Mo4+-Doped CuS Nanosheet-Assembled Hollow Spheres for CO2 Electroreduction to Ethanol in a Flow Cell.

Electrocatalytic CO2 reduction reaction (CO2RR) to ethanol has been widely researched for potential commercial application. However, it still faces limited selectivity at a large current density. Herein, Mo4+-doped CuS nanosheet-assembled hollow spheres are constructed to address this issue. Mo4+ ion doping modifies the local electronic environments and diversifies the binding sites of CuS, which increases the coverage of linear *COL and produces bridge *COB for subsequent *COL-*COH coupling toward ethanol production. The optimal Mo9.0%-CuS can electrocatalyze CO2 to ethanol with a faradaic efficiency of 67.5% and a partial current density of 186.5 mA cm-2 at -0.6 V in a flow cell. This work clarifies that doping high valence transition metal ions into Cu-based sulfides can regulate the coverage and configuration of related intermediates for ethanol production during the CO2RR in a flow cell.

Development and validation of a novel 30-plex STR assay for canine individual identification and parentage testing.

Domestic dogs are helpers in outdoor human work and companions for families; thus, individual canine identification and parentage testing are crucial in certain fields, including forensics and breeding programs. In this study, a six-dye fluorescent labeling multiplex amplification system containing 29 canine short tandem repeats (STRs) and the sex-determining marker DAmel was developed. The system was called the Tronfo Canine 30-plex STR Kit and was further validated according to the Scientific Working Group on DNA Analysis Methods and the Organization of Scientific Area Committees for Wildlife Forensics guidelines, including tests for PCR conditions, precision, species specificity, sensitivity, stability, repeatability and reproducibility, a population study, and a study of 16 paternity test cases. The results indicated that the novel canine STR assay was accurate, specific, reproducible, stable, and robust. Complete profiles were obtained with 31.25 pg of canine DNA. Additionally, 500 unrelated canine individuals were investigated using this novel system, and the combined power of discrimination and exclusion values were 0.999999999999999999 and 0.999996451039850, respectively. These results suggest that the Tronfo Canine 30-plex STR Kit is highly polymorphic, informative, and suitable for individual canine identification and parentage testing.

Endoplasmic reticulum stress and death receptor-mediated apoptosis in the neuronal differentiation of adult adipose-derived stromal cells.

Apoptosis is the primary cause of cell death in the differentiation of Adipose-derived stromal cells (ADSCs) into neurons. However, the relationship between endoplasmic reticulum stress (ERS) and death receptor-mediated apoptosis in ADSC-induced neuronal differentiation is not clear. ADSCs were isolated and induced to differentiate into neurons using ß-mercaptoethanol. The expression of neuron-specific enolase (NSE), GRP94, CHOP, Fas/FasL, TNFR1/TNF-α, DR5/TRAIL, Caspase8, and Caspase3 in ADSCs was examined using immunocytochemistry and Western blotting before induction, during pre-induction, and after induction. Transmission electron microscopy (TEM) was used to observe changes in the morphology of the endoplasmic reticulum (ER), and the MTT assay was employed to measure cell viability in the uninduced and induced groups. Additionally, the number of apoptotic cells during the induction process was measured using flow cytometry with Annexin V/PI. With increasing induction time, the positive expression rates of CHOP, Fas/FasL, Caspase8, Caspase-3, and NSE gradually increased, while the positive expression rate of GRP94 decreased. TNFR1/TNF-α and DR5/TRAIL peaked at 5 h post-induction and then decreased at 8 h. TEM revealed swelling and expansion of the ER, vacuolar changes, and degranulation in cells. The MTT assay showed a gradual decrease in the absorbance of surviving cells in all groups. Flow cytometry indicated an increasing rate of apoptosis in cells. Therefore, ERS in the normal culture and growth of ADSCs, manifesting as enhanced unfolded protein response (UPR), maintains the normal survival of ADSCs. However, in the process of ADSC-induced differentiation into neurons, ERS and death receptor-mediated apoptosis are significant causes of cell death.

Forensic characteristics and genetic structure of the Chinese Tibetan population revealed by 38 X-chromosomal InDel loci.

With the increasing importance of X-chromosome (Chr-X) genotyping in kinship identification, the exploitation of X chromosome genetic marker multiplex kits is increasing. The Human X-InDels amplification kit is a novel developed system which contained 38 X-chromosomal Insertion/deletion markers (X-InDels) and Amelogenin. Herein, we investigated the genetic diversity of the 38 X-InDels in the Tibetan ethnic minority (n = 792) from seven regions and evaluated the application potential of this novel panel. The rs16368 was the least variable locus, whereas the most polymorphic locus was the rs59605609 in Tibetan population. We confirmed three linkage groups with the haplotype diversities ranged from 0.5032 to 0.5976. The overall combined power of discrimination (PD) in males and females were 0.999999999582066 and 0.999999999999993, respectively. And the overall combined mean exclusion chance (MEC) values were not lower than 0.999125526990159. In addition, we explored the genetic relationships among the Tibetans in seven different regions via series of population comparison analyses, finding that the genetic relationship between the Ngari Tibetan and Chamdo Tibetan was the farthest, which was consistent with geographical distribution.

Ultra-high-performance liquid chromatography with tandem mass spectrometry method for cellular toxicity and pharmacokinetic study of PEG1K polymers.

Polyethylene glycol (PEG) is one of the most commonly used polymers in drug delivery systems. The investigation of the pharmacokinetic behavior of PEG is important for revealing the toxicity and efficiency of PEG-related Nano-drug delivery systems. A high through-put and selective ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method coupled with collision-induced dissociation (CID) in source technique was developed and validated to determine PEG1K polymers in cellular samples in this study. The countless precursor ions of PEG1K are dissociated in the source to generate numerous product ions which have different numbers of subunits. The transition of [M+H]+ precursor ions → product ions at m/z 177.1 (four subunits)→89.1 (two subunits) was selected to determine PEG1K due to its high sensitivity. The UHPLC-MS/MS method coupled with CID in the source showed good linearity over the range of 0.1-10 µg/mL. Intra-day and inter-day accuracies and precisions of the assay were all within ± 12.39%. The assay was successfully applied to a cellular pharmacokinetic study of PEG1K in human breast cancer cells. The cytotoxicity of PEG1K polymers was also studied and the results indicated that the cytotoxicity of PEG1K was not significant in the range of 5-1200 µg/mL.

Determination of acetochlor by UPLC-MS3 in cells and its application to a cellular pharmacokinetic study.

The aim of this work was to develop a fast, simple, and reliable UPLC-MS3 method for the sensitive detection of acetochlor in biological samples. In MS3 mode, the ion transition m/z 270.1 â†’ 224.1→148.1 was chosen for quantification with butachlor as the internal standard. In the UPLC system, separation was performed on a UPLC column (2.1 × 50 mm ID, 1.7 µm) with 0.1 % FA in water and acetonitrile as mobile phases. After simple protein precipitation via acetonitrile, the method was well validated with good linearity (0.5-20 ng/mL, r > 0.995), accuracy (-3.70 %-2.98 %), and precision (<15 %). The selectivity and sensitivity were improved obviously in MS3 mode than that in MRM mode. The developed UPLC-MS3 method was successfully applied to the cellular pharmacokinetics study of acetochlor in MCF-7 cells.

A Novel HPLC Method for Quality Inspection of NRK Biosynthesized ß-Nicotinamide Mononucleotide.

ß-nicotinamide mononucleotide (NMN) has a good effect on delaying aging, repairing DNA and ameliorating metabolic disease. Biosynthesis with nicotinamide riboside kinase (NRK) takes a large part in NMN manufacture, but there is no available NMN quality standard and analytical method at present. In this study, we developed a specific high-performance liquid chromatography method for the assessment of NMN-related substances, including NMN and its potential impurities from NRK biological production and storage. Forced degradation study was performed under acid, base, oxidative, photolytic and thermal conditions. The separation of related substances was achieved on an Elite Hypersil ODS column using phosphate buffer-methanol gradient at a flow rate of 1.0 mL/min. The detection wavelength was maintained at 260 nm. The resolutions among all related substances were better than 1.5. Significant degradation was observed in basic and thermal conditions. All related substances showed good linearity with a coefficient of determination (R2) higher than 0.999. The accuracy values of all related substances were between 91.2% and 108.6%. Therefore, the validated analytical method is appropriate for inspecting the quality of NMN in its NRK biosynthetic manufacture and storage, thus further helping to unify NMN quality standards and facilitate related studies on NMN.

Tin (II) Chloride Salt Melts as Non-Innocent Solvents for the Synthesis of Low-Temperature Nanoporous Oxo-Carbons for Nitrate Electrochemical Hydrogenation.

Carbonaceous electrocatalysts offer advantages over metal-based counterparts, being cost-effective, sustainable, and electrochemically stable. Their high surface area increases reaction kinetics, making them valuable for environmental applications involving contaminant removal. However, their rational synthesis is challenging due to the applied high temperatures and activation steps, leading to disordered materials with limited control over doping. Here, a new synthetic pathway using carbon oxide precursors and tin chloride as a p-block metal salt melt is presented. As a result, highly porous oxygen-rich carbon sheets (with a surface area of 1600 m2 g-1) are obtained at relatively low temperatures (400 °C). Mechanistic studies reveal that Sn(II) triggers reductive deoxygenation and concomitant condensation/cross-linking, facilitated by the Sn(II) → Sn(IV) transition. Due to their significant surface area and oxygen doping, these materials demonstrate exceptional electrocatalytic activity in the nitrate-to-ammonia conversion, with an ammonia yield rate of 221 mmol g-1 h-1 and a Faradic efficiency of 93%. These results surpass those of other carbon-based electrocatalysts. In situ Raman studies reveal that the reaction occurs through electrochemical hydrogenation, where active hydrogen is provided by water reduction. This work contributes to the development of carbonaceous electrocatalysts with enhanced performance for sustainable environmental applications.

Electroacupuncture Ameliorates Depression-Like Behaviors in Post-Stroke Rats via Activating AMPK-Mediated Mitochondrial Function.

Background: Post-stroke depression (PSD) is one of the most common complications of stroke. Electroacupuncture (EA) is an effective traditional Chinese medicine treatment for PSD, which is widely used in clinical settings. EA has a significant therapeutic effect against PSD, but the mechanism is still unclear. This study aimed to determine whether EA ameliorates depression-like behaviors in PSD rats by regulating the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-mediated mitochondrial function. Methods: Middle cerebral artery occlusion (MCAO) and chronic unpredictable mild stress (CUMS) were used to develop a PSD rat model. To elucidate the role of AMPK in EA treatment, compound C, a selective inhibitor of AMPK, was injected into the lateral ventricle of rats before EA treatment. EA treatment was performed for 14 consecutive days for 30 min per day after PSD modeling. A modified Zea-Longa five-point scale scoring system was used to determine neurologic function in MCAO rats. Behavioral tests were conducted to evaluate depression-like phenotypes in rats. Depression-like behaviors were tested by sucrose preference test (SPT), novelty suppressed feeding test (NSFT), and open-field test (OFT). The structure and morphology of the prefrontal cortex were observed by histopathological hematoxylin-eosin (HE) and Nissl staining. The mitochondrial morphology and function were analyzed by colorimetry, chemiluminescence, Western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR). Results: EA treatment successfully ameliorated depression-like behaviors, upregulated AMPK expression, and improved mitochondrial function. However, AMPK inhibition by Compound C exacerbated depression-like behaviors and aggravated neuronal and mitochondrial injury in PSD rats. Conclusion: EA treatment improved depression-like behaviors in PSD rats and promoted mitochondrial function by activating AMPK.

Integration of MnO2 Nanosheets with Pd Nanoparticles for Efficient CO2 Electroreduction to Methanol in Membrane Electrode Assembly Electrolyzers.

It remains a challenge to design a catalyst with high selectivity at a large current density toward CO2 electrocatalytic reduction (CO2ER) to a single C1 liquid product of methanol. Here, we report the design of a catalyst by integrating MnO2 nanosheets with Pd nanoparticles to address this challenge, which can be implemented in membrane electrode assembly (MEA) electrolyzers for the conversion of CO2ER to methanol. Such a strategy modifies the electronic structure of the catalyst and provides additional active sites, favoring the formation of key reaction intermediates and their successive evolution into methanol. The optimal catalyst delivers a Faradaic efficiency of 77.6 ± 1.3% and a partial current density of 250.8 ± 4.3 mA cm-2 for methanol during CO2ER in an MEA electrolyzer by coupling anodic oxygen evolution reaction with a full-cell energy efficiency achieving 29.1 ± 1.2% at 3.2 V. This work opens a new avenue to the control of C1 intermediates for CO2ER to methanol with high selectivity and activity in an MEA electrolyzer.

Sr2+-Doped CuO Nanoribbons with the Hydrophobic Surface Enabling CO2 Electroreduction to Ethane.

Electrochemical reduction of carbon dioxide to value-added multicarbon (C2+) products is a promising way to obtain renewable fuels of high energy densities and chemicals and close the carbon cycle. However, the difficulty of C-C coupling and complexity of the proton-coupled electron transfer process greatly hinder CO2 electroreduction into specific C2+ products with high selectivity. Here, we design an electrocatalyst of Sr-doped CuO nanoribbons with a hydrophobic surface for CO2 electroreduction to ethane with high selectivity. Sr doping enhances the chemical adsorption and activation of CO2 by inducing oxygen vacancies and increasing *CO coverage by stabilizing Cu2+ active sites, thus further boosting subsequent C-C coupling. The hydrophobic surface with dodecyl sulfate anions (DS-) adsorption increases the oxophilicity of the catalyst surface, enhancing the conversion of the *OCH2CH3 intermediate to ethane. As a result, the optimized Sr1.97%-CuO exhibits a Faradaic efficiency of 53.4% and a partial current density of 13.5 mA cm-2 for ethane under a potential of -0.8 V. This study provides a strategy to design a Cu-based catalyst by alkaline earth metal ions doping with the hydrophobic surface to engineer the evolution of the intermediates for a desired product during CO2RR.

Quantification of human serum albumin by combining chymotrypsin/trypsin digestion coupled with LC-MS/MS technique.

The quantification of albumin is important in clinical medicine because the concentration of albumin in biological fluids is closely related to human health. In this study, we developed a highly selective and robust assay to determine human serum albumin (HSA) in human plasma by combining chymotrypsin/trypsin digestion coupled with targeted LC-MS/MS technique. Human plasma samples were denatured, reduced, alkylated, and digested with both chymotrypsin and trypsin to generate surrogate peptides. A unique chymotryptic peptide (NAETF) arising from human serum albumin was finally selected for targeted LC-MS/MS detection and quantification. Numerous parameters related to the targeted LC-MS/MS assay were evaluated, including lower limit of quantitation (LLOQ), linearity range, enzyme digestion efficiency, accuracy and precision. The LC-MS/MS assay was linear in the concentration range 0.05-1 mg/mL with intra-day and inter-day precision <10.2% and accuracy ranging from -3.94% to 4.89%. The assay was successfully applied to determine HSA in 148 human plasma samples.

Secreted Effector Proteins of Poplar Leaf Spot and Stem Canker Pathogen Sphaerulina musiva Manipulate Plant Immunity and Contribute to Virulence in Diverse Ways.

Fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Early clues and molecular mechanism involved in neurodegenerative diseases induced in immature mice by combined exposure to polypropylene microplastics and DEHP.

Studies have shown that exposure to either microplastics (MPs) or di-(2-ethylhexyl) phthalic acid (DEHP) alone can cause neurotoxicity in animals, but it remains uncertain whether and to what extent co-exposure to these two substances, which often occur together in reality, can also induce neurotoxicity. This study aimed to investigate the neurotoxicity and molecular mechanisms of combined exposure to DEHP and polypropylene microplastics (synthetic PP-MPs were used), the microplastics most commonly encountered by young children, in immature mice. The results showed that exposure to PP-MPs and/or DEHP did cause neurotoxic effects in immature mice, including induction of neurocognitive and memory deficits, damage to the CA3 region of the hippocampus, increased oxidative stress, and decreased AChE activity in the brain. The severity of the neurotoxicity increased with increasing concentrations of PP-MPs, combined exposure to PP-MPs and DEHP exhibited additive or synergistic effects. Transcriptomic analyses revealed that the PP-MPs and/or DEHP exposure altered the expression profiles of gene clusters involved in the stress response, and in protein processing in endoplasmic reticulum. Quantitative analyses further indicated that PP-MPs and/or DEHP exposure inhibited the activity of the heat shock response mediated by heat shock transcription factor 1, while chronically activated the unfolded protein response, consequently inducing neurotoxicity through neuronal apoptosis and neuroinflammation in the immature mice. As a pioneer study to highlight the neurotoxicity induced by combined exposure to PP-MPs and DEHP in immature mice, this research provides new insights into mitigating the health risks of PP-MPs and DEHP exposure in young children.

CmVCall: An automated and adjustable nanopore analysis pipeline for heteroplasmy detection of the control region in human mitochondrial genome.

Genetic associations between human mitochondrial DNA (mtDNA) heteroplasmy and mitochondrial diseases, aging, and cancer have been elaborated, contributing a lot to the further understanding of mtDNA polymorphic spectrum in anthropology, population, and forensic genetics. In the past decade, heteroplasmy detection using Sanger sequencing and next generation sequencing (NGS) was hampered by the former's inefficiency and the latter's inherent bias due to amplification and mapping of short reads, respectively. Nanopore sequencing stands out for its ability to yield long contiguous segments of DNA, providing a new insight into heterogeneity authentication. In addition to MinION from Oxford Nanopore Technologies, an alternative nanopore sequencer QNome (Qitan Technology) has also been applied to various biological research and the forensic applicability of this platform has been proved recently. In this study, we evaluated the performance of four commonly used variant callers in the heterogeneity authentication of the control region of human mtDNA based on simulations of different ratios generated by mixing QNome nanopore sequencing reads of two synthetic sequences. Then, an open-source and python-based nanopore analytics pipeline, CmVCall was developed and incorporated multiple programs including reads filtering, removal of nuclear mitochondrial sequences (NUMTs), alignment, optional 'Correction' mode, and heterogeneity identification. CmVCall can achieve high precision, accuracy, and recall of 100%, 99.9%, and 92.3% with a 5% heteroplasmy level in 'Correction' mode. Moreover, blood, saliva, and hair shaft samples from monozygotic (MZ) twins were used for heterogeneity evaluation and comparison with the NGS data. Results of MZ twin samples showed that CmVCall could identify more point heteroplasmy sites, revealing significant levels of inter- and intra-individual mtDNA polymorphism. In conclusion, we believe that this analysis pipeline will lay a solid foundation for the development of a comprehensive nanopore analysis pipeline targeting the whole mitochondrial genome.

Development of Pore Pressure in Cementitious Materials under Low Thermal Effects: Evidence from Optimization of Pore Structure by Incorporation of Fly Ash.

Studies on durability of cementitious materials have focused on harsh environments, but less attention has been paid to low thermal loading situations. In this paper, with the aim of exploring the evolution of internal pore pressure and microcrack extension of cementitious under low thermal environment, cement paste specimens with thermal environment slightly below 100 °C and three water-binder ratios (0.4, 0.45 and 0.5) and four fly ash admixtures (0, 10%, 20% and 30%) were designed. Firstly, the internal pore pressure of the cement paste was tested; secondly, the average effective pore pressure of the cement paste was calculated; and finally, the phase field method was used to explore the expansion of microcracks inside the cement paste when the temperature gradually increased. It was found that the internal pore pressure of the paste showed a decreasing trend as the water-binder ratio and fly ash admixture increased, and the numerical simulation found that the sprouting and development of cracks were delayed when 10% fly ash was added to the cement paste, which was consistent with the experimental results. This work provides a basis for the durability development of concrete under low thermal environment.

Unraveling the in vivo biological fate of mPEG2000-PDLLA2500-COOH diblock copolymers by LC-MS/MS based on CID in source technique.

Methoxy poly (ethylene glycol)-poly(D, L-lactic acid) (mPEG-PDLLA) is a biocompatible and amphiphilic diblock copolymer composed of a hydrophilic poly(ethylene glycol) block and a hydrophobic poly(D, L-lactic acid) block, which can self-assemble into micelles in aqueous solution. It is one of the most widely used diblock copolymers for drug delivery, drug solubilization and drug encapsulation. Fully characterizing the in vivo fate of mPEG-PDLLA diblock copolymers is important to promote the further development of polymer-based nanocarrier drug delivery systems. However, to date, a bioanalysis assay for simultaneous quantification of mPEG-PDLLA and mPEG has not been reported. In this study, we developed such a novel LC-MS/MS assay based on CID in source technique and used it to study the multiple-dose pharmacokinetic, tissue distribution and excretion of mPEG2000-PDLLA2500-COOH and mPEG2000 in rat after intravenous administration. The results indicate that mPEG2000-PDLLA2500-COOH and mPEG2000 are mainly distributed to the liver, lung, spleen and kidney after intravenous administration. mPEG2000-PDLLA2500-COOH is mostly excreted via the renal route in the form of mPEG2000. Overall, the results of this study provide a comprehensive and clear picture of the in vivo fate of mPEG2000-PDLLA2500-COOH which will be useful in evaluating the efficiency and safety of polymer-based nanocarrier drug delivery systems.

Exploring rare differences in mitochondrial genome between MZ twins using Ion Torrent semiconductor sequencing.

Monozygotic (MZ) twins are considered to be genetically identical in that they have the same genomic DNA sequences in theory, and thus cannot be differentiated using forensic standard STR-based DNA profiling. However, a recent study employed deep sequencing to explore extremely rare mutations in the nuclear genome and reported that the mutation analysis could be applied to differentiate between MZ twins. Compared with the nuclear genome, the mitochondrial DNA (mtDNA) exhibits higher mutation rates due to fewer DNA repair mechanisms in the mitochondrial genome (mtGenome) and the lack of proofreading capability of the mtDNA polymerase. In a previous study, we used Illumina ultra-deep sequencing to describe point heteroplasmy (PHP) and nucleotide variant of the mtGenomes in venous blood samples of MZ twins. In the present study, we characterized minor differences of the mtGenomes in three tissue samples from seven sets of MZ twins using Ion Torrent semiconductor sequencing (Thermo Fisher Ion S5 XL system) and commercialized mtGenome sequencing kit (Precision ID mtDNA Whole Genome Panel). PHP was observed in blood samples from one set of MZ twins and in saliva samples from two sets of twins, but it presented in hair shaft samples from all seven sets of MZ twins. Overall, the coding region of the mtGenome exhibits more PHPs than the control region. The results of this study have further attested the competence of mtGenome sequencing in differentiating between MZ twins, and that among the three kinds of samples tested, hair shaft is more likely to accumulate minor differences in the mtGenomes of MZ twins.

Cellular toxicity and pharmacokinetic study of butachlor by ultra-performance liquid chromatography-tandem mass spectrometry based on tandem mass spectrometry cubed technique.

Butachlor is an aromatic amide compound that plays a role as a herbicide, a xenobiotic, and an environmental contaminant. The aim of this work was to develop a highly selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method based on the tandem mass spectrometry cubed technique to determine butachlor in a biological matrix. Butachlor and internal standard acetochlor were separated on a Waters Acquity ultra-performance liquid chromatography BEH C18 column (2.1 × 50 mm, 1.7 µm) with gradient elution using 0.1% formic acid aqueous solution (A) and acetonitrile (B) as mobile phases. The transitions selected for tandem mass spectrometry cubed quantitative analysis in positive ion mode were: for butachlor, mass-to-charge ratio 312.2→238.1→162.1; for acetochlor, mass-to-charge ratio 270.1→224.0→148.1. The total running time for each sample was 5.5 min. The ultra-performance liquid chromatography-tandem mass spectrometry cubed method showed a linear relationship (R2 ≥ 0.995) in the concentration range of 0.5-100 ng/ml. The intra and interday accuracies are within the range of -10.6%-4.3% and precisions are between 4.48% and 13.14%. The novelty of the method is the use of tandem mass spectrometry cubed scanning mode, which improves selectivity and sensitivity. The results indicated that butachlor was cellular toxic. The safety of butachlor should be considered when it is used as a herbicide.

Development and validation of an LC-MS/MS assay with multiple stage fragmentation for the quantification of alachlor in McF-7 cells.

Alachlor is one of the most widely used herbicides and can also be a carcinogenic compound. It is of great significance to establish a sensitive analytical method for the determination of alachlor in the environment and organisms. In this study, a high-performance liquid chromatography tandem mass spectrometry cubed (LC/MS3) method was developed and validated to quantify alachlor in human breast cancer cells (McF-7 cells). The cell samples were processed by simple protein precipitation with acetonitrile, then the analytes were separated on a Waters AcQuity® UPLC BEH (2.1 × 50 mm I.D, 1.7 µm) column using the gradient elution with solvent A (0.1 % formic acid) and solvent B (acetonitrile) at a flow rate of 0.5 mL/min. MS3 detection in positive ion mode was used to detect the analytes. The MRM3 transitions at m/z 270.1 â†’ 238.0 â†’ 162.1 and 312.2 â†’ 238.1 â†’ 147.2 were used to determine alachlor and butachlor, respectively. The run time for each sample was only 4 min. This method was validated for various parameters including accuracy, precision, selectivity, linearity, lower limit of quantitation (LLOQ), etc. The LC/MS3 assay was linear in the concentration range 0.5-50 ng/mL (R2 ≥ 0.995). For all concentrations, the precision is < 9.49 %, and the intra-day and intra-day accuracy is < 13.05 %. Cytotoxic potential of alachlor against McF-7 cell lines was measured by MTT method after 48 h of incubation. For alachlor, half maximal inhibitory concentration (IC50) on McF-7 cells was 87.95 µg/mL. This method was successfully applied to cellular pharmacokinetic study of alachlor in McF-7 cells after administration with a dose of 20 µg/mL.