Reversed-phase Chromatography Research Articles

Chemotactic activity of products of Elizabethkingia anophelis derived from Aedes albopictus against RAW264 murine macrophage cell line.

Dengue viruses enter dermal macrophages which are derived from other tissues after mosquito bites. We examined chemotactic factors derived from the dengue vector mosquito, Aedes albopictus, toward a RAW264 murine macrophage cell line. We found that Elizabethkingia anophelis that was isolated from mosquitoes exhibited migration-inducing activity toward RAW264 cells. The active substances were extracted using ethyl acetate to induce chemotactic movements. Chemotactic activity was eluted in the several fractions using the reversed-phase chromatography, suggesting that multiple substances were responsible for the activity. We isolated three bacterial colonies from the wild A. albopictus mosquitoes collected in Toyama Park, Tokyo prefecture, Japan. The bacterial 16S rRNA gene sequences were the most similar to those of Lonsdalea quercina. These bacteria also exhibited migration-inducing activity toward RAW264 cells. The migration-inducing activity of mosquito bacteria might be a new aspect of mosquito-mediated viral infections.

Intraspecific venom variation in the medically important puff adder (Bitis arietans): Comparative venom gland transcriptomics, in vitro venom activity and immunological recognition by antivenom.

Variation in snake venoms is well documented, both between and within species, with intraspecific venom variation often correlated with geographically distinct populations. The puff adder, Bitis arietans, is widely distributed across sub-Saharan Africa and into the Arabian Peninsula where it is considered a leading cause of the ~310,000 annual snakebites across the region, with its venom capable of causing substantial morbidity and mortality. Despite its medical importance and wide geographic distribution, there is little known about venom variation between different B. arietans populations and the potential implications of this variation on antivenom efficacy. We applied a range of analyses, including venom gland transcriptomics, in vitro enzymatic assays and reverse phase chromatography to comparatively analyse B. arietans venoms originating from Nigeria, Tanzania, and South Africa. Immunological assays and in vitro enzymatic neutralisation assays were then applied to investigate the impact of venom variation on the potential efficacy of three antivenom products; SAIMR Polyvalent, EchiTAb-Plus and Fav-Afrique. Through the first comparison of venom gland transcriptomes of B. arietans from three geographically distinct regions (Nigeria, Tanzania, and South Africa), we identified substantial variation in toxin expression. Findings of venom variation were further supported by chromatographic venom profiling, and the application of enzymatic assays to quantify the activity of three pathologically relevant toxin families. However, the use of western blotting, ELISA, and in vitro enzymatic inhibition assays revealed that variation within B. arietans venom does not appear to substantially impact upon the efficacy of three African polyvalent antivenoms. The large distribution and medical importance of B. arietans makes this species ideal for understanding venom variation and the impact this has on therapeutic efficacy. The findings in this study highlight the likelihood for considerable venom toxin variation across the range of B. arietans, but that this may not dramatically impact upon the utility of treatment available in the region.

Integrated strategy for high-confident global profiling of the histidine phosphoproteome

BackgroundHistidine phosphorylation (pHis) plays a key role in signal transduction in prokaryotes and regulates tumour initiation and progression in mammals. However, the pHis substrates and their functions are rarely known due to the lack of effective analytical strategies. ResultsHerein, we provide a strategy for unbiased enrichment and assignment of the pHis peptides. First, the entire procedure was designed under alkaline conditions to maintain the stability of the N–P bond of pHis and high-pH reverse-phase chromatography was used to efficiently separate the pHis peptides. Second, exploiting the coelution benefits of diethyl labelling, the ratios of light- and heavy-labelled peptides were accurately quantified, and the sites of phosphorylated histidine were assigned. Finally, Cu-IDA bead enrichment and data-independent acquisition mass spectrometry analysis were used to improve the coverage of the histidine phosphoproteome. With this novel strategy, 768 and 1125 potential pHis peptides were identified from lysates of E. coli and HeLa cells, respectively. And these values represent the highest coverage of the histidine phosphoproteome for both cell types. SignificanceThese data strongly support the presumption that pHis modifications are widely present in bacteria. The study provides an efficient strategy and can lead to a better understanding of pHis-modified substrates and their biological functions.

Leveraging HILIC/ERLIC Separations for Online Nanoscale LC-MS/MS Analysis of Phosphopeptide Isoforms from RNA Polymerase II C-terminal Domain.

The eukaryotic RNA polymerase II (Pol II) multi-protein complex transcribes mRNA and coordinates several steps of co-transcriptional mRNA processing and chromatin modification. The largest Pol II subunit, Rpb1, has a C-terminal domain (CTD) comprising dozens of repeated heptad sequences (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), each containing five phospho-accepting amino acids. The CTD heptads are dynamically phosphorylated, creating specific patterns correlated with steps of transcription initiation, elongation, and termination. This CTD phosphorylation 'code' choreographs dynamic recruitment of important co-regulatory proteins during gene transcription. Genetic tools were used to engineer protease cleavage sites across the CTD (msCTD), creating tryptic peptides with unique sequences amenable to mass spectrometry analysis. However, phosphorylation isoforms within each msCTD sequence are difficult to resolve by standard reversed phase chromatography typically used for LC-MS/MS applications. Here, we use a panel of synthetic CTD phosphopeptides to explore the potential of hydrophilic interaction and electrostatic repulsion hydrophilic interaction (HILIC and ERLIC) chromatography as alternatives to reversed phase separation for CTD phosphopeptide analysis. Our results demonstrate that ERLIC provides improved performance for separation of singly- and doubly-phosphorylated CTD peptides for sequence analysis by LC-MS/MS. Analysis of native yeast msCTD confirms that phosphorylation on Ser5 and Ser2 represents the major endogenous phosphoisoforms. We expect this methodology will be especially useful in the investigation of pathways where multiple protein phosphorylation events converge in close proximity.

Effective removal of host cell-derived nucleic acids bound to hepatitis B core antigen virus-like particles by heparin chromatography.

Virus-like particles (VLPs) show considerable potential for a wide array of therapeutic applications, spanning from vaccines targeting infectious diseases to applications in cancer immunotherapy and drug delivery. In the context of hepatitis B core antigen (HBcAg) VLPs, a promising candidate for gene delivery approaches, the naturally occurring nucleic acid (NA) binding region is commonly utilized for effective binding of various types of therapeutic nucleic acids (NAther). During formation of the HBcAg VLPs, host cell-derived nucleic acids (NAhc) might be associated to the NA binding region, and are thus encapsulated into the VLPs. Following a VLP harvest, the NAhc need to be removed effectively before loading the VLP with NAther. Various techniques reported in literature for this NAhc removal, including enzymatic treatments, alkaline treatment, and lithium chloride precipitation, lack quantitative evidence of sufficient NAhc removal accompanied by a subsequent high VLP protein recovery. In this study, we present a novel heparin chromatography-based process for effective NAhc removal from HBcAg VLPs. Six HBcAg VLP constructs with varying lengths of the NA binding region and diverse NAhc loadings were subjected to evaluation. Process performance was thoroughly examined through NAhc removal and VLP protein recovery analyses. Hereby, reversed phase chromatography combined with UV/Vis spectroscopy, as well as silica spin column-based chromatography coupled with dye-based fluorescence assay were employed. Additionally, alternative process variants, comprising sulfate chromatography and additional nuclease treatments, were investigated. Comparative analyses were conducted with LiCl precipitation and alkaline treatment procedures to ascertain the efficacy of the newly developed chromatography-based methods. Results revealed the superior performance of the heparin chromatography procedure in achieving high NAhc removal and concurrent VLP protein recovery. Furthermore, nuanced relationships between NA binding region length and NAhc removal efficiency were elucidated. Hereby, the construct Cp157 surpassed the other constructs in the heparin process by demonstrating high NAhc removal and VLP protein recovery. Among the other process variants minimal performance variations were observed for the selected constructs Cp157 and Cp183. However, the heparin chromatography-based process consistently outperformed other methods, underscoring its superiority in NAhc removal and VLP protein recovery.

Recommendations for Accurate Lipid Annotation and Semi-absolute Quantification from LC-MS/MS Datasets.

Recent developments in LC-MS instrumentation and analytical technologies together with bioinformatics tools supporting high-throughput processing of large omics datasets significantly enhanced our capabilities and efficiency of identification and quantification of lipids in diverse biological materials. However, each biological matrix is characterized by its unique lipid composition, thus requiring optimization of analytical and bioinformatics workflows for each studied lipidome. Here, we describe an integrated workflow for deep lipidome profiling, accurate annotation, and semi-absolute quantification of complex lipidomes based on reversed phase chromatography and high resolution mass spectrometry. This chapter provides details on selection of the optimal extraction protocol, acquisition of LC-MS/MS data for accurate annotation of lipid molecular species, and design of lipidome-specific mixtures of internal standards to assist quantitative analysis of complex, native lipidomes.

B-161 Liquid chromatography/mass spectrometry analysis of ethyl glucuronide and ethyl sulfate using a perfluorophenyl stationary phase

Abstract Background Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor metabolites of ethanol that are excreted in urine. These two metabolites are often tested for to determine if an individual has recently consumed alcohol. EtG and EtS are very polar analytes, which make them difficult to chromatograph using reversed-phase chromatography. These analytes often elute early, which can lead to irreproducible results. When EtG and EtS are analyzed by LC/MS with a mobile phase that contains formic acid, the signal of these two analytes can be suppressed. This leads to increased LOQ and cutoffs. We examined the perfluorophenyl (PFP) stationary phase for its utility in analyzing EtG and EtS. This stationary phase has been shown to have greater retention of polar compounds than C18 reversed-phase columns. In addition, we compared the effect of formic acid to acetic acid on the separation. Methods Urine samples containing EtG and EtS were diluted 10:1, using 50mM acetic acid or 0.1% formic acid, into a 96-well microtiter filter plate containing a 0.2micron filter. Internal standards were added to the microtiter plate containing the urine samples. The samples were filtered through the filter plate and analyzed by LC/MS using the PFP column (50 x 2.7micron) with a 50mM acetic acid or 0.1% formic acid/acetonitrile mobile phase. The analytes were detected using negative mode ionization. The total analysis time was 4.5minutes. Results EtG and EtS showed improved retention using the PFP column. Retention times for EtG and EtS were 0.75 and 1.20 minutes respectively using the PFP column, compared to 0.4 and 0.3 minutes using the C18 column. The greater retention time improved the LOQ for both analytes.The LOQ for EtS and EtG were determined to be 125 and 250 ng/mL, respectively. Greater retention of EtG was observed by switching the modifier from formic acid to acetic acid. The retention time increased from 0.45 to 0.75 minutes. There was less signal suppression using acetic acid in place of formic acid. Improvement in peak shape was observed when the injection volume was reduced to 5microliters. The method was validated over a range of 250 to 10000 ng/mL for EtG and 125 to 5000 ng/mL for EtS. Acceptable linearity was obtained for both analytes. Accuracy was performed at three concentration levels. All three levels demonstrated acceptable accuracy with the difference between the measured and expected concentration being with in 20% of each other. Conclusions The PFP stationary can be used to test EtG and EtS. This stationary phase gave greater retention of EtS and EtG. The use of acetic acid improved the limit of detection by decreasing the ion suppression, which decreased the limit of detection. The assay was accurate and precise for both analytes, demonstrated in the validation.

Global Metabolomics Using LC-MS for Clinical Applications.

Metabolomics can be used for a multitude of purposes, including monitoring of treatment effects and for increasing the knowledge of the pathophysiology of a wide range of diseases. Global (commonly referred to as "untargeted") metabolomics is hypothesis-generating and provides the opportunity to discover new biomarkers. Being versatile and having a high degree of selectivity and sensitivity, liquid chromatography-mass spectrometry (LC-MS) is the most common technique applied for metabolomics. We here present our global metabolomics LC-electrospray ionization-MS/MS method. The sample preparation procedures for plasma, serum, dried blood spots, urine, and cerebrospinal fluid are simple and nonspecific to reduce the risk of analyte loss. The method is based on reversed-phase chromatography using a diphenyl column. The high-resolution Q Exactive Orbitrap MS with data-dependent acquisition provides MS/MS spectra of a wide range of analytes. Our method covers a large part of the metabolome regarding hydrophobicity and compound class.

Development of deep learning software to improve HPLC and GC predictions using a new crown-ether based mesogenic stationary phase and beyond

The application of AI to analytical and separative sciences is a recent challenge that offers new perspectives in terms of data prediction. In this work, we report an AI-based software, named Chrompredict 1.0, which based on chromatographic data of a novel mesogenic crown ether stationary phase (CESP). Its molecular design represents a significant advancement due to the unique combination of properties and binding capabilities, including the formation of a cavity, mesogenic behavior via mobile chains, and a range of polar and non-polar interactions (aromatic rings, N=N and O=O double bonds, alkyl chains, π–π interactions, and hydrogen bonding). The mesogenic phase is effective in both normal and reversed-phase chromatography, enhancing the software's adaptability across diverse datasets.Here we introduce for the first time an unprecedented scientific approach, integrating deep learning techniques with the novel CESP, which demonstrates exceptional thermal and analytical performance in both liquid chromatography modes, especially in the separation of complex hydrocarbon isomers. This ability enables the results obtained with CESP to extend across various types of stationary phases.Leveraging these insights, a comprehensive chromatographic dataset on a series of aromatic and polyaromatic molecules interacting with our CESP was used to train a Deep Learning Model (DLM). This model is embedded within a user-friendly software, Chrompredict 1.0, designed for predicting chromatographic parameters (MAE = 0.042, R² = 0.95) by selecting chemical descriptors directly from SMILES notation. It offers a deeper understanding of molecular structure and interactions through exploratory data analysis, identifying key factors affecting model accuracy and chromatographic behavior.Users can configure hyperparameters, choose from six machine learning models, and compare their performance with DLM. Chrompredict 1.0 excels in retention behavior prediction for compounds with known structures, and it accurately predicts chromatographic retention and thermal characteristics for different temperatures in HPLC and GC. The model has been successfully tested with METLIN database of 1,023 small molecules of diverse structures and polarities (R² > 0.75, error range ±7.8 s). Overall, the CESP, combined with Chrompredict 1.0, offers a robust tool for intelligent chromatographic analysis, encompassing chemo-informatics, statistical analysis, and graphical capabilities across a broad range of compounds and stationary phases.

A Differential Protein Study on Bronchoalveolar Lavage Fluid at Different Stages of Silicosis.

In this study, by comparing the difference in protein expression in bronchoalveolar lavage fluid between silicosis patients in different stages and healthy controls, the pathogenesis of pneumoconiosis was discussed, and a new idea for the prevention and treatment of pneumoconiosis was provided. The lung lavage fluid was pretreated by 10 K ultrafiltration tube, Agilent 1100 conventional liquid phase separation, strong cation exchange column (SCX) HPLC pre-separation, and C18 reverse phase chromatography desalting purification, and protein was labeled with isotope. GO, KEGG pathway, and PPI analysis of differential proteins were conducted by bioinformatics, and protein types and corresponding signal pathways were obtained. Thermo Q-Exactive mass spectrometry identified 943 proteins. T-test analysis was used to evaluate the different significance of the results, and the different protein of each group was obtained by screening with the Ratio≥1.2 or Ratio≤0.83 and P<0.05. We found that there are 16 kinds of protein throughout the process of silicosis. There are different expressions of protein in stages Ⅲ/control, stages Ⅱ/control, stage Ⅰ/control, stages Ⅲ/ stages Ⅱ, stages Ⅲ/ stage Ⅰ and stages Ⅱ/ stage Ⅰ groups. The results of ontology enrichment analysis of total differential protein genes show that KEGG pathway enrichment analysis of differential protein suggested that there were nine pathways related to silicosis. The main biological changes in the early stage of silicosis are glycolysis or gluconeogenesis, autoimmunity, carbon metabolism, phagocytosis, etc., and microfibril-associated glycoprotein 4 may be involved in the early stage of silicosis. The main biological changes in the late stage of silicosis are autoimmunity, intercellular adhesion, etc. Calcium hippocampus binding protein may participate in the biological changes in the late stage of silicosis. It provides a new idea to understand the pathogenesis of silicosis and also raises new questions for follow-up research.

Measurement of atmospheric amines and aminoamides by column adsorption/extraction and hydrophilic liquid chromatography-electrospray-tandem mass spectrometry.

Sampling and chromatography-mass spectrometry methods were investigated to measure atmospheric amines and aminoamides. Amines and their amide derivatives play significant roles in new particle formation (NPF) in the atmosphere, especially diamines and aminoamides have higher NPF potentials compared to monoamines. For amine sampling, silica gel tube collection and formic acid extraction gave good overall recoveries (>93 ± 8%) for mono-, di-, tri-, tetramines, and aminoamides. Two chromatography methods were subjected to analyze the extracted amines. One involved direct analysis using hydrophilic interaction liquid chromatography with carboxyl or diol group functioned separation column (carboxyl-HILIC or diol-HILIC), and the other utilized derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and subsequent reversed-phase chromatography (HPLC). Separated amines were detected by electrospray ionization and tandem mass spectrometry in both cases. DBD-F-HPLC method provided good sensitivity for mono- and all polyamines (limit of detection (LOD) < 4.6nM, relative standard deviation (RSD) for 100nM < 9.2%). However, aminoamides could not be detected by DBD-F-HPLC. Carboxyl-HILIC provided good sensitivities for mono- and diamines and aminoamides (LOD < 1.6nM, RSD < 4.8%). Forest air measurement was performed and data obtained by carboxyl-HILIC and DBD-F-HPLC showed good agreement for 1,3-diaminopropane, 1,4-diaminobutane (putrescine) and 1,5-diaminopentane (cadaverine) (R2 = 0.9215-0.9739, n = 7-14). Carboxyl-HILIC method was the best for the amine analysis, and combination with silica gel tube sampling provides atmospheric monitoring available. The developed method can be used not only to study atmospheric chemistry of diamines and aminoamides but also to analyze flavor/odor of foods, flowers and wastes.

Simultaneous determination of some coccidiostat antibiotics in food by liquid chromatography tandem mass spectrometry (LC-MS/MS)

Coccidiostats (anticoccidial antibiotics) are veterinary drugs widely used for the prevention and treatment of coccidiosis, especially in poultry farming. The illegal use of anticoccidial antibiotics, in the wrong dosage and duration, can lead to antibiotic residues in animal products and harm the health of consumers. In this study, a highly sensitive and accurate liquid chromatography-tandem mass spectrometry method was developed for the simultaneous analysis of anticoccidial antibiotics (diclazuril, nicarbazin, robenidin and salinomycin) in food. Samples were extracted using the QuEChERS method with acetonitrile (ACN) as the extraction solvent and cleaned with C18 adsorbent. The cleaned extracts were analyzed on an LC-MS/MS system with a C18 reversed-phase chromatography column (2.1 x 150 mm, 3.5 &amp;micro;m) and corresponding pre-column. The mobile phase used was 0.1% HCOOH in water and methanol (MeOH), the flow rate was 0.5 mL/min. The method had good specificity, the linear range was established in the range of 10-100 ng/mL, the correlation coefficient R2 &amp;gt; 0.998. The method recovery was in the range of 81-109% and the relative standard deviation (RSD%) was in the range of 1.77-10.13% for milk and meat samples. The limit of quantification and limit of detection of the method for four coccidiostat antibiotics were 3.0 &amp;micro;g/kg and 10 &amp;micro;g/kg, respectively. The method was applied to analyze the content of these four antibiotics in 31 food samples (meat and meat products, milk and dairy products) randomly purchased in Hanoi. The results did not detect four coccidiostat antibiotics in all 31 samples, initially showing that the residual contamination of coccidiostat antibiotics is basically safely controlled.

Fabrication of a Heptapeptide-Modified Poly(glycidyl Methac-Rylate) Nanosphere for Oriented Antibody Immobilization and Immunoassay

Oriented antibody immobilization has been widely employed in immunoassays and immunodiagnoses due to its efficacy in identifying target antigens. Herein, a heptapeptide ligand, HWRGWVC (HC7), was coupled to poly(glycidyl methacrylate) (PGMA) nanospheres (PGMA-HC7). The antibody immobilization behavior and antigen recognition performance were investigated and compared with those on PGMA nanospheres by nonspecific adsorption and covalent coupling via carbodiimide chemistry. The antibodies tested included bovine, rabbit, and human immunoglobulin G (IgG), while the antigens included horseradish peroxidase (HRP) and β-2-Microglobulin (β2-MG). The nanospheres were characterized using zeta potential and particle size analyzers, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and reversed-phase chromatography, proving each synthesis step was succeeded. Isothermal titration calorimetry assay demonstrated the strong affinity interaction between IgG and PGMA-HC7. Notably, PGMA-HC7 achieved rapid and extremely high IgG adsorption capacity (~3 mg/mg) within 5 min via a specific recognition via HC7 without nonspecific interactions. Moreover, the activities of immobilized anti-HRP and anti-β2-MG antibodies obtained via affinity binding were 1.5-fold and 2-fold higher than those of their covalent coupling counterparts. Further, the oriented-immobilized anti-β2-MG antibody on PGMA-HC7 exhibited excellent performance in antigen recognition with a linear detection range of 0–5.3 μg/mL, proving its great potential in immunoassay applications.

Revisiting Protein Reversed-Phase Chromatography for Bottom-Up Proteomics.

We revisited protein reversed-phase chromatography (RP), using state-of-the-art RP columns developed for biopharmaceuticals, such as monoclonal antibodies, in order to evaluate the suitability of this methodology as a prefractionation step for bottom-up proteomics. The protein RP prefractionation (Prot-RP) method was compared with two other widely used prefractionation methods, SDS-PAGE and high-pH peptide RP (Pept-RP) by using cell lysates as samples. The overlap between fractions of Prot-RP was comparable to that of SDS-PAGE, and the protein recovery was approximately 2-fold higher. On the other hand, the overlap between fractions of Prot-RP was slightly larger than that of Pept-RP, but Prot-RP was able to identify more protein termini and more isoform-specific peptides than Pept-RP. Our results indicate that the combination of highly efficient protein prefractionation with modern mass spectrometers is particularly effective for proteoform profiling from cellular samples.

Thermoregulation Effects of Phoneutria nigriventer Isolated Toxins in Rats.

Body temperature is primarily regulated by the hypothalamus, ensuring proper metabolic function. Envenomation by Phoneutria nigriventer can cause symptoms such as hypothermia, hyperthermia, sweating, and shivering, all related to thermoregulation. This study aims to analyze and identify components of the venom that affect thermoregulation and to evaluate possible mechanisms. Rats were used for thermoregulation analysis, venom fractionation by gel filtration and reverse-phase chromatography (C18), and sequencing by Edman degradation. The venom exhibited hypothermic effects in rats, while its fractions demonstrated both hypothermic (pool II) and hyperthermic (pool III) effects. Further separations of the pools with C18 identified specific peaks responsible for these effects. However, as the peaks were further purified, their effects became less significant. Tests on U87 human glioblastoma cells showed no toxicity. Sequencing of the most active peaks revealed masses similar to those of the Tachykinin and Ctenotoxin families, both known to act on the nervous system. The study concludes that molecules derived from venom can act synergistically or antagonistically. Additionally, toxins that affect thermoregulation are poorly studied and require further characterization. These toxins could potentially serve as sources for the development of new thermoregulatory drugs.

Evaluation of greenness, blueness, and whiteness with analytical quality by design assisted instrumental reversed-phase thin-layer chromatography‒densitometric approach to estimate monosodium glutamate in commercial food samples using green-ultrasound-assisted extraction technique

Evaluation of greenness, blueness, and whiteness with analytical quality by design assisted instrumental reversed-phase thin-layer chromatography‒densitometric approach to estimate monosodium glutamate in commercial food samples using green-ultrasound-assisted extraction technique

Bioactive secondary metabolites from an endophytic fungus Aspergillus sp. CCH-1E from Catharanthus roseus

This study focused on the bioactive secondary metabolites of an endophytic fungus Aspergillus sp. CCH-1E from Catharanthus roseus. The secondary metabolites from Aspergillus sp. CCH-1E were isolated by using various chromatographic methods [such as normal-phase and reversed-phase chromatography and high-performance liquid chromatography(HPLC)], and their structures were identified by various spectroscopic methods [e.g., ultraviolet(UV) spectroscopy, infrared(IR) spectroscopy, nuclear magnetic resonance(NMR) spectroscopy, and high-resolution electrospray ionization mass spectrometry(HR-ESI-MS)]. Twelve compounds were yielded and identified from Aspergillus sp. CCH-1E, which are chermesinone H(1), chermesinone I(2), chermesinone B(3), 8,11-didehydrochermesinone B(4), chermesinone C(5), chermesinone A(6), chevalone B(7), barbacenic acid(8), 3,6,8-trihydroxy-3,5,7-trimethyl-3,4-dihydroisocoumarin(9), 5-hydroxy-2-methoxy-7-methyl-1,4-naphthoquinone(10), 1-hydroxy-6,8-dimethoxy-3-methylanthracene-9,10-dione(11), and 7-drimen-9α,11,12-triol(12). Among them, compounds 1 and 2 are new compounds. The growth inhibition effects of all compounds were evaluated against non-small cell lung cancer cell lines A549 and NCI-H1650, as well as human cervical cancer cell line HeLa by using methylthiazolyldiphenyl-tetrazolium bromide(MTT). Compound 7 significantly inhibited the growth of three tumor cells with the IC_(50) values of 1.22-2.43 μmol·L~(-1), respectively. Compounds 1-6 showed moderate cell growth inhibition with the IC_(50) values of 16.24-35.28 μmol·L~(-1).

Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography

Nucleic acid duplexes are typically analyzed in non-denaturing conditions. Melting temperature (Tm) is the property used to measure duplex stability; however, it is not known how the chromatographic conditions and mobile phase composition affect the duplex stability. We employed differential scanning calorimetry (DSC) method to measure the melting temperature of chemically modified silencing RNA duplex (21 base pairs, 0.15 mM duplex concentration) in mobile phases commonly used in reversed-phase, ion-pair reversed-phase, size exclusion and hydrophilic interaction chromatography. We investigated mobile phases consisting of ammonium acetate, alkylammonium ion-pairing reagents, alkali-ion chlorides, magnesium chloride, and additives including methanol, ethanol, acetonitrile and hexafluoroisopropanol. Increasing buffer concentration enhanced the duplex stability (Tm was 67.1 – 78.2 °C for 10–100 mM [Na+] concentration). The melting temperature decreases with the increase in cation size (70.2 °C in 10 mM [Li+], 68.1 °C in 10 mM [NH4+], 65.6 °C in 10 mM [Cs+], and 56.6 °C in 10 mM [triethylammonium+] solutions). Inclusion of 20 % of organic solvent in buffer reduced the melting temperature by 1–3 °C, and denaturation power increases in the order MeOH<EtOH<MeCN. Next, we investigated the RNA duplex melting using selected chromatographic techniques and within the 10–90 °C column temperature range. Melting temperature obtained with size exclusion chromatography in 25 mM sodium phosphate buffer was ∼ 70 °C, about 5 °C lower compared to DSC values. The apparent melting temperature for a reversed-phase chromatography experiment was < 10 °C for 10 mM ammonium acetate mobile phase with acetonitrile eluent. Ion-pair reversed-phase with 10 mM triethylamine, 100 mM hexafluoroisopropanol and MeCN as eluent yielded Tm 49.3–54.9 °C. Hydrophilic interaction chromatography experiments with 10 mM ammonium acetate and ∼ 50 % acetonitrile mobile phase showed high duplex stability (Tm ∼ 80 °C). The observed chromatographic Tm values suggest that the formation of an organic/aqueous solvent layer on the chromatographic sorbent surface affects the duplex stability more significantly than the composition of the mobile phase alone.

Charge Variants Characterization of Co-Formulated Antibodies by Three-Dimensional Liquid Chromatography-Mass Spectrometry.

Co-formulated antibodies can bring clinical benefits to patients by combining two or more antibodies in a single dosage form. However, the quality analysis of co-formulated antibodies raises additional challenges, compared to individual antibodies, due to the need for accurate analysis of multiple antibodies in one solution. It is extremely difficult to effectively separate the charge variants of the two co-formulated antibodies using one ion exchange chromatography (IEC) method because of their similar characteristics. In this study, a novel method was developed for the charge variants characterization of co-formulated antibodies using three-dimensional liquid chromatography-mass spectrometry (3D-LC-MS). Hydrophobic interaction chromatography (HIC) was used as the first dimension to separate and collect the two co-formulated antibodies. The two collections were then injected into the second-dimension IEC separately for charge variants separation and analysis. Subsequently, the separated charge variants underwent on-line desalting in the third-dimension reverse-phase chromatography (RPC) and subsequent mass spectroscopy analysis. The novel method could simultaneously provide a charge variants ratio and post-translational modification (PTM) data for the two co-formulated antibodies. Therefore, it could be used for release testing and stability studies of co-formulated antibodies, making up for the shortcomings of the existing approaches. It was the first time that charge variants of co-formulated antibodies were characterized by the 3D-LC-MS method, to the best of our knowledge.

A Review Article on Analytical Method on Sitageliptin and Metformin

An overview about simultaneous estimation of the combination drugs, metformin and sitagliptin using RP-HPLC method. Reverse phase chromatography is the most commonly used separation technique in HPLC, common reasons being its simplicity, versatility and its ability to handle compounds of a diverse polarity and molecular mass.Good knowledge about different types of mobile phases and their combination are required for highly precise and accurate method development. The retention time and linearity of metformin and sitagliptin are found to be determined underdifferent chromatographic conditions such as column, mobile phase, elution mode, flow rate and wavelength detected using UV detector. In this article, we will be reviewing different developed methods for estimating the given combination drugs by RP-HPLC.