Sample Preparation Research Articles

The evolving role of interventional cytopathology from thyroid FNA to NGS: Lessons learned at Federico II University of Naples.

Fine-needle aspiration (FNA) guided by ultrasound (US) has emerged as a highly precise diagnostic method for managing thyroid nodules, significantly diminishing unnecessary surgeries. The effectiveness of US-guided FNA is high when a single specialist performs the FNA procedure and the microscopy. This paradigm has paved the way for the evolution of interventional cytopathology, a specialist with a pivotal role in the preoperative diagnostic process, encompassing patient history review, clinical examination, FNA execution under US guidance, preparation, and microscopic interpretation of cytological samples. As the landscape of precision medicine unfolds, molecular testing assumes greater importance in thyroid cytopathology, particularly in refining the risk of malignancy for indeterminate nodules. The updated Bethesda classification system underscores the clinical significance of molecular tests, emphasizing their role in refining diagnostic accuracy. With this evolving landscape, interventional cytopathologists must adapt by acquiring expertise in molecular technologies and addressing ongoing challenges in workflow harmonization and optimization. This paper delves into our decade-long experience as interventional cytopathologists, focusing on recent endeavours to ensure adequate samples not only for microscopic diagnosis but also for molecular testing. Additionally, here we review the challenges of integrating next-generation sequencing (NGS) technology into clinical practice, highlighting the importance of integrating clinically meaningful molecular data into comprehensive molecular cytology reports.

Understanding emulsifier influence on complex coacervation: Essential oils encapsulation perspective.

The objective of this research was to explore the viability of pea protein as a substitute for gelatin in the complex coacervation process, with a specific focus on understanding the impact of incorporating an emulsifier into this process. The study involved the preparation of samples with varying polymer mixing ratios (1:1, 1:2, and 2:1) and emulsifier content. As core substances, black pepper and juniper essential oils were utilized, dissolved beforehand in grape seed oil or soybean oil, to minimize the loss of volatile compounds. In total, 24 distinct samples were created, subjected to freeze-drying to produce powder, and then assessed for their physicochemical properties. Results revealed the significant impact of emulsifier addition on microcapsule parameters. Powders lacking emulsifiers exhibited higher water solubility (57.10%-81.41%) compared to those with emulsifiers (24.64%-40.13%). Moreover, the emulsifier significantly decreased thermal stability (e.g., without emulsifier, Ton=137.21°C; with emulsifier, Ton=41.55°C) and adversely impacted encapsulation efficiency (highest efficiency achieved: 67%; with emulsifier: 21%).

NMR side-chain assignments of the Crimean–Congo hemorrhagic fever virus glycoprotein n cytosolic domain

Abstract. We assigned the side-chain resonances of the Crimean–Congo hemorrhagic fever virus (CCHFV) glycoprotein n (Gn) cytosolic domain that is 69 amino acids long to complete the backbone resonances previously published by Estrada et al. (2011). The process was facilitated by three factors. First, sample preparation using cell-free protein synthesis (CFPS) was completed in less than 2 d and allowed for correct zinc finger formation by adding zinc ions to the reaction. Second, access to NMR platforms with standardized pulse sequences allowed for data acquisition in 18 d. Third, data analysis using the online platform NMRtist allowed sequential resonance assignments to be made in a day, and assignments were verified and finalized in less than a week. Our work thus provides an example of how NMR assignments, including side chains, of small and well-behaved proteins can be approached in a rapid routine, at protein concentrations of 150 µM.

Automated single-cell proteomics providing sufficient proteome depth to study complex biology beyond cell type classifications

The recent technological and computational advances in mass spectrometry-based single-cell proteomics have pushed the boundaries of sensitivity and throughput. However, reproducible quantification of thousands of proteins within a single cell remains challenging. To address some of those limitations, we present a dedicated sample preparation chip, the proteoCHIP EVO 96 that directly interfaces with the Evosep One. This, in combination with the Bruker timsTOF demonstrates double the identifications without manual sample handling and the newest generation timsTOF Ultra identifies up to 4000 with an average of 3500 protein groups per single HEK-293T without a carrier or match-between runs. Our workflow spans 4 orders of magnitude, identifies over 50 E3 ubiquitin-protein ligases, and profiles key regulatory proteins upon small molecule stimulation. This study demonstrates that the proteoCHIP EVO 96-based sample preparation with the timsTOF Ultra provides sufficient proteome depth to study complex biology beyond cell-type classifications.

STED Imaging of Vesicular Endocytosis in the Synapse.

Endocytosis is a fundamental biological process that couples exocytosis to maintain the homeostasis of the plasma membrane and sustained neurotransmission. Super-resolution microscopy enables optical imaging of exocytosis and endocytosis in live cells and makes an essential contribution to understanding molecular mechanisms of endocytosis in neuronal somata and other types of cells. However, visualization of exo-endocytic events at the single vesicular level in a synapse with optical imaging remains a great challenge to reveal mechanisms governing the synaptic exo-endocytotic coupling. In this protocol, we describe the technical details of stimulated emission depletion (STED) imaging of synaptic endocytosis at the single-vesicle level, from sample preparation and microscopy calibration to data acquisition and analysis.

Mass spectrometry-intensive top-down proteomics: an update on technology advancements and biomedical applications.

Proteoforms are all forms of protein molecules from the same gene because of variations at the DNA, RNA, and protein levels, e.g., alternative splicing and post-translational modifications (PTMs). Delineation of proteins in a proteoform-specific manner is crucial for understanding their biological functions. Mass spectrometry (MS)-intensive top-down proteomics (TDP) is promising for comprehensively characterizing intact proteoforms in complex biological systems. It has achieved substantial progress in technological development, including sample preparation, proteoform separations, MS instrumentation, and bioinformatics tools. In a single TDP study, thousands of proteoforms can be identified and quantified from a cell lysate. It has also been applied to various biomedical research to better our understanding of protein function in regulating cellular processes and to discover novel proteoform biomarkers of diseases for early diagnosis and therapeutic development. This review covers the most recent technological development and biomedical applications of MS-intensive TDP.

Applications of Raman Microscopy/Spectroscopy-Based Techniques to Plant Disease Diagnosis

Plant diseases pose a significant threat to plant and crop health, leading to reduced yields and economic losses. The traditional methods for diagnosing plant diseases are often invasive and time-consuming and may not always provide accurate results. In recent years, there has been growing interest in utilizing Raman microscopy as a non-invasive and label-free technique for plant disease diagnosis. Raman microscopy is a powerful analytical tool that can provide detailed molecular information about samples by analyzing the scattered light from a laser beam. This technique has the potential to revolutionize plant disease diagnosis by offering rapid and accurate detection of various plant pathogens, including bacteria and fungi. One of the key advantages of Raman microscopy/spectroscopy is its ability to provide real-time and in situ analyses of plant samples. By analyzing the unique spectral fingerprints of different pathogens, researchers can quickly identify the presence of specific diseases without the need for complex sample preparation or invasive procedures. This article discusses the development of a Raman microspectroscopy system for disease diagnosis that can accurately detect and identify various plant pathogens, such as bacteria and fungi.

Structure and Properties of Bioactive Titanium Dioxide Surface Layers Produced on NiTi Shape Memory Alloy in Low-Temperature Plasma

Background: The NiTi alloy, known for its shape memory and superelasticity, is increasingly used in medicine. However, its high nickel content requires enhanced biocompatibility for long-term implants. Low-temperature plasma treatments under glow-discharge conditions can improve surface properties without compromising mechanical integrity. Methods: This study explores the surface modification of a NiTi alloy by oxidizing it in low-temperature plasma. We examine the impact of process temperatures and sample preparation (mechanical grinding and polishing) on the structure of the produced titanium oxide layers. Surface properties, including topography, morphology, chemical composition, and bioactivity, were analyzed using TEM, SEM, EDS, and an optical profilometer. Bioactivity was assessed through the deposition of calcium phosphate in simulated body fluid (SBF). Results: The low-temperature plasma oxidization produced titanium dioxide layers (29–55 nm thick) with a predominantly nanocrystalline rutile structure. Layer thickness increased with extended processing time and higher temperatures (up to 390 °C), though the relationship was not linear. Higher temperatures led to thicker layers with more precipitates and inhomogeneities. The oxidized layers showed increased bioactivity after 14 and 30 days in SBF. Conclusions: Low-temperature plasma oxidation produces bioactive titanium oxide layers on NiTi alloys, with a structure and properties that can be tuned through process parameters. This method could enhance the biocompatibility of NiTi alloys for medical implants.

Heterogeneous Integration of Acoustic Microextraction with an Optoelectronic Sensor on Glass for Nucleic Acid Testing.

Lab-on-a-chip systems (LOCs), characterized by their high sensitivity, low sample consumption, and portability, have significantly advanced the field of on-site testing. Despite the evolution of integrated LOCs from qualitative to quantitative analyses, on-chip full integration of sample preparation, purification, and multiplexed detection remains a challenge. Here, we propose a strategy for the heterogeneous integration of a set of complementary metal oxide semiconductor-compatible devices including acoustic resonator, thin-film resistors, and temperature/photosensors as a new type of LOC for nucleic acid testing (NAT). Programmed acoustic streaming-based particles and fluid manipulations largely simplify the nucleic acid extraction process including cell lysis, nucleic acid capture, and elution. The design of the acoustic microextraction module and extraction process was thoroughly studied. Benefitted by the microelectromechanical system approach, the conventional mechanical actions and complex flow control are avoided, which enables a compact hand-held NAT instrument without complicated peripherals. Validation experiments conducted on plasma-harboring mutations in the epidermal growth factor receptor (EGFR) gene confirmed the robustness of the system, achieving an impressive nucleic acid (NA) extraction efficiency of approximately 90% within 5 min and a limit of detection of the target NA in the plasma of 1 copy/μL.

Development and validation of a sensitive and simultaneous liquid chromatography tandem mass spectrometry method for the determination of eight phytocannabinoids in various CBD products

In this study, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of eight phytocannabinoids in various cannabidiol (CBD) products from Japanese market. This method was combined with electrospray ionization in positive mode and sample preparation with QuEChERS. Three types of commercial products such as honey, chocolate, and gummies were used to perform accurate quantification with unified protocol of LC-MS/MS and QuEChERS. The limit of detection and quantification were 5–20 µg g−1 and 10–40 µg g−1, respectively. Reproducibility was ensured using matrices free of target foods, resulting in an accuracy within ±10 % and a precision with a relative standard deviation of less than 5 % for all targets. Finally, this analytical method was applied to 8 series of commercial samples from the Japanese market. This unified protocol will serve as a reference as an official method in Japan.

A single-sample workflow for joint metabolomic and proteomic analysis of clinical specimens

Understanding the interplay of the proteome and the metabolome helps to understand cellular regulation and response. To enable robust inferences from such multi-omics analyses, we introduced and evaluated a workflow for combined proteome and metabolome analysis starting from a single sample. Specifically, we integrated established and individually optimized protocols for metabolomic and proteomic profiling (EtOH/MTBE and autoSP3, respectively) into a unified workflow (termed MTBE-SP3), and took advantage of the fact that the protein residue of the metabolomic sample can be used as a direct input for proteome analysis. We particularly evaluated the performance of proteome analysis in MTBE-SP3, and demonstrated equivalence of proteome profiles irrespective of prior metabolite extraction. In addition, MTBE-SP3 combines the advantages of EtOH/MTBE and autoSP3 for semi-automated metabolite extraction and fully automated proteome sample preparation, respectively, thus advancing standardization and scalability for large-scale studies. We showed that MTBE-SP3 can be applied to various biological matrices (FFPE tissue, fresh-frozen tissue, plasma, serum and cells) to enable implementation in a variety of clinical settings. To demonstrate applicability, we applied MTBE-SP3 and autoSP3 to a lung adenocarcinoma cohort showing consistent proteomic alterations between tumour and non-tumour adjacent tissue independent of the method used. Integration with metabolomic data obtained from the same samples revealed mitochondrial dysfunction in tumour tissue through deregulation of OGDH, SDH family enzymes and PKM. In summary, MTBE-SP3 enables the facile and reliable parallel measurement of proteins and metabolites obtained from the same sample, benefiting from reduced sample variation and input amount. This workflow is particularly applicable for studies with limited sample availability and offers the potential to enhance the integration of metabolomic and proteomic datasets.Graphical

Rapid, Portable, and Electricity-free Sample Extraction Method for Enhanced Molecular Diagnostics in Resource-Limited Settings.

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.

Genome analysis of multidrug resistant Enterococcus faecium and Enterococcus faecalis circulating among hospitalized patients in uMgungundlovu District, KwaZulu-Natal, South Africa

BackgroundVancomycin-resistant enterococci (VRE) are important pathogens categorized as high-priority bacteria in the Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics published by the World Health Organization. The aim of this study was to determine the risk factors, resistance, virulence, mobilomes associated with multidrug-resistant and clonal lineages of Enterococcus faecium and faecalis circulating among hospitalized patients following the health system in South Africa, using whole genome sequencing (WGS).MethodsA cross-sectional study was conducted during a two-month periods among hospitalized patients in 2017. Rectal swabs were collected from patients admitted to medical and surgical wards in an urban tertiary hospital, and a rural district hospital in uMgungundlovu district, South Africa. Enterococci were screened for vancomycin resistance on bile esculin azide agar supplemented with 6 mg/L of vancomycin and confirmation of VRE was done using ROSCO kits. Conventional and real-time PCR methods were used to ascertain the presence of VanA, VanB, VanC-2/3 and VanC-1 genes. All six multidrug-resistant Enterococcus faecalis and faecium selected were identified using multiplexed paired-end libraries (2 × 300 bp) with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and genome sequencing was done using Illumina MiSeq instrument with 100× coverage at the National Institute of Communicable Diseases Sequencing Core Facility, South Africa. Antibiotic resistance genes, virulence factors, plasmids, integrons and CRISPR were characterized using RAST, ResFinder, VirulenceFinder, PlasmidFinder, PHAST and ISFinder respectively.ResultsSequencing analysis revealed that these strains harbouring numerous resistance genes to glycopeptides (vanC[100%], vex3[100%], vex2[83,33%] and vanG[16,66%]), macrolides, lincosamides, sterptogramine B (ermB[33,32%], Isa[16,66%], emeA[16,66%]) and tetracyclines (tetM[33,32%]) in both district and tertiary hospitals. Multidrug efflux pumps including MATE, MFS and pmrA conferring resistance to several classes of antibiotics were also identified. The main transposable elements observed were in the Tn3 family, specifically Tn1546. Four single sequence types (STs) were identified among E. faecium in the district hospital, namely ST822, ST636, ST97 along with a novel ST assigned ST1386, while one lineage, ST29 was detected in the tertiary hospital.ConclusionThe study reveals the genetic diversity and high pathogenicity of multidrug-resistant Enterococcus faecalis and faecium circulating among hospitalized patients. It underlines the necessity to implement routine screening of admitted patients coupled with infection control procedures, antimicrobial stewardship and awareness should be strengthened to prevent and/or contain the carriage and spread of multidrug resistant E. faecium and E. faecalis in hospitals and communities in South Africa.

Profiling the volatile compounds of Peganum harmala L. Based on multiple sample preparation coupled with gas chromatography-mass spectrometry analysis and explored its antidepressants-like activity

Peganum harmala L., a traditional medicinal plant in China, is renowned for its significant alkaloid content in seeds and roots exhibiting a wide range of pharmacological activities, including antidepressant, antiseptic, and antiviral. However, the volatile composition of the herb remained unclear. Apart from that, the extraction of volatile compounds through essential oil presents challenges due to the low yield and the degradation of volatile active compounds at high temperatures. This study used multiple sample preparation methods including headspace (HS), needle trap device (NTD), and liquid–liquid extraction (LLE) coupled with gas chromatography-mass spectrometry (GC–MS) to analyze the volatile compounds from the areal part of P. harmala L.. A total of 93 compounds were identified with NTD facilitating the first detection of harmine among the volatile organic compounds. Through network pharmacology and protein interaction analysis, the compounds’ potential therapeutic targets of the compounds were explored, and 23 key targets were obtained (AKT1, ALB, PTGS2, MAOA, etc). KEGG pathway enrichment analysis indicated significant involvement in neuroactive ligand-receptor interactions and serotonergic synapses. The results enhanced the understanding of P. harmala’s pharmacological mechanisms and supported its ethnopharmacological use.

Simultaneous measurement of 17 endogenous steroid hormones in human serum by liquid chromatography-tandem mass spectrometry without derivatization

Mass spectrometric-based steroidomics is a valuable analytical approach that gives a comprehensive understanding of the interlinked steroid biosynthetic pathways. Here, we describe a rapid and versatile liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed to accurately quantify endogenous steroids in human serum. Sample preparation involved liquid-liquid extraction with methyl tert-butyl ether (MTBE) from 180µL serum. The targeted steroids for quantification included androgens: dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), 11-oxyandrogens: 11β-hydroxy-androstenedione (11OHA4), 11-keto-androstenedione (11KA4), 11β-hydroxy-testosterone (11OHT), 11-keto-testosterone (11KT), progestogens: 17α-hydroxy-progesterone (17OHP4), progesterone (P4), 11β-hydroxy-progesterone (11OHP4), 11-keto-progesterone (11KP4), mineralocorticoids: aldosterone, corticosterone, and glucocorticoids: 11-deoxycortisol, cortisol, and cortisone. The lower limits of quantification (LLOQ) were 0.05ng/mL for A4, T, 11KA4, P4, and cortisone, 0.1ng/mL for DHT, 11OHA4, 11OHT, 11KT, 17OHP4, 11OHP4, 11KP4, corticosterone, aldosterone, 11-deoxycortisol, and cortisol, and 0.5ng/mL for DHEA. Accuracy, precision, reproducibility, and recovery fell within acceptable limits for bioanalytical method validation. Using serum samples from 29 premenopausal women in different menstrual phases, we demonstrated the clinical utility of our method, which showed sufficient sensitivity to reliably quantify all targeted steroids at levels typically found in circulation, except for 11OHP4 and 11KP4.

Recent Advances in the Determination of Major and Trace Elements in Plants Using Inductively Coupled Plasma Optical Emission Spectrometry

Interest in measuring major and trace elements in plants has increased in recent years because of growing concerns about the elements’ contribution to daily intakes or the health risks posed by ingesting vegetables contaminated by potentially toxic elements. The recent advances in using inductively coupled plasma atomic emission spectrometry (ICP-OES) to measure major and trace elements in plant samples are reviewed in the present work. The sample preparation before instrumental determination and the main advantages and limitations of ICP-OES are described. New trends in element extraction in liquid solutions using fewer toxic solvents and microextractions are observed in recently published literature. Even though ICP-OES is a well-established and routine technique, recent innovations to increase its performance have been found. Validated methods are needed to ensure the obtaining of reliable results. Much research has focused on assessing principal figures of merit, such as limits of detection, quantification, selectivity, working ranges, precision in terms of repeatability and reproducibility, and accuracy through spiked samples or certified reference materials analysis. According to the published literature, the ICP-OES technique, 50 years after the release of the first commercially available equipment, remains a powerful and highly recommended tool for element determination on a wide range of concentrations.

O-117 Advances in PGT diagnostics - technologies beyond copy number

Abstract Genetic abnormalities are well recognised as the main factor contributing to the high rate of infertility, pregnancy loss and congenital abnormalities in humans. A prime example of this can be seen from recent studies showing that embryos identified as uniformly aneuploid following PGT-A have little to no reproductive potential. However, embryos shown to have no gross chromosomal abnormalities after PGT are not a guarantee of healthy pregnancy and many other undetected genetic issues are likely to influence embryo competence. Developments in both sample preparation and methods for DNA analysis are driving ever-increasing data yield from PGT platforms, permitting not only DNA quantitation, but also allowing qualitative evaluation of genotyping data in parallel. This opens the door to the possibility of overcoming some of the previous limitations of PGT, leading to a more comprehensive assessment of the genetic health and reproductive potential of embryos. How to use this increasing amount of information, and if it can tangibly improve test accuracy and clinical outcomes, remains open to debate. There is, for instance, no consensus on how to interrogate genotyping data for better distinction of clinically relevant aneuploidy as well as genome wide (ploidy) abnormalities and exogenous DNA contamination, which could impact the correct reporting of PGT results. There is also no consensus on the potential benefit of genotyping data to be used for the accurate distinction of meiotic recombination by linkage analysis, nor for fingerprinting of embryos to identify rare, but catastrophic, lab error events as a routine part of treatment. Finally, there is no clear path on how to utilise these improvements in embryo genotyping, to detect sub-chromosomal de novo genetic abnormalities that could impact embryo viability beyond the scope of current testing for chromosomal abnormalities alone. The aim of this presentation is therefore to focus on the current state of the ART and address the current and potential future uses of genotyping in PGT. Topics covered will include identification of biopsy sample contamination to determine the truth from the noise, through to how we can rescue embryos by utilising PGT as a molecular fertilisation check and how fingerprinting of embryo can be an invaluable tool in the clinic. The origin of aneuploidy will be explored and how genotyping is reshaping our understanding of aneuploidy landscape of the preimplantation embryo. From a more basic science perspective, the potential of genotyping to identify meiotic recombination will be discussed and how this fundamental element of human reproduction could be an important biomarker for reproductive competence. Lastly future perspectives on how the evolution of PGT technologies can bring us closer to the accurate detection of de novo genomic changes will be explored and how this is taking PGT-A beyond just aneuploidy detection. It is no secret that PGT for embryo selection continues to be a controversial area of reproductive medicine, and the field of PGT-A especially continues to show disquieting inconsistency in data analysis and reporting. This new era of genotyping brings with it information that can be used as a standardization tool helping to focus and facilitate improvements in the field of PGT for better and more consistent clinical reporting and outcomes.

Skin Surface Sebum Analysis by ESI-MS

The skin surface is an important sample source that the metabolomics community has only just begun to explore. Alterations in sebum, the lipid-rich mixture coating the skin surface, correlate with age, sex, ethnicity, diet, exercise, and disease state, making the skin surface an ideal sample source for future noninvasive biomarker exploration, disease diagnosis, and forensic investigation. The potential of sebum sampling has been realized primarily via electrospray ionization mass spectrometry (ESI-MS), an ideal approach to assess the skin surface lipidome. However, a better understanding of sebum collection and subsequent ESI-MS analysis is required before skin surface sampling can be implemented in routine analyses. Challenges include ambiguity in definitive lipid identification, inherent biological variability in sebum production, and methodological, technical variability in analyses. To overcome these obstacles, avoid common pitfalls, and achieve reproducible, robust outcomes, every portion of the workflow—from sample collection to data analysis—should be carefully considered with the specific application in mind. This review details current practices in sebum sampling, sample preparation, ESI-MS data acquisition, and data analysis, and it provides important considerations in acquiring meaningful lipidomic datasets from the skin surface. Forensic researchers investigating sebum as a means for suspect elimination in lieu of adequate fingerprint ridge detail or database matches, as well as clinical researchers interested in noninvasive biomarker exploration, disease diagnosis, and treatment monitoring, can use this review as a guide for developing methods of best-practice.

Estimating LoD-s Based on the Ionization Efficiency Values for the Reporting and Harmonization of Amenable Chemical Space in Nontargeted Screening LC/ESI/HRMS.

Nontargeted LC/ESI/HRMS aims to detect and identify organic compounds present in the environment without prior knowledge; however, in practice no LC/ESI/HRMS method is capable of detecting all chemicals, and the scope depends on the instrumental conditions. Different experimental conditions, instruments, and methods used for sample preparation and nontargeted LC/ESI/HRMS as well as different workflows for data processing may lead to challenges in communicating the results and sharing data between laboratories as well as reduced reproducibility. One of the reasons is that only a fraction of method performance characteristics can be determined for a nontargeted analysis method due to the lack of prior information and analytical standards of the chemicals present in the sample. The limit of detection (LoD) is one of the most important performance characteristics in target analysis and directly describes the detectability of a chemical. Recently, the identification and quantification in nontargeted LC/ESI/HRMS (e.g., via predicting ionization efficiency, risk scores, and retention times) have significantly improved due to employing machine learning. In this work, we hypothesize that the predicted ionization efficiency could be used to estimate LoD and thereby enable evaluating the suitability of the LC/ESI/HRMS nontargeted method for the detection of suspected chemicals even if analytical standards are lacking. For this, 221 representative compounds were selected from the NORMAN SusDat list (S0), and LoD values were determined by using 4 complementary approaches. The LoD values were correlated to ionization efficiency values predicted with previously trained random forest regression. A robust regression was then used to estimate LoD values of unknown features detected in the nontargeted screening of wastewater samples. These estimated LoD values were used for prioritization of the unknown features. Furthermore, we present LoD values for the NORMAN SusDat list with a reversed-phase C18 LC method.

Advances in targeted liquid chromatography-tandem mass spectrometry methods for endocannabinoid and N-acylethanolamine quantification in biological matrices: A systematic review.

Liquid chromatography paired with tandem mass spectrometry (LC-MS/MS) is the gold standard in measurement of endocannabinoid concentrations in biomatrices. We conducted a systematic review of literature to identify advances in targeted LC-MS/MS methods in the period 2017-2024. We found that LC-MS/MS methods for endocannabinoid quantification are relatively consistent both across time and across biomatrices. Recent advances have primarily been in three areas: (1) sample preparation techniques, specific to the chosen biomatrix; (2) the range of biomatrices tested, recently favoring blood matrices; and (3) the breadth of endocannabinoid and endocannabinoid-like analytes incorporated into assays. This review provides a summary of the recent literature and a guide for researchers looking to establish the best methods for quantifying endocannabinoids in a range of biomatrices.