Mass Spectrometry Detection Research Articles

Assessment of bioequivalence of veterinary drugs “Hollicalm” and “Serenia” in the form of tablets in the body of dogs

This paper describes the results of a study of the bioequivalence of the generic drug “Hollicalm” in comparison with the reference drug “Sereniya”. The experiment was carried out on 6 dogs. For this experiment, a sequential bioequivalence study design was used. The period between the administration of reference and generic drugs to animals was 72 hours. The drugs were administered to dogs once, individually, orally. The study drugs contained 60 mg of maropitant per tablet. The dose of the active substance that was administered to each animal was 8 mg of maropitant per 1 kg of animal weight. Blood samples were taken from dogs before drug administration, then at 14 time points within 48 hours to subsequently obtain blood serum. The content of maropitant was determined in the samples of blood serum using high-performance liquid chromatography coupled with mass-spectrometry detection. The obtained concentrations of the active substance served as the basis for calculating its pharmacokinetic parameters in dogs. The statistical analysis showed that two-sided confidence intervals for the Cmax and AUC0-t ratios were in the range of 80–125%. The results of the study allowed us to draw a conclusion about the bioequivalence of the drugs “Hollicalm” and “Sereniya”.

Aruncus dioicus var. kamtschaticu: A Newly Identified Source of Lactucaxanthin (ε,ε-Carotene-3,3′-diol)

Green leafy vegetables are rich in lipophilic metabolites such as carotenoids, phytosterols, tocopherols, and fatty acids, known for their significant health benefits. Goat’s beard (Aruncus dioicus var. kamtschaticus), a wild leafy vegetable native to Ulleungdo Island, South Korea, is reported to possess various biological activities and bioactive compounds. However, the content and profiles of lipophilic metabolites, especially carotenoids, have not been reported. This study investigated the composition of lipophilic metabolites in the foliage of goat’s beard using liquid chromatography–diode-array detection–mass spectrometry, gas chromatography–mass spectrometry, and gas chromatography–flame ionization detection. Five carotenoids (violaxanthin, neoxanthin, lactucaxanthin, lutein, β-carotene) and α-tocopherol were identified and quantified using liquid chromatography–diode-array detection–mass spectrometry. Lactucaxanthin, previously discovered in lettuce, was identified for the first time in goat’s beard foliage, with a higher concentration (45.42 ± 0.80 µg/g FW) compared to red lettuce (19.05 ± 0.67 µg/g FW). Furthermore, total carotenoids and α-tocopherol contents were higher in goat’s beard than in red lettuce. Gas chromatography–mass spectrometry analysis showed the presence of three phytosterols, namely, campesterol (3.53 ± 0.20 µg/g FW), stigmasterol (65.30 ± 4.87 µg/g FW), and β-sitosterol (89.54 ± 2.46 µg/g FW). Gas chromatography–flame ionization detection analysis revealed the presence of five essential fatty acids, with α-linolenic acid (57.03 ± 0.47%) being the most abundant, contributing to a favorable polyunsaturated FA/saturated FA ratio. These findings underscore the nutritional potential of a goat’s beard, suggesting its promising use in dietary supplements and the commercial extraction of valuable lipophilic antioxidants, particularly lactucaxanthin, for nutraceuticals and functional foods.

Complementary Nanoparticle Characterization by Resistive-Pulse Sensing, Electron Microscopy, and Charge Detection Mass Spectrometry.

Nanotechnology has provided novel modalities for the delivery of therapeutic and diagnostic agents. In particular, nanoparticles (NPs) can be engineered at a low cost for drug loading and delivery. For example, silica NPs have proven useful as a controlled release platform for anti-inflammatory drugs. Despite the wide-ranging potential applications for NPs, robust characterization across all size ranges remains elusive. Electron microscopy (EM) is the conventional tool for measuring NP diameters. However, imitations in throughput and the inability to provide comprehensive information on physical properties, such as mass and density, without underlying assumptions, hinder a complete analysis. In addition, assessing sample heterogeneity, aggregation, or coalescence in solution by traditional EM analysis is not possible. Resistive-pulse sensing (RPS) provides a high throughput, solution-phase method for characterizing particle heterogeneity based on volume. Complementing these methods, charge detection mass spectrometry (CD-MS), a single particle technique, provides accurate mass information for heterogeneous samples including NPs. By combining EM, RPS and CD-MS, accurate volume, mass, and densities were obtained for silica NPs of various sizes. The results show that the density for 20 nm silica NPs is close to the density of fused silica (2.2 g/cm3). Larger silica NPs were found to have densities that were either smaller or larger, while also falling outside the range of densities usually found for silica colloids and NPs (1.9-2.3 g/cm3). Lower densities are attributed to pores (i.e., porous particles). For one sample, the mass distribution showed two components attributed to two populations of particles in the sample with different densities. The synergistic combination of EM, RPS, and CD-MS measurements outlined here for NP samples, allows much more extensive information to be obtained than from any of the techniques alone.

An Integrated Inertial-Magnetophoresis Microfluidic Chip Online-Coupled with ICP-MS for Rapid Separation and Precise Detection of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are recognized as promising targets for liquid biopsy, which play an important role in early diagnosis and efficacy monitoring of cancer. However, due to the extreme scarcity of CTCs and partial size overlap between CTCs and white blood cells (WBCs), the separation and detection of CTCs from blood remain a big challenge. To address this issue, we fabricated a microfluidic chip by integrating a passive contraction-expansion array (CEA) inertial sorting zone and an active magnetophoresis zone with the trapezoidal groove and online coupled it with inductively coupled plasma mass spectrometry (ICP-MS) for rapid separation and precise detection of MCF-7 cells (as a model CTC) in blood samples. In the integrated microfluidic chip, most of the small-sized WBCs can be rapidly removed in the circular CEA inertial sorter, while the rest of the magnetically labeled WBCs can be further captured in the trapezoidal groove under the magnetic field. As a result, the rapid separation of MCF-7 cells from blood samples was achieved with an average recovery of 91.6% at a sample flow rate of 200 μL min-1. The developed online integrated inertial-magnetophoresis microfluidic chip-ICP-MS system has been applied for the detection of CTCs in real clinical blood samples with a fast analysis speed (5 min per 1 mL blood). CTCs were detected in all 24 blood samples from patients with different types of cancer, exhibiting excellent application potential in clinical diagnosis.

Parallel coupling of gas chromatography to mass spectrometry and solid deposition Fourier transform infrared spectroscopy: an innovative approach to address challenging identifications.

The request for novel hyphenated instruments and techniques, capable of affording exhaustive information and results, is a focus continuously watched out. In this context, the present work aimed at the development of an integrated system combining gas chromatographic (GC) separation with mass spectrometry (MS) and (solid deposition) Fourier transform infrared spectroscopy (FTIR) detection. An external transfer line was designed inthelab for the parallel coupling of the two detectors, in such a way toobtain complementary analytical information consisting of anMS spectrum, anIR spectrum and linear retention indices (LRI), within a single analysis. The instrument performance was demonstrated for the analysis of a commercial mixture consisting of 139 hydrocarbons, comprising linear, branched, unsaturated and aromatic compounds. A 100-m poly(dimethylsiloxane) column was employed for the separation, and the outlet flow was split 95:5 between the IR and MS detectors using two uncoated capillaries. The IR spectra were acquired from solid deposits on a zinc selenide disc (-90 °C), over a spot (detector area) of about 0.1 mm2, in the range of 4000-700 cm-1 and at a resolution of 4 cm-1. Final identification of the separated compounds by alibrary search was achieved by excluding incorrect results, sequentially using a three-filter approach (85% similarity against reference MS and IR library spectra and ±10 LRI unit tolerance). Based on these preliminary results, the GC-MS/sd-FTIR system is a promising tool for the characterization of complex matrix constituents, for which identification is cumbersome, by using only one detection technique.

Charge Detection Mass Spectrometry Enables Molecular Characterization of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) are increasingly used in preclinical investigations as delivery vectors. Tools that can characterize assembly and assess quality will accelerate their development and clinical translation. Standard techniques used to characterize NANPs, like gel electrophoresis, lack the resolution for precise characterization. Here, we introduce the use of charge detection mass spectrometry (CD-MS) to characterize these materials. Using this technique, we determined the mass of NANPs varying in size, shape, and molecular mass, NANPs varying in production quality due to formulations lacking component oligonucleotides, and NANPs functionalized with protein and nucleic acid-based secondary molecules. Based on these demonstrations, CD-MS is a promising tool to precisely characterize NANPs, enabling more precise assessments of the manufacturing and processing of these materials.

Pharmacokinetics of two triterpenoid saponins and three flavonoids in Astragalus membranaceus leaves by UHPLC-MS/MS

Astragalus membranaceus (A. membranaceus) leaves can be used both as a medicine and food material. Their main chemical components are flavonoids and triterpenoid saponins. The pharmacokinetics of A. membranaceus leaves are rarely reported in the literature. This study aimed to investigate the pharmacokinetics of five major bioactive components of A. membranaceus leaves [rhamnocitrin 3-glucoside (RCG), tiliroside (TIL), rhamnocitrin 3-neohesperidoside (RNH), huangqiyenin R (HuR), and huangqiyenin I (HuI)]. Simultaneously using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. The extract of A. membranaceus leaves was administered orally to rats, and the rat plasma was subjected to a fast, sensitive, and specific UHPLC-MS/MS method. Butylparaben served as the internal standard. The plasma samples were pretreated using isopropanol/ethyl acetate (1:1, v/v) liquid–liquid extraction. Chromatographic separations were performed at a flow rate of 0.3 mL/min on a Waters ACQUITY HSS T3 Column (2.1 mm × 100 mm, 1.8 μm) using mobile phases of 0.1 % formic acid/water and 0.1 % formic acid/acetonitrile. Mass spectrometry detection was performed using an electrospray ionization ion source in the negative-ion mode and the multiple reaction monitoring mode. All analytes had an intraday and interday relative standard deviation of less than 14.10 %. The range of accuracy was –11.94–6.920 % and –15.22–5.800 %. The lower limits of quantification for RCG, TIL, RNH, HuR, HuI was 10.24, 10.27, 10.12, 5.137, and 5.841 ng/mL, respectively. The criteria were met by stability, matrix effects, and extraction recovery. The pharmacokinetic parameters of A. membranaceus leaf extract were ultimately obtained using this analytical method. The study provides a theoretical basis for future pharmacological research, clinical application, and development of healthy food from A. membranaceus leaves.

“Welcome to the jungle”: TLC-SERS to wade through real complex mixtures of synthetic dyes

The finding of numerous synthetic strategies between the 19th and 20th century fostered the production of an extensive spectrum of organic dyes for artistic and industrial applications. Shortly after, challenges emerged related to the rapid deterioration of these products. Nowadays, the characterization of coloured pieces of art produced in the first half of the XXth century is further complicated by the absence of reference materials with an unambiguous composition, which are frequently a mixture between the starting dyes and their degradations products. Here we demonstrate as Surface Enhanced Raman Spectroscopy coupled with the simplest separation technique, i.e. Thin Layer Chromatography, is able beyond to recognize several synthetic dyes also to identify their isomers and degradation products. The occasion was the study of a catalogue of dyed textile swatches, produced in 1951 by the well-known Italian industry ACNA (Azienda Coloranti Nazionali e Affini). An apparently easy task turned out to be, indeed, not at all obvious as we found out that the actual composition of the reference samples seldomly corresponded to the declared one. TLC-SERS investigation of historical textile swatches was complemented by the integration of liquid chromatography coupled with a diode array detector and high-resolution mass spectrometry (HPLC-DAD-HRMS). This approach allowed us to validate the findings of TLC-SERS and to distinctively characterise synthetic by-products and degradation markers resulting from the deterioration of the dyes.

Realization of Higher Resolution Charge Detection Mass Spectrometry.

Charge detection mass spectrometry (CD-MS) allows mass distributions to be measured for heterogeneous samples that cannot be analyzed by conventional MS. With CD-MS, the m/z and charge are measured for individual ions using a detection cylinder embedded in an electrostatic linear ion trap (ELIT). Imprecision in both the m/z and charge measurements contribute to the mass resolution. However, if the charge can be measured with a precision of <0.2 e the charge state can be assigned with a low error rate and the mass resolving power only depends on the m/z resolution. Prior to this work, the best resolving power demonstrated experimentally for CD-MS was 700. Here we demonstrate a resolving power of >14,600, 20-times higher than the previous best. Trajectory simulations were used to optimize the geometry and electrostatic potentials of the ELIT. We found conditions where the energy dependence of the oscillation frequency becomes parabolic, and then operated with a nominal ion energy at the minimum of the parabola. The 14,600 resolving power was obtained with a beam collimator before the ELIT. With the collimator removed, the resolving power of the optimized ELIT is 7300, which is still an order of magnitude higher than the previous best. The resolving power was demonstrated by resolving the isotope distributions for peptides and proteins. High resolution CD-MS measurements were then used to resolve the glycans on a monoclonal antibody and applied to the analysis of hepatitis B virus capsids. The results indicate that procedures for adduct removal need to be improved for the full benefit of the higher resolving power to be realized for higher mass species. However, these results represent a key step toward using CD-MS to analyze very complex protein mixtures where charge states are not well resolved in the m/z spectrum because of congestion from numerous overlapping peaks.

Automated Immunoprecipitation, Sample Preparation, and Individual Ion Mass Spectrometry Platform for Proteoforms.

Charge detection mass spectrometry (CDMS) is a well-established technique that provides direct mass spectral outputs regardless of analyte heterogeneity or molecular weight. Over the past few years, it has been demonstrated that CDMS can be multiplexed on Orbitrap analyzers utilizing an integrated approach termed individual ion mass spectrometry (I2MS). To further increase adaptability, robustness, and throughput of this technique, here, we present a method that utilizes numerous integrated equipment components including a Kingfisher system, SampleStream platform, and Q Exactive mass spectrometer to provide a fully automated workflow for immunoprecipitation, sample preparation, injection, and subsequent I2MS acquisition. This automated workflow has been applied to a cohort of 58 test subjects to determine individualized patient antibody responses to SARS-CoV-2 antigens. Results from a range of serum donors include 37 subject I2MS spectra that contained a positive COVID-19 antibody response and 21 I2MS spectra that contained a negative COVID-19 antibody response. This high-throughput automated I2MS workflow can currently process over 100 samples per week and is general for making immunoprecipitation-MS workflows achieve proteoform resolution.

Charge Detection Mass Spectrometry Reveals Favored Structures in the Assembly of Virus-Like Particles: Polymorphism in Norovirus GI.1.

The main capsid protein (CP) of norovirus, the leading cause of gastroenteritis, is expected to self-assemble into virus-like particles with the same structure as the wild-type virus, a capsid with 180 CPs in a T = 3 icosahedron. Using charge detection mass spectrometry (CD-MS), we find that the norovirus GI.1 variant is structurally promiscuous, forming a wide variety of well-defined structures, some that are icosahedral capsids and others that are not. The structures that are present evolve with time and vary with solution conditions. The presence of icosahedral T = 3 and T = 4 capsids (240 CPs) under some conditions was confirmed by cryo-electron microscopy (cryo-EM). The cryo-EM studies also confirmed the presence of an unexpected prolate geometry based on an elongated T = 4 capsid with 300 CPs. In addition, CD-MS measurements indicate the presence of well-defined peaks with masses corresponding to 420, 480, 600, and 700 CPs. The peak corresponding to 420 CPs is probably due to an icosahedral T = 7 capsid, but this could not be confirmed by cryo-EM. It is possible that the T = 7 particles are too fragile to survive vitrification. There are no mass peaks associated with the T = 9 and T = 12 icosahedra with 540 and 720 CPs. The larger structures with 480, 600, and 700 CPs are not icosahedral; however, their measured charges suggest that they are hollow shells. The use of CD-MS to monitor virus-like particles assembly may have important applications in vaccine development and quality control.

Compositional Analysis of Cutin in Maize Leaves.

The cuticle is a lipid barrier that covers the air-exposed surfaces of plants. It consists of waxes and cutin, a cell wall-attached lipid polyester of oxygenated fatty acids and glycerol. Unlike waxes, cutin is insoluble in organic solvents, and its composition is typically studied by chemical depolymerization followed by monomer analysis by gas chromatography (GC). Here, we describe a method for the chemical depolymerization of cutin in maize leaves and subsequent compositional analysis of the constituent lipid monomers. The method has been adapted from protocols for cutin analysis developed for Arabidopsis, by both optimizing the amount of leaf tissue used and including a data analysis process specific to the monomers present in maize cutin. The approach uses base-catalyzed transmethylation, which produces fatty acid methyl esters, and silylation, which gives trimethylsilyl ether derivatives of hydroxyl groups for gas chromatographic analysis. For monomer identification, a few representative samples are first analyzed by GC-mass spectrometry (GC-MS). This is then followed by analysis of all replicates by gas chromatography coupled to a flame ionization detector (GC-FID) for monomer quantification, because the flame ionization detector provides a linear response over a wide mass range, is relatively simple to operate, and is more cost-effective to maintain compared to mass spectrometry detectors. Although the protocol bypasses time-consuming cuticle isolation steps by using whole-leaf samples, this means that a fraction of the compounds in the chromatographic profiles do not derive from cutin. Accordingly, we discuss some considerations for the interpretation of the resulting depolymerization products. Our protocol offers specific guidance on preparing maize leaf samples, ensuring reproducible results, and enabling the detection of subtle variations in cutin monomer composition among plant genotypes or developmental stages.

Water as a green solvent for sustainable sample preparation: single drop microextraction of N-nitrosamines from losartan tablets.

Water, renowned for its sustainability and minimal toxicity, is an ideal candidate for environmentally friendly solvent-based microextraction. However, its potential as an extractant solvent in miniaturized sample preparation remains largely unexplored. This paper pioneers using water as the extraction solvent in headspace single-drop microextraction (HS-SDME) for N-nitrosamines from losartan tablets. Autonomous HS-SDME is executed by an Arduino-controlled, lab-made Cartesian robot, using water for the online preconcentration of enriched extracts through direct injection into a column-switching system. Critical experimental parameters influencing HS-SDME performance are systematically explored through univariate and multivariate experiments. While most previously reported methods for determining N-nitrosamines in pharmaceutical formulations rely on highly selective mass spectrometry detection techniques to handle the strong matrix effects typical of pharmaceutical samples, the water-based HS-SDME method efficiently eliminates the interfering effects of a large amount of the pharmaceutical active ingredient and tablet excipients, allowing straightforward analysis using high-performance liquid chromatography with ultraviolet detection (HPLC-UV-Vis). Under optimized conditions, the developed method exhibits linear responses from 100 to 2400ngg-1, demonstrating appropriate detectability, precision, and accuracy for the proposed application. Additionally, the environmental sustainability of the method is assessed using the AGREEprep methodology, positioning it as an outstanding green alternative for determining hazardous contaminants in pharmaceutical products.

(Invited) Analytical Validation of Aptamer-Based Blood Drug Measurements Against Clinical Benchmark Methods

The practice of monitoring therapeutic drug concentrations in patient biofluids can significantly improve clinical outcomes while simultaneously minimizing adverse side effects. Although undeniably important to achieve effectiveness, therapeutic drug monitoring remains inconvenient in practice, due primarily to the lengthy process of sample collection, transport to a centralized facility, and analysis using costly instrumentation. Adding to this workflow is the possibility of backlogs at centralized clinical laboratories, which is not uncommon and may result in additional delays between biofluid sampling and concentration measurement, which can negatively affect clinical outcomes. In this talk I explore the possibility of using point-of-care, aptamer-based sensors to minimize the time delay between biofluid sampling and drug measurement. I will discuss analytical validations of aptamer-based blood measurements for two drugs, the antibiotic vancomycin and the antiretroviral drug emtricitabine. We conducted clinical agreement studies comparing the measurement outcomes of electrochemical and optical aptamer-based sensors to the benchmark (1) automated competitive immunoassays for vancomycin monitoring in serum, and (2) liquid chromatography coupled to mass spectrometry detection (LC-MS) for plasma emtricitabine. Our results demonstrate that aptamer-based sensors are selective for the free form of these drugs, and can achieve up to 95% positive correlation and 100% negative correlation with the benchmark assays. Given these results, aptamer sensors offer a promising path for point-of-care drug monitoring that is instantaneous and overcomes current barriers for broad implementation of drug monitoring in clinical settings.

Use of p-methoxy cinnamaldehyde in label-assisted laser desorption/ionization mass spectrometry (LALDI-MS) for detection of different classes of primary amines, amino acids and neurotransmitters

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), useful for the detection of macromolecules, is generally not suitable for recording the mass spectrum of small molecules, especially within the range of m/z 500, because of the appearance of matrix cluster peaks in the small molecular weight range. To circumvent this problem, a matrix-free variant of MALDI-MS, namely Label-assisted laser desorption/ionization mass spectrometry (LALDI-MS) has been developed in recent years where analytes get covalently attached to a laser absorbing label which induces the desorption/ionization process thus facilitating the recording the mass spectra and avoiding the problem of formation of cluster peaks. In general, the probes used for such detections are all pyrene-based scaffolds which despite being a good laser-active chromophore have both reactivity and solubility problems. Inspired by the right chromophoric nature, we have undertaken a systematic study to design and optimize cinnamaldehyde derivatives and have found that p-methoxy cinnamaldehyde (PMC) as a simpler, cost-effective, less hydrophobic laser-active label, which can detect a series of primary amines, including the neurotransmitters and amino acids. The label PMC can also discriminate between aliphatic and aromatic primary amines.

Metabolomic profiling of pheochromocytomas in dogs: Catecholamine phenotype and tricarboxylic acid cycle metabolites.

In humans with pheochromocytomas (PCCs), targeted metabolomics is used to determine the catecholamine phenotype or to uncover underlying pathogenic variants in tricarboxylic acid (TCA) cycle genes such as succinate dehydrogenase subunits (SDHx). To analyze catecholamine contents and TCA cycle metabolites of PCCs and normal adrenals (NAs). Ten healthy dogs, 21 dogs with PCC. Prospective observational study. Dogs diagnosed with PCC based on histopathological and immunohistochemical confirmation were included. Tissue catecholamine contents and TCA metabolites in PCCs and NAs were measured by liquid chromatography with mass spectrometry or electrochemical detection. Compared to NAs, PCCs had significantly higher tissue proportion of norepinephrine (88% [median: range, 38%-98%] vs 14% [11%-26%]; P < .001), and significantly lower tissue proportion of epinephrine (12% [1%-62%] vs 86% [74%-89%]; P < .001). Pheochromocytomas exhibited significantly lower fumarate (0.4-fold; P < .001), and malate (0.5-fold; P = .008) contents than NAs. Citrate was significantly higher in PCCs than in NAs (1.6-fold; P = .015). One dog in the PCC group had an aberrant succinate : fumarate ratio that was 25-fold higher than in the other PCCs, suggesting an SDHx mutation. This study reveals a distinct catecholamine content and TCA cycle metabolite profile in PCCs. Metabolite profiling might be used to uncover underlying pathogenic variants in TCA cycle genes in dogs.

Benzotriazoles and bisphenols in wastewater from the food processing industry and the quantitative changes during mechanical/biochemical treatment processes

Benzotriazoles (BTRs) and bisphenols (BPs), categorized as contaminants of emerging concern (CECs), pose significant risks to human health and ecosystems due to their endocrine-disrupting properties and environmental persistence. This study investigates the occurrence and behavior of nine BTRs and ten BPs in wastewater generated in a large-scale meat processing plant, evaluating the effectiveness of a modern mechanical-biological industrial on-site treatment plant in removing these contaminants, and based on the concentration levels from eleven sampling points at different stages of the treatment process. The method used to determine these micropollutants' concentration was ultrasound-assisted emulsification-microextraction for analytes isolation and gas chromatography–mass spectrometry for detection (USAEME-GC/MS). The results indicate that the rigorous quality control processes in the meat processing facility effectively limit the presence of these micropollutants, especially concerning BPs, which are absent or below detection limits in raw wastewater. While the concentrations of some of these micropollutants increased at different points in the treatment process, these values were relatively low, typically below one microgram per liter. Among the compounds analyzed, the only one present after completing the treatment was 5Cl-BTR (maximum concentration: 3007 ng/L), and these contamination levels are around seven times lower than the reference value associated with non-cancer health risk for drinking water. This study contributes to understanding these CECs in industrial wastewater and highlights the importance of effective treatment systems for environmental protection.

Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

Investigation of the phytochemical profiling and antioxidant, anti-diabetic, anti-inflammatory, and MDA-MB-231 cell line antiproliferative potentials of extracts from Ephedra alata Decne

Ephedra is one of the many medicinal herbs that have been used as folk/traditional medicine in Jordan and other countries to cure various illnesses. Plants of this genus are well known for their antioxidant and antibacterial properties. In this study, three different solvents were used to obtain Ephedra extracts. When evaluated, the Ephedra alata Decne ethanolic extract reportedly had the greatest levels of total phenolic compounds (TPC) and total flavonoid compounds (TFC). The aqueous extracts displayed the highest antioxidant activity in the DPPH and ABTS assays, demonstrating their considerable capacity to neutralize free radicals. However, when evaluated using the FRAP method, the acetone extracts showed the strongest antioxidant activity, indicating their high reducing power. LC–MS/MS, a potent method of analysis that combines the liquid chromatographic separation properties with mass spectrometry detection and identification capabilities, was used in this study to detect and measure phytochemical content of a total of 24 phenolic compounds and 16 terpene compounds present in the extracts of Ephedra alata Decne. Various concentrations of these chemicals were found in these extracts. The extracts’ inhibitory effects on albumin denaturation and alpha-amylase activity were also assessed; the findings demonstrated the potentials of these extracts as anti-inflammatory and anti-diabetic medicines, with the acetone extract having the lowest IC50 values in the concomitant tests (306.45 µg/ml and 851.23 µg/ml, respectively). Furthermore, the lowest IC50 value (of 364.59 ± 0.45 µg/ml) for the 80% ethanol extract demonstrated that it has the strongest antiproliferative impact regarding the MDA-MB-231 breast cancer cell line. This finding indicates that this particular extract can be potentially used to treat cancer.

Selective Estrogen Receptor Modulators' (SERMs) Influence on TET3 Expression in Breast Cancer Cell Lines with Distinct Biological Subtypes.

Tamoxifen, a selective estrogen receptor modulator (SERM), exhibits dual agonist or antagonist effects contingent upon its binding to either G-protein-coupled estrogen receptor (GPER) or estrogen nuclear receptor (ESR). Estrogen signaling plays a pivotal role in initiating epigenetic alterations and regulating estrogen-responsive genes in breast cancer. Employing three distinct breast cancer cell lines-MCF-7 (ESR+; GPER+), MDA-MB-231 (ESR-; GPER-), and SkBr3 (ESR-; GPER+)-this study subjected them to treatment with two tamoxifen derivatives: 4-hydroxytamoxifen (4-HT) and endoxifen (Endox). Through 2D high-performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS), varying levels of 5-methylcytosine (5-mC) were found, with MCF-7 displaying the highest levels. Furthermore, TET3 mRNA expression levels varied among the cell lines, with MCF-7 exhibiting the lowest expression. Notably, treatment with 4-HT induced significant changes in TET3 expression across all cell lines, with the most pronounced increase seen in MCF-7 and the least in MDA-MB-231. These findings underscore the influence of tamoxifen derivatives on DNA methylation patterns, particularly through modulating TET3 expression, which appears to be contingent on the presence of estrogen receptors. This study highlights the potential of targeting epigenetic modifications for personalized anti-cancer therapy, offering a novel avenue to improve treatment outcomes.