Sample Preparation Research Articles

The Bridge: Supernatant Derived From Cytological Sample Preparations.

The scope and extent of molecular cytopathology in the era of precision medicine has been expanding in recent years. The versatility of cytology specimen preparations has provided ample opportunity for the cytopathology community to evolve, innovate and 'do more with less' using limited amounts of tissue. More recently, cytology-derived supernatant liquid biopsy samples have been identified as a substantial source of high-quality genomic material that can be interrogated for genotyping for therapeutic decision-making, as well as other roles in cancer screening for early-stage disease, longitudinal monitoring for therapeutic response and disease prognostication. These novel substrates, including supernatants from body fluids such as urine, pleural effusion, ascitic fluid, cerebrospinal fluid, as well as fine-needle aspiration (FNA) specimens, serve as a bridge between tissue-based testing and conventional liquid biopsy testing from the patient's plasma. Cytologically derived liquid biopsy samples can only be used insituations where the tissue sample is inadequate for genotyping, or when plasma-based liquid biopsy fails to identify an oncogenic driver alteration, but they can be used as a stand-alone complementary specimen source that can provide reliable genomic information for therapeutic decisions. This review aims to highlight some of the advances in the field and the clinical applications of the cytology-derived supernatant specimen.

A novel method for semi-quantitative detection of HPV16 and HPV18 mRNA with a low-cost, open-source fluorimeter.

Despite global calls to eliminate cervical cancer, rates of cervical cancer incidence and mortality remain high in resource-limited settings, where it is challenging to implement and sustain screening, diagnosis, and treatment programs. The presence of high-risk HPV mRNA in cervical cells is a sensitive and specific biomarker of cervical precancer. Yet, current testing methods are too costly and complex for use in resource-limited settings. Here, we present a novel method for semi-quantitative detection of HPV16 and HPV18 mRNA with minimal infrastructure requirements. The assay relies on isothermal reverse transcription recombinase polymerase amplification (RT-RPA) with real-time fluorescence readout, demonstrated on rugged, portable, and affordable instruments. We demonstrate adapting the assay from DNA detection to RNA detection, characterizing the test with samples of increasing biological complexity, and ultimately establishing a limit of detection of 1000 HPV16 or HPV18 transcripts per reaction with RNA extracted from cell lines. HPV16 and HPV18 mRNA assays were used to test total RNA from 11 patient samples; results for 10 samples (91%) agreed with the gold standard of RT-qPCR. To reduce cost, the assay was demonstrated with multiplexed detection of HPV16 and HPV18 DNA, validated with a reaction volume that was reduced from 50 to 5 µL with DNA and RNA, and performed using a low-cost, portable reader with DNA and RNA. With incorporation of point-of-care-friendly sample preparation and detection of additional genotypes, this test has the potential to expand global access to HPV testing.

Non-contact SThM thermal conductivity measurements of GeSbTe thin films sputtered on silicon and glass substrates

Abstract Chalcogenide materials based on germanium-antimony-tellurium (GST) are typically used in phase change memory applications. The thermal conductivity of GST films is an important factor in predicting the temperature evolution and the structural alterations of the material in response to rapid thermal transitions inherent in memory operations. While several techniques have been used to determine the thin film thermal conductivity of GSTs, they require fabricating metal heaters or thermometers on test samples. In this paper we report using a non-contact scanning thermal microscopy (SThM) technique to measure the thermal conductivity of GST thin-films while circumventing the need for sample preparation. A Wollaston wire probe of a 5-µm diameter was used as a Joule-heated thermometer to measure the probe thermal resistance in air far away and at 100 nm away from the sample surface. Detailed heat transfer modeling between the probe, sample, and ambient, which considers the non-classical heat transfer across the gap between the SThM probe and sample surface was used to determine the thermal resistance of several GST films sputtered with different powers on glass and silicon substrates. The thermal conductivity of GST thin film shows a reducing trend from 0.7 to 0.2 W m-1 K-1 when the thickness reduces from 159 nm to 24 nm. The reasons for thermal conductivity reduction are discussed based on analytical heat transfer modeling.

Cloud Point Extraction as an Environmentally Friendly Technique for Sample Preparation

Cloud point extraction is a sample preparation technique that involves using surfactants that are not harmful to the environment. It is based on micelle formation in which the extracted compound is encapsulated in the hydrophobic core of the micelles, which are the extracting agent. The most commonly used surfactants are nonionic. The others are anionic, cationic, or zwitterionic. The effectiveness of cloud point extraction might be enhanced by the addition of neutral salts, the application of proper pH, as well as acidic conditions and temperature. This sample preparation technique may be applied to extract analytes from the following matrices, such as biological and environmental samples. Cloud point extraction may be combined with various analytical techniques and detectors such as HPLC-UV, HPLC-MS, HPLC-FLD, inductively coupled plasma–optical emission spectrometry, gas chromatography, and flame atomic absorption spectrometry. When it is combined with electrothermal atomic absorption spectrometry, the limit of quantitation is low—even of the order of ng/L. The recovery of the analyte may reach the value of 100%.

Analysis of vertical loading forces on three different sagittal split ramus osteotomy modifications.

The study aimed to compare and analyze the in vitro mechanical behavior of vertical load on three different sagittal split osteotomy designs proposed by Epker, Wolford, and Wyatt, focusing on the implications of each design on mandibular stability. Synthetic polyurethane hemi-mandibular models were used to replicate the osteotomies according to the designs suggested by Epker, Wolford, and Wyatt. Each model group was subjected to linear vertical loading until system failure, with peak load and deformation recorded. The study utilized a controlled sample preparation and loading test to ensure standardization across all groups. Analysis of variance (ANOVA) and the Tukey test were applied to compare the mechanical responses among the different osteotomy designs. The findings indicated no significant difference in displacement and vertical loading resistance between Groups 1 and 2; however, differences were found in Group 3 (Wyatt), where increased mandibular fragility was observed when screws were placed in thinner bone areas. Statistical analysis showed that the modifications in the osteotomy design led to significant differences in mechanical behavior, particularly in Group 3, highlighting the importance of bone thickness and osteotomy technique on postoperative early stability and mechanical stress distribution. The study concludes that the choice of sagittal split osteotomy design significantly impacts the mechanical behavior under vertical loading, with particular emphasis on the importance of bone thickness at fixation points and the technique used. The findings suggest a preference for the modification proposed in group 1 and 2 in cases where increased mandibular stability and minimized postoperative complications are desired.

Three-Dimensional Reconstruction-Characterization of Polymeric Membranes: A Review.

Polymeric membranes serve as vital separation materials in diverse energy and environmental applications. A comprehensive understanding of three-dimensional (3D) structures of membranes is critical to performance evaluation and future design. Such quantitative 3D structural information is beyond the limit of most employed conventional two-dimentional characterization techniques such as scanning electron microscopy. In this review, we summarize eight types of 3D reconstruction-characterization techniques for membrane materials. Originated from life and materials science, these techniques have been optimized to reveal the 3D structures of membrane materials in the separation field. We systematically introduce the theories of each technique, summarize the sample preparation procedures developed for membrane materials, and demonstrate step-by-step data processing, including 3D model reconstruction and subsequent characterization. Representative case studies are introduced to show the progress of this field and how technical challenges have been overcome over the years. In the end, we share our perspectives and believe that this review can serve as a useful reference for 3D reconstruction-characterization techniques developed for membrane materials.

Adsorption Studies of Salmonella Enteritidis and Escherichia coli on Chitosan-Coated Magnetic Nanoparticles

One of the challenges of microbiological testing is the complex and lengthy sample preparation, causing delays in getting the final result. Immunomagnetic separation is one of the sample preparation techniques recently used to overcome this complexity. However, it is expensive, fragile, and requires cold storage. This study aimed to use chitosan-coated magnetic nanoparticles (cMNP) to capture bacterial cells from a simulated matrix and understand the interaction between the bacteria and the cMNP using batch adsorption studies. To illustrate the concept, Salmonella Enteritidis and Escherichia coli were used. Results showed that the adsorption of Salmonella Enteritidis and E. coli fitted the pseudo-second-order kinetic model (R2 = 0.939 and 0.968, respectively) and the Freundlich isotherm model (R2 = 0.999 and 0.970, respectively). The increased ionic strength enhanced bacterial adsorption, and the highest capture efficiency was observed at pH 4 (32.8% and 98.1% for Salmonella Enteritidis and E. coli, respectively). These results show that chemisorption plays a significant role in bacterial adsorption to cMNP. Furthermore, increasing ionic strength and acidic pH (pH 4) significantly affects the adsorption of Salmonella Enteritidis and E. coli on cMNP, making them crucial for enhancing the performance of cMNP-based sample preparation methods.

Quantification of mycophenolic acid in plasma by isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method.

Accurate measurements of plasma mycophenolic acid (MPA) are essential for therapeutic drug monitoring in transplant recipients and autoimmune diseases. The performance of plasma mycophenolic acid routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of plasma mycophenolic acid measurements. In this study, sample preparation was based on protein precipitation with methanol followed by further dilution. The mycophenolic acid was quantified by the isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with electrospray ionization in positive ion mode. According to the Clinical and Laboratory Standards Institute (CLSI) documents C62-A and C50-A, the basic analytical performance of the candidate reference method was verified, such as linearity, limit of quantification, matrix effect, precision, accuracy, and uncertainty. Moreover, the candidate reference measurement procedure was compared with the routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a clinical laboratory. Based on the data, the mycophenolic acid in human plasma was well detected by ID-LC-MS/MS. No apparent interferences were found with the mycophenolic acid measurement. The calibration curve for the mycophenolic acid was linear in the concentration range of 0.1-50μg/mL with a correlation coefficient of 0.9999 under the optimum experimental conditions. This method was sensitive because the low limit of quantitation (LOQ) was 0.05μg/mL. The recoveries of MPA were 98.11-98.95%. The intra-day and inter-day coefficients of variations (CV) of our method were ≤ 1.53% and ≤ 0.51%, respectively. No obvious matrix effect was observed. There was a good correlation between this method and the clinical routine LC-MS/MS method. To sum up, we established and validated a reliable plasma MPA method using ID-LC/MS/MS. The desirable accuracy and precision of this method enable it to serve as a promising cRMP to improve the standardization for plasma MPA routine measurements.

Rapid, Sensitive Detection of Protein Biomarkers in Minimally-Processed Blood Products with a Monolithic Sandwich Immunoassay Reagent.

For more than fifty years, the enzyme-linked immunosorbent assay (ELISA) serves as the gold standard for protein biomarker detection. However, conventional ELISA requires considerable sample preparation including reagent addition, incubation, and washing steps, limiting its usefulness at the point-of-care. In this work, the "instant ELISA" (fluorophore-linked immunosorbent assay) biosensor that can measure protein biomarkers in the picomolar range within 15min in undiluted plasma or serum with no sample preparation is described. The sensor leverages a synthetic reagent termed the "monolithic dual-antibody clamp" (MDAC) which preserves the specificity, sensitivity, and generalizability of an ELISA, but produces a fluorescence signal as two surface-tethered antibodies form a "sandwich" by binding to two distinct epitopes on the target. As exemplars, picomolar quantification of tumor necrosis factor alpha (TNFα) and monocyte chemotactic protein (MCP)-1, the latter of which is a useful prognostic indicator of cytokine release syndrome in patient plasma samples during chimeric antigen receptor T cell therapy are demonstrated.

Development and Validation of Targeted Metabolomics Methods Using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) for the Quantification of 235 Plasma Metabolites

Plasma contains metabolites with diverse physicochemical properties, ranging from highly polar to highly apolar, and concentrations spanning at least nine orders of magnitude. Plasma metabolome analysis is valuable for monitoring health and evaluating medical interventions but is challenging due to the metabolome’s diversity and complexity. This study aims to develop and validate targeted LC-MS/MS methods for quantifying 235 mammalian metabolites from 17 compound classes in porcine plasma without prior derivatization. Utilizing reversed-phase and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry, each analyte is identified and quantified using two selected reaction monitoring (SRM) transitions. Fast polarity switching and scheduled SRM enhance the metabolome coverage and throughput, enabling the analysis of one sample in about 40 min. A simple “dilute and shoot” sample preparation protocol was employed, with samples injected at two dilution levels to align metabolite concentrations within calibration curve ranges. Validation in porcine plasma included assessments of carryover, linearity, detection and quantification limits, repeatability and recovery. The method was further applied to plasma samples from various animal species, demonstrating its applicability to human and animal studies. This study establishes two robust LC-MS/MS methods for comprehensive porcine plasma metabolome quantification, advancing large-scale targeted metabolomics in biomedical research.

Fabrication and characterization approach to enhance the mechanical performance of zirconia‐coated Multi‐walled carbon nanotubes reinforced High density polyethylene composites

AbstractThis study presents a comprehensive investigation into the fabrication and characterization of high‐density polyethylene (HDPE) composites reinforced with zirconia (ZrO2)‐coated multi‐walled carbon nanotubes (MWCNTs). HDPE, a widely used polymer for pressure pipe applications, was selected for its superior mechanical properties, while ZrO2‐coated MWCNTs were employed as nanofillers to enhance these properties further. The acid functionalization of MWCNTs, followed by the application of a ZrO2 coating, was executed to ensure optimal dispersion and interfacial bonding within the HDPE matrix. The functionalized MWCNTs (f‐MWCNTs) were synthesized via an acid treatment process using 8 M HNO3, which significantly eased agglomeration and facilitated a stable ZrO2 coating. Subsequent hydrothermal processing at 200°C for 18 h yielded uniformly coated MWCNTs. These nanofillers were then melt‐blended with HDPE, followed by injection molding. Mechanical testing revealed substantial enhancements in the tensile, flexural, and hardness properties of the composites. The tensile strength exhibited a marked increase, peaking at a 3 wt.% ZrO2‐coated MWCNT concentration, with a 50% improvement over pristine HDPE. Flexural modulus and hardness values also saw significant increments, with the highest enhancements recorded at 4 wt.% filler content. Impact testing showed improved toughness, although excessive filler loading led to diminished returns due to nanoparticle aggregation. FTIR and XRD analyses supported the successful integration of ZrO2‐coated MWCNTs within the HDPE matrix, highlighting the presence of characteristic peaks associated with both MWCNTs and HDPE. These findings provide the potential of ZrO2‐coated MWCNTs as effective nanofillers in enhancing the mechanical performance of HDPE composites, making suitable for advanced engineering applications.Highlights Coating of ZrO2 on MWCNTs using Hydrothermal method. Melt blending of ZrO2 coated MWCNT with HDPE using twin‐screw extruder. Preparation of samples through injection molding as per ASTM standard. Mechanical properties and characterization analysis of the composite.

Validated HPLC method for simultaneous determination of azelastine hydrochloride fluticasone propionate and oxymetazoline in nasal mucosa and nasopharyngeal swabs from real human samples

A combination of three co-administrated drugs, such as azelastine hydrochloride (AZT), fluticasone propionate (FP), and oxymetazoline (OXY), is more effective than single therapy for the treatment of seasonal allergy and COVID-19. We established an efficient methodology for the determination of those analytes in spiked nasal mucosa and nasopharyngeal swabs from real human samples. A simple and quick protein precipitation method was used for sample extraction, using acetonitrile. RP-HPLC/DAD method was performed using an Exsil 100 ODS C18 (250 × 4.6 mm, 5 μm) column with an acetonitrile: water (70:30 v/v) solvent system at a flow rate of 0.7 mL/min. A photodiode array detector was applied at 240 nm. A good separation of the three proposed analytes with a short run time of 10 min was noted. Our method was validated according to FDA guidelines for bioanalytical validation methods. Calibration curves were linear in nasal mucosa samples at concentration ranges of 8–125, 10–100, and 10–125 µg/mL, with average recoveries ± SD of 101.56%±0.39, 102.45%±0.86, and 104.61%±4.52 for AZT, FP, and OXY; respectively. The results of precision and accuracy are within acceptable limits. According to stability assays, the three analytes under investigation were stable throughout sample preparation, storage, and injection. Our method was applied to real nasopharyngeal swabs. It shows that the results of the swabs were not affected by gender or age. Good recoveries with low % RSD were observed: 99.03% ± 0.75, 100.02% ± 0.94, and 100.94% ± 1.98 for both genders, and 100.45% ± 0.96, 100.69% ± 1.08, and 100.32% ± 1.53 for different ages for AZT, FP, and OXY; respectively. Moreover, the amount of those drugs in the nasal mucosa was observed for seven hours, and a constant concentration with a low% RSD was noted for the first four hours. Therefore, this method can be applied to monitor the therapeutic dose in the nasal mucosa for the determination of those analytes.

Simple steps to achieve harmonisation and standardisation of dried blood spot phenylalanine measurements and facilitate consistent management of patients with phenylketonuria.

Management of phenylketonuria (PKU) relies upon life-long monitoring of phenylalanine (Phe) in dried blood spots (DBS), thus comparability of measurements is important. The lack of harmonisation and standardisation between laboratories, combined with the variable quality of patient-collected DBS specimens, are currently preventing this from being achieved. A traceable, matrix-matched Phe certified reference material, common methodology and means to ensure patient collected DBS specimens are of consistent quality would improve comparability between laboratories. Baseline inter-laboratory (n=15) variation of DBS Phe was determined by triplicate measurement of four DBS materials, on three days. Laboratories prepared and analysed these samples using their routine method of analysis. A sub-set of laboratories (n=5) repeated the process using a common sample preparation and instrument methodology (LC-MS/MS), and three different calibration approaches. Samples prepared on dried blood spot microsampling cards (DBS-MCs) from whole blood, value assigned for Phe concentration by National Measurement Laboratories (NML), were then analysed using the harmonised methodology. Inter-laboratory co-efficient of variation (CV) differed with calibration approach; internal calibration 27.7 %; in-house aqueous calibration 4.7 %; centrally distributed aqueous calibration, 2.1 %. Inter-laboratory CV was reduced from 8.7 to 2.1 % by using common sample preparation and LC-MS/MS methodology. No significant difference was observed between consensus and assigned values for Phe in the four materials (p>0.05). This study demonstrates a simple approach to harmonising and standardising DBS Phe measurements, traceable to value assigned materials. Combined with the introduction of DBS-MCs to ensure specimen quality, clinical laboratories can achieve comparability of patient results over time.

Direct observations of nucleation and early-stage growth of Au-catalyzed GaAs nanowires on Si(111)

Developing a reliable procedure for the growth of III-V nanowires (NW) on silicon (Si) substrates remains a significant challenge, as current methods rely on trial-and-error approaches with varying interpretations of critical process steps such as sample preparation, Au-Si alloy formation in the growth reactor, and NW alignment. Addressing these challenges is essential for enabling high-performance electronic and optoelectronic devices that combine the superior properties of III-V NW semiconductors with the well-established Si-based technology. Combining conventional scalable growth methods, such as metalorganic chemical vapor deposition (MOCVD) within situcharacterization using environmental transmission electron microscopy (ETEM-MOCVD) enables a deeper understanding of the growth dynamics, if that knowledge is transferable to the scalable processes. We report on successful epitaxial growth of Au-catalyzed GaAs NWs on Si(111) substrates using micro-electromechanical system chips with monocrystalline Si-cantilevers in both conventional MOCVD and ETEM-MOCVD systems. The conventional MOCVD provided a framework for initial parameter tuning, while ETEM-MOCVD offered valuable insights into early nucleation and catalyst-substrate interactions. Our findings show that nucleation is significantly influenced by the removal of native oxide layers and the initial formation of the Au-Si alloy. Ourin situstudies revealed different NW-substrate interfaces, essential for optimizing the epitaxial growth process. We identified three typical configurations of NW 'roots', each impacted by growth conditions and preparation steps, affecting the structural and potentially the optical properties of the NWs. Similarly, doping from the Si-substrate may affect both optical and electrical properties; however, compositional analysis revealed no traces of Si in NWs post-nucleation and a small amount in the catalytic droplet. Our research highlights the importance ofin situstudies for a comprehensive understanding of nucleation mechanisms, paving the way for optimizing III-V NW growth on Si substrates and developing high-performance III-V/Si devices.

The Promise of Infrared Spectroscopy in Liquid Biopsies for Solid Cancer Detection

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy has shown significant promise in the context of liquid biopsy, offering a potential tool for cancer diagnostics. Unlike traditional tissue biopsies, which may not fully capture the clonal heterogeneity of tumors, liquid biopsy reflects the dynamic state of the disease and its progression more comprehensively. Biofluids such as serum and plasma are low-cost, minimally invasive diagnostic media with well-established clinical uses. This review assesses the use of ATR-FTIR spectroscopy to detect biochemical changes in biofluids linked to various malignancies, including breast, ovarian, endometrial, prostate, bladder, kidney, pancreatic, colorectal, hepatic, esophageal, gastric, lung, and brain cancers. While ATR-FTIR offers the advantages of rapid, minimally invasive detection and real-time disease monitoring, its integration into clinical practice faces challenges, particularly in terms of reproducibility due to variability in sample preparation, spectral acquisition, and data processing. The translation of ATR-FTIR into routine diagnostics will require validation through large-scale cohort studies and multicenter trials to ensure its clinical reliability and effectiveness.

European Society of Toxicologic Pathology-Pathology 2.0 Mass Spectrometry Imaging Special Interest Group: Mass Spectrometry Imaging in Diagnostic and Toxicologic Pathology for Label-Free Detection of Molecules-From Basics to Practical Applications.

Mass Spectrometry Imaging (MSI) is a powerful tool to understand molecular pathophysiology and therapeutic and toxicity mechanisms, as well as for patient stratification and precision medicine. MSI, a label-free technique offering detailed spatial information on a large number of molecules in different tissues, encompasses various techniques including Matrix-Assisted Laser Desorption Ionization (MALDI), Desorption Electrospray Ionization (DESI), and Secondary Ion Mass Spectrometry (SIMS) that can be applied in diagnostic and toxicologic pathology. Given the utmost importance of high-quality samples, pathologists play a pivotal role in providing comprehensive pathobiology and histopathology knowledge, as well as information on tissue sampling, orientation, morphology, endogenous biomarkers, and pathogenesis, which are crucial for the correct interpretation of targeted experiments. This article introduces MSI and its fundamentals, and reports on case examples, determining the best suited technology to address research questions. High-level principles and characteristics of the most used modalities for spatial metabolomics, lipidomics and proteomics, sensitivity and specific requirements for sample procurement and preparation are discussed. MSI applications for projects focused on drug metabolism, nonclinical safety assessment, and pharmacokinetics/pharmacodynamics and various diagnostic pathology cases from nonclinical and clinical settings are showcased.

Contact and Non-Contact Measurements of Chlorophyll-a in Water Based on Laser Spectroscopy

Chlorophyll-a fluorescence detection is an important technique for monitoring water quality. In this paper, we proposed an approach that employs the ratio of fluorescence to Raman obtained from contact and non-contact laser-induced fluorescence detection methods as the key for the detection of Chlorophyll-a in water. At first, extracted Chlorophyll-a solutions were prepared, and each sample was tested using two detection methods. The true values of the samples were measured in the laboratory using spectrophotometry. Then, the detection system was calibrated through the linear fitting of Chlorophyll-a and the fluorescence–Raman ratio. The linear correlation coefficients of contact and non-contact detection were 0.9453 and 0.9401, respectively. Finally, we tested the actual water samples in two ways, and compared the test results with the value measured using the national standard method. The root mean square error (RMSE) of contact and non-contact detection was 0.16 and 0.23, respectively. The results show that the two detection methods have high accuracy and sensitivity, and preliminary preparation of samples is not required. Compared with contact detection, the non-contact detection results had higher accuracy and stronger anti-interference, but the maintenance cost was higher because the probe is vulnerable to wear. The advantage of non-contact detection is that it avoids sample contamination and is easy to apply over large areas of water. In the future, it can be used for the real-time monitoring of algal biomass in water by selecting the appropriate detection method according to the requirements of the application.

Pure Shift NMR with Solvent Suppression: A Robust and General Method for Determining Quantitative Metabolic Profiles in Biofluids.

Ultrahigh-resolution pure shift NMR has recently been shown as a promising approach for providing quantitative metabolic profiles that can be used to study the metabolic footprint left by cancer cells in their aqueous growth medium. In this approach, a library of reference 1H pure shift spectra with water suppression was implemented to determine metabolite concentrations from the NOESY-presat-PSYCHE-SAPPHIRE spectrum recorded on the extracellular medium. This achievement clearly called for a generalization of a quantification method relying on ultrahigh-resolution data to other biological samples of interest (urine, plasma, tissue extracts, etc.), which requires evaluating the robustness of the analytical workflow. We have first addressed the influence of sample preparation on the quality of metabolite quantification. The quantification performed on a model mixture of metabolites prepared under different conditions shows good linearity, trueness, and precision, which highlights the high reproducibility of the proposed analytical protocol regardless of the physicochemical conditions in the sample. Second, we have successfully implemented this quantification protocol to determine metabolite levels in real urine and plasma samples, thereby paving the way for the use of the library of pure shift reference spectra for accurate and quantitative metabolic profiling of a broad range of aqueous samples.

Enabling high-throughput quantitative wood anatomy through a dedicated pipeline.

Throughout their lifetime, trees store valuable environmental information within their wood. Unlocking this information requires quantitative analysis, in most cases of the surface of wood. The conventional pathway for high-resolution digitization of wood surfaces and segmentation of wood features requires several manual and time consuming steps. We present a semi-automated high-throughput pipeline for sample preparation, gigapixel imaging, and analysis of the anatomy of the end-grain surfaces of discs and increment cores. The pipeline consists of a collaborative robot (Cobot) with sander for surface preparation, a custom-built open-source robot for gigapixel imaging (Gigapixel Woodbot), and a Python routine for deep-learning analysis of gigapixel images. The robotic sander allows to obtain high-quality surfaces with minimal sanding or polishing artefacts. It is designed for precise and consistent sanding and polishing of wood surfaces, revealing detailed wood anatomical structures by applying consecutively finer grits of sandpaper. Multiple samples can be processed autonomously at once. The custom-built open-source Gigapixel Woodbot is a modular imaging system that enables automated scanning of large wood surfaces. The frame of the robot is a CNC (Computer Numerical Control) machine to position a camera above the objects. Images are taken at different focuspoints, with a small overlap between consecutive imagesin the X-Y plane, and merged by mosaic stitching, into a gigapixel image. Multiple scans can be initiated through the graphical application, allowing the system to autonomously image several objects and large surfaces. Finally, a Python routine using a trained YOLOv8 deep learning network allows for fully automated analysis of the gigapixel images, here shown as a proof-of-concept for the quantification of vessels and rays on full disc surfaces and increment cores. We present fully digitized beech discs of 30-35cm diameter at a resolution of 2.25 m, for which we automatically quantified the number of vessels (up to 13 million) and rays. We showcase the same process for five 30cm length beech increment cores also digitized at a resolution of 2.25 m, and generated pith-to-bark profiles of vessel density. This pipeline allows researchers to perform high-detail analysis of anatomical features on large surfaces, test fundamental hypotheses in ecophysiology, ecology, dendroclimatology, and many more with sufficient sample replication.

Robust Detection of Ecstasy-Like and Adulterants Through ASAP-MS and DD-SIMCA.

Ecstasy is a complex and hazardous substance, and its identification is increasingly challenging. Conventional analytical methods have limitations in terms of sensitivity and selectivity, and more precise techniques are time-consuming and necessitate sample preparation. ASAP-MS and DD-SIMCA are two methods that have the potential to address these issues. This research delves into the efficacy of ASAP-MS and DD-SIMCA as a rapid and dependable approach for detecting ecstasy and its adulterants. PCA was conducted as an initial exploration, with the first three principal components (PCs) capturing 69% of the overall data variability. The score plot of PC1 × PC3 revealed the distribution of samples containing MDA and MDMA. The DD-SIMCA model exhibited high sensitivity in identifying the target samples (MDA) and relatively high specificity in training and test sets. These results underscore the effectiveness of ASAP-MS, PCA, and DD-SIMCA for precise identification of ecstasy and its adulterants, indicating their potential in drug identification and analysis. We observed that the chemometric model associated with ASAP-MS was able to accurately identify, when compared to the results obtained by the standard technique, the constituents of ecstasy tablets, even in the presence of adulterants. Furthermore, the method could detect emerging psychoactive substances that are typically not targeted by traditional analytical approaches. These findings suggest that ASAP-MS and DD-SIMCA could be valuable tools in forensic drug analysis laboratories. The method is rapid, reliable, and versatile for identifying a wide range of ecstasy and its adulterants.