Clean-up Step Research Articles

Optimization of the Combined Use of Z-Sep Plus and EMR-Lipid in QuEChERS Procedure for the Analysis of Eight Pesticides in Real Milk Samples

AbstractOne of the most applied procedures for the determination of trace analytes in complex matrices is QuEChERS (an acronym for Quick, Easy, Cheap, Effective, Rugged, and Safe). QuEChERS procedures include an extraction step followed by a dispersive solid-phase extraction (dSPE) for analytes cleaning-up from the matrix components. A challenging task in QuEChERS procedures is extracting and determining pesticides from samples of high fat such as milk samples. This challenge induced the innovation of new adsorbents for the clean-up step such as Z-Sep Plus® and EMR-Lipid® to enable removal of fatty matrix components without affecting the recovery of hydrophobic analytes. This work aims to apply experimental design to optimize the combined application of both QuEChERS clean-up adsorbents; Z-Sep Plus® and EMR-Lipid® in addition to other QuEChERS parameters in the determination of eight pesticides: hexachlorocyclohexane, dichlorodiphenyldichloroethane, dichlorodiphenyltrichloroethane, primiphos ethyl, diazinon, malathion, endrin, and dimethoate in milk matrix. This was augmented by optimization of GC–MS/MS and UPLC-MS/MS to detect and determine analytes in extracts. The experimental design of QuEChERS procedure enabled the optimization of Z-Sep Plus®- and EMR-Lipid®-added adsorbent amounts with other method parameters to enable the maximum recovery of analytes. Furthermore, the optimized methods enabled low detection limits of the studied pesticides within a short analysis time (28 min for GC and 12 min for LC methods, respectively). The procedure was validated according to European SANTE/11312/2021 Guideline. Quantitation limit ranged from 1.7 to 3.2 ng/mL for GC–MS/MS method and from 1.7 to 3 ng/mL for UPLC-MS/MS method. Greenness assessment of the methods followed four approaches indicating an excellent value of greenness for the proposed methods. Furthermore, 45 real milk samples collected from the Egyptian market were tested with the developed procedure for the presence of pesticides.

Insights into type 2 diabetes mellitus in atrial fibrillation patients: a proteomics approach

Abstract Introduction Atrial fibrillation (AF) and Type 2 Diabetes Mellitus (T2DM) are two closely interconnected conditions, sharing a complex relationship within cardiac pathophysiology. Common risk factors such as obesity, hypertension, inflammation, and endothelial dysfunction contribute to their coexistence. T2DM is known to drive cardiac fibrosis and promote electrical and structural remodeling of the heart, creating a substrate conducive to AF onset, while AF in T2DM patients increases the risk of adverse cardiovascular outcomes, such as stroke. Understanding this complex relationship is pivotal for improving the therapeutic and preventive strategies for these patients. This study aims to examine the proteomic differences between AF patients with and without T2DM, providing novel insights into pathophysiological mechanisms and identifying novel therapeutic targets and biomarkers. Methods In total, 14 AF patients undergoing open-heart surgery, comprising of 6 T2DM individuals and 8 non-T2DM individuals have been included. The mean age of the T2DM patients was 69.5 ± 10 years, while the non-T2DM was 69.3 ± 8.2 and BMI was 29.2 ± 2.9 and 29.5 ± 3 respectively. Right atrial appendages (RAA) from these patients were obtained in an RNA stabilization solution during open-heart surgery and processed using the phenol-based method for protein extraction. The proteins were digested with trypsin to generate peptides, followed by an additional clean-up step using C18 pipette tips. The prepared peptide samples were analyzed by employing a label-free-based quantitative (LFQ) approach, using a C18 (75cmx75cm) column. Proteomic analysis was performed using a Nano liquid chromatography system coupled to tandem mass spectrometer (nano LC-MS/MS), followed by a comprehensive bioinformatics analysis, using appropriate software. Results A total of 1548 proteins were identified, of which 899 could be quantified using LFQ analysis. Applying strict significance criteria, including FDR <0.1 and Abundance ratio (AR)>2 or <0.5, we identified 100 differential expressed proteins, with 21 upregulated and 79 downregulated in T2DM (Fig.1). Most notably, DPP9 and ATP1A2 were overexpressed in hearts of AF patients with T2DM (AR=100), while INPPL1 and ARID5A were underexpressed (AR=0.01), thereby representing attractive therapeutic targets. In pathway enrichment analysis (Fig.2), the dysregulated proteins participate in noteworthy pathways, including Non-alcoholic fatty liver disease (NAFLD), Type II diabetes mellitus, and Insulin signaling pathway. Conclusion Given the increased morbidity associated with the coexistence of AF and T2DM, there is a pressing need for novel therapeutic strategies tailored to this patient population. While AF and T2DM share certain molecular characteristics in the heart, this study emphasizes the necessity for further evaluation and validation of the distinct proteomic signatures identified.Fig.1:Volcano PlotFig.2:Protein Enrichment Network

ELIME-IMS hybrid assay for Salmonella detection in swine mesenteric lymph nodes at slaughterhouse

Salmonella contamination in pig slaughterhouses is linked to infection rate on farms. Accurate diagnosis in heavy pigs relies on isolating pathogens from the gut wall or lymph nodes. A key technique is Immunocapture using Magnetic Beads (IMS), which purifies target bacteria from Salmonella enrichment broths. This is followed by an Enzyme-Linked Immunomagnetic Electrochemical (ELIME) assay for rapid detection. In our study, we developed an ELIME-IMS hybrid assay to detect Salmonella in swine mesenteric lymph nodes (MNL), involving a clean-up with N-acetylcysteine and centrifugation. Detection limits for S. Typhimurium and S. Derby were estimated at 2.80 and 3.52 Log CFU/ml, respectively. We analysed 103 MNL samples from a northern Italy slaughterhouse. Additionally, we examined 15 carcass swabs. Both the ELIME assay and the IMS-based culture method showed strong agreement with the ISO 6579–1:2017 method, especially after 20 h of enrichment (89.47% concordance). The clean-up step significantly influenced the results, as samples processed without it showed higher variability. A logistic regression model indicated high classification accuracy for negative samples using ELIME values. The ELIME-IMS assay facilitates rapid Salmonella screening and isolation in swine mesenteric lymph nodes.

Establishment and Application of a Multiplex PCR NGS Method for the Genotyping of HLA-Class I and HPA.

Selecting compatible HLA-Class I and/or HPA platelets based on genotyping could alleviate immune platelet transfusion refractoriness (PTR). A fast and reliable method of HLA-Class I and HPA genotyping is necessary to construct a platelet donor bank with known HLA-Class I and HPA genotypes. Ten pairs of specific primers for HLA-A, HLA-B, HLA-C, HPA-1 through HPA-6w, HPA-15 and HPA-21w were designed. The appropriate fragments were optimised for amplification in a single multiplex reaction. After a cleanup step using paramagnetic beads, the amplicon library was prepared and sequenced. To validate the accuracy of the developed method, commercial NGS kits for the genotyping of HLA-A, HLA-B and HLA-C and the TaqMan real-time PCR method in-house for the genotyping of HPA-1 through HPA-6w, HPA-15 and HPA-21w were used to detect all the specimens in parallel. A total of 386 specimens were detected and the results of genotyping HLA-A, HLA-B, HLA-C and HPA-1 through HPA-6w, HPA-15 and HPA-21w were obtained simultaneously, which is 100% consistent between the two methods. Four new HLA alleles, HLA-A*11:451, HLA-A*30:01:26, HLA-B*39:201 and HLA-B*40:538, were also reconfirmed. Two novel SNVs, c.2671C > T and c.2681T > G, in the coding region of ITGA2B were detected, all of which are heterozygous in individuals. A novel NGS method based on multiplex PCR was established to detect HLA-Class I and HPA simultaneously, which is high-throughput, rapid and accurate and could be applied to build a platelet donor bank.

Systematic Comparison of Extract Clean-Up with Currently Used Sorbents for Dispersive Solid-Phase Extraction

Dispersive solid-phase extraction (dSPE) is a crucial step for multiresidue analysis used to remove matrix components from extracts. This purification prevents contamination of instrumental equipment and improves method selectivity, sensitivity, and reproducibility. Therefore, a clean-up step is recommended, but an over-purified extract can lead to analyte loss due to adsorption to the sorbent. This study provides a systematic comparison of the advantages and disadvantages of the well-established dSPE sorbents PSA, GCB, and C18 and the novel dSPE sorbents chitin, chitosan, multi-walled carbon nanotube (MWCNT), and Z-Sep® (zirconium-based sorbent). They were tested regarding their clean-up capacity by visual inspection, UV, and GC-MS measurements. The recovery rates of 98 analytes, including pesticides, active pharmaceutical ingredients, and emerging environmental pollutants with a broad range of physicochemical properties, were determined by GC-MS/MS. Experiments were performed with five different matrices, commonly used in food analysis (spinach, orange, avocado, salmon, and bovine liver). Overall, Z-Sep® was the best sorbent regarding clean-up capacity, reducing matrix components to the greatest extent with a median of 50% in UV and GC-MS measurements, while MWCNTs had the largest impact on analyte recovery, with 14 analytes showing recoveries below 70%. PSA showed the best performance overall.

Development and validation of a multiresidue method for the determination of antibiotic residues in crawfish using LCMS/MS

ABSTRACT This study developed a rapid multi-class method for determining 29 antibiotic residues in crawfish using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A quick and affordable extraction method was employed, and sample preparation was performed using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) technique. The extraction process involved adding acetonitrile to crawfish samples. The sample was homogenised with a solution of sodium citrate buffer and sodium EDTA, followed by a cleanup step. The method was validated according to European Union (EU) guidelines 2002/657/EC. The limit of quantitation (LOQ) for all antibiotics was 5 µg/kg, except for flumequine and trimethoprim, which had LOQs of 2.5 µg/kg. The LOQ for the tetracycline group was 25 µg/kg, and for chloramphenicol, it was 0.015 µg/kg. The average recoveries at different concentration levels varied between 60–120%. The precision, expressed as repeatability and reproducibility, was calculated to be less than 16%. Thirty crawfish samples collected from local Egyptian market were monitored for antibiotic residues. The results revealed contamination with antibiotics belonging to three different groups. Of the thirty samples, 27% were contaminated with ciprofloxacin and enrofloxacin, 17% with chlortetracycline and oxytetracycline, 7% with oxolinic acid, and 3% with sulphamethazine. The applicability of the developed method was proven through successful proficiency testing. The developed method met performance criteria and is suitable for monitoring these antibiotics in crawfish.

Determination of pesticide residues in five different corn-based products using a single and simple solid–liquid extraction without clean-up steps followed by comprehensive two-dimensional liquid chromatography coupled to tandem mass spectrometry

Pesticide residues in food products are a critical issue concerning food safety. Recent studies have increasingly focused on developing selective, rapid, innovative, and environmentally friendly analytical methods for detecting pesticides in foodstuffs. This study presents a novel analytical method utilizing comprehensive two-dimensional liquid chromatography-tandem mass spectrometry for the simultaneous determination of one hundred and thirteen multiclass pesticides in corn products. The method simplifies the process with a single-step solid–liquid extraction and benefits from two-dimensional separation to enhance resolution and minimize matrix effects. The methodology was validated according to European Commission DG-SANTE (SANTE/11312/2021) and European Committee for Standardization (EN 15662, 2018) guidelines, obtaining acceptable analytical parameters. Limits of detection ranged from 0.2 µg/L to 30.2 µg/L and limits of quantification from 0.3 to 91.6 μg/L, all bellow regulatory limits. Accuracy, ranged from 60 to 120 % and precision, estimated by relative standard deviation, was consistently below 15.1 % in all cases. The applicability of the method was demonstrated by analyzing pesticides in five representative corn products (tortilla, corn flakes, corn cake, starch, and polenta). Sixteen pesticides were detected, all within the regulatory residue limits. Three metric tools were applied to assess the analytical process. Environmental sustainability was evaluated using the AGREE metric, while analytical performance and method applicability were assessed with the Red-Green-Blue and Blue Applicability Grade Index models, respectively, yielding promising scores (0.5, ≥ 0.55, and 0.65). These findings and the comparison to previous methods indicated that this approach represents a promising, alternative and user-friendly strategy for pesticide determination, offering effective productivity and reliable analytical performance.

High-Throughput Covalent Modifier Screening with Acoustic Ejection Mass Spectrometry.

Interests in covalent drugs have grown in modern drug discovery as they could tackle challenging targets traditionally considered "undruggable". The identification of covalent binders to target proteins typically involves directly measuring protein covalent modifications using high-resolution mass spectrometry. With a continually expanding library of compounds, conventional mass spectrometry platforms such as LC-MS and SPE-MS have become limiting factors for high-throughput screening. Here, we introduce a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for the rapid screening of a covalent modifier library comprising ∼10,000 compounds against a 50 kDa-sized target protein─Werner syndrome helicase. The screening samples were arranged in a 1536-well format. The sample buffer containing high-concentration salts was directly analyzed without any cleanup steps, minimizing sample preparation efforts and ensuring protein stability. The entire AEMS analysis process could be completed within a mere 17 h. An automated data analysis tool facilitated batch processing of the sample data and quantitation of the formation of various covalent protein-ligand adducts. The screening results displayed a high degree of fidelity, with a Z' factor of 0.8 and a hit rate of 2.3%. The identified hits underwent orthogonal testing in a biochemical activity assay, revealing that 75% were functional antagonists of the target protein. Notably, a comparative analysis with LC-MS showcased the AEMS platform's low risk of false positives or false negatives. This innovative platform has enabled robust high-throughput covalent modifier screening, featuring a 10-fold increase in library size and a 10- to 100-fold increase in throughput when compared with similar reports in the existing literature.

PANAMA-enabled high-sensitivity dual nanoflow LC-MS metabolomics and proteomics analysis

High-sensitivity nanoflow liquid chromatography (nLC) is seldom employed in untargeted metabolomics because current sample preparation techniques are inefficient at preventing nanocapillary column performance degradation. Here, we describe an nLC-based tandem mass spectrometry workflow that enables seamless joint analysis and integration of metabolomics (including lipidomics) and proteomics from the same samples without instrument duplication. This workflow is based on a robust solid-phase micro-extraction step for routine sample cleanup and bioactive molecule enrichment. Our method, termed proteomic and nanoflow metabolomic analysis (PANAMA), improves compound resolution and detection sensitivity without compromising the depth of coverage as compared with existing widely used analytical procedures. Notably, PANAMA can be applied to a broad array of specimens, including biofluids, cell lines, and tissue samples. It generates high-quality, information-rich metabolite-protein datasets while bypassing the need for specialized instrumentation.

Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study.

Amino acids (AAs) and their metabolites are important building blocks, energy sources, and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Therefore, robust, reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification are required to identify candidate biomarkers and to improve screening methods. We developed a validated semi-automated robotic liquid extraction and processing workflow and a rapid method for absolute quantification of 20 free, underivatized AAs and six TRP metabolites using dual-column U(H)PLC-MRM-MS. The extraction and sample preparation workflow in a 96-well plate was optimized for robust, reproducible high sample throughput allowing for transfer of samples to the U(H)PLC autosampler directly without additional cleanup steps. The U(H)PLC-MRM-MS method, using a mixed-mode reversed-phase anion exchange column with formic acid and a high-strength silica reversed-phase column with difluoro-acetic acid as mobile phase additive, provided absolute quantification with nanomolar lower limits of quantification within 7.9 min. The semi-automated extraction workflow and dual-column U(H)PLC-MRM-MS method was applied to a human prostate cancer study and was shown to discriminate between treatment regimens and to identify metabolites responsible for discriminating between healthy controls and patients on active surveillance.

Liquid-liquid and solid-liquid extractions with low-temperature partitioning – A review

The paper represents the first review of solvent extraction techniques utilizing the low-temperature partitioning/purification (LTP) approach. Initially conceived in the 1960s to purify extracts from fatty matrices, it wasn't until the 2000s that this approach received increasing attention for its efficacy in extracting organic compounds from diverse samples, often without additional cleanup steps. This review covers a brief history and proposes a mechanism for LTP-based solvent extraction. Furthermore, the principal practical issues of the technique are spotlighted, elucidating the factors influencing extraction efficiency. The advantages, limitations, and potential combinations with other extraction techniques of the LTP-based solvent extractions are analyzed. The versatility of the LTP approach is demonstrated by its applications in extracting various compounds from food, environmental, and biological samples, emphasizing its potential for rapid sample preparation with minimal steps, few chemicals, and minimal analyst intervention.

A novel CNN-based image segmentation pipeline for individualized feline spinal cord stimulation modeling

Objective. Spinal cord stimulation (SCS) is a well-established treatment for managing certain chronic pain conditions. More recently, it has also garnered attention as a means of modulating neural activity to restore lost autonomic or sensory-motor function. Personalized modeling and treatment planning are critical aspects of safe and effective SCS (Rowald and Amft 2022 Front. Neurorobotics 16 983072, Wagner et al 2018 Nature 563 65–71). However, the generation of spine models at the required level of detail and accuracy requires time and labor intensive manual image segmentation by human experts. This study aims to develop a maximally automated segmentation routine capable of producing high-quality anatomical models, even with limited data, to facilitate safe and effective personalized SCS treatment planning. Approach. We developed an automated image segmentation and model generation pipeline based on a novel convolutional neural network (CNN) architecture trained on feline spinal cord magnetic resonance imaging data. The pipeline includes steps for image preprocessing, data augmentation, transfer learning, and cleanup. To assess the relative importance of each step in the pipeline and our choice of CNN architecture, we systematically dropped steps or substituted architectures, quantifying the downstream effects in terms of tissue segmentation quality (Jaccard index and Hausdorff distance) and predicted nerve recruitment (estimated axonal depolarization). Main results. The leave-one-out analysis demonstrated that each pipeline step contributed a small but measurable increment to mean segmentation quality. Surprisingly, minor differences in segmentation accuracy translated to significant deviations (ranging between 4% and 13% for each pipeline step) in predicted nerve recruitment, highlighting the importance of careful workflow design. Additionally, transfer learning techniques enhanced segmentation metric consistency and allowed generalization to a completely different spine region with minimal additional training data. Significance. To our knowledge, this work is the first to assess the downstream impacts of segmentation quality differences on neurostimulation predictions. It highlights the role of each step in the pipeline and paves the way towards fully automated, personalized SCS treatment planning in clinical settings.

Optimization of an analytical method based on miniaturized matrix solid-phase dispersion combined with gas chromatography-mass spectrometry for the determination of bisphenols in mussel samples

In this study, a new method has been optimized to determinate nine bisphenols (BPs) in mussel samples. This approach is based on miniaturized matrix solid-phase dispersion combined with gas chromatography-mass spectrometry determination after trimethylsilylation with 99% N,O-Bis-(trimethylsilyl)trifluoroacetamide + 1 % trimethylchlorosilane. The optimization process investigated the derivatization (derivatizing agent volume, time and temperature) and sample preparation (C18 co-column packed and C18 dispersant agent amounts and elution volume) parameters. A second clean-up step was performed using EMR-Lipid cartridges in order to efficiently remove lipids from mussel, the optimized parameters were Captiva EMR-Lipid sorbent amount and elution volume. The whole method was validated and very good linearity (r2 > 0.99) was obtained. The recoveries (accuracy) at two concentration levels ranged from 51.2 to 109 % and the coefficients of variation (precision) were between 0.55 and 13 %. The limits of quantification were between 0.014 μg/Kg dry weight for BPM and 4.0 μg/Kg dry weight for BPS. The analytical procedure was applied to four mussel samples from Galician Rías. Except for one of four samples whose BPAF level was below the limit of quantification, all BPs were found in concentrations that ranged from 0.39 to 301 μg/Kg dry weight.

Investigating the capability of UA-DLLME and DART-HRMS in the analysis of benzodiazepines in whole human blood

Benzodiazepine (BZD) misuse has increased in the last decade, making its occurrence in criminal cases more commonplace. The detection of BZD in complex biological samples is challenging since they are usually found in small concentrations, requiring the development of sensitive and fast-to-execute methods. In this study, the application of direct analysis in real time – high-resolution mass spectrometry (DART-HRMS) was evaluated in the detection of 10 benzodiazepines (diazepam, oxazepam, chlordiazepoxide, temazepam, alprazolam, flunitrazepam, bromazepam, clonazepam, lorazepam, and midazolam) in ante and postmortem blood samples. Moreover, an ultrasound-assisted dispersive liquid–liquid microextraction (UA-DLLME) approach was developed by full factorial design as a clean-up step before the DART-HRMS analysis. DART-HRMS was capable of qualitative detection of all evaluated BZD in raw antemortem blood samples at concentrations as low as 10 µg mL−1. The UA-DLLME DART-HRMS approach was linear for the 10 BZD in the range of 1 to 10 µg mL−1, with recoveries ranging from 78.5 to 119.5 %, a precision lower than 36 % at 1 µg mL−1, and limits of detection varying between 0.25 and 0.50 µg mL−1. Moreover, the UA-DLLME DART-HRMS method was efficiently applied to postmortem blood samples from criminal cases, enabling the detection of BZD. The developed method facilitated the analysis of 10 BZD in ante and postmortem blood samples, offering a quick sample extraction that linked to the DART-HRMS can be used as a fast and reliable triage method for regulatory screening purposes and could be readily integrated into routine forensic analysis workflows in a high throughput manner.

Application of a novel deep eutectic solvent modified carbon nanotube for pipette-tip micro solid phase extraction of 6-mercaptopurine

Background6-mercaptopurine (6-MP) is a chemotherapy drug mainly used to treat leukemia. It is a persistent organic pollutant and can remain in the environment for a long period of time. The presence of 6-MP in the environment poses a number of hazards and needs to be assessed to monitor its potential risk to human health and the environment. However, due to its trace amount in complicated matrices, a clean-up and preconcentration step before its determination is compulsory.ResultsAs a highly efficient adsorbent for the extrication of 6-mercaptopurine (6-MP), a novel carbon nanotube doped with camphor: decanoic acid deep eutectic solvent was synthesized and applied as a packing material for the pipette-tip micro solid phase extraction sorbent of 6-MP from tap, wastewater and seawater samples before its spectrophotometric determination. Characteristics and structure of this adsorbent was fully investigated. Factors affecting extraction, including type and volume of the eluent, ionic strength and pH of the sample solution, amount of adsorbent, and number of extraction and elution cycles were optimized using one-factor-at-a-time and response surface methodologies. The method was found to be linear in the range of 1 to 1000 µg/L with a limit of detection and quantification of 0.2 and 0.7 µg/L, respectively. Reproducibility as relative standard deviation was better than 4.6%.ConclusionApplication of deep eutectic solvent modified carbon nanotube indicated suitable microextraction results and good potential for rapid extraction of trace amounts of 6-MP from different aqueous samples. The amount of sample required for the analysis was less than 10 mL and only 1.5 mg of the adsorbent was used. The total analysis time, including extraction was less than 15 min and the adsorbent could be used for at least 10 times, without significantly losing its adsorption ability. Compared to using unmodified usual carbon nanotubes, deep eutectic solvent doped carbon nanotubes showed 19.8% higher extraction ability.

Development of Green and Facile Sample Preparation Method for Determination of Seven Neonicotinoids in Fresh Vegetables, and Dissipation and Risk Assessment of Imidacloprid and Dinotefuran.

A facile procedure for extracting and determining seven neonicotinoids was developed. Water was the only extraction solvent without phase separation and cleanup steps. The method was validated according to European Union standards, and the values obtained were compared with the criteria. The accuracy values were between 99.8% (thiamethoxam) and 106.8% (clothianidin) at the spiking levels of 0.01, 0.1, and 1 mg/kg in the tested matrices. The precision as pooled RSD values was ≤6.1% (intra-day) and ≤6.9% (inter-day). The limit of quantification was set and tested at 0.01 mg/kg. The matrix effect was evaluated, and all matrices had a suppressive effect. The matrix of the cucumber was the most effective, with -20.9% for dinotefuran and an average of -9.8% for all compounds, while the tomato matrix had the slightest effect. Real marketed samples were analyzed using the developed and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methods; the results were not significantly different. A supervised field trial was conducted in the open field to study the dissipation patterns of imidacloprid and dinotefuran in tomatoes. The dissipation of both compounds followed first-order kinetics. The half-life (T½) values were 3.4 and 2.5 days, with dissipation rates k of 0.2013 and 0.2781 days, respectively. Following the EU-MRL database, the calculated pre-harvest interval (PHI) values were 7 and 14 days for imidacloprid and dinotefuran, respectively, and 3 days for both compounds following Codex Alimentarius regulations. The risk of imidacloprid and dinotefuran residues was estimated from chronic and acute perspectives. The risk factors of dinotefuran were lower than those of imidacloprid. Nonetheless, the highest expected residues of both compounds were below the tolerance limits.

Heat 'n Beat: A Universal High-Throughput End-to-End Proteomics Sample Processing Platform in under an Hour.

Proteomic analysis by mass spectrometry of small (≤2 mg) solid tissue samples from diverse formats requires high throughput and comprehensive proteome coverage. We developed a nearly universal, rapid, and robust protocol for sample preparation, suitable for high-throughput projects that encompass most cell or tissue types. This end-to-end workflow extends from original sample to loading the mass spectrometer and is centered on a one-tube homogenization and digestion method called Heat 'n Beat (HnB). It is applicable to most tissues, regardless of how they were fixed or embedded. Sample preparation was divided into separate challenges. The initial sample washing and final peptide cleanup steps were adapted to three tissue sources: fresh frozen (FF), optimal cutting temperature (OCT) compound embedded (FF-OCT), and formalin-fixed paraffin embedded (FFPE). Third, for core processing, tissue disruption and lysis were decreased to a 7 min heat and homogenization treatment, and reduction, alkylation, and proteolysis were optimized into a single step. The refinements produced near doubled peptide yield when compared to our earlier method ABLE delivered a consistently high digestion efficiency of 85-90%, reported by ProteinPilot, and required only 38 min for core processing in a single tube, with the total processing time being 53-63 min. The robustness of HnB was demonstrated on six organ types, a cell line, and a cancer biopsy. Its suitability for high-throughput applications was demonstrated on a set of 1171 FF-OCT human cancer biopsies, which were processed for end-to-end completion in 92 h, producing highly consistent peptide yield and quality for over 3513 MS runs.

An Optimized and Validated QuEChERS-Based Method for the Determination of PCBs in Edible Aquatic Species

AbstractIn the present research, a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was optimized and validated for the determination of 14 selected PCB congeners in mussel (Mytilus galloprovincialis) and fish samples (Sparus aurata). The optimization included the selection of different QuEChERS procedures, extraction solvent, sample weight, and suitable sorbents for the clean-up step to achieve high sensitivity and minimal matrix interferences simultaneously. The identification and quantification of the selected PCBs were carried out using GC–MS. The method was validated providing in all cases excellent linearity (r2 above 0.99). Recoveries were estimated at three different fortification levels (10, 50, and 100 ng g−1) and ranged from 72.5 to 109.5% and 72.1 to 88.4% for mussel and fish samples, respectively. In addition, for both matrices, the LOQs ranged from 1 to 2.5 ng g−1. The matrix effect was in all cases < 29%, while the expanded uncertainty U%, which was estimated at all the fortification levels, was found below 53% in all cases. Eventually, the optimized and validated method was applied to mussel and fish samples acquired from aquacultures in NW Greece, revealing the absence of the selected congeners in all cases.

A Multi-Residue Analytical Method for Assessing the Effects of Stacking Treatment on Antimicrobial and Coccidiostat Degradation in Broiler Litter.

Antimicrobial drugs and coccidiostat compounds are commonly used in poultry farming. These compounds are subsequently excreted and released into the environment via broiler litter (BL) and can re-enter the food chain as fertilizer or animal feed. Such residue in animal feed can encourage the appearance of antibiotic-resistant bacteria as well as toxicity. Most analytical methods used to identify and quantitate these drug residues are traditional, and are specific to some antimicrobials and present limitations in assessing complex matrixes like BL. The aim of this study was to develop a multi-residue analytic method for assessing 30 antimicrobial drugs and coccidiostats associated with BL. We investigated the presence and the effects of biotic stack treatment on the degradation of drug residue in BL. Liquid-liquid extraction (LLE) and solid phase extraction (SPE) were replaced by Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) clean-up steps and detected by liquid chromatography mass spectrometry (LC/MS/MS). Results show that a wide spectrum of residues were detected from 0.4 to 8.9 mg kg-1. Following lab-scale stacking treatment, tilmicosin and eight coccidiostats persisted in BL (26-100%). This research supports the need for better understanding, regulation, and management of the use of BL that might carry a high risk of residue drugs.

One step cleanup of 160 pesticides and veterinary drugs in aquatic products using melamine-based automatic pressure filtration purification method combined with HPLC-MS/MS

A 15-channel pressure filtration purification method was presented for high throughput sample preparation of aquatic products. A cost-effective device was constructed and melamine sponge was selected as the cleanup sorbent. Upon interfacing with HPLC-MS/MS, the analytical procedure demonstrated its suitability for quantifying 160 pesticides and veterinary drug residues in aquatic products such as fish, shrimp, and crab. The method achieved sample recoveries ranging from 61.3 to 124.9 %. The detection limits were established between 0.5 and 1.0 μg/kg, while the quantitation limits were confirmed to be within the range of 1.0–2.0 μg/kg. The method was applied to quantify the pesticide and veterinary drug residues in mostly consumed aquatic products from five coastal provinces in China. The results showed significant differences between different aquatic products in the concentrations of pesticide and veterinary drug residues, implying the necessity of supervision for the accurate determination of pesticides and veterinary drugs.