MS Method Research Articles

Metabolism characterization and toxicity of N-hydap, a marine candidate drug for lung cancer therapy by LC–MS method

N-Hydroxyapiosporamide (N-hydap), a marine product derived from a sponge-associated fungus, has shown promising inhibitory effects on small cell lung cancer (SCLC). However, there is limited understanding of its metabolic pathways and characteristics. This study explored the in vitro metabolic profiles of N-hydap in human recombinant cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs), as well as human/rat/mice microsomes, and also the pharmacokinetic properties by HPLC–MS/MS. Additionally, the cocktail probe method was used to investigate the potential to create drug-drug interactions (DDIs). N-Hydap was metabolically unstable in various microsomes after 1 h, with about 50% and 70% of it being eliminated by CYPs and UGTs, respectively. UGT1A3 was the main enzyme involved in glucuronidation (over 80%), making glucuronide the primary metabolite. With a favorable bioavailability of 24.0%, N-hydap exhibited a higher distribution in the lungs (26.26%), accounting for its efficacy against SCLC. Administering N-hydap to mice at normal doses via gavage did not result in significant toxicity. Furthermore, N-hydap was found to affect the catalytic activity of drug metabolic enzymes (DMEs), particularly increasing the activity of UGT1A3, suggesting potential for DDIs. Understanding the metabolic pathways and properties of N-hydap should improve our knowledge of its drug efficacy, toxicity, and potential for DDIs.Graphical

Quality evaluation and identification of Houttuynia cordata bleached with sodium metabisulfite based on whole spectrum metabolomics

Houttuynia Cordata (HC) is a widely distributed plant in Asia and is used extensively for both food and medicinal purposes. A preliminary investigation found that HC is often bleached with sodium metabisulfite solution during its field processing, leading to health risks. In this study, the effects of sodium metabisulfite on the quality of HC were comprehensively evaluated using volatile and non-volatile targeted metabolomic methods. The results revealed a positive correlation between the extent of chemical composition changes and the bleaching time. These notable changes mainly occurred at the initial stage of bleaching. Subsequently, an untargeted UPLC/Q-TOF MS method was used to explore the potential chemical bleaching markers in bleached HC. The marker 1-hydroxy-3-oxodecane-1-sulfonic acid was subsequently prepared, isolated, and identified. Market sample verification further validated the accuracy and effectiveness of this marker.

DNA Walker-Driven Mass Nanotag Assembly System for Simultaneously Profiling Dual Markers of Oxidative Stress at Different Cellular Locations.

Simultaneous profiling of redox-regulated markers at different cellular sublocations is of great significance for unraveling the upstream and downstream molecular mechanisms of oxidative stress in living cells. Herein, by synchronizing dual target-triggered DNA machineries in one nanoentity, we engineered a DNA walker-driven mass nanotag (MNT) assembly system (w-MNT-AS) that can be sequentially activated by oxidative stress-associated mucin 1 (MUC1) and apurinic/apyrimidinic endonuclease 1 (APE1) from plasma membrane to cytoplasm and induce recycled assembly of MNTs for multiplex detection of the two markers by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In the working cascade, the sensing process governs the separate activation of w-MNT-AS by MUC1 and APE1 in diverse locations, while the assembly process contributes to the parallel amplification of the ion signal of the characteristic mass tags. In this manner, the differences between MCF-7, HeLa, HepG2, and L02 cells in membrane MUC1 expression and cytoplasmic APE1 activation were fully characterized. Furthermore, the oxidative stress level and dynamics caused by exogenous H2O2, doxorubicin, and simvastatin were comprehensively demonstrated by tracking the fate of the two markers across different cellular locations. The proposed w-MNT-AS coupled MS method provides an effective route to probe multiple functional molecules that lie at different locations while participating in the same cellular event, facilitating the mechanistic studies on cellular response to oxidative stress and other disease-related cellular processes.

Multiple-component covalent organic frameworks for simultaneous extraction and determination of multitarget pollutants in sea foods

Persistent organic pollutants (POPs), such as perfluoroalkyl and polyfluoroalkyl substances (PFASs), polychlorinated biphenyls (PCBs), and bisphenols (BPs), have been raising global concerns due to their toxic effects on environment and human health. The monitoring of residues of POPs in seafood is crucial for assessing the accumulation of these contaminants in the study area and mitigating potential risks to human health. However, the diversity and complexity of POPs in seafood present significant challenges for their simultaneous detection. Here, a novel multi-component fluoro-functionalized covalent organic framework (OH-F-COF) was designed as SPE adsorbent for simultaneous extraction POPs. On this basis, the recognition and adsorption mechanisms were investigated by molecular simulation. Due to multiple interactions and large specific surface area, OH-F-COF displayed satisfactory coextraction performance for PFASs, PCBs, and BPs. Under optimized conditions, the OH-F-COF sorbent was employed in a strategy of simultaneous extraction and stepwise elution (SESE), in combination with HPLC−MS/MS and GC−MS method, to effectively determined POPs in seafood collected from coastal areas of China. The method obtained low detection limits for BPs (0.0037 −0.0089 ng/g), PFASs (0.0038 −0.0207 ng/g), and PCBs (0.2308 −0.2499 ng/g), respectively. This approach provided new research ideas for analyzing and controlling multitarget POPs in seafood. Environmental implicationsPersistent organic pollutants (POPs), such as perfluoroalkyl and polyfluoroalkyl substances (PFASs), polychlorinated biphenyls (PCBs), and bisphenols (BPs), have caused serious hazards to human health and ecosystems. Hence, there is a need to develop a quantitative method that can rapidly detect POPs in environmental and food samples. Herein, a novel multi-component fluorine-functionalized covalent organic skeletons (OH-F-COF) were prepared at room temperature, and served as adsorbent for POPs. The SESE-SPE strategy combined with chromatographic techniques was used to achieve a rapid detection of POPs in sea foods from the coastal provinces of China. This method provides a valuable tool for analyzing POPs in environmental and food samples.

Mass spectrometry–based assessment of M protein in peripheral blood during maintenance therapy in multiple myeloma

AbstractMass spectrometry (MS) can detect multiple myeloma–derived monoclonal proteins in the peripheral blood (PB) with high sensitivity, potentially serving as a PB assay for measurable residual disease (MRD). This study evaluated the significance of PB MS MRD negativity during posttransplant therapy in patients with newly diagnosed multiple myeloma. Serum samples from 138 patients treated in the phase 3 ATLAS trial of posttransplant maintenance with either carfilzomib, lenalidomide, and dexamethasone, or with lenalidomide alone were analyzed using EXENT MS methodology. We established feasibility of measuring MRD by MS in the PB in the posttransplant setting, despite unavailability of pretreatment calibration samples. There was high agreement between MRD by MS in the PB and paired bone marrow (BM) MRD results at the 10–5 threshold, assessed by either next-generation sequencing (NGS) or multiparameter flow cytometry (MFC) (70% and 67%, respectively). Agreement between PB MS and both BM MRD methods was lowest early after transplant and increased with time. MS negativity was associated with improved progression-free survival (PFS), which, in landmark analysis, reached statistical significance after 18 cycles after transplant. Combined PB/BM MRD negativity by MFC or NGS was associated with superior PFS compared with MRD negativity by only 1 modality. Sustained MS negativity carried similar prognostic performance to sustained BM MRD negativity at the 10–5 threshold. Overall, posttransplant MS assessment was feasible and provided additional prognostic information to BM MRD negativity. Further studies are needed to confirm the role and optimal timing of MS in disease evaluation algorithms. The ATLAS trial is registered at www.clinicaltrials.gov as #NCT02659293.

Interleukin-13 Treatment of Living Lung Tissue Model Alters the Metabolome and Proteome-A Nano-DESI MS Metabolomics and Shotgun Proteomics Study.

Asthma is a chronic respiratory disease with one of the largest numbers of cases in the world; thus, constant investigation and technical development are needed to unravel the underlying biochemical mechanisms. In this study, we aimed to develop a nano-DESI MS method for the in vivo characterization of the cellular metabolome. Using air-liquid interface (ALI) cell layers, we studied the role of Interleukin-13 (IL-13) on differentiated lung epithelial cells acting as a lung tissue model. We demonstrate the feasibility of nano-DESI MS for the in vivo monitoring of basal-apical molecular transport, and the subsequent endogenous metabolic response, for the first time. Conserving the integrity of the ALI lung-cell layer enabled us to perform temporally resolved metabolomic characterization followed by "bottom-up" proteomics on the same population of cells. Metabolic remodeling was observed upon histamine and corticosteroid treatment of the IL-13-exposed lung cell monolayers, in correlation with alterations in the proteomic profile. This proof of principle study demonstrates the utility of in vivo nano-DESI MS for characterizing ALI tissue layers, and the new markers identified in our study provide a good starting point for future, larger-scale studies.

Impact of genetic κ-casein variants (A, B, E) on chymosin-induced milk coagulation properties: Application of a new LC–MS-based genotyping method

This study evaluated the impact of κ-casein (κ-CN) protein phenotype (AA (n = 18), AB (n = 18), AE (n = 5), BB (n = 3)) in the raw milk of 44 individual Holstein cows from a single farm on coagulation properties using diffusion wave spectroscopy. κ-CN variants were analyzed using a newly developed LC–MS method in individual milk samples, and relative milk protein fractions were quantified by LabChip-analysis. κ-CN fractions expressed in the percentage of total milk protein, total casein, and β- and κ-CN were then calculated. The gross composition of the individual milk samples and variation between individual cows was analyzed and was rather large, as expected. Traits were analyzed using an ANCOVA model with pH as a covariate.Our results showed that mean percentages of κ-CN fractions increased for κ-CN phenotypes in the order AA < AE < AB < BB. Rennet coagulation time was slightly affected, but curd firmness was significantly affected by κ-CN phenotype (BB > AE > AB > AA) when controlling for pH.

Identifying organisms directly from BacT/Alert FAN plus blood culture bottles using Vitek MS in a state-wide laboratory network

Identifying organisms directly from positive blood culture bottles using matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has many advantages to patients, clinical services, and laboratories. However, few published methods have demonstrated good performance using the current BioMérieux culture bottles and MALDI-TOF system: BacT/Alert FAN plus and Vitek MS. The effect of transporting bottles on test performance has not been assessed for any direct-from-bottle MS method. In this study, 802 positive blood culture bottles were analysed including 234 requiring inter-laboratory transport, using a method involving protein extraction with formic acid and acetonitrile. Correct identification rates were high for Staphylococcus aureus (58/58 of new diagnostic samples), Enterococcus faecalis (27/27), Gram-negative bacilli (160/176, 90.1%), and coagulase-negative Staphylococcus species (108/132, 81.8%). Three false identifications were made, none with clinical significance. For Gram-positive cocci in pairs or chains, more correct identifications were made from bottles analysed immediately compared to transported bottles (67% vs 44%, p=0.016), and longer transport time was associated with slightly lower probability of correct identification (OR 0.984 per additional hour, p=0.040). Transportation was not associated with a difference for other organism types. This technique is a vastly more cost-effective alternative to molecular techniques for rapid identification of bacteraemia isolates, and performance is minimally affected by inter-laboratory transport of bottles at ambient temperature.

Analysis and health risk assessment of polychlorinated biphenyls (PCB) in meat products (sausage) by using GC/MS

In this research, the amount of PCB (polychlorinated biphenyls) in sausage meat products was analyzed using magnetic adsorbent of Fe3O4@SiO2@PSS/PDDA/MWCNTs (Fe3O4@SiO2@poly(sodium4-styrenesulfonate)/poly diallyl dimethyl ammonium chloride/Multi walled carbon nanotubes) and GC–MS (Gas chromatography–mass spectrometry) method. The recovery (%), LODs (limit of detections) and LOQs (limit of quantifications) were 93.40–102.54 %, 0.03–0.12 ng/g fat and 0.10–0.36 ng/g fat, respectively. Also, the results indicated that the total mean concentrations of different PCB compounds, in sausages was ranged 0.08 (PCB 153) to 0.88 (PCB 138) ng/g fat. Additionally, PCB 52 had the highest concentration (10.72 ng/g fat) in sausage 80 %. The highest permissible level of PCB in meat products is 40 ng/g fat, which were lower than this limit in the evaluated samples. Finally, the outcomes of Monte Carlo simulation exhibited that the estimated daily intake of PCBs from sausage samples was below the existing tolerable daily intake of PCB compounds (Tolerable Daily Intake (TDI) < 10 ng/kg BW (body weight) per day). The ILCR (Incremental Lifetime Cancer Risk) was in the safe range (less than 6–10) for consumers of this product in Iran.

Comparison of methods for DMSP measurements in dinoflagellate cultures

AbstractA comparison of three analytical methods (the indirect GC‐FPD and MIMS, and direct LC‐MS/MS) for dimethylsulfoniopropionate (DMSP) measurements was conducted to assess their accuracy and reliability. The three methods showed a linear response but are distinguished by their linearity range, the largest being for MIMS. All three methods showed good precision on Alexandrium minutum samples (2–12%). The variability between the three methods when comparing analyses of A. minutum replicates was 11%, with the DMSP measurements by LC‐MS/MS being the highest. This result also confirms that indirect DMSP measurement after hydrolysis for GC or MIMS methods does not lead to an overestimation of DMSP values in A. minutum. A special focus was made on the more recent LC‐MS/MS method including further assays in sample preparation and storage from cultures of the dinoflagellate A. minutum. Dinoflagellate cells should be harvested by gentle filtration (&lt; 5 cm Hg) or slow centrifugation (500 × g) to retrieve the largest DMSP pool. For the LC‐MS/MS method, MeOH used for cell extraction should be added prior to freezing (to prevent DMSP degradation). Samples will then be stable in frozen storage for at least 2 months. Finally, direct and indirect methods are complementary for identifying the exact DMSP fraction among dimethylsulfide‐producing compounds that compose total and particulate DMSP pools issued from newly screened organisms or environmental samples.

Silane Gas Production Through Hydrolysis of Magnesium Silicide by Hydrochloric Acid

Monosilane (SiH4) is a common precursor for the production of high-purity silicon for solar PV applications. As an alternative to carbothermic reduction of silica to produce metallurgical grade silicon with subsequent conversion to silane, an alternative route over magnesiothermic reduction of silica to Mg2Si has been explored in our earlier work. In the current work, silane gas production through hydrolysis of Mg2Si in HCl acid solution was studied. Two sources of Mg2Si were chosen: a commercial Mg2Si source and a Mg2Si source produced through magnesiothermic reduction of high-purity natural quartz. Effects of various parameters on the hydrolysis of Mg2Si, including different experimental setups, temperature of the acid solution, acid concentration, reaction time, and relative amounts of reactants were studied. The evolution of produced gases was determined by two different methods: firstly, by passing the produced gas through a KOH solution to capture Si with subsequent analysis of the Si content in the KOH solution by inductively coupled plasma mass spectrometry and secondly, on-line gas analysis by GC–MS. The silane distribution between different silane species with reaction time was evaluated and the activation energy of silane formation was calculated. The results indicated comparable silane yields obtained from the on-line GC–MS method and KOH solution analysis method, as well as for commercial Mg2Si and the Mg2Si–MgO mixture produced through magnesiothermic reduction. Furthermore, adding HCl acid to Mg2Si in water led to higher SiH4 formation yield than adding Mg2Si to acid. However, the total silane yield for the two methods was similar at approximately 32%.Graphical

High-Performance Thin-Layer Chromatography Coupled with Single Quadrupole: Application the Identification and Differentiation of Rehmanniae Radix and Its Different Processing Products from Raw Materials to Commercial Products.

The authentication of ingredients in formulas is crucial yet challenging, particularly for constituents with comparable compositions but vastly divergent efficacy. Rehmanniae Radix and its derivatives are extensively utilized in food supplements, which contain analogous compositions but very distinct effects. Rehmanniae Radix, also a difficult-to-detect herbal ingredient, was chosen as a case to explore a novel HPTLC-QDa MS technique for the identification of herbal ingredients in commercial products. Through systematic condition optimization, including thin layer and mass spectrometry, a stable and reproducible HPTLC-QDa MS method was established, which can simultaneously detect oligosaccharides and iridoids. Rehmannia Radix and its processed products were then analyzed to screen five markers that could distinguish between raw and prepared Rehmannia Radix. An HPTLC-QDa-SIM method was further established for formula detection by using the five markers and validated using homemade prescriptions and negative controls. Finally, this method was applied to detect raw and prepared Rehmannia Radix in 12 commercial functional products and supplements.

Antioxidant capacity sources of soils under different land uses

Antioxidants (AOX) in soils originate mainly from secondary plant metabolites and are pivotal in many redox processes in environment, maintaining soil quality. Still, little is known about the influence of land uses on their accumulation in soil. The aim of the paper was to determine the content of these redox-active compounds in the extracts of A horizons of abandoned fallows, arable and woodland soils. Total antioxidant capacity (TAC) of soils under various uses and vegetation was evaluated in different soil extracts using Folin-Ciocâlteu method. The contribution of humic acids to TAC was determined and antioxidant profiles estimated using the chromatographic GC–MS method. Forest soils exhibited the highest TAC (15.5 mg g−1) and AOX contents (4.34 mg g−1), which were positively correlated with soil organic carbon content. It was estimated that humic acids contribute to over 50% of TAC in soils. The main phenolics in woodland A horizons were isovanillic and p-hydroxybenzoic acid (p-HA), while esculetin and p-HA predominated in the abandoned fallows due to the prevalence of herbaceous vegetation. Cultivated soils were the most abundant in p-HA (56.42%). In the studied topsoils, there were considerable amounts of aliphatic organic matter, which role in redox processes should be further evaluated.

Sustentaculum fracture fixation with lateral plate or medial screw fixation are equivalent

BackgroundFixation of sustentaculum tali fractures is important to maintain the biomechanical function of the subtalar joint. A common method of fixation is securing the sustentacular fragment by way of a laterally based locking plate (LP). A medial approach with a single screw (MS) has been proposed as an alternative method of fixation. MethodsFive pairs of formalin-preserved cadaveric ankles with the subtalar joint and interosseous ligaments intact (“osseous cadavers”) and four pairs of fresh-frozen cadaveric ankles with soft-tissue preserved dissected from mid-tibia down (“soft tissue cadavers”) were used in the study. The left ankle was randomly assigned to one of the two fixation methods (LP or MS), while the right ankle was the opposite. These same steps for fixation were repeated for six synthetic ankle models. All models were loaded with a body mass of 80 kg. Statistical differences between LP and MS stiffness were determined using a paired t-test in cadavers and un-paired t-tests in synthetic ankles. ResultsFor osseous cadaveric ankles, LP demonstrated a mean stiffness of 232.95(SD: 59.96) N/mm, while MS was 239.72(SD:131.09) N/mm (p = 0.9293). For soft tissue cadaveric ankles, LP mean stiffness was 133.58(SD:37.84) N/mm, while MS was 134.88(SD:20.75) N/mm (p = 0.9578). For synthetic ankles, LP mean stiffness was 220.40(SD:81.93) N/mm, while MS was 261.50(SD:100.21) N/mm (p = 0.6116). ConclusionsAcross all three models, there was no significant difference between LP and MS methods. Retrospective observational studies are recommended to assess patient outcomes from each of the methods.

Characterization of four peptides from milk fermented with kombucha cultures and their metal complexes-in search of new biotherapeutics.

The most common skin diseases include eczema, psoriasis, acne, and fungal infections. There is often no effective cure for them. Increasing antimicrobial drug resistance prompts us to search for new, safe, and effective therapeutics. Among such interesting candidates are peptides derived from milk fermented with specific lactic acid bacteria or with kombucha cultures, which are a potential treasure trove of bioactive peptides. Four of them are discussed in this article. Their interactions with zinc and copper ions, which are known to improve the well-being of the skin, were characterized by potentiometry, MS, ITC, and spectroscopic methods, and their cytostatic potential was analyzed. The results suggest that they are safe for human cells and can be used alone or in complexes with copper for further testing as potential therapeutics for skin diseases.

Demonstration of Interaction between Carbapenem Group Antibiotics and Different Immunosuppressant Drugs by Molecular Docking

Background: It has been shown that drugs used parenterally cause errors in immunosuppressant concentrations measured by LC-MS / MS method. It is yet unknown whether this measurement error is due to drug-drug interaction or analytical interference. Objective: The aim of this study is to investigate the possible interaction and inhibition concentrations of broad-spectrum antibiotics (ertapenem, meropenem, imipenem) with 4 different immunosuppressants (tacrolimus, sirolimus, everolimus, cyclosporine A) by molecular docking. Method: The docking results of ertapenem, meropenem, and imipenem-cilastatin drugs, which are frequently used in intensive care units and wards, were analyzed with the Autodock 4.2 program. Binding energy levels and inhibition concentrations were recorded. Results: The highest binding energies of the most stable conformations, providing the best compatibility among the active ingredients, belong to cilastatin. The interaction energy of cilastatin with sirolimus in 320 conformations was calculated as -4.08 kcal/mol. Sirolimus interacted with ertapenem at -3.43, imipenem at -2.53, and meropenem at -3.84 kcal/mol. According to these values, the receptor, which is the most compatible host with all ligand molecules, is sirolimus. The least interaction energy value was calculated between cyclosporine and imipenem (-1.12 kcal / mol). Conclusion: Concerning the most stable conformations of models docked with Autodock tools, it has been determined that carbapenems interact with immunosuppressants. Since the detected inhibition concentration levels can be seen in blood samples taken immediately after carbapenem injection, immunosuppressant measurement is recommended before the use of carbapenem in immunosuppressant monitoring of transplant patients.

Molecularly Imprinted Heterostructure-Assisted Laser Desorption Ionization Mass Spectrometry Analysis and Imaging of Quinolones.

Quinolone residues resulting from body metabolism and waste discharge pose a significant threat to the ecological environment and to human health. Therefore, it is essential to monitor quinolone residues in the environment. Herein, an efficient and sensitive matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) method was devised by using a novel molecularly imprinted heterojunction (MIP-TNs@GCNs) as the matrix. Molecularly imprinted titanium dioxide nanosheets (MIP-TNs) and graphene-like carbon nitrides (GCNs) were associated at the heterojunction interface, allowing for the specific, rapid, and high-throughput ionization of quinolones. The mechanism of MIP-TNs@GCNs was clarified using their adsorption properties and laser desorption/ionization capability. The prepared oxygen-vacancy-rich MIP-TNs@GCNs heterojunction exhibited higher light absorption and ionization efficiencies than TNs and GCNs. The good linearity (in the quinolone concentration range of 0.5-50 pg/μL, R2 > 0.99), low limit of detection (0.1 pg/μL), good reproducibility (n = 8, relative standard deviation [RSD] < 15%), and high salt and protein resistance for quinolones in groundwater samples were achieved using the established MIP-TNs@GCNs-MALDI/MS method. Moreover, the spatial distributions of endogenous compounds (e.g., amino acids, organic acids, and flavonoids) and xenobiotic quinolones from Rhizoma Phragmitis and Rhizoma Nelumbinis were visualized using the MIP-TNs@GCNs film as the MALDI/MS imaging matrix. Because of its superior advantages, the MIP-TNs@GCNs-MALDI/MS method is promising for the analysis and imaging of quinolones and small molecules.

A Targeted Approach for the Metabolome Analysis of E. coli Biofilms

AbstractBiofilms of pathogenic bacteria are responsible for persistent infections in humans, therefore investigations of biofilm formation and treatment strategies are required. The gram‐negative enterobacterium Escherichia (E.) coli is the most common pathogen causing chronic or recurring urinary tract infections. Metabolomics approaches targeted the bacterium to investigate specific metabolic patterns of biofilms and regulatory influences on biofilm formation. In this study, we aimed to investigate the metabolome of biofilms formed by the multidrug‐resistant extended‐spectrum beta‐lactamase‐producing (ESBL) E. coli PBIO729. For this purpose, a protocol for fast sampling of the macrocolony biofilms and efficient extraction of metabolites was optimized. Validation of an LC‐MS/MS method confirmed its usability for the analysis of nucleotides and other phosphorylated metabolites. A GC‐MS approach was used to monitor nutrient uptake from the medium in addition to the analysis of amino acid content and metabolites of glycolysis and TCA cycle in E. coli biofilms.

Profile of Phenolic Compounds and Antioxidant Activity of Celery (Apium graveolens) Juices Obtained from Pulp after α-Amylase Treatment from Aspergillus oryzae.

The purpose of this study was to determine the content of certain phenolic compounds, antioxidant activity, pressing efficiency, extract content, and sugars in celeriac juices obtained from the pulp after α-amylase treatment from Aspergillus oryzae. The test material consisted of peeled and unpeeled celery pulp kept at a temperature of 25 °C with and without the enzyme for a period of 30 and 60 min. The juices obtained from them were analyzed for the content of selected phenolic acids and flavonoids using the UPLC-PDA-ESI-MS/MS method, for antioxidant activity measured using the ABTS˙+ and DPPH˙ method, and for the total polyphenol content using the F-C method. Additionally, the juice pressing efficiency, the extract content using the refractometer method, and the sugar content using the HPLC method were checked. Significantly higher antioxidant activity, pressing yield, and average content of caffeic acid glucoside, quinic acid, kaempferol-3,7-di-O-glucoside, and chrysoeriol-7-O-apiosylglucoside were obtained in juices from peeled celery. Maceration of the pulp with amylase resulted in a significant reduction in antioxidant activity compared to control samples. An is-total increase of 17-41% in total flavonoid content was observed in all juices tested after treatment with the enzyme for 30 and 60 min, and the phenolic acid content increased by 4-41% after treatment of the pulp with amylase for 60 min. The 60 min holding of the pulp at 25 °C, including with the enzyme, was shown to decrease the antioxidant activity and the content of quinic acid, ferulic acid, and chrysoriol-7-O-apiose-glucoside in the juices tested compared to the samples held for 30 min, while the content of other phenolic acids and flavonoids increased. In addition, after 60 min of enzymatic maceration, the pressing yield of the juices increased.

A fully validated LC‑MS/MS method for quantifyingbevacizumab in plasma samples from patients with NSCLC and its implications in therapeutic drug monitoring.

Given the increasing use of bevacizumab in combinatorial drug therapy for a multitude of different cancer types, there is a need for therapeutic drug monitoring to analyze the possible correlation between drug trough concentration, and therapeutic effect and adverse reactions. An ultra-performance liquid chromatography tandem-mass spectrometry method was then developed and validated to determine bevacizumab levels in human plasma samples. Chromatographic separation was achieved on a Shimadzu InertSustainBio C18 HP column, whereas subsequent mass spectrometric analysis was performed using a Shimadzu 8050CL triple quadrupole mass spectrometer equipped with an electro-spray ionization source in the positive ion mode. In total, three multiple reaction monitoring transitions of each of the surrogate peptides were chosen with 'FTFSLDTSK' applied as the quantification peptide whereas 'VLIYFTSSLHSGVPSR' and 'STAYLQMNSLR' were designated as the verification peptides using the Skyline software. This analytical method was then fully validated, with specificity, linearity, lower limit of quantitation, accuracy, precision, stability, matrix effect and recovery calculated. The linearity of this method was developed to be within the concentration range 5-400 µg/ml for bevacizumab in human plasma. Subsequently, eight patients with non-small cell lung cancer (NSCLC) were recruited and injected with bevacizumab over three periods of treatment to analyze their steady-state trough concentration and differences. To conclude, the results of the present study suggest that bevacizumab can be monitored in a therapeutic setting in patients with NSCLC.