Impact of four different extraction methods and three different reconstitution solvents on the untargeted metabolomics analysis of human and rat urine samples.

Unsuitable sample preparation may result in loss of important analytes and consequently affect the outcome of untargeted metabolomics. Due to species differences, different sample preparations may be required within the same biological matrix. The study aimed to compare the in-house sample preparation method for urine with methods from literature and to investigate the transferability of sample preparation from human urine to rat urine. A total of 12 different conditions for protein precipitation were tested, combining four different extraction solvents and three different reconstitution solvents using an untargeted liquid-chromatography high resolution mass spectrometry (LC-HRMS) metabolomics analysis. Evaluation was done based on the impact on feature count, their detectability, as well as the reproducibility of selected compounds. Results showed that a combination of methanol as extraction and acetonitrile/water (75/25) as reconstitution solvent provided improved results at least regarding the total feature count. Additionally, it was found that a higher amount of methanol was most suitable for extraction of rat urine among the tested conditions. In comparison, human urine requires significantly less volume of extraction solvent. Overall, it is recommended to systematically optimize both, the extraction method, and the reconstitution solvent for the used biofluid and the individual analytical settings.

Organic-solvent ditch overlap in reversed-phase liquid chromatography: A molecular dynamics simulation study in cylindrical 6-12 nm-diameter pores.

Mass transport through the mesopore space of a reversed-phase liquid chromatography (RPLC) column depends on the properties of the chromatographic interface, particularly on the extent of the organic-solvent ditch that favors the analyte surface diffusivity. Through molecular dynamics simulations in cylindrical RPLC mesopore models with pore diameters between 6 and 12 nm we systematically trace the evolution of organic-solvent ditch overlap due to spatial confinement in the mesopore space of RPLC columns for small-molecule separations. Each pore model of a silica-based, endcapped, C18-stationary phase is equilibrated with two mobile phases of comparable elution strength, namely 70/30 (v/v) water/acetonitrile and 60/40 (v/v) water/methanol, to consider the influence of the mobile-phase composition on the onset of organic-solvent ditch overlap. The simulations show that, as the pore diameter decreases from 9 to 6 nm, the bonded-phase density extends and compacts towards the pore center, which leads to increased accumulation of organic-solvent excess and thus enhanced organic-solvent diffusivity in the ditch. Because the acetonitrile ditch is more pronounced than the methanol ditch, acetonitrile ditch overlap sets in at less severe spatial confinement than methanol ditch overlap. The pore-averaged methanol and acetonitrile diffusivities are considerably raised by ditch overlap in the 6 nm-diameter pore, but also benefit from the ditch (without overlap) in the 7 to 12 nm-diameter pores, whereby local and pore-averaged effects are generally larger for acetonitrile than methanol.

Activity and resistance to KRASG12C inhibitors in non-small cell lung cancer and colorectal cancer.

Non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) are associated with a high mortality rate. Mutations in the V-Ki-ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) proto-oncogene GTPase (KRAS) are frequently observed in these cancers. Owing to its structural attributes, KRAS has traditionally been regarded as an "undruggable" target. However, recent advances have identified a novel mutational regulatory site, KRASG12C switch II, leading to the development of two KRASG12C inhibitors (adagrasib and sotorasib) that are FDA-approved. This groundbreaking discovery has revolutionized our understanding of the KRAS locus and offers treatment options for patients with NSCLC harboring KRAS mutations. Due to the presence of alternative resistance pathways, the use of KRASG12C inhibitors as a standalone treatment for patients with CRC is not considered optimal. However, the combination of KRASG12C inhibitors with other targeted drugs has demonstrated greater efficacy in CRC patients harboring KRAS mutations. Furthermore, NSCLC and CRC patients harboring KRASG12C mutations inevitably develop primary or acquired resistance to drug therapy. By gaining a comprehensive understanding of resistance mechanisms, such as secondary mutations of KRAS, mutations of downstream intermediates, co-mutations with KRAS, receptor tyrosine kinase (RTK) activation, Epithelial-Mesenchymal Transitions (EMTs), and tumor remodeling, the implementation of KRASG12C inhibitor-based combination therapy holds promise as a viable solution. Furthermore, the emergence of protein hydrolysis-targeted chimeras and molecular glue technologies has been facilitated by collaborative efforts in structural science and pharmacology. This paper aims to provide a comprehensive review of the recent advancements in various aspects related to the KRAS gene, including the KRAS signaling pathway, tumor immunity, and immune microenvironment crosstalk, as well as the latest developments in KRASG12C inhibitors and mechanisms of resistance. In addition, this study discusses the strategies used to address drug resistance in light of the crosstalk between these factors. In the coming years, there will likely be advancements in the development of more efficacious pharmaceuticals and targeted therapeutic approaches for treating NSCLC and CRC. Consequently, individuals with KRAS-mutant NSCLC may experience a prolonged response duration and improved treatment outcomes.

Time-resolved keto-enol tautomerization of the medicinal pigment curcumin.

Curcumin is a medicinal agent that exhibits anti-cancer and anti-Alzheimer's disease properties. It has a keto-enol moiety that gives rise to many of its chemical properties including metal complexation and acid-base equilibria. A previous study has shown that keto-enol tautomerization at this moiety is implicated in the anti-Alzheimer's disease effect of curcumin, highlighting the importance of this process. In this study, tautomerization of curcumin in methanol, acetone and acetonitrile was investigated using time-resolved 1H nuclear magnetic resonance spectroscopy. Curcumin undergoes hydrogen-deuterium exchange with the solvents and the proton resonance peak corresponding to the hydrogen at the α-carbon position (Cα) decays as a function of time, signifying deuteration at this position. Because tautomerization is the rate limiting step in the deuteration of curcumin at the Cα position, the rate of tautomerization is inferred from the rate of deuteration. The rate constant of tautomerization of curcumin shows a temperature dependence and analysis using the Arrhenius equation revealed activation energies (Ea) of tautomerization of (80.1 ± 5.9), (64.1 ± 1.0) and (68.3 ± 5.5) kJ mol-1 in methanol, D2O/acetone and D2O/acetonitrile, respectively. Insight into the role of water in tautomerization of curcumin was further offered by density functional theory studies. The transition state of tautomerization was optimized in the presence of water molecules. The results show a hydrogen-bonded solvent bridge between the diketo moiety and Cα of curcumin. The Ea of tautomerization of curcumin shows a strong dependence on the number of water molecules in the solvent bridge, indicating the critical role played by the solvent bridge in catalyzing tautomerization of curcumin.

Targeted therapeutic options in early and metastatic NSCLC-overview.

The complex therapeutic strategy of non-small cell lung cancer (NSCLC) has changed significantly in recent years. Disease-free survival increased significantly with immunotherapy and chemotherapy registered in perioperative treatments, as well as adjuvant registered immunotherapy and targeted therapy (osimertinib) in case of EGFR mutation. In oncogenic-addictive metastatic NSCLC, primarily in adenocarcinoma, the range of targeted therapies is expanding, with which the expected overall survival increases significantly, measured in years. By 2021, the FDA and EMA have approved targeted agents to inhibit EGFR activating mutations, T790 M resistance mutation, BRAF V600E mutation, ALK, ROS1, NTRK and RET fusion. In 2022, the range of authorized target therapies was expanded. With therapies that inhibit KRASG12C, EGFR exon 20, HER2 and MET. Until now, there was no registered targeted therapy for the KRAS mutations, which affect 30% of adenocarcinomas. Thus, the greatest expectation surrounded the inhibition of the KRAS G12C mutation, which occurs in ∼15% of NSCLC, mainly in smokers and is characterized by a poor prognosis. Sotorasib and adagrasib are approved as second-line agents after at least one prior course of chemotherapy and/or immunotherapy. Adagrasib in first-line combination with pembrolizumab immunotherapy proved more beneficial, especially in patients with high expression of PD-L1. In EGFR exon 20 insertion mutation of lung adenocarcinoma, amivantanab was registered for progression after platinum-based chemotherapy. Lung adenocarcinoma carries an EGFR exon 20, HER2 insertion mutation in 2%, for which the first targeted therapy is trastuzumab deruxtecan, in patients already treated with platinum-based chemotherapy. Two orally administered selective c-MET inhibitors, capmatinib and tepotinib, were also approved after chemotherapy in adenocarcinoma carrying MET exon 14 skipping mutations of about 3%. Incorporating reflex testing with next-generation sequencing (NGS) expands personalized therapies by identifying guideline-recommended molecular alterations.

Affinity Measurement of Non-covalent Interactions of the Covalent KRAS G12C GDP Inhibitor MRTX849 to RAS Isoforms Using Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is an optical effect at an electron-rich surface that enables affinity measurements of biomolecules in real time. It is label free and versatile, not limited to proteins, nucleic acids, and small molecules. SPR is a widely accepted method to measure not only affinity of molecular interactions but also association and dissociation rates of such interactions. In this chapter, we describe a general method to measure the affinity of a small molecule drug, MRTX849, to GDP bound HRAS, KRAS, and NRAS.

[Determination of 222 pesticide residues in olive oil by fully automatic QuEChERS pre-treatment instrument coupled with gas chromatography-quadrupole-time-of-flight mass spectrometry].

Pesticide residues may be present in olive oil because pesticides are applied to olive trees during their cultivation and growth for pest prevention and some of these pesticides are not easily degraded. Studies on pesticide residues in olive oil have mainly focused on the detection of single types of pesticide residues, and reports on the simultaneous detection of multiple pesticide residues are limited. At present, hundreds of pesticides with different polarities and chemical properties are used in practice. In this study, an analytical method based on fully automatic QuEChERS pretreatment instrument coupled with gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOF-MS) was established for the rapid determination of 222 pesticide residues in olive oil. The effects of acetonitrile acidification concentration, n-hexane volume, oscillation time, centrifugation temperature, and purification agent on the determination of the 222 pesticide residues were investigated. First, ions with good responses and no obvious interference were selected for quantification and characterization. The purification process was then developed by setting the parameters of the fully automatic QuEChERS pretreatment instrument to optimal values. The sample was extracted with acetonitrile containing 2% formic acid, and the supernatant was purified by centrifugation in a centrifuge tube containing 400 mg N-propylethylenediamine (PSA), 400 mg octadecylsilane-bonded silica gel (C18), and 1200 mg anhydrous magnesium sulfate. The purified solution was blown dry with nitrogen and then fixed with ethyl acetate for instrumental analysis. Finally, a matrix standard solution was used for quantification. The method was validated in terms of matrix effects, linear ranges, limits of detection (LODs) and quantification (LOQs), accuracies, and precisions. The results showed that 86.04% of the 222 pesticides had linear ranges of 0.02-2.00 µg/mL, 10.81% had linear ranges of 0.10-2.00 µg/mL, and 3.15% had linear ranges of 0.20-2.00 µg/mL. The pesticide residues showed good relationships within their respective linear ranges, and the correlation coefficients (R2) were greater than 0.99. The LODs of all tested pesticides ranged from 0.002 to 0.050 mg/kg, and their LOQs ranged from 0.007 to 0.167 mg/kg. Among the 222 pesticides determined, 170 pesticides had LOQs of 0.007 mg/kg while 21 pesticides had LOQs of 0.017 mg/kg. At the three spiked levels of 0.2, 0.5, and 0.8 mg/kg, 79.58% of all tested pesticides had average recoveries of 70%-120% while 65.92% had average recoveries of 80%-110%. In addition, 93.54% of all tested pesticides had relative standard deviations (RSDs, n=6)<10% while 98.35% had RSDs (n=6)<20%. The method was applied to 14 commercially available olive oil samples, and seven pesticides were detected in the range of 0.0044-0.0490 mg/kg. The residues of fenbuconazole, chlorpyrifos, and methoprene did not exceed the maximum limits stated in GB 2763-2021. The maximum residual limits of molinate, monolinuron, benalaxyl, and thiobencarb have not been established. The method utilizes the high mass resolution capability of TOF-MS, which can improve the detection throughput while ensuring good sensitivity. In addition, high-resolution and accurate mass measurements render the screening results more reliable, which is necessary for the high-throughput detection of pesticide residues. The use of a fully automatic QuEChERS instrument in the pretreatment step reduces personnel errors and labor costs, especially when a large number of samples must be processed, thereby offering significant advantages over other approaches. Moreover, the method is simple, rapid, sensitive, highly automatable, accurate, and precise. Thus, it meets requirements for the high-throughput detection of pesticide residues in olive oil and provides a reference for the development of detection methods for pesticide residues in other types of oils as well as the automatic pretreatment of complex matrices.

Anti-Inflammatory Effects of a Novel Acetonitrile-Water Extract of Lens Culinaris against LPS-Induced Damage in Caco-2 Cells.

Natural compounds like flavonoids preserve intestinal mucosal integrity through their antioxidant, anti-inflammatory, and antimicrobial properties. Additionally, some flavonoids show prebiotic abilities, promoting the growth and activity of beneficial gut bacteria. This study investigates the protective impact of Lens culinaris extract (LE), which is abundant in flavonoids, on intestinal mucosal integrity during LPS-induced inflammation. Using Caco-2 cells as a model for the intestinal barrier, the study found that LE did not affect cell viability but played a cytoprotective role in the presence of LPS. LE improved transepithelial electrical resistance (TEER) and tight junction (TJ) protein levels, which are crucial for barrier integrity. It also countered the upregulation of pro-inflammatory genes TRPA1 and TRPV1 induced by LPS and reduced pro-inflammatory markers like TNF-α, NF-κB, IL-1ß, and IL-8. Moreover, LE reversed the LPS-induced upregulation of AQP8 and TLR-4 expression. These findings emphasize the potential of natural compounds like LE to regulate the intestinal barrier and reduce inflammation's harmful effects on intestinal cells. More research is required to understand their mechanisms and explore therapeutic applications, especially for gastrointestinal inflammatory conditions.

Integrated transcriptomic and metabolomic analysis of the hepatotoxicity of dichloroacetonitrile.

Dichloroacetonitrile (DCAN), an emerged nitrogenous disinfection by-product (N-DBP) in drinking water, has garnered attention owing to its strong cytotoxicity, genotoxicity, and carcinogenicity. However, there are limited studies on its potential hepatotoxicity mechanisms. Understanding hepatotoxicity is essential in order to identify and assess the potential risks posed by environmental pollutants on liver health and to safeguard public health. Here, we investigated the viability, reactive oxygen species (ROS) levels, and cell cycle profile of DCAN-exposed HepG2 cells and analyzed the mechanism of DCAN-induced hepatotoxicity using both transcriptomic and metabolomic techniques. The study revealed that there was a decrease in cell viability, increase in ROS production, and increase in the number of cells in the G2/M phase with an increase in the concentration of DCAN. Omics analyses showed that DCAN exposure increased cellular ROS levels, leading to oxidative damage in hepatocytes, which further induced DNA damage, cell cycle arrest, and cell growth impairment. Thus, DCAN has significant toxic effects on hepatocytes. Integrated analysis of transcriptomics and metabolomics offers new insights into the mechanisms of DCAN-induced hepatoxicity.

Preparation of high-efficiency HILIC capillary columns utilizing slurry packing at 2100 bar.

Complex mixture analysis requires high-efficiency chromatography columns. Although reversed phase liquid chromatography (RPLC) is the dominant approach for such mixtures, hydrophilic interaction liquid chromatography (HILIC) is an important complement to RPLC by enabling the separation of polar compounds. Chromatography theory predicts that small particles and long columns will yield high efficiency; however, little work has been done to prepare HILIC columns longer than 25 cm packed with sub-2 µm particles. In this work, we tested the slurry packing of 75 cm long HILIC columns with 1.7 µm bridged-ethyl-hybrid amide HILIC particles at 2,100 bar (30,000 PSI). Acetonitrile, methanol, acetone, and water were tested as slurry solvents, with acetonitrile providing the best columns. Slurry concentrations of 50-200 mg/mL were assessed, and while 50-150 mg/mL provided comparable results, the 150 mg/mL columns provided the shortest packing times (9 min). Columns prepared using 150 mg/mL slurries in acetonitrile yielded a reduced minimum plate height (hmin) of 3.3 and an efficiency of 120,000 theoretical plates for acenaphthene, an unretained solute. Para-toluenesulfonic acid produced the lowest hmin of 1.9 and the highest efficiency of 210,000 theoretical plates. These results identify conditions for producing high-efficiency HILIC columns with potential applications to complex mixture analysis.

Detection of trace components in Xiangdan injection of Dalbergia odorifera based on microextraction and back-extraction along with bar-form-diagram strategy.

Xiangdan Injection are commonly used traditional Chinese medicine formulations for the clinical treatment of cardiovascular diseases. However, the trace components of Dalbergia odorifera in Xiangdan Injection pose a challenge for evaluating its quality due to the difficulty of detection. This study proposes a technology combining dispersive liquid-liquid microextraction and back-extraction (DLLME-BE) along with Bar-Form-Diagram (BFD) to address this issue. The proposed combination method involves vortex-mixing tetradecane, which has a lower density than water, with the sample solution to facilitate the transfer of the target components. Subsequently, a new vortex-assisted liquid-liquid extraction step is performed to enrich the components of Dalbergia odorifera in acetonitrile. The sample analysis was performed on HPLC-DAD, and a clear overview of the chemical composition was obtained by integrating spectral and chromatographic information using BFD. The combination of BFD and CRITIC-TOPSIS strategies was used to optimize the process parameters of DLLME-BE. The determined optimal sample pre-treatment process parameters were as follows: 200 µL extraction solvent, 60 s extraction time, 50 µL back-extraction solvent, and 90 s back-extraction time. Based on the above strategy, a total of 29 trace components, including trans-nerolidol, were detected in the Xiangdan Injection. This combination technology provides valuable guidance for the enrichment analysis of trace components in traditional Chinese medicines.

Preparation and evaluation of a piperidinium-sulfonate based zwitterionic monolith for HILIC separation.

In this study, a novel piperidinium-sulfonate based zwitterionic hydrophilic monolith was prepared through thermally initiated co-polymerization of a piperidinium-sulfonate monomer 3-(4-((methacryloyloxy)methyl)-1-methylpiperidin-1-ium-1-yl)propane-1-sulfonate (MAMMPS), and a hydrophilic crosslinker N,N'-methylenebisacrylamide (MBA) using n-propanol and H2O as porogenic system. Satisfactory mechanical and chemical stabilities, good repeatability and high column efficiency (120,000 N/m) were obtained on the optimal monolith. The resulting poly(MAMMPS-co-MBA) monolith showed a typical HILIC retention behavior over an ACN content range between 5 and 95 %. Furthermore, this column exhibited good separation performance for various polar compounds. Compared to quaternary ammonium-sulfonate based zwitterionic hydrophilic monolith, i.e. poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine-co-MBA), the poly(MAMMPS-co-MBA) monolith displayed stronger retention and better selectivity for the tested phenolic and amine compounds at different pH conditions. Finally, this column was applied for the separation of six sulfonamide antibiotics, and the analytical characteristics of the method were evaluated in terms of precision, repeatability, limits of detection (LOD) and quantitation (LOQ). Overall, this study not only developed a novel HILIC monolithic column, but also proved the potential of piperidinium-sulfonate based zwitterionic chemistry as stationary phase, which further increased the structure diversity of zwitterionic HILIC stationary phases.

Scandium Ion-Promoted Electron-Transfer Disproportionation of 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-Oxide (PTIO•) in Acetonitrile and Its Regeneration Induced by Water.

2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO•), a persistent nitronyl nitroxide radical, has been used for the detection and trapping of nitric oxide, as a redox mediator for batteries, for the activity estimation of antioxidants, and so on. However, there is no report on the reactivity of PTIO• in the presence of redox-inactive metal ions. In this study, it is demonstrated that the addition of scandium triflate, Sc(OTf)3 (OTf = OSO2CF3), to an acetonitrile (MeCN) solution of PTIO• resulted in an electron-transfer disproportionation to generate the corresponding cation (PTIO+) and anion (PTIO-), the latter of which is suggested to be stabilized by Sc3+ to form [(PTIO)Sc]2+. The decay of the absorption band at 361 nm due to PTIO•, monitored using a stopped-flow technique, obeyed second-order kinetics. The second-order rate constant for the disproportionation, thus determined, increased with increasing the Sc(OTf)3 concentration to reach a constant value. A drastic change in the cyclic voltammogram recorded for PTIO• in deaerated MeCN containing 0.10 M Bu4NClO4 was also observed upon addition of Sc(OTf)3, suggesting that the large positive shift of the one-electron reduction potential of PTIO• (equivalent to the one-electron oxidation potential of PTIO-) in the presence of Sc(OTf)3 may result in the disproportionation. When H2O was added to the PTIO•-Sc(OTf)3 system in deaerated MeCN, PTIO• was completely regenerated. It is suggested that the complex formation of Sc3+ with H2O may weaken the interaction between PTIO- and Sc3+, leading to electron-transfer comproportionation to regenerate PTIO•. The reversible disproportionation of PTIO• was also confirmed by electron paramagnetic resonance (EPR) spectroscopy.

Separation of ochratoxins by centrifugal partition chromatography.

The present research work was dedicated to developing an efficient method based on liquid-liquid chromatography (centrifugal partition chromatography, CPC) applicable to routine purifications of ochratoxins (OT) from the liquid culture of the strain A. albertensis SZMC 2107. The crude extract contained numerous components in addition to OTA (90.1 %,) and OTB (1.1 %,) according to HPLC examinations. For the separation of OTs by CPC, several tertiary systems based on acetonitrile, acetone, and short-chain alcohols were examined to find the most applicable biphasic system. The hexane/i-propanol/water 35:15:50 system supplemented with 0.1 % acetic acid was found to be the most efficient for use in CPC separation. Using liquid-liquid instrumental separation, the two OTs, namely OTA (2.23 mg) and OTB (0.031 mg), were successfully isolated with 96.3 % and-72.8 % purity, respectively, from 1 L ferment broth. The identities and purities of the purified components were confirmed and the performance parameters of each separation step and the whole procedure were determined. The developed method could be used effectively to purify OTs for analytical or toxicological applications.

Adagrasib Treatment After Sotorasib-Related Hepatotoxicity in Patients With KRASG12C-Mutated Non-Small Cell Lung Cancer: A Case Series and Literature Review.

PURPOSE: KRAS is the most commonly mutated driver oncogene in non-small cell lung cancer (NSCLC). Sotorasib and adagrasib, KRASG12C inhibitors, have been granted accelerated US approval; however, hepatotoxicity is a common side effect with higher rates in patients treated with sotorasib proximal to checkpoint inhibitor (CPI) therapy. The aim of this study was to assess the feasibility and safety of adagrasib after discontinuation of sotorasib because of treatment-related grade 3 hepatotoxicity through real-world and clinical cases. METHODS: Medical records from five patients treated in real-world settings were retrospectively reviewed. Patients had locally advanced or metastatic KRASG12C-mutated NSCLC and received adagrasib after sotorasib in the absence of extracranial disease progression. Additional data were collected for 12 patients with KRASG12C-mutated NSCLC enrolled in a phase Ib cohort of the KRYSTAL-1 study and previously treated with sotorasib. The end points associated with both drugs included timing and severity of hepatotoxicity, best overall response, and duration of therapy. RESULTS: All patients were treated with CPIs followed by sotorasib (initiated 0-64 days after CPI). All five real-world patients experienced hepatotoxicity with sotorasib that led to treatment discontinuation, whereas none experienced treatment-related hepatotoxicity with subsequent adagrasib treatment. Three patients from KRYSTAL-1 transitioned from sotorasib to adagrasib because of hepatotoxicity; one experienced grade 3 ALT elevation on adagrasib that resolved with therapy interruption and dose reduction. CONCLUSION: Adagrasib may have a distinct hepatotoxicity profile from sotorasib and is more easily combined with CPIs either sequentially or concurrently. These differences may be used to inform clinical decisions regarding an initial KRASG12C inhibitor for patients who recently discontinued a CPI or experience hepatotoxicity on sotorasib.

Comprehensive study on the differential extraction and comparison of bioactive health potential of the Broccoli (Brassica oleracea).

Introduction: Broccoli is a cruciferous vegetable that has been shown to have numerous potential therapeutic benefits because of its bioactive compounds. Methods: In this study, we compared the bioactive efficacy of cooked and uncooked (fresh) stems and florets of broccoli extracted with three different solvents: acetonitrile, methanol, and aqueous extracts. The extraction yield and antioxidant and antibacterial potential of different broccoli extracts were examined. Results: Fresh and boiled floret stem extracts increased the extraction yield. The extraction yields were higher for the methanol and acetonitrile extracts than for the aqueous extracts. The antioxidant efficacy of the different extracts was studied using ABTS, DPPH, and metal ion reduction assays. The acetonitrile and aqueous extracts exhibited higher antioxidant activities than the methanolic extracts in different antioxidant assays. In addition, increased antioxidant activity was observed in fresh florets and boiled broccoli stems. TPC and TFC contents were higher in the methanolic extracts than in the aqueous extracts. Similar to antioxidant activities, anti-inflammatory activities were found to be higher in the acetonitrile and aqueous extracts, particularly in boiled stems and fresh florets. Broccoli extracts have been shown to be active against Bacillus subtilis and moderately effective against Pseudomonas aeruginosa and Staphylococcus aureus. Conclusions: Acetonitrile and aqueous extraction of broccoli might be an ideal choice for extraction methods, which show increased extraction yield and antioxidant and anti-inflammatory potentials. Utilization of phytomolecules from natural sources is a promising alternative approach to synthetic drug development.

[Determination of tetrodotoxin in urine by liquid chromatography-tandem mass spectrometry with internal standard calibration].

OBJECTIVE: An analytical method was developed for tetrodotoxin(TTX) in urine by liquid chromatography-tandem mass spectrometry(LC-MS/MS) with internal standard calibration. METHODS: TTX in the sample was extracted with the mixture of acetic acid/methanol/acetonitrile(0.005 mL/0.8 mL/1.8 mL), cleaned by solid phase extraction(SPE) with cation exchange cartridge, eluted with 50% acetonitrile/water containing 0.3% hydrochloric acid, and neutralized with ammonia. The extract was separated by a Waters XBridge~(TM) BEH Amide column(150 mm×3.0mm, 1.7 µm) and measured by MS/MS. By optimizing sample extraction and SPE cleanup conditions, the problems of low recovery and strong suppression effects of MS signal for TTX in urine were resolved when cleaned with cation exchange cartridge. RESULTS: Quantitatively calibrated by the internal standard of Kasugamycin, good linear relationship was found for TTX in urine at the range of 0.2-200 µg/L with the correlation coefficient(r~2) of 0.997. The limits of detection and quantitation for TTX in sample matrix were 0.1 and 0.2µg/L, respectively. The average recoveries at three spiking levels(0.2, 10.0 and 200 µg/L) were 89.3%-95.3% with relative standard deviation(n=6) less than 5.1%. The concentrations of TTX in urine from 11 poisoning patients were 0.4-138 µg/L. The detection rate was 100% in urine collected within 3 days after poisoning. CONCLUSION: The established method was simple, accurate and sensitive. It can provide reliable technical support for the rapid treatment of TTX poisoning events and the study of toxin metabolism in vivo.

[Analytical Method for Melengestrol Acetate in Livestock Products Using LC-MS/MS].

The present study verified that it is possible to analyze melengesterol acetate using the existing multi-residue method. Melengestrol acetate was extracted from livestock products using acidic acetonitrile acidified with acetic acid in the presence of n-hexane and anhydrous sodium sulfate. The crude extracts were cleaned up using an octadecylsilanized silica gel cartridge column. Separation by HPLC was performed using an octadecylsilanized silica gel column with linear gradient elution of 0.1 vol% formic acid and acetonitrile containing 0.1 vol% formic acid. For the determination of the analyte, tandem mass spectrometry with positive ion electrospray ionization was used. In recovery tests using four livestock products fortified with maximum residue limits levels of melengestrol acetate (0.001-0.02 mg/kg), the truenesses ranged from 82% to 100%, and the repeatabilities for the entire procedure ranged from 0.5 RSD% to 5.6 RSD%. In recovery tests using 11 livestock products fortified with 0.0005 mg/kg of melengestrol acetate, the truenesses ranged from 88% to 99%, and the repeatabilities ranged from 1.3 RSD% to 5.4 RSD%. The limit of quantification for melengestrol acetate in livestock products was 0.0005 mg/kg.

Simultaneous Determination of Enantiomeric Purity and Organic Impurities of Dexketoprofen Using Reversed-Phase Liquid Chromatography-Enhancing Enantioselectivity through Hysteretic Behavior and Temperature-Dependent Enantiomer Elution Order Reversal on Polysaccharide Chiral Stationary Phases.

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of the potential impurities of dexketoprofen, including the distomer R-ketoprofen. After screening the separation capability of four polysaccharide columns (Lux Amylose-1, Lux Amylose-2, Lux Cellulose-1 and Lux Cellulose-2) in polar organic and in reversed-phase modes, appropriate enantioseparation was observed only on the Lux Amylose-2 column in an acidified acetonitrile/water mixture. A detailed investigation of the mobile phase composition and temperature for enantio- and chemoselectivity showed many unexpected observations. It was observed that both the resolution and the enantiomer elution order can be fine-tuned by varying the temperature and mobile phase composition. Moreover, hysteresis of the retention times and enantioselectivity was also observed in reversed-phase mode using methanol/water mixtures on amylose-type columns. This could indicate that the three-dimensional structure of the amylose column can change by transitioning from a polar organic to a reversed-phase mode, which affects the enantioseparation process. Temperature-dependent enantiomer elution order and rare enthalpic/entropic controlled enantioseparation in the operative temperature range were also observed in reversed-phase mode. To find the best methodological conditions for the determination of dexketoprofen impurities, a full factorial optimization design was performed. Using the optimized parameters (Lux Amylose-2 column with water/acetonitrile/acetic acid 50/50/0.1 (v/v/v) at a 1 mL/min flow rate at 20 °C), baseline separations were achieved between all compounds within 15 min. Our newly developed HPLC method was validated according to the current guidelines, and its application was tested on commercially available pharmaceutical formulations. According to the authors' knowledge, this is the first study to report hysteretic behavior on polysaccharide columns in reversed-phase mode.

Degrading surface-water-based natural organic matter and mitigating haloacetonitrile formation during chlorination: Comparison of UV/persulfate and UV/hydrogen peroxide pre-treatments.

Haloacetonitriles (HANs) are unregulated disinfection by-products that are more toxic than regulated species. Therefore, efficient decomposition of HAN precursors prior to disinfection is crucial for allaying the potential HAN-induced health risks. This study investigated the key roles of ultraviolet-activated persulfate (UV/PS) treatment in alleviating HAN formation. The effects of UV/PS treatment were evaluated by correlating with the characteristics of organic matter in surface water and comparing with conventional UV/H2O2 treatment. Upon irradiating raw water samples and a Suwannee River humic acid solution spiked with 10 mM PS or H2O2 with 254 nm UV light, UV/PS treatment was found to be more potent than UV/H2O2 in mitigating the HAN production and degrading organic substances; moreover, UV/PS treatment effectively decreased the dissolved organic nitrogen (DON) content. In contrast, UV/H2O2 treatment did not induce any noticeable reduction in DON level. Furthermore, both UV/PS and UV/H2O2 treatments reduced the dichloroacetonitrile (DCAN) formation potential (FP), leading to strong correlations with the degradation of aromatic and humic-acid-like compounds. Notably, UV/PS treatment efficiently decreased the FP of bromochloroacetonitrile (BCAN) and dramatically reduced that of dibromoacetonitrile (DBAN) after a sharp increase; however, UV/H2O2 treatment gradually increased the DBAN-FP. Bromide was activated by sulfate radicals during UV/PS treatment, negatively correlating with the BCAN-FP and DBAN-FP, indicating that the formation of reactive bromine species increased the DBAN-FP; however, excessive oxidation possibly led to the recovery of inorganic bromine for decreasing the BCAN-FP and DBAN-FP. Additionally, UV/PS treatment effectively suppressed toxicity owing to its high reduction rate for brominated HANs; in contrast, UV/H2O2 treatment resulted in less significant BCAN and DBAN reductions, leading to minimal net reduction in toxicity. Overall, UV/PS treatment was remarkably effective at diminishing the toxicity of brominated HANs, underscoring its potential to mitigate drinking-water-related health risks.