Clean-up Step Research Articles

A loop‐mediated isothermal amplification assay to detect insect‐associated Serratia spp. in the critically endangered insect Dryococelus australis

Abstract Point‐of‐care diagnostic methods are being increasingly employed to monitor infection risks in animal populations. These approaches are particularly valuable for ex situ invertebrate conservation programmes that often involve large numbers of individuals and where the rapid return of results supports timely risk mitigation decisions. Insect frass is a useful substrate to screen for the presence of pathogens transmitted by the faecal‐oral route in live insects but can result in false negatives when using common nucleic acid detection techniques, such as polymerase chain reaction (PCR), because of poor DNA extraction yields and the presence of inhibitors. We developed a diagnostic test to detect potentially entomopathogenic Serratia spp. in the frass of captive Dryococelus australis, using a cleanup step to improve DNA extraction by separating and concentrating bacterial cells, and a loop‐mediated isothermal amplification (LAMP) assay targeting the ureD gene. The LAMP assay had 1000 times greater analytical sensitivity than quantitative PCR when testing frass, did not yield false positive reactions when used to test a panel of 102 non‐Serratia spp., achieved results in 30 min and required only basic equipment. We applied the assay in real‐life conditions to screen captive insects, yielding information about the enclosures, life stages and insect species shedding potential entomopathogens, to inform the biosecurity and management practices of the captive breeding and conservation programme for D. australis. This diagnostic test could be modified for use in field conditions and adapted to detect other pathogens of concern in invertebrate conservation programmes and for insects reared in captivity for research or agricultural purposes.

Solid-Liquid Extraction with Low-Temperature Partitioning as an Alternative to Determinate Pesticide Residues in Teas

Methods used to determine pesticide residues in teas typically require several laborious steps for sample preparation before analysis. In this study, a simple method based on solid-liquid extraction with low-temperature partitioning (SLE/LTP) and analysis by gas chromatography coupled to mass spectrometry (GC-MS) was optimized and validated for the determination of pesticide residues (pirimiphos-methyl, flutriafol, cyproconazole, and bifenthrin) in dehydrated green tea leaves. After low-temperature partitioning (LTP), the extract in acetonitrile was subjected to a clean-up step using primary secondary amine (PSA) and octadecylsilane (C18) as sorbents for chlorophyll removal. The optimized SLE/LTP-GC-MS method was validated, and it proved to be effective and selective for extracting pesticides from green tea samples, presenting limit of detection (LOD) and limit of quantification (LOQ) of 0.015 and 0.050 mg kg-1, respectively. Recovery ranged between 81-111%, coefficients of variation were less than 16% and coefficients of determination (R2 ) were greater than 0.990. The optimized and validated method was applied to green tea and 13 other tea varieties sampled in South and Southeast regions of Brazil. The pesticides under study were detected in some of those samples at values higher than the maximum residue limits (MRLs) allowed by the European Union.

Analysis of Plasticizer Contamination Throughout Olive Oil Production.

This study monitored the contamination of 32 plasticizers in olive oil throughout the production and storage process. Samples were collected at different stages of production from three olive oil production lines in distinct regions of Portugal and analyzed for 23 phthalates and 9 phthalates substitutes to identify contamination sources. The developed analytical method employed liquid-liquid extraction with hexane/methanol (1:4, v/v), followed by centrifugation, extract removal, and freezing as a clean-up step. Analysis was conducted using gas chromatography tandem mass spectrometry (GC-MS/MS), with detection limits ranging from 0.001 to 0.103 mg/kg. The results revealed that plasticizer concentrations progressively increased at each stage of the production process, although unprocessed olives also contained contaminants. Di-isononyl phthalate (DINP) was the most prevalent compound, but all phthalates regulated by the European Union for food contact materials were detected, as well as some unregulated plasticizers. In a few packaged olive oils, DINP concentrations exceeded the specific migration limits established by European regulations. Samples stored in glass and plastic bottles showed no significant differences in plasticizer concentrations after six months of storage. However, higher concentrations were observed in plastic-packaged samples after 18 months of storage. Our findings indicate that the primary source of plasticizer contamination in olive oil originates from the production process itself, except for prolonged storage in plastic bottles, which should be avoided.

Environmentally friendly LLME-LTP of the bisphenol A from bovine milk samples

This paper developed a novel and environmental microextraction for bisphenol A from milk samples using liquid-liquid microextraction with low-temperature partitioning, which is a sample clean-up step for endocrine disruptors, being a suitable alternative. Furthermore, the microextraction and determination steps were optimized to determine and quantify the bisphenol A from milk. Thus, the determination of it was made using HPLC with fluorescence detection, obtaining the analytical with a linear range from 5.0 to 150 µg L−1 bisphenol (Area = 11,894 C (µg L−1) + 22,595) with good linearity (r = 0.9983), quantification (5.0 µg L−1) limit, and variation coefficient (4.4%), which were according to the established by regulatory agencies. The bisphenol A microextraction employed only 500 µL of the bovine milk sample and 1000 µL of acetonitrile, obtaining excellent recovery values for (89 − 105%) in UHT and pasteurized milk. This proposed methodology had several advantages in determining the bisphenol A, such as quick analysis, miniaturization of the process, decreasing the solvent and sample volume, and following the Green Analytical Chemistry.

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.

Study on the Determination of Polycyclic Aromatic Hydrocarbons (PAHs) in Fingernail and Hair Samples by Gas Chromatography Mass Spectrometry (GC/MS)

Information about human exposure to polycyclic aromatic hydrocarbons (PAHs) in Vietnam is still very limited. In this study, an accurate method for the determination of 16 PAHs in human hair and fingernail samples was developed. The nail and hair samples were digested in sodium hydroxide solution. PAHs in the digested solutions were extracted using organic solvents such as hexane and dichloromethane. The extracts were cleaned up by using solid phase extraction (SPE) cartridges containing silica gel with dichloromethane/hexane mixture as elution solvent. PAHs were quantified by a gas chromatography/mass spectrometry (GC/MS) system operated in electron impact ionization and selected ion monitoring (EI/SIM) mode. The liquid-liquid extraction and SPE clean-up steps were investigated to obtain acceptable recovery of PAHs before application to nail and hair samples. Recovery of PAHs in spiked nail and hair samples ranged from 66% to 112%, indicating acceptable method accuracy. Concentrations of 16 PAHs in the nail and hair samples were 160 and 733 ng/g, respectively. Major PAHs found in the nail and hair samples were naphthalene, phenanthrene, fluoranthene, and pyrene. Additional studies on human biomonitoring of PAHs and their related compounds should be conducted in Vietnam.

Enhanced analysis of PAHs in tea using GC–MS/MS with hydrogen carrier gas

A green multiresidue method for PAH analysis was developed using hydrogen as the carrier gas, avoiding harmful solvents and integrating waste-reducing strategies such as µSPE. The use of hydrogen carrier gas in gas chromatography coupled with tandem mass spectrometry (GC–MS/MS) was explored for the enhanced detection of polycyclic aromatic hydrocarbons (PAHs) in black tea. The study demonstrated that hydrogen, as a carrier gas, offers better chromatographic properties than helium or nitrogen. This leads to improved separation of critical pairs using a short run time method. A fast extraction method using micro solid-phase extraction (µSPE) as a clean-up step was developed to analyze 24 PAHs. The extraction method was optimized using a 4 mL 1:1 mixture of ethyl acetate and acetone. The method was validated and demonstrated good recoveries, ranging from 73 % to 113 % for most PAHs, with lower recoveries (50–63 %) observed for high molecular weight PAHs. Reproducibility was also high, with relative standard deviation (RSD) values below 15 %. Linearity and matrix effects were also evaluated, yielding excellent results, with no PAH exhibiting strong matrix effects. Real sample analysis of black tea from various brands and origins revealed the presence of multiple PAHs with concentrations ranging 9.5–39.2 µg/Kg, some exceeding the maximum residue levels set by the European Union. These findings highlight the need for efficient methodologies to monitor PAHs in food products.

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.