Identification of Volatiles in Tomato Fruit Using Headspace Solid-Phase-Micro-Extraction (HS-SPME) Coupled with Gas Chromatography-Mass Spectrometry (GC-MS).

Plant volatile organic compounds (VOCs) are organic chemicals that plants release as part of their natural biological processes. Various plant tissues produce VOCs, including leaves, stems, flowers, and roots. VOCs are essential in plant communication, defense against pests and pathogens, aroma and flavor, and attracting pollinators. The study of plant volatiles has become an increasingly important area of research in recent years, as scientists have recognized these compounds' important roles in plant physiology. As a result, there has been a growing interest in developing methods for collecting and analyzing plant VOCs. HS-SPME-GC-MS (headspace solid-phase microextraction-gas chromatography-mass spectrometry) is commonly used for plant volatile analysis due to its high sensitivity and selectivity. This chapter describes an efficient method for extracting and identifying volatile compounds by HS-SPME coupled with GC-MS in tomato fruits.

From molecules to visuals: Empowering drug discovery and development with mass spectrometry imaging.

Over the past three decades, mass spectrometry imaging (MSI) has emerged as a valuable tool for the spatial localization of drugs and metabolites directly from tissue surfaces without the need for labels. MSI offers molecular specificity, making it increasingly popular in the pharmaceutical industry compared to conventional imaging techniques like quantitative whole-body autoradiography (QWBA) and immunohistochemistry, which are unable to distinguish parent drugs from metabolites. Across the industry, there has been a consistent uptake in the utilization of MSI to investigate drug and metabolite distribution patterns, and the integration of MSI with omics technologies in preclinical investigations. To continue the further adoption of MSI in drug discovery and development, we believe there are two key areas that need to be addressed. First, there is a need for accurate quantification of analytes from MSI distribution studies. Second, there is a need for increased interactions with regulatory agencies for guidance on the utility and incorporation of MSI techniques in regulatory filings. Ongoing efforts are being made to address these areas, and it is hoped that MSI will gain broader utilization within the industry, thereby becoming a critical ingredient in driving drug discovery and development.

Innovative direct introduction-ion mobility-mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix: Application to 2'-fucosyllactose (2'-FL) in breast milk.

Identification and specific quantification of isomers in a complex biological matrix by mass spectrometry alone is not an easy task due to their identical chemical formula and therefore their same mass-to-charge ratio (m/z). Here, the potential of direct introduction combined with ion mobility-mass spectrometry (DI-IM-MS) for rapid quantification of isomers as human milk oligosaccharides (HMOs) was investigated. Differences in HMO profiles between various analyzed breast milk samples were highlighted using the single ion mobility monitoring (SIM2) acquisition for high ion mobility resolution detection. Furthermore, the Se+ (secretor) or Se- (non-secretor) phenotype could be assigned to breast milk samples studied based on their HMO contents, especially on the response of 2'-fucosyllactose (2'-FL) and lacto-N-fucopentaose I (LNFP I). The possibility of quantifying a specific isomer in breast milk by DI-IM-MS was also investigated. The standard addition method allowed the determination of the 2'-FL despite the presence of other oligosaccharides, including 3-fucosyllactose (3-FL) isomer in breast milk. This proof-of-concept study demonstrated the high potential of such an approach for the rapid and convenient quantification of isomers in complex mixtures.

Metabolomic-Based Comparison of Daphnia magna and Japanese Medaka Responses After Exposure to Acetaminophen, Diclofenac, and Ibuprofen.

Pharmaceuticals are found in aquatic environments due to their widespread use and environmental persistence. To date, a range of impairments to aquatic organisms has been reported with exposure to pharmaceuticals; however, further comparisons of their impacts across different species on the molecular level are needed. In the present study, the crustacean Daphnia magna and the freshwater fish Japanese medaka, common model organisms in aquatic toxicity, were exposed for 48 h to the common analgesics acetaminophen (ACT), diclofenac (DCF), and ibuprofen (IBU) at sublethal concentrations. A targeted metabolomic-based approach, using liquid chromatography-tandem mass spectrometry to quantify polar metabolites from individual daphnids and fish was used. Multivariate analyses and metabolite changes identified differences in the metabolite profile for D. magna and medaka, with more metabolic perturbations for D. magna. Pathway analyses uncovered disruptions to pathways associated with protein synthesis and amino acid metabolism with D. magna exposure to all three analgesics. In contrast, medaka exposure resulted in disrupted pathways with DCF only and not ACT and IBU. Overall, the observed perturbations in the biochemistry of both organisms were different and consistent with assessments using other endpoints reporting that D. magna is more sensitive to pollutants than medaka in short-term studies. Our findings demonstrate that molecular-level responses to analgesic exposure can reflect observations of other endpoints, such as immobilization and mortality. Thus, environmental metabolomics can be a valuable tool for selecting sentinel species for the biomonitoring of freshwater ecosystems while also uncovering mechanistic information. Environ Toxicol Chem 2024;00:1-13. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

In situ fabrication of covalent organic frameworks on solid-phase microextraction probes coupled with electrospray ionization mass spectrometry for enrichment and determination of androgens in biosamples.

Solid-phase microextraction (SPME) coupled with electrospray ionization mass spectrometry (ESI-MS) was developed for rapid and sensitive determination of endogenous androgens. The SPME probe is coated with covalent organic frameworks (COFs) synthesized by reacting 1,3,5-tri(4-aminophenyl)benzene (TPB) with 2,5-dioctyloxybenzaldehyde (C8PDA). This COFs-SPME probe offers several advantages, including enhanced extraction efficiency and stability. The analytical method exhibited wide linearity (0.1-100.0 µg L-1), low limits of detection (0.03-0.07 µg L-1), high enrichment factors (37-154), and satisfactory relative standard deviations (RSDs) for both within one probe (4.0-14.8%) and between different probes (3.4-12.7%). These remarkable performance characteristics highlight the reliability and precision of the COFs-SPME-ESI-MS method. The developed method was successfully applied to detect five kinds of endogenous androgens in female serum samples, indicating that the developed analytical method has great potential for application in preliminary clinical diagnosis.

Characterization of banned colorants in cosmetics: A tandem mass-based molecular networking approach.

Colorants have been a staple in the cosmetics industry for a considerable time, although certain varieties have been banned owing to health risks. Detecting and confirming these banned colorants simultaneously poses several challenges when employing LC-MS/MS. Molecular networking is a promising analytical technology that can be used to predict the structure of components and the correlation between them using structural and MS/MS spectral similarities. Molecular networking entails assessing the number of fragmented ions and the cosine score (the closer it is to one, the higher the similarity). In this study, we developed and verified a method for the simultaneous quantitative analysis of the 26 banned colorants in cosmetics using LC-MS/MS. Additionally, we propose a novel approach that combines LC-Q-TOF-MS and molecular networking technology to detect banned colorants in cosmetics. For successful molecular networking, a minimum of six fragment ions with cosine scores exceeding 0.5 is required. We developed a screening method for characterizing banned colorants using molecular networking based on LC-TOF-MS results for 26 banned colorants. Furthermore, we demonstrated that our established method can be used for screening by analyzing actual cosmetics (eyebrow tattoo, lipstick tattoo, and hair tint) spiked with three non-targeted banned colorants with similar structures (m/z 267.116, 315.149, and 345.157) in cosmetics. The combination of molecular networking techniques and LC-MS/MS proves highly advantageous for the swift characterization and screening of non-targeted colorants in cosmetics.

Evaluation of pollution levels in sediments from Lake Edku, Egypt using laser ablation inductively coupled plasma mass spectrometry.

The concentrations of 11 heavy metals in sediments from Lake Edku, Egypt were determined using LA-ICP-MS. The average concentrations of elements occurred in the order of Fe > V > Cr > Zn > Ni > Cu > Co > Pb > As > Sn > Mo with respective values of 4.67 %, 104.8, 77.9, 76.6, 59.2, 52, 27.8, 19.8, 4.14, 2.24, and 1.45 µg/g. Several pollution indices were used to evaluate individual and cumulative contamination levels. All HMs were found to be in the deficiency to minimal enrichment range based on the enrichment factor. The contamination factor indicated low contamination levels of Cr and As, low to moderate contamination levels of Fe, Ni, Zn, Mo, Sn, and Pb, and moderate contamination levels of Co and Cu. The pollution load index and contamination degree indicated the sediments to be polluted and moderately polluted, respectively.

Structural basis for the interaction of Redß single strand annealing protein with Escherichia coli single-stranded DNA binding protein.

Redß is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ exonuclease (λ-exo), Redß is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redß can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redß with E. coli single-stranded DNA binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redß and SSB involves the C-terminal domain (CTD) of Redß and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redß CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redß and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redß-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology.

Volumetric dried blood spots for determination of phosphatidylethanol: Validation of a liquid chromatography tandem masspectrometry method and clinical application.

Phosphatidylethanol (PEth) measurement in whole blood samples is established as a specific alcohol biomarker with clinical and forensic applications. Establishment of dried blood spots (DBSs) as a specimen for PEth determination offers several advantages and was the focus of this work. A liquid chromatography tandem mass spectrometry method using a 96-well format for sample preparation was developed and validated. PEth was extracted from DBSs by using isopropanol containing PEth-d5 as internal standard. The blood sampling used a commercial volumetric DBS device having a phosholipase D inhibitor incorporated to stop continuous PEth formation. The method quantified PEth in the range of 0.05-10 µmol/L, with a bias and imprecision of less than 15%. In a clinical study (n = 25) using fingerprick blood, the volumetric device offered more precise quantifications (CV 4.6%) compared with the Whatman 903 Protein Saver card device (CV 16.6%). In another clinical study (n = 48), the use of dried venous and capillary blood, and liquid venous blood was compared under real-life conditions with samples sent by postal service. The capillary and venous DBS samples gave identical results while the liquid blood gave slightly higher values. Calculation of elimination half-life (PEth 16:0/18:1) in 31 cases based on two consecutive samples with 2-9 days in between gave results (mean 6.2 days) that agree with literature but several cases with values over 10 days. In conclusion, this study demonstrates that volumetric DBS is a valid specimen for determination of PEth blood concentrations, offering several advantages.

A comprehensive dataset for assessing the impact of ammonium salts and zeolite on anaerobic digestion performance, microbial dynamics, and metabolomic profiles.

This article presents comprehensive data derived from lab-scale batch anaerobic digesters that were subjected to inhibition by various sources of ammonia. To counter this inhibition, zeolite was introduced into selected digesters. The provided dataset offers a detailed depiction of degradation performance dynamics over time, as well as insights into both microbial and metabolic changes during the inhibition. In detail, 10 conditions were tested in triplicate. In a first series of 15 bioreactors ammonia was introduced to achieve a TAN concentration of 8 g/L, utilizing NH3 solution, NH4Cl salt, (NH4)2CO3 salt, or (NH4)2PO4 salt as inhibitors. A control condition without ammonia was also set up. A second series of 15 bioreactors was set up exactly as the first one, with the addition of zeolite at a concentration of 15 g/L. The data provided includes information on operational conditions, degradation performance measurements throughout the entire process (using biogas production and composition, dissolved organic and inorganic carbon, volatile fatty acids, pH, free and total ammonia nitrogen, apparent isotopic fractionation of biogas as indicators), microbial community analysis using 16S rRNA gene sequencing (50 samples analysed), and metabolomic analysis through liquid chromatography-mass spectrometry (LC-MS) (108 samples analysed). Sequencing data were generated by using IonTorrent PGM sequencer. The sequencing data have been deposited with links to project PRJEB52324, in ENA database from EBI (https://www.ebi.ac.uk/ena/browser/view/PRJEB52324). Sample accession numbers go from SAMEA14277573 to SAMEA14277621. The metabolomic data were generated using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, MA, US). The metabolomic data have been deposited to the EMBL-EBI MetaboLights database with the identifier MTBLS7859 (https://www.ebi.ac.uk/metabolights/MTBLS7859). This data can be used as a source for comparisons with other studies focusing on the inhibition of anaerobic digestion by ammonia, particularly in the context of exploring microbial or metabolomic dynamics during inhibition. Additionally it provides a multi-omic dataset (metataxonomic and metabolomic) with detailed associated metadata describing anaerobic digesters. The dataset is directly is associated to the research article titled "Inhibition of anaerobic digestion by various ammonia sources resulted in subtle differences in metabolite dynamics." [1].

Targeted metabolomics profiling in pregnancy associated with vitamin D deficiency.

BACKGROUND: Vitamin D deficiency is common in pregnancy, however, its effects has not been fully elucidated. Here, we conducted targeted metabolomics profiling to study the relationship. METHODS: This study enrolled 111 pregnant women, including sufficient group (n = 9), inadequate group (n = 49) and deficient group (n = 53). Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based targeted metabonomics were used to characterize metabolite profiles associated with vitamin D deficiency in pregnancy. RESULTS: Many metabolites decreased in the inadequate and deficient group, including lipids, amino acids and others. The lipid species included fatty acyls (FA 14:3, FA 26:0; O), glycerolipids (MG 18:2), glycerophospholipids (LPG 20:5, PE-Cer 40:1; O2, PG 29:0), sterol lipids (CE 20:5, ST 28:0; O4, ST 28:1; O4). Decreased amino acids included aromatic amino acids (tryptophan, phenylalanine, tyrosine) and branched-chain amino acids (valine, isoleucine, leucine), proline, methionine, arginine, lysine, alanine, L-kynurenine,5-hydroxy-L-tryptophan, allysine. CONCLUSIONS: This targeted metabolomics profiling indicated that vitamin D supplementation can significantly affect lipids and amino acids metabolism in pregnancy.

Modern approaches for detection of volatile organic compounds in metabolic studies focusing on pathogenic bacteria: Current state of the art.

Pathogenic microorganisms produce numerous metabolites, including volatile organic compounds (VOCs). Monitoring these metabolites in biological matrices (e.g., urine, blood, or breath) can reveal the presence of specific microorganisms, enabling the early diagnosis of infections and the timely implementation of targeted therapy. However, complex matrices only contain trace levels of VOCs, and their constituent components can hinder determination of these compounds. Therefore, modern analytical techniques enabling the non-invasive identification and precise quantification of microbial VOCs are needed. In this paper, we discuss bacterial VOC analysis under in vitro conditions, in animal models and disease diagnosis in humans, including techniques for offline and online analysis in clinical settings. We also consider the advantages and limitations of novel microextraction techniques used to prepare biological samples for VOC analysis, in addition to reviewing current clinical studies on bacterial volatilomes that address inter-species interactions, the kinetics of VOC metabolism, and species- and drug-resistance specificity.

The effects of immortalization on the N-glycome and proteome of CDK4-transformed lung cancer cells.

Biological experiments are often conducted in vitro using immortalized cells due to their accessibility and ease of propagation compared to primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from the primary cell source due to the introduction of genes that enhance proliferation (e.g. CDK4) or enable telomere lengthening. To demonstrate the changes in phenotype upon CDK4-transformation, we performed LC-MS/MS glycomic and proteomic characterizations of a human lung cancer primary cell line (DTW75) and a CDK4-transformed cell line (GL01) derived from DTW75. We observed that the primary and CDK4-transformed cells expressed significantly different levels of sialylated, fucosylated, and sialofucosylated N-glycans. Specifically, the primary cells expressed higher levels of hybrid- and complex-type sialylated N-glycans, while CDK4-transformed cells expressed higher levels of complex-type fucosylated and sialofucosylated N-glycans. Further, we compared the proteomic differences between the cell lines and found that CDK4-transformed cells expressed higher levels of RNA-binding and adhesion proteins. Further, we observed that the CDK4-transformed cells changed N-glycosylation after 31 days in cell culture, with a decrease in high-mannose and increase in fucosylated, sialylated, and sialofucosylated N-glycans. Identifying these changes between primary and CDK4-transformed cells will provide useful insight when adapting cell lines that more closely resemble in vivo physiological conditions.

Drivers of the In-Mouth Interaction between Lupin Protein Isolate and Selected Aroma Compounds: A Proton Transfer Reaction-Mass Spectrometry and Dynamic Time Intensity Analysis.

Plant proteins often carry off-notes, necessitating customized aroma addition. In vitro studies revealed protein-aroma binding, limiting release during consumption. This study employs in vivo nose space proton transfer reaction-time-of-flight-mass spectrometry and dynamic sensory evaluation (time intensity) to explore in-mouth interactions. In a lupin protein-based aqueous system, a sensory evaluation of a trained "green" attribute was conducted simultaneously with aroma release of hexanal, nonanal, and 2-nonanone during consumption. Results demonstrated that enlarging aldehyde chains and relocating the keto group reduced maximum perceived intensity (Imax_R) by 71.92 and 72.25%. Protein addition decreased Imax_R by 30.91, 36.84, and 72.41%, indicating protein-aroma interactions. Sensory findings revealed a perceived intensity that was lower upon protein addition. Aroma lingering correlated with aroma compounds' volatility and hydrophobicity, with nonanal exhibiting the longest persistence. In vitro mucin addition increased aroma binding four to 12-fold. Combining PTR-ToF-MS and time intensity elucidated crucial food behavior, i.e., protein-aroma interactions, that are pivotal for food design.

Rapid determination of glyphosate and glufosinate in human blood by probe electrospray ionization tandem mass spectrometry.

In forensic science, glyphosate (GLYP) and glufosinate (GLUF), a class of non-selective broad-spectrum herbicides, have been frequently encountered in many fatal poisoning and suicide cases due to their widespread availability. Therefore, it is essential to develop an effective method for detecting these compounds. Some conventional methods, such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), have been reported to detect these compounds. However, these methods are not ideal for their time-consuming and non-sensitive feature. Herein, probe electrospray ionization (PESI) tandem mass spectrometry (MS/MS), a fast and sensitive technique, was applied for the determination of GLYP and GLUF in human blood, which can obtain analytical results within 0.5 min without derivatization and chromatographic separation. After protein precipitation of blood samples, the supernatant was mixed with isopropanol and ultra-pure water (1:1 v/v). Then, 8 µL of the mixture was introduced into the plastic sample plate for PESI-MS/MS analysis. The limits of detection (LODs) of the method were 0.50 µg/mL and 0.25 µg/mL for two analytes, and the limits of quantitation (LOQs) were both 1.00 µg/mL, which are higher than the concentration of reported poisoning and fatal cases. In the linear range of 1-500 µg/mL, the regression coefficients (r2) for GLYP and GLUF were over 0.99. The matrix effects ranged from 94.8 % to 119.5 %, and the biases were below 4.3 %. The recoveries ranged between 84.8 % and 107.4 %, and the biases were below 7.6 %. Meanwhile, the method was effectively utilized to detect and quantify the blood, urine, and other samples. Consequently, the results suggest that PESI-MS/MS is a straightforward, fast, and sensitive method for detecting GLUF and GLYP in forensics. In the future, PESI-MS/MS will become an indispensable technique for polar substances in grassroots units of public security where rapid detection is essential.

Advanced Tandem Mass Spectrometric Analysis of Complex Mixtures of Triacylglycerol Regioisomers: A Case Study of Bovine Milk Fat.

Comprehensive analysis of triacylglycerol (TAG) regioisomers is extremely challenging, with many variables that can influence the results. Previously, we reported a novel algorithmic method for resolving regioisomers of complex mixtures of TAGs. In the current study, the TAG Analyzer software and its mass spectrometric fragmentation model were further developed and validated for a much wider range of TAGs. To demonstrate the method, we performed for the first time a comprehensive analysis of TAG regioisomers of bovine milk fat, a very important and one of the most complex TAG mixtures in nature containing FAs ranging from short to long carbon chains. This analysis method forms a solid basis for further investigation of TAG regioisomer profiles in various natural fats and oils, potentially aiding in the development of new and healthier foods and nutraceuticals with targeted lipid structures.

m-nitrobenzyl alcohol supercharging reagent enhances the chromatographic separation and the charging of disulfide bond linked and His-tag peptides.

The linkages of disulfide bond (DSB) play important roles in protein stability and activity. Mass spectrometry-based (MS-based) techniques become accepted tools for DSB analysis in the recent decade. In the bottom-up approach, after enzyme digestion, the neighbouring amino acids of cysteines have great impacts on the physicochemical properties of resulting disulfide bond peptides, determining their retention behaviour on liquid chromatography (LC) and their MS ionization efficiency. In this study, the addition of supercharging reagent in LC mobile phase was used to examine the impact of supercharging reagent on the charge states of disulfide-bond peptides. The results showed that 0.1 % m-nitrobenzyl alcohol (m-NBA) in LC mobile phase increased the sensitivity and charge states of DSB peptides from our model protein, equine Interleukin-5 (eIL5), as well as the resolution of reversed-phase chromatography. Notably, also the sensitivity of C-terminal peptide with His-tag significantly improved. Our findings highlight the effectiveness of employing m-NBA as a supercharging reagent when investigating disulfide-linked peptides and the C-terminal peptide with a His-tag through nano-liquid chromatography mass spectrometry.

Systematic evaluation of benzoylation for liquid chromatography-mass spectrometry analysis of different analyte classes.

LC-MS is an indispensable tool for small molecule analysis in many fields; however, many small molecules require chemical derivatization to improve retention on commonly used reversed-phase columns and increase ionization. Benzoyl chloride (BzCl) derivatization is commonly used for derivatization of primary and secondary amines and phenolic alcohols, though evidence exists that with proper reaction conditions (i.e., specific bases), other hydroxyl groups may be derivatized too. Previous studies have examined BzCl concentration, reaction times, and reaction temperatures for derivatization of amines and phenols for LC-MS analysis; however, use of different bases, base concentration, and extending to conditions to hydroxyl groups for LC-MS analysis has not been well-studied. To address this understudied area and identify reaction conditions for both amino and hydroxyl groups, we performed a systematic study of reaction conditions on multiple classes of potential targets. For selected derivatization methods, detection limits and performance in a variety of biological matrices were assessed. Results highlight the importance of tailoring derivatization methods for a given application as they varied by molecule and/or molecule class. Compared to the standard BzCl method commonly used, alternative methods were identified to better derivatize challenging analytes (glucosamine, choline, cortisol, uridine, cytidine) with detection limits reaching 1100, 9, 38, 170, and 67 nM compared to undetectable, 170, 86, 1000, and 86 nM respectively. Sub-nanomolar detection limits were achieved for norepinephrine with alternative derivatization approaches. Improved derivatization methods for several classes and molecules including nucleosides, steroids, and molecules containing hydroxyl groups were also identified.

Multicenter evaluation of the hemostatic activity of emicizumab in patients with severe hemophilia A.

BACKGROUND: Emicizumab has been approved for the prophylaxis of patients with hemophilia A with or without inhibitors. However, spontaneous and trauma-induced breakthrough bleeds have been reported in patients on emicizumab prophylaxis, and no laboratory assay has been validated to evaluate the hemostatic activity of emicizumab. OBJECTIVES: The thrombin generation assay (TGA) could be a surrogate marker of the hemostatic efficacy of emicizumab. The correlation between TGA and the methods used to measure emicizumab blood concentration was evaluated in this study. METHODS: TGA was modified by the use of a trigger reagent combining a very low concentration of tissue factor and activated factor (F)XI. Emicizumab quantification was performed by 3 methods: the modified 1-step FVIII assay and 2 methods based on liquid chromatography and mass spectrometry (LC-MS). RESULTS: Using tissue factor/activated FXI-triggered TGA and platelet-poor plasma, a relationship was observed between the area under the thrombin generation curve (endogenous thrombin potential [ETP]) and the clinical response of patients to emicizumab. The ultrastructure of fibrin clots was consistent with ETP results and showed that emicizumab had a hemostatic activity equivalent to 20 to 30 IU/dL of FVIII. Finally, pharmacokinetic/pharmacodynamic analyses showed no correlation between ETP and LC-MS nor with modified 1-stage FVIII assay, but a statistically significant correlation between the LC-MS methods and the time-to-peak results of the TGA. CONCLUSION: Using a modified TGA, this study showed that patients who experienced breakthrough bleeds while on emicizumab had a lower thrombin-generating capacity compared with others with good clinical response to emicizumab.

Optimization and uncertainty assessment of a gas chromatography coupled to Orbitrap mass spectrometry method to determine organic contaminants in blood: A case study of an endangered seabird.

Birds are excellent bioindicators of environmental pollution, and blood provides information on contaminant exposure, although its analysis is challenging because of the low volumes that can be sampled. The objective of the present study was to optimize and validate a miniaturized and functional extraction and analytical method based on gas chromatography coupled to Orbitrap mass spectrometry (GCOrbitrap-MS) for the trace analysis of contaminants in avian blood. Studied compounds included 25 organochlorine pesticides (OCPs), 6 polychlorinated biphenyls (PCBs), 8 polybrominated diphenyl ethers (PBDEs) and 15 polycyclic aromatic hydrocarbons (PAHs). Four extraction and clean-up conditions were optimized and compared in terms of efficiency, accuracy, and uncertainty assessment. Extraction with hexane:dichloromethane and miniaturized Florisil pipette clean-up was the most adequate considering precision and accuracy, time, and costs, and was thereafter used to analyse 20 blood samples of a pelagic seabird, namely the Bermuda petrel (Pterodroma cahow). This species, endemic to the Northwest Atlantic, is among the most endangered seabirds of the region that in the '60 faced a decrease in the breeding success likely linked to a consistent exposure to dichloro-diphenyl-trichloroethane (DDT). Indeed, p,p'-DDE, the main DDT metabolite, was detected in all samples and ranged bewteen 1.13 and 6.87 ng/g wet weight. Other ubiquitous compounds were PCBs (ranging from 0.13 to 6.76 ng/g ww), hexachlorobenzene, and mirex, while PAHs were sporadically detected at low concentrations, and PBDEs were not present. Overall, the extraction method herein proposed allowed analysing very small blood volumes (∼ 100 µL), thus respecting ethical principles prioritising the application of less-invasive sampling protocols, fundamental when studying threatened avian species.