form new and unstable complexes through intermolecular hydrogen bond. This reactivity was also confirmed by the presence of irregular peaks in reverse-phase high-performance liquid chromatography (RP-HPLC) besides spectroscopic data. Therefore, we inferred that the number of carboxyl groups may increase through the successive oxidative polymerization of the TFs oxidation products. The high-molecular polymer could also interact with biomacromolecules in a similar manner to their interaction with protic solvents. This interaction might be one of the main factors contributing to the broad hump of thearubigins (TRs) on the RP-HPLC baseline. Additionally, these findings lay a solid foundation for interpreting the structures of TRs and understanding their generation mechanism.

Two Novel Angiotensin-Converting Enzyme (ACE) Inhibitory and ACE2 Upregulating Peptides from the Hydrolysate of Pumpkin (Cucurbita moschata) Seed Meal.

Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 μM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.

Fermentation Kinetics, Potability, and Sensory Compounds of Joubidwi: An Ethnic Alcoholic Beverage

Background: Fermentation is a metabolic pathway of the breakdown of carbohydrate molecules like glucose, fructose to ethanol and various volatile and non-volatile congeners. The production of alcoholic beverages from foodstuffs like rice, cereals, fruits, maize etc., is an age-old traditional protocol. In this study, we adapted the traditional protocol of preparation of the ethnic beverage Joubidwi, with a fermentation time of 6 days, i.e., aliquots from the fermented mash (cooked rice) were collected on a daily basis from day 1 to day 6. Objectives: This study aimed to get insight into fermentation kinetics, ethanol content, carbohydrate content, antioxidant activities, bitterness, protein content, pH, colour, and turbidity of the ethnic alcoholic beverage Joubidwi and to identify the sensory compounds and polyphenol molecules. Method: UV-Visible spectroscopic methods, GC-FID and GC-MS analytics methods were used for the estimation and identification of carbohydrates, proteins, ethanol contents, and sensory compounds, respectively. Reverse phase HPLC was adapted for the identification of polyphenol molecules in Joubidwi. Results: A pseudo-first-order fermentation kinetics was observed from day 1 to day 6 fermentation of Joubidwi. Apart from ethanol content (60.48 ± 1.36 mg/mL after 72 h fermentation to 78.80 ± 1.48 mg/mL after 144 h fermentation), the beverage had nutritional values and good antioxidant properties with 22.38 to 35.11% Radical Scavenging Effect (RSE) from 1 to 6th day of fermented Joubidwi. The beverage also contained 12 sensory compounds. Gallic acid was identified as polyphenol in Joubidwi samples. Conclusion: Joubidwi is an alcoholic beverage prepared by Bodos using rice and certain herbs through a traditional fermentation process. The fermentation process follows pseudo-first-order kinetics. The beverage is potable.

Subcutaneous administration of ceftazidime at 20 and 40 mg/kg produces theoretically therapeutic plasma concentrations for at least 120 hours in red-eared sliders (Trachemys scripta elegans).

To evaluate and compare the pharmacokinetic parameters of SC ceftazidime administered at 20 and 40 mg/kg to red-eared sliders. 8 adult red-eared sliders (Trachemys scripta elegans). In a sequential, 2-period study with a 3-week washout period between treatments, ceftazidime was administered SC to turtles at 20 and 40 mg/kg. Blood samples were collected from the subcarapacial sinus at 0, 24, 48, 72, 96, and 120 hours after ceftazidime administration. Plasma ceftazidime concentrations were quantified using reversed-phase HPLC. Mean plasma half-life after 20- and 40-mg/kg dosing was 39.75 ± 8.0 hours and 33.03 ± 6.56 hours, respectively. Mean maximum plasma concentration after 20- and 40-mg/kg dosing was 71.0 ± 15.93 µg/mL and 120.0 ± 30.62 µg/mL, respectively. Mean plasma ceftazidime concentrations remained ≥ 8 µg/mL, the theoretical MIC for various reptile pathogens for all time points. Results indicate that ceftazidime dosed at either 20 or 40 mg/kg produces plasma concentrations exceeding the theoretical MIC of various reptile pathogens for at least 120 hours. An ideal dosing interval could not be determined, as all plasma concentrations remained above the threshold of interest for all time points. Follow-up studies should focus on establishing a dosing interval and more rigorous monitoring for potential adverse effects.

A greener RP-HPLC method for quaternary estimation of caffeine, paracetamol, levocetirizine, and phenylephrine acquiring AQbD with stability studies

Abstract The main objective of this article was to develop method and validate quaternary estimation of caffeine (CFE), paracetamol (PCM), levocetirizine (LEV), and phenylephrine (PHE) and to conduct degradation experiments using reverse-phase high-performance liquid chromatography (RP-HPLC). This was the first innovative approach to this drug combination, combining analytical quality by design with green analytical chemistry. This method was developed using HPLC Agilent 1220 Infinity II, a binary solvent delivery pump, an automatic sampling device injector, and a photodiode array detector. Agilent Inertsil ODS 3 (250 mm × 4.6 mm, 5 μm) was used for separation. The mobile phase is composed of ethanol and 10 mM phosphate buffer (pH 3). To adjust the pH, 1% of orthophosphoric acid was used. The flow rate was set to 0.8 ml·min−1, the injection volume was 10 μl, and the detecting wavelength was 220 nm. The analytes were eluted via gradient elution. The retention time for PCM was 3.5 min, CFE was 8.1 min, PHE was 15.9 min, and LEV was 20.5 min. Green evaluation tools used in this research include Green Analytical Procedure Index, analytical eco-scale, analytical greenness, analytical method greenness score, and carbon footprint analysis. The developed method was greener than the previously reported method, as per the results of the greenness evaluation tools.

Development and characterisation of buccal dispersible film, syrup and effervescent granules of extemporaneous antigout remedy

Homemade medicinal remedies used to treat both acute and chronic ailments for centuries need to be transformed into modern evidence-based dosage forms. Therefore, the present study aimed to develop buccal dispersible film, syrup and effervescent granules of an extemporaneous antigout homemade remedy. The dosage forms were prepared using holistic remedy (HR) as an active entity and excipients masking disagreeable features. The dosage forms and HR were subjected to physicochemical evaluation, and standardised by a reversed-phase HPLC method using chlorogenic acid, caffeic acid, vanillin, and ferulic acid as analytical standards. The standardised dosage forms were investigated for antigout activity using an appropriate model. The dosage forms were found to be physically elegant and phytochemically aligned with HR. The developed dosage forms contained the selected markers and possessed antigout activity. Hence, these standardised and evidence-based dosage forms may have wider acceptance in the market compared to HR.

Comprehensive Analysis of Antioxidant and Phenolic Profiles of Thai Medicinal Plants for Functional Food and Pharmaceutical Development.

Medicinal plants, are resources of traditional medicines, have played a significant role in human culture throughout history. The connect of traditional knowledge with contemporary scientific findings, display the potential of these plants for nutritional and pharmaceutical applications. This study aimed to investigates the antioxidant activity and phenolic compositions of seven medicinal plants (Kaempferia parviflora, Hibiscus sabdariffa, Phyllanthus emblica, Terminalia chebula, Terminalia bellirica, Gynostemma pentaphyllum, and Stevia rebaudiana) from Kalasin Province, Thailand. 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays were used to evaluate the the antioxidant activities. The qualitative method was used for the phytochemical screening. Additionally, reverse-phase high-performance liquid chromatography (RP-HPLC) analysis reveals their phenolic compositions. Results revealed that phenols, flavonoids, and terpenoids were found in all medicinal plants studied, with flavonoids and phenols most predominant. Notably, P. emblica and T. bellirica show significant antioxidant properties and high levels of phenolics and flavonoids. P. emblica had the highest phenolic acid concentrations, with ρ-coumaric (79.43µg/g), chlorogenic (45.22µg/g), and ferulic acid (29.58µg/g) being dominant. The research emphasizes the value of these plants in developing functional foods and pharmaceuticals, underlining the importance of conserving biodiversity and traditional knowledge for future health product innovation.

Investigation of liquid chromatography-mass spectrometry analysis of a peptide aldehyde SJA6017 with identifying its hemiacetal, gem-diol, and enol ether.

The quantitative analysis of SJA6017, a peptide aldehyde inhibitor of calpain (Calpain Inhibitor VI), has encountered challenges in preclinical drug studies. The complex reverse-phase HPLC chromatographic behavior exhibits two peaks, each containing multiple species. An liquid chromatography-mass spectrometry (LC-MS/MS) study proposed an explanation for this phenomenon, caused by the amide aldehyde structure of SJA6017. Four chemical species corresponding to the two HPLC peaks have been identified as SJA6017 and its methyl hemiacetal, methyl enol ether, and gem-diol. In many instances of preclinical studies, methanol is favored as a substitute for DMSO. The hemiacetal is formed when the amide-activated peptide aldehyde reacts with methanol, which can then be further dehydrated in the mass spectrometer ion source under high temperature to form the methyl enol ether. The hemiacetal and gem-diol can also be decomposed to SJA6017 in the ion source. Additionally, the amide-activated peptide aldehyde can easily hydrate to the gem-diol of SJA6017 during sample incubation or sample preparation. The hemiacetal and gem-diol of SJA6017 are stable enough to have different retention times in the liquid chromatography, which explains why SJA6017 appears as two peaks, each containing multiple species. An LC-MS/MS tandem quadrupole mass spectrometer quantitative analysis method is proposed, enabling the analysis of these types of samples. This work serves as both an illustrative example and a cautionary note for mass analysis, sample incubations, and sample preparations involving compounds of peptide aldehyde, including similar aldehyde-containing metabolites, especially when methanol is present. This study provides the information needed to understand peptide aldehyde behavior at various steps of preclinical in vitro studies in the presence of methanol. It has assisted in the development of the SJA6017 bioanalysis method and will also aid in the development of bioanalysis methods for similar peptide aldehydes.

Optimization of HPLC Method by Using Central Composite Design for Simultaneous Estimation of Montelukast and Ebastine Dosage Form

In this work, a novel method for the simultaneous estimate of Montelukast (MONT) and Ebastine (EBAS) in bulk drugs was developed using Central Composite Design (CCD).A novel reverse-phase high-performance liquid chromatography (RP-HPLC) approach for the simultaneous measurement of Montelukast and Ebastine in bulk and formulation is what this work aims to create and test.A novel reverse-phase high-performance liquid chromatography (RP-HPLC) approach for the simultaneous measurement of Montelukast and Ebastine in bulk and formulation is what this work aims to create and test.With isocratic elution at a flow rate of 0.8 ml min-1 and a diode array detector operating at 241 nm, the separation was accomplished using methanol and 0.02M ammonium acetate buffer (pH 5.5 adjusted with diluted acetic acid) in the ratio of 80:20 v/v. Methanol composition (percent) (A), pH (B), and flow rate (C) were three independent variables that were examined.This method showed good linearity over a range of 2.5–25 μg/ml for both drugs. MONT limit of detection (LOD) and limit of quantitation (LOQ) were 0.298 and 0.905μg/ml while LOD and LOQ for EBAS were 0.594 and 1.80For both drugs, this technique demonstrated high linearity throughout a range of 2.5–25 μg/ml. Limits of detection (LOD) and quantitation (LOQ) for MONT were 0.298 and 0.905 μg/ml and 0.594 and 1.80 μg/ml for EBAS, respectively.The statistical data analysis determined that the Methanol composition and pH were the two independent variables that were most significant, with P 0.001. The suggested technique worked well and was optimised for the CCD model-based simultaneous estimate of both drugs.

Development and Validation of Stability indicating RP-HPLC Assay Method for Trospium Chloride in Tablet Dosage Form

A simple, rapid, accurate and sensitive method was developed for quantitative analysis of Trospium Chloride in tablet dosage form using Reverse Phase high performance liquid chromatography (RP-HPLC) with DAD detection. The chromatography separation was achieved Xterra RP18, (250 x 4.6mm, 5µParticle Size)), at column temperature 40°C±2, in the isocratic mobile phase mode using Acetonitrile (ACN): Buffer (65:35; v/v) at a flow rate of 1.0mL/min. The determination was performed using Buffer HPLC with DAD detector set at 215nm. Samples were prepared with diluent [Buffer: ACN (200:100; v/v)], and the volume injected was 50µL. The retention time for Trospium Chloride was 3.275 minute. The analytical curve was linear (r2 0.9993) over a wide concentration range (50.0-150.0 µg/mL). The presence of components of the tablet did not interfere in the results of the analysis. The method showed adequate precision with a relative standard deviation (RSD) less than 2%.

Novel RP-HPLC method for estimation of a newly developed combination of tizanidine and etoricoxib in rat plasma: Eight criteria for greens evaluation.

A novel environmentally friendly reversed-phase high-performance liquid chromatography (RP-HPLC) method has been effectively validated for simultaneously measuring a prospective conjunction of tizanidine (TIZ) and etoricoxib (ETC), the combined medicine, in rat plasma. The technique employs diclofenac potassium as the internal standard, guaranteeing dependable and precise outcomes. This study aimed to assess the impact of the suggested combination therapy on treating inflammation resulting from rheumatoid arthritis (RA) in a rat model. The procedure was performed using an Agilent series 1200 model HPLC apparatus. The chromatographic conditions consist of isocratic elution mode, C18 column with dimensions of 150 mm × 4.6 mm × 5 µm, flow rate of 1.5 mL/min, wavelength of 230 nm, temperature of 50°C, and injection volume of 10 µL. The elution was performed using a mobile phase consisting of a phosphate buffer with a pH of 3.5 and acetonitrile in a ratio of 80:20 v/v. Calibration curves were conducted for TIZ and ETC within the 1-50 µg/mL range, demonstrating linear trends with R2 values over 0.999. The effectiveness and eco-friendliness of the proposed method were evaluated using eight separate environmentally conscious metrics. The addition of TIZ and ETC to arthritic rodents amplified these effects significantly. Furthermore, TIZ and ETC significantly reduced serum levels in arthritic rodents, and safety investigations revealed normal complete blood count, liver, and renal functions. TIZ and ETC appear to have antiarthritic, anti-inflammatory, and safe combinations, making them viable future treatment options for RA that are also safe and efficacious. Following validation by United States Food and Drug Administration(US-FDA) rules, all goods met the criteria.

Novel post-functionalized polymer microspheres containing capsaicin as reversed-phase chromatography stationary phase separation for hydrophilic compounds

Here, we created for the first time that highly stable polymer microspheres copolymerized with capsaicin derivative, divinyl benzene, and N-(Hydroxymethyl)acrylamide could be efficient for reversed-phase high-performance liquid chromatography (RP-HPLC) by using a simple “one-pot cooking” method, which had the potential to be produced in large quantities. Then, the spheres were post-functionalized with two different monomers, N-vinyl formamide and 4-acrylomorpholine by double coating, which had ideal mechanical strength and efficiency as stationary phase. In order to reach best separation condition, the effects of buffer concentration, pH, column temperature, and mobile phase polarity on the retention rate were studied. Specifically, the interaction mechanism showed that the stationary phase had hydrophobic and electrostatic interaction with the water-soluble vitamins. It is of significant interest that all columns had excellent precision and accuracy (RSD < 4 %). Taken together, this study played a specific role in promoting the development of a new reverse-phase mode for separating hydrophilic compounds.

Chromatographic Determination of Denatonium Benzoate in Colognes

Denatonium benzoate, commercially known as Bitrex, is an ionic compound containing a quaternary ammonium cation (denatonium) and negatively charged benzoate. It is commonly used as a bittering agent in various alcohol-based products to deter ingestion. The national regulations regarding the production and domestic-foreign trade of ethyl alcohol and methanol states that ethyl alcohols for domestic use should contain 0.8 g denatonium benzoate per 100 L of absolute alcohol. This leads to the necessity of quantitative analysis of denatonium benzoate in alcoholic products. Cologne samples of different brands were analyzed using reversed-phase high performance liquid chromatography (RP-HPLC) coupled with UV/vis detector. Isocratic elution was performed on an ODS column with a mobile phase containing 75% MeOH:25% H2O. Since the proposed method does not require any pre-treatment step, the procedure is simple and rapid. The limit of quantitation was found to be 0.17 mg/L. The linearity of the method was tested in the range 0.26 – 15.75 mg/L with a limit of detection of 0.050 mg/L (signal-to-noise ratio of 3).

Comparison between Conventional Ageing Process in Barrels and a New Rapid Aging Process Based on RSLDE: Analysis of Bioactive Compounds in Spirit Drinks

“Aging” is a practice that allows alcoholic beverages to mature and gives them particular flavors and colors. In this context, oak or durmast wooden barrels are used in this process, thus providing different types of aging. This conventional process produces a slow enrichment of organic compounds in the spirit inside the barrels. Organic substances present in the internal part of the barrels slowly undergo the phenomenon of extraction by the liquid phase (solid–liquid extraction). In this work, a new procedure based on rapid solid–liquid dynamic extraction (RSLDE) was used to evaluate the potential of obtaining the effects of aging in spirits in shorter times than conventional methods. For this purpose, a comparison between two solid–liquid extraction techniques, RSLDE and conventional maceration, was made. Four water/ethanol 60:40 (v/v) model solutions were prepared and put in contact with medium-toasted chips using the two extraction procedures (conventional and non-conventional) and determining dry residue and total polyphenol content. Reversed phase high-performance liquid chromatography (RP-HPLC) analyses allowed the identification and quantification of furfural, ellagic acid and phenolic aldehydes (vanillin, syringaldehyde, coniferaldehyde and sinapaldehyde). The aging procedure with medium-toasted chips was tested on a young commercial grappa using maceration and RLSDE.