Liquid Chromatography Technique Research Articles

Turkish population-based screening for first identified changes of BRCA1 and BRCA2 genes in breast and/or ovarian cancer patients

IntroductionIt is known that BRCA1 and BRCA2 genes’ mutation carriers are predisposed to breast and ovarian cancers and other organ cancers such as prostate, colon and cervix. In the previous study performed at X University, all coding exons of both genes were screened by denaturing gradient gel electrophoresis (DGGE). In addition to various nonsense, missense mutations, polymorphisms and intronic region changes, seven novel missense mutations, including H513L, H816P and S1517Y in BRCA1 and S326R, G258P, E2903K and N2742S in BRCA2, had been identified.MethodsTo determine whether these unclassified variants are pathogenic, DNA samples of 150 healthy individuals without a known cancer history in the family were screened in this study for these seven novel missense mutations. These DNA samples were recruited from archives of previous polymorphism studies. PCR performed DNA amplifications, and denaturing high-performance liquid chromatography (DHPLC) techniques did mutation screenings.ResultsPeak patterns suggestive of a change in DNA fragments were considered for sequencing analyses. Analyses revealed that none of the 150 DNA samples had any change in the seven screened fragments. As a result, it is assumed that these seven mutations might be novel pathogenic mutations described in the Turkish population.ConclusionIn conclusion, these carriers must be informed about the mutation and given appropriate genetic counseling by their physicians. In addition, genetic testing must be offered to high-risk individuals (men/women) in the family so that it would be possible for other family members to have genetic counseling and contribute to disease prevention. On the other hand, these findings would contribute to current literature with novel results and shed light on future research.

Validation of Novel High-Performance Liquid Chromatography Method for Meropenem Quantification in Plasma

Eminent societies of clinicians worldwide advise the implementation of therapeutic monitoring of meropenem. Our aim was to validate the new high-performance liquid chromatography (HPLC) technique for the measurement of meropenem in plasma that we developed. The validation of the method was performed in accordance with the official European Medicines Agency (EMA) guideline for bioanalytical method validation through the assessment of the following validation parameters: linearity and limit of detection/quantification, trueness and precision, recovery, selectivity, matrix-effect assessment, dilution integrity, carry-over assessment, and stability. Our calibration curve was found to be linear over the concentration ranges of 1.25–100 μg/mL, which covers the therapeutic range of meropenem in patients. From the calibration curve, the limits of detection and quantification were calculated to be 0.4 μg/mL and 1.2 μg/mL, respectively. Recovery ranged from 81.7 to 95.9 percent. Intra-day truennes ranged from −1.9 to 2.6% and inter-day trueness ranged from −4.7 to 3.8%. Intra-day precision was less than 4 percent, while inter-day precision was less than 7 percent. Meropenem remained stable in the patient plasma sample for one week at −20°C. Our HPLC technique can be applied in clinical practice for the therapeutic monitoring of meropenem in critically ill and other vulnerable patients since it is simple, rapid, and reliable.

Concomitant investigation of crustacean amphipods lipidome and metabolome during the molting cycle by Zeno SWATH data-independent acquisition coupled with electron activated dissociation and machine learning

BackgroundDIA (Data-Independent Acquisition) is a powerful technique in Liquid Chromatography coupled with high-resolution tandem Mass Spectrometry (LC-MS/MS) initially developed for proteomics studies and recently emerging in metabolomics and lipidomics. It provides a comprehensive and unbiased coverage of molecules with improved reproducibility and quantitative accuracy compared to Data-Dependent Acquisition (DDA). Combined with the Zeno trap and Electron-Activated Dissociation (EAD), DIA enhances data quality and structural elucidation compared to conventional fragmentation under CID. These tools were applied to study the lipidome and metabolome of the freshwater amphipod Gammarus fossarum, successfully discriminating stages and highlighting significant biological features. Despite being underused, DIA, along with the Zeno trap and EAD, holds great potential for advancing research in the omics field. ResultsDIA combined with the Zeno trap enhances detection reproducibility compared to conventional DDA, improving fragmentation spectra quality and putative identifications. LC coupled with Zeno-SWATH-DIA methods were used to characterize molecular changes in reproductive cycle of female gammarids. Multivariate data analysis including Principal Component Analysis and Partial Least Square Discriminant Analysis successfully identified significant features. EAD fragmentation helped to identify unknown features and to confirm their molecular structure using fragmentation spectra database annotation or machine learning. EAD database matching accurately annotated five glycerophospholipids, including the position of double bonds on fatty acid chain moieties. SIRIUS database predicted structures of unknown features based on experimental fragmentation spectra to compensate for database incompleteness. SignificanceReproducible detection of features and confident identification of putative compounds are pivotal stages within analytical pipelines. The DIA approach combined with Zeno pulsing enhances detection sensitivity and targeted fragmentation with EAD in positive polarity provides orthogonal fragmentation information. In our study, Zeno-DIA and EAD thereby facilitated a comprehensive and insightful exploration of pertinent biological molecules associated with the reproductive cycle of gammarids. The developed methodology holds great promises for identifying informative biomarkers on the health status of an environmental sentinel species.

New Stability Indicating HPLC Method for Related Substances in Dapagliflozin

The requirements established by the International Council for Harmonization (ICH) for the measurement of dapagliflozin from tablets were satisfied by the development and successful implementation of an reversed-phase high performance liquid chromatography (RP-HPLC) technique that is characterized by its speed, precision, and accuracy. In order to separate the samples, an isocratic mode was used on a Princeton C18 column that was operating at a flow rate of 1-mL per minute. The phase was composed of acetonitrile and 0.1% triethylamine in a volume-to-volume ratio of 50:50, with a pH of 5.0 and a detection wavelength of 224 nm. 5.163 minutes was the retention time that was determined to be best under the circumstances of the chromatographic analysis. Based on the correlation value of 0.999, it can be concluded that the procedure adhered to Beer-Lambert’s law within the measurement range of 10 to 70 μg/mL. The findings not only verified the claim that was made on the label, but they also validated the mean percent drug concentration that was specified for the commercial tablet formulation, which was 100.57%. Following the completion of an investigation, it was discovered that the approaches that were proposed have sufficient proportions of precision measurements, robustness, accuracy, range, and linearity. As part of the technique for stress testing, a number of criteria were evaluated and evaluated overall. The elements that were considered to be among these were light, humidity, temperature, oxidation, and pH (acid/base).

Novel HPLC Method Validation for Quantification of Dapagliflozin in Bulk Drug and Formulation

A new, steady, easy-to-use, and straightforward high-performance liquid chromatography (HPCL) technique was also tested to determine dapagliflozin concentration in medicinal and API forms. For the chromatographic extraction study, a Phenomenex Gemini-NX C18 Column was used. This column was filled with a mobile phase that was 70:30 v/v methanol to sodium 1-octanesulphonate. This experiment used a 20-liter syringe amount and a flow rate of 1-mL per minute. A bunch of factors were used to make sure the new method worked. The drug dapagliflozin was released at a wavelength of 203 nm after 6.5 ± 0.5 minutes. A correlation coefficient (r2) of 0.9994 between 15 to 55 μg/mL shows that the planned process is linear. The theoretical plates and peak tailing were found to be 4129 and 1.03, respectively. In terms of healing, the percent accuracy that was reached was within the acceptable range of 98 to 100. New suggestions say that the regular testing of dapagliflozin in large amounts must be done using a proven method that is unique and easy to repeat. Forced breakdown tests with drugs were used to show that the proposed method is stable. The product was tested according to the steps laid out in the ICH Q2R1 Guidelines. The test looked at many chemical processes, such as oxidation, photolysis, heat stress, and acids and alkaline hydrolysis.

Green Analytical HPLC Method Development of Acyclovir Loaded Floating Raft Formulation in Spiked Rabbit Plasma

Objective: The need of this investigation is to design eco-friendly, sensitive, and precise bioanalytical high-performance liquid chromatography (HPLC) techniques that leverage green analytical tools such as green analytical procedure index (GAPI), analytical eco scale (AES), and analytical greenness metric (AGREE) to analyze ACV-spiked rabbit plasma and ACV-loaded floating raft formulation (FRF) because The literature survey revealed insufficient data for the HPLC approach to FRF. Method: Methods involving plasma spikes mirroring the in-vivo models provide insights into potential real-life scenarios and the functionality of floating raft formulations loaded with ACV. The Design Expert tool optimized critical process parameters and identified significant factors influencing the chromatographic technique. Results: The CCD was used to optimize the flow rate (0.6 mL/min) and buffer solvent ratio (99:1) for separation. The C18 column (250 X 4.6 mm) was utilized to achieve the elution of ACV. The developed method was thoroughly validated through ICH guidelines. The ACV, lower limit of detection (LLoD), and lower limit of quantitation (LLoQ) retention times were established at 2.3 minutes, 15, and 20 ng/mL, respectively. Discussion: In summary, the study demonstrated the exceptional performance sensitivity, specificity, and robustness of the bioanalytical method. The method proved highly effective in quantifying ACV in plasma samples in terms of revalidatability, transferability, reproducibility, and the product development cycle. The developed method proves that using both design of experiments (DoE) and green analytical chemistry together creates a valid, eco-friendly, and reliable way to analyze the making of an ACV-loaded floating raft formulation.

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

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

Formulation and optimisation of Ozenoxacin topical nano-emulgel including a comprehensive methodology to qualify and validate the critical parameters of an in-vitro release test method and ex-vivo permeation test

Objective The purpose of this study was to formulate, optimize Ozenoxacin topical nano-emulsion using factorial design followed by to prepare and evaluate nano-emulgel using validated in-vitro release testing (IVRT) technique for determination of Ozenoxacin release rate along with ex-vivo permeation testing (EVPT). Significance: Nano-emulgel is a proven delivery system for poorly soluble substances works by enhancing the solubility and bioavailability. Factorial design provides a systematic and efficient means to study the effect of multiple factors on responses. IVRT is an USP compendia technique utilized for performance analysis of semi-solid formulations. Methods Nano-emulsion formulation optimization was done with factorial design, evaluated for globule size and % entrapment efficiency (EE). Nano-emulgels were characterized for assay, organic impurities, rheological behavior, IVRT, EVPT, and skin retention studies. IVRT validation was executed using vertical diffusion cells (VDCs). Results Ozenoxacin nano-emulsion was optimized with 1:1 ratio of Oil: Smix, 3:1 ratio of Surfactant:Co-Surfactant, and 15000 RPM of homogenization speed which resulted 414.6 ± 5.2 nm globule size and 92.8 ± 2.1% entrapment efficiency. Results confirmed that IVRT and Reversed Phase – High Performance Liquid Chromatographic techniques were validated as per regulatory guidelines. In-vitro, ex-vivo drug release, and skin retention from the optimized nano-emulgel formulation was comparatively higher (∼1.5 times) than that from the innovator (OZANEXTM) formulation. Conclusions Based on these results, Ozenoxacin nano-emulgel can be considered an effective alternative and was found to be stable at 40 °C/75% RH and 30 °C/75% RH storage condition for 6 months.

Electrostatic Repulsion Hydrophilic Interaction Liquid Chromatography (ERLIC) for the Quantitative Analysis of Polyamines

Highly polar low molecular weight organic molecules are still very challenging to analyze by liquid chromatography. Yet, with the steadily increasing application of metabolomics and similar approaches in chemical analysis, separating polar compounds might be even more important. However, almost all established liquid chromatography techniques (i.e., normal and reversed phase, hydrophilic interaction liquid chromatography (HILIC), ion chromatography) struggle with either carry-over, low sensitivity, or a lack of retention. For improving these shortcomings, electrostatic repulsion hydrophilic interaction chromatography (ERLIC) might be an alternative. By combining a HILIC mobile phase, that is highly organic with a low water content, and an ion exchange column, a distinct layer system develops. When the analyte's charge is of the same direction as the stationary phase, retention and elution are determined by two antagonistic forces: electrostatic repulsion and hydrophilicity. One prominent group of challenging polar analytes are the polyamines cadaverine, putrescine, spermidine, and spermine. Carrying charges from +2 to +4 at physiological pH, these compounds are essential cell constituents and found in all living organisms. However, they are still notoriously challenging to analyze via the established liquid chromatography methods. In the present work, an ERLIC tandem mass spectrometry method has been exemplarily developed, optimized, and validated for the quantitative determination of cadaverine, putrescine, spermidine, and spermine. This method enables symmetrical peak shapes and good separation of analytes with different charges while simultaneously selectively detecting the co-eluting diamines by MS/MS. Furthermore, high linearity (R > 0.998) and sensitivity (LODs ≤ 2 ng/mL) have been proven. Thus, ERLIC may be interesting for both targeted and untargeted analysis approaches of highly charged low molecular weight organic molecules.

A comparative study on the nutritional composition, protein structure and effects on gut microbiota of 5 fermented soybean products (FSPs)

In this study, we conducted an analysis of the differences in nutrient composition and protein structure among various fermented soybean products and their impacts on the gut microbiota of rats. Conventional physicochemical analysis was employed to analyze the fundamental physicochemical composition of the samples. Additionally, we utilized high-performance liquid chromatography and ELISA techniques to quantify the presence of antinutritional compounds. Fourier infrared spectroscopy was applied to delineate the protein structure, while 16 s rRNA gene sequencing was conducted to evaluate alterations in gut microbiota abundance. Subsequently, KEGG was utilized for metabolic pathway analysis. Our findings revealed that fermented soybean products improved the nutritional profile of soybeans. Notably, Douchi exhibited the highest protein content at 52.18 g/100 g, denoting a 26.58 % increase, whereas natto showed a 24.98 % increase. Douchi and natto demonstrated the most substantial relative amino acid content, comprising 50.86 % and 49.04 % of the total samples, respectively. Moreover, the levels of antinutritional factors markedly decreased post-fermentation. Specifically, the α-helix content in doujiang decreased by 13.87 %, while the random coil content in soybean yogurt surged by 132.39 %. Rats that were fed FSP showcased notable enhancements in gut microbiota and associated metabolic pathways. A strong correlation was observed between nutrient composition, protein structure, and gut microbiota abundance. This study furnishes empirical evidence supporting the heightened nutritional attributes of FSPs.

Growth adaptation of Nitraria sibirica in drought: Hormone-mediated flavonoid accumulation, altered metabolic pathways, and decreased network complexity

Drought is a severe abiotic stress condition that impedes plant growth and development. During adaptation to drought conditions, plants release numerous small-molecule compounds that enhance their adaptive capacity. Nitraria sibirica Pall. is an ecologically valuable plant; however, its metabolic adaptation mechanisms in different soil–water habitats have not been clearly reported. In this study, high-performance liquid chromatography (HPLC) and non-targeted metabolomic techniques were combined to investigate the growth adaptation mechanisms of N. sibirica in different soil environments. The results showed that the response of N. sibirica leaves to drought mainly manifested as a significant increase in abscisic acid (ABA) levels and a significant decrease in gibberellin (GA) levels (except during flowering). Drought led to the production of more metabolites during the growth and development of N. sibirica but reduced the complexity of the metabolite network. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed significant differences in the growth adaptation pathways of metabolites between the two habitats. In riparian habitats (RH), the phenylpropane biosynthesis pathway was significantly upregulated, while the synthesis of cutin, suberin, wax, α-linolenic acid metabolism, and linoleic acid metabolism was significantly downregulated. However, the tryptophan metabolism pathway was consistently downregulated in desert habitats (DH), with flavonoid and flavonol biosynthesis pathways playing important roles in drought resistance. The random forest model showed that the soil environment explained 3.21–94.34 % of the metabolite variation. Furthermore, structural equation modeling demonstrated that soil water had an indirect and positive effect on flavonoid metabolites by regulating ABA. This study revealed the adaptive mechanisms of hormones and metabolites in N. sibirica under drought conditions. It identified certain hormones that could potentially be harnessed for the development of plant additives, providing potential opportunities for exploring new stress adaptation strategies and developing new plant drought resistance products.

A Multi-step High-Performance Liquid Chromatography (HPLC) Technique for the Extraction and Purification of Medical-Grade Cyclosporine A

Cyclosporine A (CsA), a potent immunosuppressive drug, is used to treat autoimmune diseases and prevent organ transplant recipients from rejecting their new organs. Maintaining CsA's uniformity and purity is essential to maintaining its therapeutic efficacy and safety. Impurities and contaminants in CsA formulations can exacerbate side effects and patient toxicity. Purification processes can improve the safety profile of the drug and reduce the likelihood of adverse effects by eliminating impurities. Using High-performance liquid chromatography (HPLC) methods, CsA has been isolated and refined from crude in recent years. However, after the procedure, the CsA purity was not at its best. To solve this issue, this work employs a multi-step HPLC method to increase purity levels to above 95%. When exploring the HPLC detection conditions of CsA, we found that the method from the Chinese pharmacopoeia: acetonitrile-water-tert-butyl methyl ether-phosphoric acid (430 : 520 : 50 : 1) was a better mobile phase solution, our further research, including the substitution of tert-butyl methyl ether with other buffer solvents, showed no significant improvement. At the same time, we established that the key factor affecting its separation was temperature. When the temperature is lower than 70 ℃, the HPLC separation effect worsens, the retention time increases, and the peak width becomes longer. Finally, the utilization of acetonitrile-water-tert-butyl methyl ether-phosphoric acid (430 : 520 : 50 : 1) as the mobile phase with a column temperature of 70 °C, petroleum ether: acetic acid Ethyl ester (70 : 30) as the mobile phase, and filling the φ 40 mm × 500 mm chromatography column using a 40~60 μm silica gel so that a 10 : 1 height-to-diameter ratio of the packed part is obtained, under normal temperature conditions, resulted in an average cyclosporine A yield of 80.1%. The corresponding average purity was 97.2%, of which the first isolation yield was 82.3%, and the purity was 98.1%.

A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

Toward a More Comprehensive Approach for Dissolved Organic Matter Chemical Characterization Using an Orbitrap Fusion Tribrid Mass Spectrometer Coupled with Ion and Liquid Chromatography Techniques.

Dissolved organic matter (DOM) represents one of the largest active organic carbon pools in the global carbon cycle. Although extensively studied, only <10% of DOM has been chemically characterized into individual dissolved compounds due to its molecular complexity. This study introduced a more comprehensive DOM characterization method by coupling both ion chromatography (IC) and liquid chromatography (LC) with high mass accuracy and resolution mass spectrometry. We presented a new on-the-fly mass calibration of the Orbitrap technique by utilizing the "lock mass" function in the Orbitrap Fusion Tribrid mass spectrometer (OT-FTMS), which assures high mass accuracy at every scan by a postcolumn introduction of internal labeled standards. With both IC and LC, tested unlabeled standards of amino acids, small peptides, and organic acids were consistently below 1.0 ppm mass error, giving the OT-FTMS the potential of reaching mass accuracy of the Fourier-transform ion cyclotron resonance mass spectrometer. In addition to mass accuracy, a pooled quality control sample (QC) was used to increase reproducibility by applying systematic error removal using random forest (SERRF). Using an untargeted mass spectrometry approach, estuarine DOM samples were analyzed by OT-FTMS coupled to IC in negative mode and LC in positive mode detection to cover a wide range of highly cationic to highly anionic molecules. As a proof of concept, we focused on elucidating the structures of three distinct DOM compound classes with varied acidities and basicities. In UPLC-OT-FTMS, a total of 915 compounds were detected. We putatively elucidated 44 small peptides and 33 deaminated peptides of these compounds. With IC-OT-FTMS, a total of 1432 compounds were detected. We putatively elucidated 20 peptides, 268 deaminated peptides, and 188 organic acids. Except for five compounds, all putatively elucidated compounds were uniquely detected in their corresponding chromatography technique. These results highlight the need for combining these two techniques to provide a more comprehensive method for DOM characterization. Application of the combined IC and LC techniques is not limited to DOM chemical characterization. It can analyze other complex compound mixtures, such as metabolites, and anthropogenic pollutants, such as pesticides and endocrine-disrupting chemicals, in environmental and biological samples.

Evaluation of stability of (1R,2 S)-(−)-2-methylamino-1-phenyl-1-propanol hydrochloride in plasma and urine samples-inoculated with Escherichia coli using high-performance liquid chromatography (HPLC)

The preservation of drug stability in biological evidence during the processes of collection and storage poses a substantial obstacle to the progress of forensic investigations. In conjunction with other constituents, the microorganisms present in the samples play a vital role in this investigation. The present investigation employed the high-performance liquid chromatography (HPLC) technique to assess the stability of (1R,2 S)-(–)-2-methylamino-1-phenyl-1-propanol hydrochloride in plasma and urine samples that were inoculated with Escherichia coli. These samples were subjected to storage conditions of 37 °C for 48 h and − 20 °C for a duration of 6 months. Minimal inhibitory concentration (MIC) and Minimal bactericidal concentration (MBC) of MPPH against E. coli were determined using microdilution method. The stability of MPPH in plasma and urine samples inoculated with E. coli was investigated using HPLC method. The results showed the MIC and MBC of MPPH were 87.5 ± 25 ppm and 175 ± 50 ppm, respectively. While MPPH remained stable in plasma for 48 h at 37 °C, it showed a notable decrease of about 11% in stability when stored in urine for the same period and temperature. From the beginning of the first month, a decrease in the stability of the compound appeared in all samples that were stored at − 20 °C, and the decrease reached 7% for plasma samples and about 11% for urine samples. The decrease in the stability reached its peak in the sixth month, reaching more than 30% and 70% of plasma and urine samples preserved at − 20 °C. This work concluded that E. coli can negatively affect the stability of MPPH in plasma and urine samples. This may lead to incorrect conclusions regarding the analysis of biological samples in criminal cases.

Investigating the Merits of Microfluidic Capillary Zone Electrophoresis-Mass Spectrometry (CZE-MS) in the Bottom-Up Characterization of Larger RNAs.

Established bottom-up approaches for the characterization of nucleic acids (NAs) rely on the strand-cleavage activity of nucleotide-specific endonucleases to generate smaller oligonucleotides amenable to gas-phase sequencing. The complexity of these hydrolytic mixtures calls for the utilization of a front-end separation to facilitate full mass spectrometric (MS) characterization. This report explored the merits of microfluidic capillary zone electrophoresis (CZE) as a possible alternative to common liquid chromatography techniques. An oligonucleotide ladder was initially employed to investigate the roles of fundamental analyte features and experimental parameters in determining the outcome of CZE-MS analyses. The results demonstrated the ability to fully resolve the various rungs into discrete electrophoretic peaks with full-width half-height (FWHH) resolution that was visibly affected by the overall amount of material injected into the system. Analogous results were obtained from a digestion mixture prepared by treating yeast tRNAPhe (75 nt) with RNase T1, which provided several well-resolved peaks in spite of the increasing sample heterogeneity. The regular shapes of such peaks, however, belied the fact that most of them contained sets of comigrating species, as shown by the corresponding MS spectra. Even though it was not possible to segregate each species into an individual electrophoretic peak, the analysis still proved capable of unambiguously identifying a total of 29 hydrolytic products, which were sufficient to cover 96% of the tRNAPhe's sequence. Their masses accurately reflected the presence of modified nucleotides characteristic of this type of substrate. The analysis of a digestion mixture obtained from the 364 nt HIV-1 5'-UTR proved to be more challenging. The electropherogram displayed fewer well-resolved peaks and significantly greater incidence of product comigration. In this case, fractionating the highly heterogeneous mixture into discrete bands helped reduce signal suppression and detection bias. As a result, the corresponding MS data enabled the assignment of 248 products out of the possible 513 predicted from the 5'-UTR sequence, which afforded 100% sequence coverage. This figure represented a significant improvement over the 36 total products identified earlier under suboptimal conditions, which afforded only 57% coverage, or the 83 observed by direct infusion nanospray-MS (72%). These results provided a measure of the excellent potential of the technique to support the bottom-up characterization of progressively larger NA samples, such as putative NA therapeutics and mRNA vaccines.

Sesquiterpene Lactones and Flavonoid from the Leaves of Basin Big Sagebrush (Artemisia tridentata subsp. tridentata): Isolation, Characterization and Biological Activities.

This research is an exploratory study on the sesquiterpenes and flavonoid present in the leaves of Artemisia tridentata subsp. tridentata. The leaf foliage was extracted with 100% chloroform. Thin-layer chromatography (TLC) analysis of the crude extract showed four bands. Each band was purified by column chromatography followed by recrystallization. Three sesquiterpene lactones (SLs) were isolated-leucodin, matricarin and desacetylmatricarin. Of these, desacetylmatricarin was the major component. In addition, a highly bio-active flavonoid, quercetagetin 3,6,4'-trimethyl ether (QTE), was also isolated. This is the first report on the isolation of this component from the leaves of Artemisia tridentata subsp. tridentata. All the components were identified and isolated by TLC, high-performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques. Likewise, the structure and stereochemistry of the purified components were characterized by extensive spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) studies. The antioxidant activities of crude extract were analyzed, and their radical-scavenging ability was determined by Ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The crude extract showed antioxidant activity of 18.99 ± 0.51 and 11.59 ± 0.38 µmol TEg-1 FW for FRAP and DPPH assay, respectively, whereas the activities of matricarin, leucodin, desacetylmatricarin and QTE were 13.22, 13.03, 14.90 and 15.02 µmol TEg-1 FW, respectively, for the FRAP assay. The antitumor properties were probed by submitting the four isolated compounds to the National Cancer Institute (NCI) for NCI-60 cancer cell line screening. Overall, the results of the one-dose assay for each SL were unremarkable. However, the flavonoid's one-dose mean graph demonstrated significant growth inhibition and lethality, which prompted an evaluation of this compound against the 60-cell panel at a five-dose assay. Tests from two separate dates indicate a lethality of approximately 75% and 98% at the log-4 concentration when tested against the melanoma cancer line SK-Mel 5. This warrants further testing and derivatization of the bioactive components from sagebrush as a potential source for anticancer properties.

Simple Determination of Bosentan in Plasma Samples by Reversed-Phase High-Performance Liquid Chromatography.

In order to measure the plasma levels of Losartan and Bosentan, a sensitive Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) technique was developed. To compare bioavailability, the Area Under the Curve (AUC), peak plasma concentration (Cmax), and time to Cmax (Tmax) were employed. The standard curve (150-2400 ng/ml) was linear (R2=0.999), relative errors were between 2.4 to 10.05% and the coefficient of variation (CV%) ranged from 1.52 to 10.88. A single dosage (test and reference) was used for the in vivo investigation, which involved 16 healthy individuals. The AUC0-48, AUC0-, Cmax, and Tmax of the test and reference had no statistically significant differences. The Cmax and 95% confidence intervals of the ratio of Cmax of the two formulations were 0.93-0.96 and 97.6-135%, respectively. Therefore, it was established that generic Bosentan was equivalent to Bosentan from Actelion and that both medications could be regarded as equally effective in clinical settings. The blood level of Bosentan could be measured using this straightforward procedure in all hospital laboratories.

Phytocompounds-based therapeutic approach: Investigating curcumin and green tea extracts on MCF-7 breast cancer cell line

BackgroundBreast cancer (BC) has transcended lung cancer as the most common cancer in the world. Due to the disease's aggressiveness, rapid growth, and heterogeneity, it is crucial to investigate different therapeutic approaches for treatment. According to the World Health Organization (WHO), Plant-based therapeutics continue to be utilized as safe/non-toxic complementary or alternative treatments for cancer, even in developed countries, regardless of how cutting-edge conventional therapies are. Despite their low bioavailability, curcumin (CUR) and green tea (GT) represent safer therapeutic options. Due to their potent molecular-modulating properties on various cancer-related molecules and signaling pathways, they are considered gold-standard therapeutic agents and have been incorporated into the development of one or more therapeutic strategies of BC treatment. MethodsWe investigated the modulatory role of CUR and GT extracts on significant multi molecular targets in MCF-7 BC cell line to assess their potential as BC multi-targeting agents. We analyzed the phytocompounds in GT leaves using High-performance liquid chromatography (HPLC) and Gas chromatography-mass spectrometry (GC-MS) techniques. The mRNA expression levels of Raf-1, Telomerase, Tumor necrosis factor alpha (TNF-α) and Interleukin-8 (IL-8) genes in MCF-7 cells were quantified using quantitative real-time PCR (qRT-PCR). The cytotoxicity of the extracts was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the released Lactate dehydrogenase (LDH), a valuable marker for identifying the programmed necrosis (necroptosis). Additionally, the concentrations of the necroptosis-related proinflammatory cytokines (TNF-α and IL-8) were measured using enzyme-linked immunosorbent assay (ELISA). ResultsIn contrast to the GT, the results showed the anticancer and cytotoxic properties of CUR against MCF-7 cells, with a relatively higher level of released LDH. The CUR extract downregulated the oncogenic Raf-1, suppressed the Telomerase and upregulated the TNF-α and IL-8 genes. Results from the ELISA showed a notable increase in IL-8 and TNF-α cytokines levels after CUR treatment, which culminated after 72 h. ConclusionsAmong both extracts, only CUR effectively modulated the understudy molecular targets, achieving multi-targeting anticancer activity against MCF-7 cells. Moreover, the applied dosage significantly increased levels of the proinflammatory cytokines, which represent a component of the cytokines-targeting-based therapeutic strategy. However, further investigations are recommended to validate this therapeutic approach.

A comprehensive exploration of diverse green analytical techniques and their influence in different analytical fields

AbstractThe primary goals of green analytical chemistry (GAC) involve the development of novel analytical technologies or the modification of existing methods to incorporate procedures with reduced reliance on hazardous chemicals. Several approaches can be employed to achieve these objectives, including the utilization of environmentally friendly solvents and reagents, shortening chromatographic separation times, and the miniaturization of analytical devices. The review summarizes the evolution of GAC with its specific principles and with special emphasis on recent applications in modern era. A detailed exploration of the choice of green solvents and their practical utilization in green liquid chromatography (LC) techniques for environmentally friendly analysis of pharmaceuticals has been discussed. An ordinary description with a tabular illustration of various modern green analytical techniques like micellar LC, superheated water chromatography, high‐speed LC, enhanced fluidity LC, two‐dimensional LC, and aspects of gas chromatography has been thoroughly provided for concepts with their mechanism and limitations. GAC provides potential applications for food analysis, environmental analysis of pharmaceutically active compounds in the aquatic environment with special emphasis on strategies for greening LC and impurity profiling, and so on. Numerous benefits, limitations, and challenges that are linked to the field of GAC are highlighted in this review. We pay special attention to new green approaches, such as automation, miniaturization, direct analysis techniques, and some initiatives like the National Environmental Methods Index Label for Analytical Eco‐Scale assessment. In spite of many promising advancements, the understanding is still limited and challenging.