Mass Spectrometry Detection Research Articles

Advances in Single Particle Mass Analysis.

Single particle mass analysis methods allow the measurement and characterization of individual nanoparticles, viral particles, as well as biomolecules like protein aggregates and complexes. Several key benefits are associated with the ability to analyze individual particles rather than bulk samples, such as high sensitivity and low detection limits, and virtually unlimited dynamic range, as this figure of merit strictly depends on analysis time. However, data processing and interpretation of single particle data can be complex, often requiring advanced algorithms and machine learning approaches. In addition, particle ionization, transfer, and detection efficiency can be limiting factors for certain types of analytes. Ongoing developments in the field aim to address these challenges and expand the capabilities of single particle mass analysis techniques. Charge detection mass spectrometry is a single particle version of mass spectrometry in which the charge (z) is determine independently from m/z. Nano-electromechanical resonator mass analysis relies on changes in a nanoscale device's resonance frequency upon deposition of a particle to directly derive its inertial mass. Mass photometry uses interferometric video-microscopy to derive particle mass from the intensity of the scattered light. A common feature of these approaches is the acquisition of single particle data, which can be filtered and concatenated in the form of a particle mass distribution. In the present article, dedicated to our honored colleague Richard Cole, we cover the latest technological advances and applications of these single particle mass analysis approaches.

Upconversion-Linked Immunosorbent Assay for the Biomimetic Detection of the Mycotoxin Cyclopiazonic Acid.

The neurotoxin α-cyclopiazonic acid (CPA) is an emerging mycotoxin produced as a secondary metabolite by several fungi species (i.e., Penicillium spp. and Aspergillus spp.). CPA commonly contaminates maize, crops, cheese, and wine. In this work, CPA detection in foodstuff was accomplished by the innovative integration of two strategies: upconversion nanoparticles (UCNPs) and epitope-mimicking peptides, to develop a competitive upconversion-linked immunosorbent assay (ULISA). We have applied UCNPs (type NaYF4:Yb3+, Er3+) as background-free optical labels due to their anti-Stokes shift with excitation in the near-infrared region and emission in the ultraviolet-visible region. Moreover, a CPA epitope-mimicking cyclic peptide (A2) was used as a substitute for the toxin-conjugates traditionally applied to competitive assays. UCNPs were decorated with an anti-CPA fragment antigen-binding antibody (UCNP-Fab), and CPA detection was achieved through competition with a biotinylated CPA epitope-mimicking cyclic peptide (A2-biotin, ACNWWDLTLC-GGGSK (Biotin)-NH2), anchored to a streptavidin-coated microtiter plate, for antibody binding. The ULISA platform offers ultrasensitive detection of CPA (limit of detection of 1.3 pg mL-1 and IC50 value of 15 pg mL-1), and no cross-reactivity was observed with other coproduced mycotoxins. These results substantially outperformed the analytical features of conventional heterogeneous immunoassays based on enzymatic detection. Additionally, the use of advanced computational tools, such as MOE and Alphafold AI, proved advantageous in elucidating the molecular interactions between the antibody and the epitope, providing insights that enhance the rational design of immunoassays. The proposed ULISA was applied to detect CPA in spiked maize samples, and the results were validated by high-performance liquid chromatography coupled to a tandem mass spectrometry detector (HPLC-MS/MS).

Advances in High-Performance Liquid Chromatography (HPLC) and Ultra-Performance Liquid Chromatography (UPLC)

High-Performance Liquid Chromatography (HPLC) and Ultra-Performance Liquid Chromatography (UPLC) are fundamental analytical techniques that have revolutionized chemical analysis across various fields. These chromatographic methods offer exceptional separation capabilities, high resolution, and precise quantification of complex mixtures. HPLC has evolved significantly since its inception, with improvements in column technology, detection systems, and automation. UPLC, introduced as an advancement to conventional HPLC, operates at significantly higher pressures using sub-2-μm particle sizes, resulting in enhanced resolution, speed, and sensitivity. Both techniques have found extensive applications in pharmaceutical analysis, environmental monitoring, food safety, clinical diagnostics, and biomedical research. The implementation of various detection methods, including UV-visible, fluorescence, mass spectrometry, and electrochemical detection, has expanded their analytical capabilities. Recent developments in column technology, such as core-shell particles and monolithic columns, have further improved separation efficiency. The integration of artificial intelligence and machine learning has enhanced method development and data analysis. This review discusses the fundamental principles, technological advancements, and diverse applications of HPLC and UPLC. Additionally, it discusses current challenges, emerging trends, and future prospects in chromatographic science, including green chromatography initiatives and miniaturization efforts. The continuous evolution of these techniques contributes significantly to analytical chemistry's advancement, promising even more sophisticated applications in various scientific disciplines.

Chemical composition, antimicrobial and antioxidant activities of Vateria indica L. oleo-gum resin (white dammar) essential oil

White dammar is an oleo-gum resin obtained from Vateria indica L. (Dipterocarpaceae) and is known to possess diverse biological activities. This study examines the chemical composition, antimicrobial, and antioxidant activities of essential oil extracted from white dammar. The chemical constituents of essential oil were identified by gas chromatography coupled with mass spectrometry (GC-MS) and flame ionization detection (FID). A total of 18 compounds constituting 97.15% of the oil were identified, which consist of β-caryophyllene (36.32%), α-humulene (14.31%), β-humulene (9.73%), caryophyllene oxide (8.28%), allo-aromadendrene (5.63%) and junenol (5.1%). The antimicrobial activity of the essential oil obtained was measured by means of disc diffusion assay, minimum inhibitory concentration (MIC), and minimum bactericidal/fungal concentrations (MBC/MFC). The highest antimicrobial activity was shown against Bacillus cereus (MIC/MBC = 31.2/62.5 μg/mL) and also Staphylococcus aureus (MIC/MBC = 62.5/125 μg/mL), whereas only a moderate level of activity was recorded against Salmonella typhi (MIC/MBC = 125/250 μg/mL), Vibrio cholerae, MRSA (MIC/MBC = 250/500 μg/mL) and Candida albicans (MIC/MFC = 125/250 μg/mL). Antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays; IC50 value, of 29.42 μg/mL and FRAP value, of 166.5 ± 2.2 μmol Fe2+/g indicated that the essential oil exhibited adequate antioxidant activity. These results substantiate the possible medicinal application of white dammar as an antimicrobial and antioxidant agent.

Exploring Trial-and-Error in Deep Learning: Initial Application to Isotope Detection in Mass Spectrometry

Exploring Trial-and-Error in Deep Learning: Initial Application to Isotope Detection in Mass Spectrometry

Upcycling of post-industrial multilayer plastic packaging waste through gasoline-equivalent hydrocarbon-targeted pyrolysis by an environmentally friendly catalyst

This study’s novelty lies in investigating the performance of calcined cement kiln dust (CCKD) as a new, low-cost catalyst in the flash pyrolysis of EVOH-containing multilayer plastic packaging waste (MLPW). An analytical pyrolyzer, connected with gas chromatographic separation and mass spectrometry detection (Py–GC/MS), was employed to investigate the catalytic pyrolysis of MLPW. Py-GC/MS experiments have shown that condensable volatiles from catalytic pyrolysis of MLPW exhibit higher yields of aromatics (24.7 %) and cyclic aliphatic compounds (26.3 %). The catalytic pyrolysis of MLPW yields C5–C12 hydrocarbons (76.2 %), while the non-catalytic conversion yields C8–C24 hydrocarbons (95.4 %). These results underscore the efficacy of catalytic pyrolysis in recovering gasoline-range hydrocarbons from MLPW, selectively enhancing the yield of cyclic aliphatics and aromatic compounds. Thus, within the circular economy’s horizon, flash pyrolysis can be scaled up to process large amounts of MLPW using CCKD as a low-cost catalyst, converting MLPW into a potential gasoline-range transportation fuel.

Covalent triazine frameworks as particle electrode for three-dimensional photoelectrocatalytic degradation of oxytetracycline: Synergy effects, pathway, and mechanism.

Photoelectrocatalysis has been widely employed for degrading antibiotics due to its high efficiency. However, the application is significantly impeded by the rapid recombination of photogenerated charge carriers and the limited surface areas of photoelectrodes. In the study, high crystallinity covalent triazine frameworks were fabricated at low temperature of 150°C and firstly used as particle photoelectrode in the three-dimensional photoelectrochemical reactor to degrade oxytetracycline (OTC). SEM, TEM, XRD, XPS, and FT-IR confirmed the successful synthesis of high crystallinity covalent triazine frameworks. Compared to CTF-120 (71.2%) and CTF-180 (46.9%), CTF-150 exhibited excellent OTC removal. Electrochemical impedance, UV-vis absorption spectra, and Mott-Schottky tests showed that CTF-150 demonstrated more wide light absorption range of 501nm and narrow bandgap of 2.52eV, and smaller Rct value under illumination, in comparing to CTF-120 and CTF-180. When the initial concentration of OTC was 50mgL-1, the 86.2% of OTC removal and 62.7% of mineralization were obtained under light irradiation (λ>420nm), current of 10mA, pH of 6.4, electrolyte of 0.1M Na2SO4. The synergy effect between photocatalytic and electrocatalytic processes of CTF-150 not only enhanced by 38.5% current efficiency but also reduced energy consumption to 1.90 kWh m-3. CTF-150 had a wide range of acid-base application and displayed resistance on coexisting ions. Electron spin resonance detection, quenching experiments, and probe experiments illustrated that h+, •O2-, 1O2, and •OH contributed to the degradation of OTC and the generation pathways of •O2-, 1O2, and •OH were verified. Moreover, •O2-, 1O2, and h+ were the main reactive species responsible for OTC removal, while 1O2 was for OTC mineralization. Based on high-performance liquid chromatography-tandem mass spectrometry detection, OTC with benzene ring was decomposed to opening ring products. The acute toxicity, developmental toxicity, bioaccumulation factor and mutagenicity of OTC and its intermediates using T.E.S.T. showed the toxicity of 82.35% degradation products decreased.

Phase I pharmacokinetic study of etmaben, a malonic acid derivative

Introduction. Etmaben is a malonic acid derivative that has shown cardiotropic activity and is a promising candidate for treating ischemic heart disease and chronic heart failure. It is currently undergoing Phase I clinical trials, and its pharmacokinetics have not yet been studied in humans.Aim. The aim of the study is to investigate the pharmacokinetics of Etmaben, film-coated tablets, 300 mg (SPCPU, Russia) in healthy volunteers after fasting administration of different doses following both single and multiple dosing over a 7-day period.Materials and methods. The open-label, non-randomized clinical trial involved 48 healthy volunteers divided into 6 cohorts. Volunteers in cohort 1 received a single dose of 600 mg of etmaben, cohort 2 received 900 mg, and cohort 3 received 1200 mg. Cohorts 4, 5, and 6 received daily doses of 600 mg, 900 mg, and 1200 mg, respectively. Plasma concentrations of etmaben were measured using high-performance liquid chromatography with tandem mass spectrometry detection. Pharmacokinetic parameters were calculated using Microsoft Excel with the Boomer extension (Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA 92606, USA).Results and discussion. Pharmacokinetic parameters were calculated for 6 cohorts of 8 volunteers after single and multiple dosing of Etmaben at doses of 600, 900, and 1200 mg. The maximum plasma concentration (Cmax) of etmaben was reached on average within 0.5 h. Cmax values ranged from 2.708 ± 1.461 µg/mL to 19.871 ± 4.415 µg/mL. The maximum half-life was 0.874 ± 0.236 h, with an elimination rate constant not exceeding 1.053 ± 0.149 h–1. The mean residence time (MRT) of etmaben in plasma did not exceed 1.527 ± 0.272 h. The average volume of distribution was above 45 L, and clearance exceeded 42 L, indicating significant tissue distribution and rapid drug elimination. Due to low area under the curve values and high elimination rates, the accumulation of etmaben is minimal.Conclusion. Pharmacokinetic parameters were calculated, and averaged pharmacokinetic profiles were constructed in linear and semilogarithmic coordinates after single and multiple dosing of various etmaben doses. This study represents the first investigation of etmaben pharmacokinetics in humans, paving the way for subsequent clinical trials.

Catalyst screening for conversion of Chlorella sp. to aromatic fuel additives: A sustainable strategy for CO2 capture

Developing biofuels with characteristics similar to current fossil fuels and compatibility with existing petroleum infrastructure (drop-in biofuels) is gaining prominence, aligning with global efforts towards carbon-neutral economies and decarbonization of the transportation sector. The originality of the current study involves two directions: first, the use of Chlorella sp. microalgae, cultivated under simulated post-combustion gas, as a raw material for producing drop-in biofuel precursors; and second, an investigation of the catalytic activity of hierarchical zeolites in the deoxygenation and denitrogenation of volatile pyrolysis products, enhancing hydrocarbon content. To accomplish this, a micro-furnace-type temperature-programmable pyrolyzer coupled with chromatographic separation and mass spectrometry detection (Py-GC/MS) was utilized to assess the upgrading effectiveness of distinct zeolites (faujasite-type, MFI-type, and mordenite-type) on volatile pyrolysis products. All tested zeolites effectively reduced oxygenated and nitrogenated compounds in the volatile pyrolysis products, enhancing their suitability for producing renewable fuel. This supports sustainable development goals by promoting affordable, clean energy and climate action. HMor yielded the highest hydrocarbon content (98.5 %), followed by HZSM-5 (97.6 %) and HY (85.5 %). Catalytic upgrading significantly increased the concentration of aromatic hydrocarbons (at least 66.3 %), with MFI-type zeolites showing the highest selectivity for valuable BETX compounds (benzene, ethylbenzene, toluene, xylene). Hydrocarbons in the gasoline range, with up to 91.7 %, predominantly align with the needs of the transportation fuel market. The principal component analysis illustrates that using MFI-type zeolites promoted the lowest selectivity for PAHs, constituting precursors for coke formation, which is advantageous for ensuring a longer catalyst lifespan. Our results are promising and encourage the conversion of microalgal biomass into renewable fuel additives for formulating drop-in biofuels, as hydrocarbon-rich volatile pyrolysis products could be directly integrated into existing biorefineries. Thus, microalgal biomass cultivated using flue gas as the carbon source can be viewed as a versatile and promising resource for producing renewable fuel additives, contributing to developing a sustainable, low-carbon future.

Determination of sacubitril and seven sartan drugs in serum samples by online solid phase extraction-liquid chromatography-tandem mass spectrometry

An online solid phase extraction–ultra performance liquid chromatography–tandem mass spectrometry detection method was developed for the simultaneous determination of sacubitril and seven sartan drugs in blood serum in this study. The compounds were separated through a C18 column. Mass spectrometry of the samples was performed using a jet stream electrospray ion source (AJS, ESI+). The samples were detected via a multiple reaction monitoring (MRM) mode and quantified via a stable isotope internal standard method. 900 μL of prepared sample was injected and a run time of 12.5 minutes was obtained in this proposed method. The eight examined target compounds showed good linearity (r2＞0.994) in the corresponding mass concentration range and a lower limit of quantitation (LLOQ) was in the range of 0.05–0.1 μg/L. The recovery of the spiked serum samples ranged from 90.90 % to 106.20 % with relative standard deviations (RSDs) of 4.57 %–9.27 %. Five target compounds were detected in the actual serum samples using this method. The proposed method is simple to use, sensitive, accurate, and suitable for the trace detection of sacubitril and seven sartan drugs present in serum samples. The method can meet the needs for clinical monitoring of blood concentrations of this type of drug, while providing detection technology support for the development of compound preparations of sacubitril and other sartan drugs.

Charge Detection Mass Spectrometry for the Analysis of Atmospheric Dust on Mars

Charge Detection Mass Spectrometry for the Analysis of Atmospheric Dust on Mars

Valorisation of Winery By-Products: Revealing the Polyphenolic Profile of Grape Stems and Their Inhibitory Effects on Skin Aging-Enzymes for Cosmetic and Pharmaceutical Applications.

Grape (Vitis vinifera L.) stems, a by-product of winemaking, possess significant potential value due to their rich polyphenolic composition, which allows their exploitation for cosmetic and pharmaceutical applications. This presents a promising opportunity for valorisation aimed at developing innovative products with potential health-promoting effects. In this study, the polyphenolic profile of extracts from grape stems of seven white grape varieties was determined using spectrophotometric and chromatographic methods, specifically high-performance liquid chromatography coupled with a photodiode array detector and electrospray ionization multi-stage mass spectrometry (HPLC-PDA-ESI-MSn), as well as on their ferric-reducing antioxidant power (FRAP) and radical scavenging capacity, using 2,2-diphenyl-1-picrylhydrazyl (DPPH●) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS●+) radicals. This study also evaluated the anti-aging activity and skin depigmenting activity of these extracts. These findings revealed a diverse polyphenolic profile, encompassing proanthocyanidins and catechin derivatives (PCDs), phenolic acids, and flavonols. Among the varieties studied, 'Códega do Larinho' exhibited the highest concentrations of six distinct polyphenols and the highest total phenolic content. It also demonstrated the highest results for antioxidant capacity and elastase and tyrosinase inhibition. Pearson's correlation analysis showed a significant positive correlation between certain PCDs with both FRAP and DPPH assays, as well as between the identified flavonols and anti-elastase activity. These results underscore the potential health benefits of grape stem extracts and emphasize the importance of their polyphenolic composition in enhancing antioxidant and anti-aging properties, thus supporting their application in different industries.

Potential source contribution function coupled with mass spectrometry detection to identify source of atmospheric polyethylene terephthalate

Source identification of atmospheric microplastics (MPs) is crucial for the development of mitigation policies. Compared with wind directions or backward trajectories of air masses, the potential source contribution function (PSCF) analysis identifies more comprehensive sources of atmospheric particles. However, conducting PSCF analysis requires hourly pollutant concentration data, which cannot be met by the atmospheric MPs abundance obtained through commonly used methods. In this study, total suspended particles (TSP) samples were collected hourly and the concentrations of atmospheric polyethylene terephthalate (PET) were detected using a liquid chromatography-tandem mass spectrometry. Atmospheric concentrations of PET MPs were 112.9 ± 39.04 ng/m³ (average ± SD). Based on the hourly backward trajectories of air masses and the varied PET concentrations at the sampling site, potential sources of atmospheric PET were identified by PSCF analysis. The backward trajectory-based method indicates that atmospheric PET of the target site in this study primarily originates from dry farmlands. In comparison, both the residential areas and the dry farmlands were identified by PSCF as major sources of atmospheric PET at the receptor site. In contrast, both the backward-trajectory based method and PSCF analysis indicate that TSP mainly originates from the dry farmlands near the sampling site. This indicates that atmospheric PET in urban areas may have different sources from those of TSP, and PSCF is a suitable method for identifying sources of atmospheric PET.

Comparative Pharmacokinetic Assessment of Curcumin in Rats Following Intratracheal Instillation Versus Oral Administration: Concurrent Detection of Curcumin and Its Conjugates in Plasma by LC-MS/MS.

To establish and validate an LC-MS/MS method for the simultaneous determination of curcumin (CUR) as well as its glucuronide conjugate (COG) and sulfate conjugate (COS) in rat plasma. The method was employed to evaluate and compare the pharmacokinetic behaviors of curcumin following oral and intratracheal administration in rats. Rat plasma samples were separated by chromatography on a C18 column after protein precipitation with acetonitrile. Gradient elution with a mobile phase of 0.5 mM ammonium acetate in acetonitrile was utilized. Mass spectrometry detection incorporated an electrospray ionization (ESI) source, multiple reaction monitoring (MRM), and dual-mode (positive and negative) scanning for quantitative analysis. A total of 12 SD rats were randomly divided into two groups and were orally (20 mg/kg) or intratracheally (10 mg/kg) administrated curcumin, respectively. CUR, COG, and COS concentrations in plasma were measured to assess pharmacokinetic disparities. The method demonstrated linearity within the ranges of 2-400 ng/mL for CUR and COS and 5-1000 ng/mL for COG. Intratracheal administration significantly elevated CUR plasma concentrations compared to oral administration. The exposure of COG was higher than COS following oral administration. Conversely, intratracheal administration resulted in markedly higher COS exposure, with no significant difference in COG exposure after dose normalization between oral and inhalation routes. The established LC-MS/MS method provides a reliable tool for the simultaneous measurement of CUR, COG, and COS in rat plasma, facilitating preclinical pharmacokinetic investigations. The study reveals distinct pharmacokinetic profiles for CUR following oral versus intratracheal administration, suggesting that inhalation may offer superior therapeutic efficacy.

An orthogonal approach for analysis of underivatized steroid hormones using ultrahigh performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS).

The crucial role of steroid hormones in health and diseases merits their high-throughput, accurate and affordable measurements in biological specimens. Despite advances in analytical methods, sensing and quantifying steroid hormones remains challenging. Immunoassays offer excellent sensitivity but are inherently labour-intensive, costly, and prone to false positives. Mass spectrometry (MS) has been increasingly utilised, with the main hurdle being the isobaric tendencies of similar analytes, which complicates their separation and accurate quantification. This study compares ultrahigh-performance supercritical fluid chromatography separation (UHPSFC) and ultra-high-performance liquid chromatography (UHPLC) for MS detection. It optimises the column chemistry, temperature, and pressure to provide an operational protocol for the resolution and quantification of analytes. It presents the systematic characterisation of UHPSFC-MS performance by investigating spiked blood samples using Solid-Phase Extraction (SPE) and describes the matrix effects associated with MS measurements. Although both separation methods showed adequate resolution, specificity, and retention time, UHPSFC-MS was superior for five out of seven columns tested. With added high-throughput capacities, UHPSFC-MS, thus, offers an optimal solution for the analysis of steroid hormones for research, medical chemistry, and clinical diagnostics.

DART isotope dilution high resolution mass spectrometry and 19F-NMR detection of fluorotelomeric alcohols in hydrolyzed food contact paper

Fluorotelomer-based acrylate polymers and surfactants used to grease-proof food contact paper (FCP) are potential sources of dietary exposure to perfluoroalkyl substances (PFAS). Food contact substances (FCS) containing polyfluorinated long-chains (≥C8) were voluntarily removed by their manufacturers from the U.S. market in 2011 due to health concerns and largely replaced with FCSs containing short-chain (≤C7) PFAS. In 2020, FDA findings of potential biopersistence of 6:2 FTOH (CF3(CF2)5CH2CH2OH) similarly prompted an additional voluntarily phase-out of FCSs containing 6:2 FTOH by their manufacturers that was completed by the end of 2023. To monitor the phase-out process, a screening method was developed to detect FCPs containing ester-linked polyfluorinated pendant chains. Direct Analysis in Real Time-Isotope Dilution-High Resolution Mass Spectrometry (DART-ID-HRMS) enabled rapid semi-quantitative detection of 6:2 FTOH in FCP saponification reaction headspace without requiring sample concentration or chromatography. 19F-NMR analysis confirmed 6:2 FTOH pendant chain identity and detection dependence on saponification. The speed and specificity of this approach arise from ester saponification in the presence of stable isotopically labeled 6:2 FTOH; high FTOH differential volatility relative to nonfluorinated matrix, and the facile production of FTOH gas-phase anions (e.g., [M + O2]·−, [M-H + CO2]−) under ambient ionization conditions. The efficiency of this simple workflow makes it well-suited for monitoring the phase-out of FCS containing ester-linked polyfluorinated chains from the U.S. marketplace.

Hydrophobic deep eutectic solvent-ferrofluid microextraction followed by liquid chromatography-mass spectrometry for the enantioselective determination of chiral agrochemicals in natural waters.

The increasing use of chiral agrochemicals sold as racemic formulations raises concern for the negative impacts that inactive enantiomers can have on aquatic life and human health. The present work just focuses on the determination of ten chiral pesticides in river water samples by applying a ferrofluid-based microextraction followed by their stereoselective liquid chromatography analysis. To develop the ferrofluid, magnetite nanoparticles were prepared and coated with oleic acid and then dispersed in a hydrophobic natural deep eutectic solvent (NaDES), composed ofL-menthol and thymol (1:1). The stable colloidal dispersion was characterised by scanning electron microscopy, thermogravimetric analysis, energy-dispersive X-ray spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy. The analyte microextraction from 5ml river samples was performed using 50µl of ferrofluid, while acidified acetonitrile (150µl) was used to break down the ferrofluid and solubilise the NaDES containing the analytes. All the extracts were analysed by high-performance liquid chromatography-tandem mass spectrometry. For each analyte, the baseline separation of isomers was achieved on a Lux i-Amylose-3 column (amylose tris(3-chloro-5-methylphenylcarbamate) working in reversed-phase mode; the combination with mass spectrometry detection allows the overall separation of 24 isomers (ten chiral analytes among which eight containing a single (one) chiral centre, one with two chiral centres and the last one existing in four stereoisomeric forms, due to the presence of two regioisomers with a chiral carbon) within 37min. The method showed very good figures of merit in terms of recoveries (77.7-97.5%), intra-day and inter-day precision (2.7-7.7% and 6.9-14.9%, respectively), limit of detection (0.01-0.35µg/L), limit of quantitation (0.03-1.20µg/L), linear dynamic range, and intra-day and inter-day accuracy (1.2-14.8% and 1.8-15.0%, respectively). The presented method was able to detect 14 out of 24 isomers at the preventive limit established by the Italian legislation for single pesticide (that for a chiral pesticide is the sum of all its isomers) in surface waters, set at 0.1µg/l. Finally, the method was evaluated using AGREEprep and ComplexGAPI metrics, compared with other ferrofluid-based methods, and applied to the analysis of water samples from two Italian rivers (the Nera River and the Tiber River), providing to be sustainable and reliable for the application to real river matrices.

Trade-Off Between Growth Regimes in Chlorella vulgaris: Impact on Carotenoid Production

With the increasing awareness of socio-environmental issues, a global trend has emerged emphasizing the valorization of natural ingredients that promote health and well-being within sustainable production systems, such as microalgae-based carotenoids. Currently, little is understood about the correlation between biomass productivity and carotenoid content, which is a fundamental parameter for facilitating the immediate expansion of microalgae bioprocesses and ensuring the availability and industrial viability of these compounds. In this context, this study aims to investigate the carotenoid profile of Chlorella vulgaris through growth curve experiments conducted under photoautotrophic and heterotrophic regimes. Additionally, a trade-off analysis was performed for the production of carotenoids from microalgae. Carotenoids were quantified using high-performance liquid chromatography coupled with diode array and mass spectrometry detectors (HPLC-PDA-MS/MS). The performance of kinetic phases and energy demands across growth regimes was assessed to provide insights into production trade-offs. The results indicated that a total of 22 different carotenoids were identified in all the extracts. The all-trans-lutein and all-trans-β-carotene were the majority compounds. The total carotenoid content of Chlorella vulgaris revealed significant differences in the kinetic phases of carotenoid production, indicating that carotenoid volumetric production is only viable if the cultures are conducted until the log and stationary phases, based on the function of the biomass volumetric production (weight.volume−1). Therefore, the best trade-off for the process was to provide photoautotrophic growth until the exponential phase (log). Under this condition, the maximum carotenoid and lutein content was 2921.70 µg.L−1, reaching a maximum cell biomass of 1.46 g.L−1. From an environmental/economic point of view, the energy demand was 7.74 kWh.L−1. Finally, the scientific advances achieved in this study provide a holistic view of the influence of the main cultivation methods on the production of microalgae carotenoids, suggesting a viable initial direction for different industrial applications.

Open-source Electrophilic Fragment Screening Platform to Identify Chemical Starting Points for UCHL1 Covalent Inhibitors

Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC50 <5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling.

Pharmacokinetic study of iptacopan and its two acyl glucuronide metabolites in monkey plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

In this study, a simple and sensitive liquid chromatography tandem mass spectrometric method was developed and validated for the determination of iptacopan and two acyl glucuronidation metabolites in monkey plasma. The plasma sample was precipitated with acetonitrile and then separated on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using 0.1% formic acid and 5 mM ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometry (MS) detection was performed in positive multiple reactions monitoring (MRM) mode with precursor-to-production transitions. The developed assay was validated over the range of 1-2000 ng/mL for three analytes with correlation coefficient (r) more than 0.99. The validation parameters including accuracy, precision, carryover effect, matrix effect, recovery, and stability were all within the acceptable limits. The validated method has been applied to investigate the pharmacokinetics of iptacopan and its two acyl glucuronidation metabolites in monkey plasma. After intravenous administration, iptacopan showed low clearance (2.75 mL/min/kg) in monkey plasma. After oral administration, the bioavailability was 55.43%. The exposure (AUC0-t) of direct acyl glucuronide (AG) of iptacopan accounts for 9.73% of the iptacopan plasma exposure. The AUC0-t of AG of dealkylated metabolite of iptacopan was present at a lower level, accounting for 0.5% of the iptacopan plasma exposure.