Reversed-phase High-performance Liquid Research Articles

Simultaneous Estimation of Quercetine and Betanine by RP-HPLC from Herbal Formulations Used in Nutritional Deficiencies

Background: Medicinal plants have curative properties due to the presence of various complex chemical substances of different compositions, which are found as secondary plant metabolites in one or more parts of the plants. Moringa oleifera from Moringaceae and Beta vulgaris root are, native to India, grows in the tropical and subtropical regions of the world. It is commonly known as 'drumstick tree' or 'horseradish tree' or 'miracle tree’. Incorporation of more herbal powder leads to much complexity. Above plants were chosen for their utmost nutritional values. Result: Herbal tablet and granules were prepared and evaluated further for various Physico-chemical parameters as a nutritional supplement. Promising results indicate that prepared formulations have potential as supplements. Conclusion: Present communication mainly focused on estimation of marker components by Reverse Phase-High Performance Liquid Chromatography. It showed the presence of enough number of secondary metabolites and minerals which can be easily consumed by all age groups.

Development and validation of a bioanalytical RP-HPLC method for quantification of ifenprodil in rat plasma

This research aimed to develop a simple, reliable, efficient, and precise Reverse phase high-performance liquid chromatography technique for measuring or determining the quantity of Ifenprodil concentration in rat plasma. Dextromethorphan served as the internal standard (IS). The separation was achieved using the isocratic method with a Luna Phenyl hexyl column (dimensions: 150 mm × 4.6 mm, particle size: 3.5 μm) on a Waters 2,695 equipment with Empower software version 2.0. The analyte was extracted utilizing a liquid–liquid extraction technique, employing acetonitrile (ACN) as an additive. For the measurement of the drug in rat plasma, a mobile phase was utilized, consisting of a mixture of ammonium formate (pH 2.5) with formic acid (FA) and ACN, in a volumetric ratio of 60:40%. The separation procedure was performed utilizing a flow rate of 1 ml/minute and a detection wavelength of 220 nm. The retention time of Ifenprodil and IS were 2.362, and 5.603 minutes, respectively. The linearity of the separation is observed within the concentration range of 200–4,000 ng/ml, exhibiting a robust correlation coefficient of 0.9998. The validation and stability study was conducted by following the guidelines outlined by the US Food and Drug Administration. The findings obtained from the study imply that the proposed approach can be effectively employed for the pharmacokinetics and bioequivalence profiling of Ifenprodil in rat plasma.

Quantification of rifampicin loaded into inhaled polymeric nanoparticles by reversed phase high-performance liquid chromatography in pulmonary nonphagocytic cellular uptake.

Rifampicin is an antibiotic effective against both Gram-negative and Gram-positive bacteria and is commonly used as a first-line treatment for tuberculosis caused by Mycobacterium tuberculosis. In this study, a reversed-phase high-performance liquid chromatography method was developed and validated to assess rifampicin, either free or combined with ascorbic acid, loaded into chitosan/Tween 80-coated alginate nanoparticles. The method utilized a reversed-phase C18 Restek column with specific chromatographic conditions: a mobile phase of 60 : 40 ratios of methanol/buffer phosphate (pH 7.0), at a flow rate of 0.8 mL min-1, and an injection volume of 15 μL. rifampicin and the internal standard (rifamycin) had retention times of 4.0 and 2.5 min, respectively, and were detected at 334 nm. The method demonstrated the stability of stored samples after freezing-thawing cycles and specificity for rifampicin, even in the presence of degradation products from stress conditions. The high-performance liquid chromatography method was found to be specific, precise, robust, and sensitive. Results indicated that rifampicin accumulation and uptake kinetics varied based on cell type, formulation (free or loaded in nanoparticles), rifampicin concentration, and incubation time. Confocal fluorescence microscopy images supported these findings, showing isothiocyanate fluorescein nanoparticles distribution in different intracellular regions depending on the cell type used. The societal impact of this research lies in its potential to advance the treatment of respiratory infectious diseases, such as tuberculosis, through the development of more effective and specific drug delivery methods. By optimizing the way drugs, particularly rifampicin in this case, interact with lung cells, we aim to achieve greater treatment efficacy and alleviate the overall burden of disease. Furthermore, our study offers novel insights into the intracellular behavior of rifampin from polymeric nanoparticles, paving the way for personalized medicine approaches in the treatment of respiratory infections. This dual focus on social impact and innovation underscores our commitment to improving global health outcomes and addressing pressing public health challenges.

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION OF THE DISSOLUTION OF BROMELAIN-A COMPLEX MIXTURE OF PROTEOLYTIC ENZYMES, IN DELAYED-RELEASE TABLETS

Objective: To develop a simple, accurate, precise and linear Reverse Phase High-Performance Liquid Chromatographic (RP-HPLC) method and verify for the quantitative estimation (Dissolution) of Bromelain in delayed-release tablets. Methods: The optimized RP-HPLC method for both acid and buffer stage dissolutions of delayed-release tablets uses Zorbax 300 SB-C8 column (150 mm X 4.6 mm; 3.5μ), a mobile phase-A of 0.1% trifluoroacetic acid in water and a mobile phase-B of 0.1% Trifluoroacetic acid in Acetonitrile in the gradient proportion, flow rate of 1.0 ml/min, injection volume of 25 µl, detection wavelength of 280 nm using a UV/PDA detector, column temperature of 40 °C, sample tray/compartment temperature of 5 °C and a run time of 20 min. Results: The developed method gave Bromelain eluting at about 6 min. Bromelain exhibited linearity in the range 53.4-800.6 μg/ml (r2=0.99992). The precision is exemplified by relative standard deviation of 1.3 and 2.3% for acid and buffer stages, respectively. Percentage recovery of the drug was found to be between 90.0 and 110.0 during accuracy studies. Conclusion: A simple, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (Dissolution) of Bromelain in tablets and hence this method can be explored for the analysis of Bromelain in tablets in various pharmaceutical industries.

Study of resistance mechanism of Alternaria blight (Alternaria brassicicola) by biochemical markers in Indian Mustard (Brassica juncea L. Czern. &Coss.).

Indian mustard (Brassica juncea) is an important oilseed crop in India. Alternaria leaf spot (Alternaria blight) is incited by the fungus Alternaria brassicicola. It majorly affects crop production leading to a yield loss of up to 70%. To circumvent this problem, the study of the resistance mechanism and identification of biochemical markers is one of the important strategies for its management. In the present study, a total of 219 genotypes of Indian mustard with check were screened for Alternaria blight over two seasons. Based on the area under the disease progress curve (AUDPC) scores, ten consistently performing genotypes were selected for the screening of biochemical and yield attributes under artificial inoculated conditions of Alternaria brassicicola (Berk) Sacc. The result showed a negative correlation between disease and yield attributes. The catalase (CAT) activity was significantly increased in resistant genotypes compared to susceptible ones, indicating the crucial role of CAT in the resistance mechanism. Pathogen infection also increases the total protein content and the Alternaria-resistant genotype showed the highest total soluble protein while the susceptible genotype showed the lowest total soluble protein. The ten genotypes were categorized by SSI (stress susceptibility index) and Varuna was identified as a tolerant genotype and Giriraj as a susceptible genotype for Alternaria brassicicola (Berk) Sacc. Varuna and Giriraj were chosen for quantitative analysis of methionine and tryptophan amino acids from seeds using RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) and there were significant differences in the levels of methionine and tryptophan between the Varuna and Giriraj genotypes. Varuna showed higher methionine and tryptophan content compared to the Giriraj genotype. Higher protein content demonstrated an increase in biotic stress-responsive amino acids, such as methionine and tryptophan, suggesting increased resistance to Alternaria diseases in these high-protein genotypes. These amino acids could be used as biochemical markers for Alternaria resistance of mustard.

Quantitation of Active Pharmaceutical Ingredients in Polymeric Drug Products Using Elemental Analysis.

Polymeric prodrugs have gained significant popularity as a strategy to enhance the bioavailability and improve the pharmacokinetic properties of active pharmaceutical ingredients (API). Since the amount of the API in a polymeric prodrug product directly impacts both safety and efficacy, there is a pressing need for robust and accurate analytical methods to quantify the API in these formulations. Presently, drug quantification methods include reversed-phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography (SEC)-based molecular weight determination. Even though these methods are highly precise and reproducible, a deep understanding of chromatography is required for complex method development, including optimization of the elution profile and selecting the appropriate column and mobile phase. In this chapter, we introduce the automated elemental analyzer for drug quantification, which is simple to use and does not require special method development.

Ion Quantitation in Liposomal Products Using RP-HPLC with Charged Aerosol Detection.

Ion concentration in liposomal drugs is important for drug stability and drug release profile. However, quantifying ion concentration in liposomal drugs is challenging due to the absence of chromophores or fluorophores of ions and the efficiency of their release from the liposome structure. To address these issues, a method based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a charged aerosol detector (CAD) has been developed to determine total, internal, and external ions in drug-loaded liposomal products. In this protocol, we focused on the quantitation of ammonium and sulfate ions in liposomal products, using generic PEGylated liposomal doxorubicin as an example. This method can be extended to calcium, acetate, and other ions in different liposomal formulations with slight modifications.

Simultaneous Method Development and Validation of Fosamprenavir Calcium and Nevirapine by Reversed-Phase High-Performance Liquid Chromatography in Dendrimer-Carbon Nanotubes Conjugates

Simultaneous Method Development and Validation of Fosamprenavir Calcium and Nevirapine by Reversed-Phase High-Performance Liquid Chromatography in Dendrimer-Carbon Nanotubes Conjugates

Separation and purification of short-, medium-, and long-stranded RNAs by RP-HPLC using different mobile phases and C18 columns with various pore sizes.

Nucleic acids, which have been employed in medicines for various diseases, are attracting attention as a new pharmaceutical model. Depending on the target substances, nucleic acid medicines with various nucleic acid chain lengths (several tens of nucleotides [nt] to several thousands of nt) exist. The purification of synthesized nucleic acids is crucial as various impurities remain in the crude product after synthesis. Presently, reversed-phase high-performance liquid chromatography (RP-HPLC) represents an effective purification method for nucleic acids. However, the information regarding the HPLC conditions for separating and purifying nucleic acids of various chain lengths is insufficient. Thus, this technical note describes the separation and purification of short-, medium-, and long-stranded nucleic acids (several tens of nt to thousands of nt) by RP-HPLC with various mobile phases and octadecyl-based columns with various pore sizes, such as normal (9-12 nm), wide (30 nm), and super wide (>30 nm) pores.

Identification and taste characteristics of novel umami peptides from Yanjin black bone chicken hydrolysates and their binding mechanism with umami receptor.

This study aimed to obtain umami peptides from Yanjin black bone chicken and to investigate the formation mechanism of umami taste. The umami peptides were purified from the enzymatic hydrolysate of chicken using ultrafiltration (UF), gel filtration chromatography (GFC), and reversed-phase high-performance liquid chromatography (RP-HPLC). Potential novel umami peptides were then identified by nano-scale liquid chromatography-tandem mass spectrometry (nano-HPLC-MS/MS). Based on the predictions of iUmami-SCM and BIOPEP-UWM databases, five umami peptides (EELK, EEEIK, EELMK, LEEEIK, DELDKYS) with high umami scores were synthesized. Sensory evaluation revealed that these umami peptides exhibited a threshold ranging from 0.12 mg mL-1 to 0.36 mg mL-1. Circular dichroism (CD) analysis indicated the presence of β-sheet structures in the umami peptides that could be associated with taste formation. In addition, molecular docking and molecular dynamics (MD) were employed to investigate the binding mechanisms between umami peptides and the umami receptor T1R1/T1R3. The findings reveal that Lys155, Arg255, and Gln250 of T1R1/T1R3 potentially play critical roles in umami peptide binding. Taken together, our results lay a foundation for researching flavor substances and for developing flavor products from Yanjin black bone chicken.

Synthetic approach to iodosulfuron-methyl and metsulfuron-methyl metabolites and their application for water analysis.

A synthetic approach to ten metabolites of iodosulfuron-methyl sodium and metsulfuron-methyl was performed and reported in this study. The compounds of interest were prepared by controlled hydrolytic degradation of active substances or by de novo synthesis from commercially available triazine precursor 10. Obtained compounds were characterized by IR, NMR, and elemental analysis techniques. Metabolites and active substances were utilized during the development of a separation and quantification method using reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry. The validated method was applied for the analysis of all studied compounds in the extracts from water samples collected from the Vistula river (Toruń, Poland).

An isocratic RP-HPLC-UV method for simultaneous quantification of tizanidine and lidocaine: application to in vitro release studies of a subcutaneous implant.

Implantable devices have been widely investigated to improve the treatment of multiple diseases. Even with low drug loadings, these devices can achieve effective delivery and increase patient compliance by minimizing potential side effects, consequently enhancing the quality of life of the patients. Moreover, multi-drug products are emerging in the pharmaceutical field, capable of treating more than one ailment concurrently. Therefore, a simple analytical method is essential for detecting and quantifying different analytes used in formulation development and evaluation. Here, we present, for the first time, an isocratic method for tizanidine hydrochloride (TZ) and lidocaine (LD) loaded into a subcutaneous implant, utilizing reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a UV detector. These implants have the potential to treat muscular spasticity while providing pain relief for several days after implantation. Chromatographic separation of the two drugs was accomplished using a C18 column, with a mobile phase consisting of 0.1% TFA in water and MeOH in a 58 : 42 ratio, flowing at 0.7 ml min-1. The method exhibited specificity and robustness, providing accurate and precise results. It displayed linearity within the range of 0.79 to 100 μg ml-1, with an R2 value of 1 for the simultaneous analysis of TZ and LD. The developed method demonstrated selectivity, offering limits of detection and quantification of 0.16 and 0.49 μg ml-1 for TZ, and 0.30 and 0.93 μg ml-1 for LD, respectively. Furthermore, the solution containing both TZ and LD proved stable under various storage conditions. While this study applied the method to assess an implant device, it has broader applicability for analysing and quantifying the in vitro drug release of TZ and LD from diverse dosage forms in preclinical settings.

Hydrolyzed Reactive Red 195 adsorption on cellulose: Experimental and theoretical investigations

In this investigation, the mechanism of dye transfer during the mixed washing of natural cellulosic fabrics was elucidated through a comprehensive study of the hydrolysis of Reactive Red 195 (RR 195) and the subsequent adsorption of its hydrolysis products onto cellulose, approached both experimentally and theoretically. Initially, Reversed-phase high-performance liquid chromatography (RP-HPLC) was employed to identify the final products of Reactive Red 195 hydrolysis, collectively referred to as the unfixed dye substance ( UDS). This data provided theoretical insight into the hydrolysis mechanism of the dye. Following this identification, an adsorption kinetic model was established to delineate the interaction of the hydrolysis products with cellulose. Adsorption exhibited its maximal rate at a temperature of 60°C, under conditions of a wash bath ratio of 120:1 and a pH value of 7. Furthering the investigation, a molecular dynamics model was constructed to detail the adsorption process of these hydrolysis products on the hydrophobic surface Iβ(1 0 0) of cellulose within an aqueous environment. This model offered precise insights into the adsorption volume, kinetics, and mechanistic interaction at a molecular scale. Conclusive evidence from both theoretical analyses and experimental results suggested that the hydrolysis products, devoid of reactive groups, were incapable of chemically interacting with the hydroxyl groups inherent to cellulose. Instead, it was discerned that these products underwent a physical adsorption process onto the cellulose surface. Such adsorptive behavior is postulated to be a predominant factor influencing the dye transfer phenomenon in mixed washing conditions.

Isolation and structural identification of a potassium ion channel Kv4.1 inhibitor SsTx-P2 from centipede venom.

To isolate a potassium ion channel Kv4.1 inhibitor from centipede venom, and to determine its sequence and structure. Ion-exchange chromatography and reversed-phase high-performance liquid chromatography were performed to separate and purify peptide components of centipede venom, and their inhibiting effect on Kv4.1 channel was determined by whole-cell patch clamp recording. The molecular weight of isolated peptide Kv4.1 channel inhibitor was identified with matrix assisted laser desorption ionization-time-of-flight mass spectrometry; its primary sequence was determined by Edman degradation sequencing and two-dimensional mass spectrometry; its structure was established based on iterative thread assembly refinement online analysis. A peptide SsTx-P2 was separated from centipede venom with the molecular weight of 6122.8, and its primary sequence consists of 53 amino acid residues NH2-ELTWDFVRTCCKLFPDKSECTKACATEFTGGDESRLKDVWPRKLRSGDSRLKD-OH. Peptide SsTx-P2 potently inhibited the current of Kv4.1 channel transiently transfected in HEK293 cell, with 1.0 μmol/L SsTx-P2 suppressing 95% current of Kv4.1 channel. Its structure showed that SsTx-P2 shared a conserved helical structure. The study has isolated a novel peptide SsTx-P2 from centipede venom, which can potently inhibit the potassium ion channel Kv4.1 and displays structural conservation.

Koncept ekološki prihvatljivih hromatografskih metoda - studija slučaja na primeru razdvajanja dronedaron hidrohlorida i njegovih degradacionih proizvoda

Recently, concern about the environmental impact of drug analysis methods has increased significantly. Reversed-phase high-performance liquid chromatography (RP-HPLC), the predominant technique in drug analysis, relies heavily on organic solvents such as acetonitrile, which is known for its chromatographic efficiency, but also for its toxicity and flammability. To address these concerns, it is essential to minimize the use of toxic organic solvents. The aim of this study is to explore greener RP-HPLC modifications and evaluate their applicability in the pharmaceutical industry. Methods were developed for the separation of dronedarone hydrochloride and its degradation products based on experimental design, including micellar liquid chromatography (MLC), b-cyclodextrin (CD) modified RP-HPLC and ultra-high performance liquid chromatography (UHPLC). The eco-friendliness of these methods was assessed using the analytical eco-scale score, green analytical procedure index (GAPI) and analytical greenness (AGREE). AGREE appears to be the most suitable, as it revealed the greatest differences between the compared methods, as well as insights into critical aspects of the methods. UHPLC and b-CD modified RP-HPLC have been shown to be superior to MLC, and both methods can be a good choice, depending on whether the ease of implementation or energy efficiency is considered to be a more important criterion.

Development of a validated RP-HPLC/PDA method for the quantitative estimation of tepotinib in tablet dosage form

A novel and effective reversed-phase high-performance liquid chromatographic (RP-HPLC) technique has been developed to determine the tepotinib, an anti-cancer drug, in a tablet formulation. Furthermore, this method has been validated in accordance with the guidelines of the International Conference on Harmonization. The mobile phase consisted of a mixture of potassium dihydrogen orthophosphate and acetonitrile in a ratio of 15:85 v/v, while the flow rate of the mobile phase was maintained at a constant rate of 1.0 ml/minute. The analysis was conducted using an isocratic approach on a C18 analytical column with dimensions of 250 × 4.4 mm and a particle size of 5 μm. To detect tepotinib, a photo diode array detector was employed, with a wavelength of 284 nm. The quantification of tepotinib was not affected by the presence of excipients. The concentration range of the calibration curve was linear, spanning from 8 to 12 μg/ml. In terms of precision, both intra-day and inter-day relative standard deviations were found to be less than 2%, thus indicating high precision and repeatability. The recovery rate of the compound ranged from 99.76%–101.34 %, with relative standard deviations not exceeding 2%. The limit of detection and quantification were determined to be 0.02 μg/ml and 0.06 μg/ml, respectively. The system suitability parameters were deemed to be within the acceptable range. Therefore, this current report proposes an innovative analytical approach for the evaluation of a recently discovered anti-cancer drug, tepotinib, through the utilization of the RP-HPLC methodology.

Rapid Reversed-Phase High-Performance Liquid Chromatography Profiling of Serotonin Receptor Ligands and their Related Compounds

Rapid Reversed-Phase High-Performance Liquid Chromatography Profiling of Serotonin Receptor Ligands and their Related Compounds

A rapid and improved method for the determination of ethyl carbamate in foodstuffs of different matrices.

This work deals with the rapid and simple determination of the probable carcinogen ethyl carbamate (EC), which is naturally present in fermented food products. An undemanding, robust, and rapid pre-column derivatization utilizing a 9-xanthydrol reagent has been developed. The resulting derivative was subsequently analysed by reversed-phase high-performance liquid chromatography coupled with fluorescence detection. As a result of the thorough optimisation of the chromatographic conditions, the run was completed in just 5 minutes, considerably speeding up the usual time of EC separation (30-60 min). Thanks to the fast separation, satisfactory yields (around 90%), negligible matrix effects, no interfering peaks, very low detection limit, and simple sample pre-treatment (for the very first time, the derivatization was performed in the presence of light and without any extraction step), the proposed method represents a significant improvement of the EC determination protocol used so far. After method validation, a total of fifty food samples were subjected to analysis without any additional sample pre-treatment despite their diverse matrix. Due to its robustness, simplicity, and low time, cost, and manual demands, this method is suitable for rapid screening of EC in both final food products and during their production.

Analytical method development and validation for the simultaneous estimation of abacavir and lamivudine by reversed-phase high-performance liquid chromatography in bulk and tablet dosage forms

Analytical method development and validation for the simultaneous estimation of abacavir and lamivudine by reversed-phase high-performance liquid chromatography in bulk and tablet dosage forms

Detection and Quantitation of Endogenous Membrane-Bound RAS Proteins and KRAS Mutants in Cancer Cell Lines Using 1D-SDS-PAGE LC-MS2.

In healthy cells, membrane-anchored wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) regulate critical cellular processes (e.g., proliferation, differentiation, survival). When mutated, RAS proteins are principal oncogenic drivers in approximately 30% of all human cancers. Among them, KRAS mutants are found in nearly 80% of all patients diagnosed with RAS-driven malignancies and are regarded as high-priority anti-cancer drug targets. Due to the lack of highly qualified/specific RAS isoform and mutant RAS monoclonal antibodies, there is a vital need for an effective antibody-free approach capable of identifying and quantifying membrane-bound RAS proteins in isoform- and mutation-specific manner. Here, we describe the development of a simple antibody-free protocol that relies on ultracentrifugation to isolate the membrane fraction coupled with single-dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate and enrich membrane-bound endogenous RAS isoforms. Next, bottom-up proteomics that utilizes in-gel digestion followed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) is used for detection and relative quantitation of all wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) and corresponding RAS mutants (e.g., G12D, G13D, G12S, G12V). Notably, this simple 1D-SDS-PAGE-HPLC-MS2-based protocol can be automated and widely applied to multiple cancer cell lines to investigate concentration changes in membrane-bound endogenous RAS proteins and corresponding mutants in the context of drug discovery.