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.

Simultaneous determination of semi-essential nutrients taurine, l-carnitine and choline in infant formulas and adult/pediatric nutritional formulas by hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry.

BACKGROUND: The nutritional intake of formula-fed newborns is often limited to a single source, so it must be supplemented with essential nutrients for the growth and proper development of infants. Taurine, l-carnitine, and choline are considered conditionally essential nutrients especially in newborns and infants. RESULTS: In this work, a simple routine hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was developed and validated for the simultaneous determination of these semi-essential nutrients in infant and adult/pediatric milk formulas. The extraction recoveries were between 90% and 114%. Precision of the method offered relative standard deviation below 5% and 7% for intra-day and inter-day precision, respectively. The proposed method was successfully applied to quantification of taurine, l-carnitine, and choline in milk formula. The contents found were in good agreement with those provided on the product label for almost all samples. CONCLUSION: In view of these results, it can be concluded that the developed method can be a useful approach for the simultaneous determination of taurine, l-carnitine and choline in powdered milk samples, so it can be useful in the routine quality control of this kind of samples. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Analysis of the polymeric mixture in hypoxen, using high-performance liquid chromatography coupled with high-resolution mass spectrometry.

RATIONALE: Hypoxen is a medication known for providing individuals with a "second wind," by lowering the threshold for muscle fatigue and enhancing the body's efficiency under challenging conditions. Athletes who have used this medication report enhanced training outcomes and increased physical endurance. It is crucial to emphasize that hypoxen is not categorized as a prohibited substance as yet and is thus assumed safe for use in competitive sports. However, the polymeric nature of hypoxen presents challenges in detection and identification. METHODS: To prevent the potential misuse of this substance as a doping agent, doping control laboratories must therefore develop a detection method. This study aimed to address this gap by developing a comprehensive detection method for the polymeric mixtures within hypoxen, employing liquid chromatography-electrospray ionization-mass spectrometry. RESULTS: Among the different columns tested, the Accucore and Syncronis HILIC columns demonstrated exceptional performance, yielding excellent separation with high-quality results. The study found that the hypoxen consisted of between 1 and 6 repetitions of 2,4-dihydroxyphenylene units. It was crucial to highlight that each unit contained multiple isomers due to the incorporation of the sulfonic acid group at both the -SH and -OH positions. CONCLUSIONS: The results of this study will significantly contribute to the precise identification of hypoxen use, thereby aiding in the scrutiny of its abuse in competitive sports.

New Meroterpenes from South China Sea Soft Coral Litophyton brassicum.

A chemical investigation of the extracts from the soft coral Litophyton brassicum led to the isolation and identification of four new meroterpenes, brassihydroxybenzoquinone A and B (1 and 2) and brassinaphthoquinone A and B (3 and 4), along with two known related meroterpenes (5 and 6). Their structures were elucidated using high-resolution electrospray ionization mass spectrometry (HRESIMS), nuclear magnetic resonance (NMR) spectroscopy, and a comparison with the literature data. All compounds were evaluated for antibacterial activity against six pathogenic bacterial strains and for cytotoxic activity against three cancer cell lines. In the cytotoxic assay, all compounds were inactive at 10 µM against the A549, HeLa, and MDA-MB-231 cell lines. In the antibacterial assay, compounds 1 and 2 exhibited moderate inhibitory activity with minimum inhibitory concentrations (MIC) ranging from 8 to 64 µg/mL.

Aligning Post-Column ESI-MS, MALDI-MS, and Coagulation Bioassay Data of Naja spp., Ophiophagus hannah, and Pseudonaja textillis Venoms Chromatographically to Assess MALDI-MS and ESI-MS Complementarity with Correlation of Bioactive Toxins to Mass Spectrometric Data.

Snakebite is a serious health issue in tropical and subtropical areas of the world and results in various pathologies, such as hemotoxicity, neurotoxicity, and local swelling, blistering, and tissue necrosis around the bite site. These pathologies may ultimately lead to permanent morbidity and may even be fatal. Understanding the chemical and biological properties of individual snake venom toxins is of great importance when developing a newer generation of safer and more effective snakebite treatments. Two main approaches to ionizing toxins prior to mass spectrometry (MS) analysis are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). In the present study, we investigated the use of both ESI-MS and MALDI-MS as complementary techniques for toxin characterization in venom research. We applied nanofractionation analytics to separate crude elapid venoms using reversed-phase liquid chromatography (RPLC) and high-resolution fractionation of the eluting toxins into 384-well plates, followed by online LC-ESI-MS measurements. To acquire clear comparisons between the two ionization approaches, offline MALDI-MS measurements were performed on the nanofractionated toxins. For comparison to the LC-ESI-MS data, we created so-called MALDI-MS chromatograms of each toxin. We also applied plasma coagulation assaying on 384-well plates with nanofractionated toxins to demonstrate parallel biochemical profiling within the workflow. The plotting of post-column acquired MALDI-MS data as so-called plotted MALDI-MS chromatograms to directly align the MALDI-MS data with ESI-MS extracted ion chromatograms allows the efficient correlation of intact mass toxin results from the two MS-based soft ionization approaches with coagulation bioassay chromatograms. This facilitates the efficient correlation of chromatographic bioassay peaks with the MS data. The correlated toxin masses from ESI-MS and/or MALDI-MS were all around 6-8 or 13-14 kDa, with one mass around 20 kDa. Between 24 and 67% of the toxins were observed with good intensity from both ionization methods, depending on the venom analyzed. All Naja venoms analyzed presented anticoagulation activity, whereas pro-coagulation was only observed for the Pseudonaja textillis venom. The data of MALDI-MS can provide complementary identification and characterization power for toxin research on elapid venoms next to ESI-MS.

Enabling high-sensitivity live single-cell mass spectrometry using an integrated electrical lysis and nano electrospray ionization interface.

BACKGROUND: Live single-cell metabolomic studies encounter inherent difficulties attributed to the limited sample volume, minimal compound quantity, and insufficient sensitivity in the Mass Spectrometry (MS) method used to obtain single-cell data. However, understanding cellular heterogeneity, functional diversity, and metabolic processes within individual cells is essential. Exploring how individual cells respond to stimuli, including drugs, environmental changes, or signaling molecules, offers insights into biology, oncology, and drug discovery. Efficient release of cell contents (lysis) is vital for accurate metabolite detection at the single-cell level. Despite this, traditional approaches in live single cell metabolomics methods do not emphasize efficient lysis to prevent sample dilution. Instead, current live single cell metabolomics methods use direct infusion to introduce the cell into the mass spectrometry without prior chromatographic separation or a lysis step, which adversely affects sensitivity and metabolic coverage. RESULTS: To address this, we developed an integrated single-cell electrical lysis and nano spray (SCEL-nS) platform coupled to an Orbitrap MS capable of efficiently lysing a single cell after being sampled with specially manufactured micropipettes. Lysis efficiency was validated by comparing live cell stain fluorescent intensities of intact and electrically lysed cells through microscopy imaging. The SCEL-nS platform successfully induced the breakdown of a single cell, significantly reducing the live cell stain's fluorescent intensity indicating cell membrane breakdown. Additionally, SCEL-nS was validated by measuring single cells spiked with the anti-cancer drug tamoxifen by MS. SCEL-nS use resulted in statistically significant increase in the peak measured by the method compared to the traditional non-lysis method. SIGNIFICANCE: Overall, our results demonstrate that the newly incorporated SCEL-nS platform achieved higher sensitivities compared to traditional live single cell analysis methods.

[Recent advances in capillary electrophoresis-mass spectrometry analysis of |trace biomolecules].

In recent years, various trace bioanalysis methods have been developed, including single-cell transcriptome analysis methods. As the sample volume and amount of biomolecules contained therein are extremely limited, development of new single-cell analysis methods require extremely high-level techniques. It is necessary to design an appropriate analysis system that integrates a highly sensitive detection system and a pretreatment protocol for minimizing sample loss, where separation method is especially important for analyzing diverse mixtures of biomolecules. Among them, capillary electrophoresis (CE) can separate biomolecules in nanoliter-scale solutions with high resolution, making it highly compatible with trace samples such as single cells. By combining with highly sensitive nano-electrospray ionization-mass spectrometry (MS), it is possible to detect nanomolar to sub-nanomolar biomolecules, which can be further improved by using online sample preconcentration methods. These highly sensitive analytical techniques have made it possible to analyze trace amounts of metabolites, proteins, lipids, etc. This review paper summarizes the research on CE-MS trace bioanalysis that has been reported to date, with a focus on single-cell analysis.

Hydrophilic interaction liquid chromatography: An efficient tool for assessing thorium interaction with phosphorylated biomimetic peptides.

In the field of nuclear toxicology, the knowledge of the interaction of actinides (An) with biomolecules is of prime concern in order to elucidate their toxicity mechanism and to further develop selective decorporating agents. In this work, we demonstrated the great potential of hydrophilic interaction liquid chromatography (HILIC) to separate polar thorium (Th) biomimetic peptide complexes, as a key starting point to tackle these challenges. Th4+ was used as plutonium (Pu4+) analogue and pS16 and pS1368 as synthetic di- and tetra-phosphorylated peptides capable of mimicking the interaction sites of these An in osteopontin (OPN), a hyperphosphorylated protein. The objective was to determine the relative affinity of pS16 and pS1368 towards Th4+, and to evaluate the pS1368 selectivity when Th4+ was in competition complexation reaction with UO22+ at physiological pH. To meet these aims, HILIC was simultaneously coupled to electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS), which allowed to identify online the molecular structure of the separated complexes and quantify them, in a single step. Dedicated HILIC conditions were firstly set up to separate the new dimeric Th2(peptide)2 complexes with good separation resolution (peptide = pS16 or pS1368). By adding pS16 and pS1368 in different proportions relatively to Th4+, we found that lower or equal proportions of pS16 with respect to pS1368 were not sufficient to displace pS1368 from Th2pS13682 and pS16 proportion higher than pS1368 led to the formation of a predominant ternary complex Th2(pS16)(pS1368), demonstrating preferential Th4+ binding to the tetra-phosphorylated peptide. Finally, online identification and quantification of the formed complexes when Th4+ and UO22+ were mixed in equimolar ratio relatively to pS1368 showed that in spite of pS1368 has been specifically designed to coordinate UO22+, pS1368 is also Th4+-selective and exhibits stronger affinity for this latter than for UO22+. Hence, the results gathered through this approach highlight the impact of Th4+ coordination chemistry on its interaction with pS1368 and more widely to its affinity for biomolecules.

Advantages of online supercritical fluid extraction and chromatography hyphenated to mass spectrometry to analyse plastic additives in laboratory gloves.

Plastic additives are introduced in plastic material formulations, along with organic polymers, to offer different properties such as stability, plasticity or color. However, plastic additives may migrate from the plastic material to the content (in case of plastic containers) or to the material in contact with the plastic, like human skin. In the case of plastic medical devices, this migration is of particular interest, as plastic additives may be deleterious to health. In the present paper, we examined the interest of combining supercritical fluid extraction (SFE) to supercritical fluid chromatography (SFC) hyphenated to mass spectrometry (MS) in an online system to characterize plastic additives in laboratory gloves, taken as samples of medical devices. A set of target compounds comprising 18 plasticizers, 4 antioxidants and 2 lubricants was defined and their detectability with MS was examined, where it appeared that electrospray ionization (ESI) provided better detectability than atmospheric pressure chemical ionization (APCI). After examining possible stationary phases with the help of Derringer desirability function, an isocratic chromatographic method (CO2:methanol 95:5) was developed on Shim-pack UC Phenyl column. The extraction method was examined with a 3-level full factorial design of experiments to optimize the extraction temperature (40 °C) and pressure (200 bar). The online SFE-SFC-MS method was compared to offline methods where the samples were extracted with liquid solvents at atmospheric pressure or high pressure then analysed with SFC-MS. In all cases, offline methods showed significant contaminants (like the oleamide lubricant) issuing from laboratory plastic materials as nitrogen drying station, syringes and filters, while the online method allowed a complete elimination of laboratory contaminations. Furthermore, the online method saved time, solvents and laboratory consumables. It will also show that transferring a compressible fluid from a loading loop is favourable to high efficiency, as the resulting chromatographic peaks are much thinner than when transferring a liquid. Compared to injecting liquid heptane, the efficiency increase was 3.4-fold, while compared to injecting liquid methanol (a common practice in SFC), the efficiency increase was 13-fold. Finally, the additive composition of different laboratory gloves was compared.

Nanopore ion sources deliver individual ions of amino acids and peptides directly into high vacuum.

Electrospray ionization is widely used to generate vapor phase ions for analysis by mass spectrometry in proteomics research. However, only a small fraction of the analyte enters the mass spectrometer due to losses that are fundamentally linked to the use of a background gas to stimulate the generation of ions from electrosprayed droplets. Here we report a nanopore ion source that delivers ions directly into high vacuum from aqueous solutions. The ion source comprises a pulled quartz pipette with a sub-100 nm opening. Ions escape an electrified meniscus by ion evaporation and travel along collisionless trajectories to the ion detector. We measure mass spectra of 16 different amino acid ions, post-translationally modified variants of glutathione, and the peptide angiotensin II, showing that these analytes can be emitted as desolvated ions. The emitted current is composed of ions rather than charged droplets, and more than 90% of the current can be recovered in a distant collector.

Slot Blot- and Electrospray Ionization-Mass Spectrometry/Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry-Based Novel Analysis Methods for the Identification and Quantification of Advanced Glycation End-Products in the Urine.

Proteins, saccharides, and low molecular organic compounds in the blood, urine, and saliva could potentially serve as biomarkers for diseases related to diet, lifestyle, and the use of illegal drugs. Lifestyle-related diseases (LSRDs) such as diabetes mellitus (DM), non-alcoholic steatohepatitis, cardiovascular disease, hypertension, kidney disease, and osteoporosis could develop into life-threatening conditions. Therefore, there is an urgent need to develop biomarkers for their early diagnosis. Advanced glycation end-products (AGEs) are associated with LSRDs and may induce/promote LSRDs. The presence of AGEs in body fluids could represent a biomarker of LSRDs. Urine samples could potentially be used for detecting AGEs, as urine collection is convenient and non-invasive. However, the detection and identification of AGE-modified proteins in the urine could be challenging, as their concentrations in the urine might be extremely low. To address this issue, we propose a new analytical approach. This strategy employs a method previously introduced by us, which combines slot blotting, our unique lysis buffer named Takata's lysis buffer, and a polyvinylidene difluoride membrane, in conjunction with electrospray ionization-mass spectrometry (ESI)/matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). This novel strategy could be used to detect AGE-modified proteins, AGE-modified peptides, and free-type AGEs in urine samples.

In-Depth Mass Spectrometry Study of Vanadium(IV) Complexes with Model Peptides.

Investigating the speciation of vanadium complexes in the presence of potential biomolecular targets under physiological conditions remains challenging, and further experimental techniques are needed to better understand the mechanism of action of potential metallodrugs. The interaction of two model peptides (angiotensin I and angiotensin II) with three well-known oxidovanadium(IV) compounds with antidiabetic and/or anticancer activity, [VIVO(pic)2(H2O)], [VIVO(ma)2], and [VIVO(dhp)2] (where pic, ma, and dhp are picolinate, maltolate, and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate anions, respectively), was investigated by ESI-MS/MS (electrospray ionization tandem mass spectrometry) and complemented by EPR (electron paramagnetic resonance) spectroscopy measurements and theoretical calculations at the DFT (density functional theory) level. The results demonstrated that vanadium-peptide bonds are preserved after HCD (higher energy collisional dissociation) fragmentation, allowing for the identification of binding sites through a detailed analysis of the fragmentation spectra. Angiotensin I (AT1) and angiotensin II (AT2) exhibited different coordination behaviors. AT1, with two His residues (His6, His9), prefers to form [AT1 + VOL] adducts with both histidine residues coordinated to the metal ion, while AT2, which has only His6, can bind the metal in a monodentate fashion, forming also [AT2 + VOL2] adducts. Insights from this study pave the way to ESI-MS/MS investigations of more complex systems, including target proteins and further development of vanadium-based drugs.

Assessment of bacterial biotransformation of alkylnaphthalene lubricating base oil component 1-butylnaphthalene by LC/ESI-MS(/MS).

Alkylnaphthalene lubricating oils are synthetic Group V base oils that are utilized in wide-ranging industrial applications and which are composed of polyalkyl chain-alkylated naphthalenes. Identification of alkylnaphthalene biotransformation products and determination of their mass spectrometry (MS) fragmentation signatures provides valuable information for predicting their environmental fates and for development of analytical methods to monitor their biodegradation. In this work, laboratory-based environmental petroleomics was applied to investigate the catabolism of the alkylnaphthalene, 1-butylnaphthalene (1-BN), by liquid chromatography electrospray ionization MS data mapping and targeted collision-induced dissociation (CID) analyses. Comparative mapping revealed that numerous catabolites were produced from soil bacterium, Sphingobium barthaii KK22. Targeted CID showed unique patterns of production of even-valued deprotonated fragments that were found to originate from specific classes of bacterial catabolites. Based upon results of CID analyses of catabolites and authentic standards, MS signatures were proposed to occur through formation of distonic radical anions from bacterially-produced alkylphenol biotransformation products. Finally, spectra interpretation was guided by CID results to propose chemical structures for twenty-two 1-BN catabolites resulting in construction of 1-BN biotransformation pathways. Multiple pathways were identified that included aromatic ring-opening, alkyl chain-shortening and production of α,ß-unsaturated aldehydes from alkylated phenols. Until now, α,ß-unsaturated aldehydes have not been a class of compounds much reported from alkylated polycyclic aromatic hydrocarbon (APAH) and PAH biotransformation. This work provides a new understanding of alkylnaphthalene biotransformation and proposes MS markers applicable to monitoring APAH biotransformation in the form of alkylated phenols, and by extension, α,ß-unsaturated aldehydes, and toxic potential during spilled oil biodegradation.

Investigating Anion Effects on Metal Ion Binding Interactions With Amyloid ß Peptide by Ion Mobility Mass Spectrometry.

The study of metal ion's role in the biological processes of Alzheimer's disease has spurred investigations into the coordination chemistry of amyloid beta peptide and its fragments. Nano-electrospray ionization mass spectrometry (nESI-MS) has been utilized to examine the stabilization of bound anions on multiprotein complexes without bulk solvent. However, the effects of anions on metal ion binding interactions with amyloid beta peptide have not been explored. This study directly examined metal-peptide complexes using nESI-MS and investigated the effects of various anions on the binding ratio and stability of these complexes from ammonium salt solutions. The results indicate that different anions have distinct effects on the binding ratio and stability of various metal-peptide complexes. Of these, the bicarbonate ion exhibits the highest binding ratios for metal-peptide complexes, while binding ratios for these complexes in phosphate are comparatively low. Our results suggest that acetate, formate, bicarbonate, and phosphate have weak affinities and act as weak stabilizers of the metal-peptide complex structure in the gas phase. Intriguingly, chloride and sulfate act as stabilizers of the metal-peptide complex in the gas phase. The rank order determined from these data is substantially different from the Hofmeister salt series in solution. Although this outcome was anticipated due to the reduced influence of anions and water solvation, our findings correlate well with expected anion binding in solution and emphasize the importance of both hydration layer and anion-metal-peptide binding effects for Hofmeister-type stabilization in solution. This approach proved useful in examining the interactions between metal ions and amyloid beta peptide, which are relevant to Alzheimer's disease, using direct ESI-MS.

Electrophoresis-Correlative Data-Independent Acquisition (Eco-DIA) Improves the Sensitivity of Mass Spectrometry for Limited Proteome Amounts.

Capillary zone electrophoresis (CE) combines high separation power, scalability, and speed to limited proteome analyses by mass spectrometry (MS). However, compressed separation in CE challenges the duty cycle of tandem MS, even during data-independent acquisition (DIA). To help remedy this limitation, we introduce the concept of electrophoresis-correlative (Eco) data acquisition for CE-MS. We recognize CE electrospray ionization (ESI) to sort peptide ions into reproducible mass-to-charge (m/z) vs migration time (MT) trends in the solution phase, before subsequent ionization and m/z analysis. We proposed that such a correlation can be leveraged to improve the economy of data acquisition. We test this hypothesis using DIA frames that are tailored to the observed m/z-MT trends. The resulting Eco-DIA method substantially improves the bandwidth utilization of tandem MS during CE-MS. In proof-of-principle studies, Eco-DIA identified and quantified ∼38% more proteins from 1 ng of the HeLa proteome digest compared to the classical DIA, without the assistance of a project-specific tandem MS spectral library. Eco-DIA was able to quantify ∼51% more proteins with <10% coefficient of variation vs the control DIA approach. Based on label-free quantification, the proteins that were exclusively measured by Eco-MS occupied the lower dynamic range of the detected proteome concentration, revealing sensitivity enhancement. In addition to marking the inception of Eco-MS, this work lays the foundation for the development of next-generation data acquisition strategies that leverage electrophoretic ion sorting for high-sensitivity proteomics.

Predicting Peptide Ionization Efficiencies for Electrospray Ionization Mass Spectrometry Using Machine Learning.

Mass spectrometry (MS) is inherently an information-rich technique. In this era of big data, label-free MS quantification for nontargeted studies has gained increasing popularity, especially for complex systems. One of the cornerstones of successful label-free quantification is the predictive modeling of ionization efficiency (IE) based on solutes' physicochemical properties. While many have studied IE modeling for small molecules, there are limited reports on peptide IEs. In this study, we leverage the stoichiometric relationship in trypsin digests of well-characterized monoclonal antibodies (mAbs) to compile a data set of relative ionization efficiencies (RIEs) for 241 peptides. From each peptide's sequence, we computed a set of physiochemical descriptors, which were then used to train machine learning regression models to predict RIEs. Peptides shorter than 20 amino acids had RIEs that were highly correlated to their molecular weight. A random forest (RF) model was able to best predict the RIEs of a test data set with a mean relative error of 23.9%. For larger peptides, a multilayer perceptron (MLP) model improved RIE prediction compared to current best practices, reducing mean relative error from 60.5% to 32.0%. Finally, we also show the application of the RF model in label-free relative protein quantification and improving the quantification of peptide post-translational modifications (PTMs). This approach to predicting peptide IEs from their sequences enables the development of accurate label-free quantification workflows for peptide and protein analysis.

Liquid Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using a Commercial Ion Source and Orbitrap Mass Analyzer.

A liquid atmospheric pressure-matrix-assisted laser desorption/ionization (LAP-MALDI) method has been developed and applied to a commercial AP-MALDI source on a hybrid orbitrap mass analyzer. It is shown that electrospray ionization (ESI)-like mass spectra of a range of peptides and proteins can be acquired by LAP-MALDI mass spectrometry (MS) as previously demonstrated on a homemade LAP-MALDI-Q-TOF setup but without the need of any modification to the commercially available MS equipment used. Multiply charged peptide ions were recorded with a resolution of around 100,000 and a mass accuracy of less than 5 ppm. The higher resolution and mass accuracy of the orbitrap analyzer compared with previously employed Q-TOF instrumentation provided high confidence in bacterial proteoform and species identification by top-down protein analysis.

Quantification of osilodrostat in horse urine using LC/ESI-HRMS to establish an elimination profile for doping control.

Aim: The use of osilodrostat, developed as a medication for Cushing's disease but categorized as an anabolic agent, is banned in horses by both the International Federation of Horseracing Authorities and the Fédération Equestre Internationale. For doping control purposes, elimination profiles of hydrolyzed osilodrostat in horse urine were established and the detectability of free forms of osilodrostat and its major metabolite, mono-hydroxylated osilodrostat (M1c), was investigated.Materials & methods: Post-administration urine samples obtained from a gelding and three mares were analyzed to establish the elimination profiles of osilodrostat using a validated method involving efficient enzymatic hydrolysis followed by LC/ESI-HRMS analysis.Results: Applying the validated quantification method with an LLOQ of 0.05 ng/ml, hydrolyzed osilodrostat could be quantified in post-administration urine samples from 48 to 72 h post-administration; by contrast, both hydrolyzed osilodrostat and M1c were detected up to 2 weeks. In addition, confirmatory analysis identified the presence of hydrolyzed osilodrostat for up to 72 h post-administration.Conclusion: For doping control purposes, we recommend monitoring both hydrolyzed M1c and osilodrostat because of the greater detectability of M1c and the availability of a reference material of osilodrostat, which is essential for confirmatory analysis.

[Box: see text].

Qualitative and quantitative characterization of elastin-like polypeptides combining low-PH/low-temperature bottom-up and intact protein liquid chromatography mass spectrometric analysis.

RATIONALE: Elastin-like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products. METHODS: ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline-specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed-phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using a time-of-flight mass spectrometer. RESULTS: Absolute quantification of ELPs was achieved by utilizing ELP-specific properties, that is, proline-rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed-phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N-terminal modifications. CONCLUSIONS: Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs.

An Integrated Strategy of UHPLC-ESI-MS/MS Combined with Bioactivity-Based Molecular Networking for Identification of Antitumoral Withanolides from Athenaea fasciculata (Vell.) I.M.C. Rodrigues & Stehmann.

BACKGROUND: Athenaea fasciculata, a Brazilian native species from the Solanaceae family, is recognized as a promising source of bioactive withanolides, particularly Aurelianolide A and B, which exhibit significant antitumoral activities. Despite its potential, research on the chemical constituents of this species remains limited. This study aimed to dereplicate extracts and partitions of A. fasciculata to streamline the discovery of bioactive withanolides. METHODS: Using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), various extracts-including n-hexane, methanol, and ethanol-were analyzed, and their mass spectrometry data were processed through the GNPS platform for the generation of molecular networking. The results indicated that crude extracts displayed comparable cytotoxicity against Jurkat cells, by treatment at 150 µg/mL, while alcoholic extracts achieved approximately 80% inhibition of K562 cells and K562-Lucena 1 at the same concentration. Notably, the dichloromethane partition exhibited the highest cytotoxicity across leukemia cell lines, particularly against Jurkat cells (IC50 = 14.34 µg/mL). A total of 22 compounds were annotated by manual inspection and different libraries, with six of them demonstrating significant cytotoxic effects. CONCLUSIONS: This research underscores the therapeutic potential of A. fasciculata and highlights the effectiveness of integrating advanced analytical methods in drug discovery, paving the way for further exploration of its bioactive compounds.