Software-assisted automated detection and identification of "unknown" fentanyl analogues.

Fentanyl and its non-pharmaceutical analogues (NPFs) are potent synthetic opioids, traditionally used for pain management, with ever-increasing illicit uses. Tightening the regulation for known fentanyls leads to new synthetic analogues in the opioid market. Furthermore, the Organization for the Prohibition of Chemical Weapons (OPCW) has recently issued a decision regarding aerosolized use of central nervous system (CNS)-acting agents, such as fentanyl and its analogues, under the concern that these materials could be misused for terror or war purposes. The ever-increasing development of new fentanyl analogues makes the task of detection and identification of these new, unknown analogues crucial. In this work, we introduce an automated tool for the detection and putative identification of "unknown" fentanyl analogues, using liquid chromatography-mass spectrometry (LC-MS) (high-resolution mass spectrometry [HRMS]) analysis, subsequently followed by data processing using the "Compound Discoverer" software. This software, in our modified use, enabled the automatic detection of various fentanyl analogues, by "digging" out components and comparing them to pre-calculated theoretical molecular ions of possible modifications or transformations on the fentanyl backbone structure (no library or database used). Subsequently, structural elucidation for the proposed component of interest is carried out by automated MS/MS data interpretation, as performed by the software. This method was explored on 12 fentanyl-based "unknown" analogues used as model examples, including chemical modifications such as fluorination and methylation. In all tested compounds, automatic detection and identification were achieved, even at concentrations as low as 1 ng/mL in an environmental soil matrix extract.

Monitoring Exposure to Five Chemical Warfare Agents Using the Dried Urine Spot Technique and Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-In Vivo Determination of Sarin Metabolite in Mice.

Dried urine spot (DUS) is a micro-sample collection technique, known for its advantages in handling, storage and shipping. It also uses only a small volume of urine, an essential consideration in working with small animals, or in acute medical situations. Alkyl-phosphonic acids are the direct and indicative metabolites of organophosphorus chemical warfare agents (OP-CWAs) and are present in blood and urine shortly after exposure. They are therefore crucially important for monitoring casualties in war and terror scenarios. We report here a new approach for the determination of the metabolites of five CWAs in urine using DUS. The method is based on a simple and rapid sample preparation, using only 50 µL of urine, spotted and dried on DBS paper, extracted using 300 µL methanol/water and analyzed via targeted LC-MS/MS. The detection limits for the five CWAs, sarin (GB), soman (GD), cyclosarin (GF), VX and RVX in human urine were from 0.5 to 5 ng/mL. Recoveries of (40-80%) were obtained in the range of 10-300 ng/mL, with a linear response (R2 > 0.964, R > 0.982). The method is highly stable, even with DUS samples stored up to 5 months at room temperature before analysis. It was implemented in a sarin in vivo exposure experiment on mice, applied for the time course determination of isopropyl methylphosphonic acid (IMPA, sarin hydrolysis product) in mice urine. IMPA was detectable even with samples drawn 60 h after the mice's (IN) exposure to 1 LD50 sarin. This method was also evaluated in a non-targeted screening for multiple potential CWA analogs (LC-Orbitrap HRMS analysis followed by automatic peak detection and library searches). The method developed here is applicable for rapid CWA casualty monitoring.

Pharmacokinetics of the Recalcitrant Drug Lamotrigine: Identification and Distribution of Metabolites in Cucumber Plants.

Treated wastewater is an important source of water for irrigation. As a result, irrigated crops are chronically exposed to wastewater-derived pharmaceuticals, such as the anticonvulsant drug lamotrigine. Lamotrigine is known to be taken up by plants, but its plant-derived metabolites and their distribution in different plant organs are unknown. This study aimed to detect and identify metabolites of lamotrigine in cucumber plants grown for 35 days in a hydroponic solution by using LC-MS/MS (Orbitrap) analysis. Our data showed that 96% of the lamotrigine taken up was metabolized. Sixteen metabolites possessing a lamotrigine core structure were detected. Reference standards confirmed two; five were tentatively identified, and nine molecular formulas were assigned. The data suggest that lamotrigine is metabolized via N-carbamylation, N-glucosidation, N-alkylation, N-formylation, N-oxidation, and amidine hydrolysis. The metabolites LTG-N2-oxide, M284, M312, and M370 were most likely produced in the roots and were translocated to the leaves. Metabolites M272, M312, M314, M354, M368, M370, and M418 were dominant in leaves. Only a few metabolites were detected in the fruits. With an increasing exposure time, lamotrigine leaf concentrations decreased because of continuous metabolism. Our data showed that the metabolism of lamotrigine in a plant is fast and that a majority of metabolites are concentrated in the roots and leaves.

Species-specific lipophilicities of fluorinated diketones in complex equilibria systems and their potential as multifaceted reversible covalent warheads.

Combined molecular, physicochemical and chemical properties of electrophilic warheads can be applied to create covalent drugs with diverse facets. Here we study these properties in fluorinated diketones (FDKs) and their multicomponent equilibrium systems in the presence of protic nucleophiles, revealing the potential of the CF2(CO)2 group to act as a multifaceted warhead for reversible covalent drugs. The equilibria compositions of various FDKs in water/octanol contain up to nine species. A simultaneous direct species-specific 19F-NMR-based log P determination of these complex equilibria systems was achieved and revealed in some cases lipophilic to hydrophilic shifts, indicating possible adaptation to different environments. This was also demonstrated in 19F-MAS-NMR-based water-membrane partitioning measurements. An interpretation of the results is suggested by the aid of a DFT study and 19F-DOSY-NMR spectroscopy. In dilute solutions, a model FDK reacted with protected cysteine to form two hemi-thioketal regioisomers, indicating possible flexible regio-reactivity of CF2(CO)2 warheads toward cysteine residues.

Evaluation of Matrix Complexity in Nontargeted Analysis of Small-Molecule Toxicants by Liquid Chromatography-High-Resolution Mass Spectrometry.

Complex mixtures, characterized by high density of compounds, challenge trace detection and identification. This is further exacerbated in nontargeted analysis, where a compound of interest may be well hidden under thousands of matrix compounds. We studied the effect of matrix complexity on nontargeted detection (peak picking) by LC-MS/MS (Orbitrap) analysis. A series of ∼20 drugs, V-type chemical warfare agents and pesticides, simulating toxic unknowns, were spiked at various concentrations in several complex matrices including urine, rosemary leaves, and soil extracts. Orbitrap "TraceFinder" software was used to explore their peak intensities in relation to the matrix (peak location in an intensity-sorted list). Average practical detection limits of nontargets were determined. While detection among the first 10,000 peaks was achieved at 0.3-1 ng/mL levels in the extract, for the more realistic "top 1000" list, much higher concentrations were required, approaching 10-30 ng/mL. A negative power law functional dependence between the peak location in an intensity-sorted suspect list and the nontarget concentration is proposed. Controlled complexity was explored with a series of urine dilutions, resulting in an excellent correlation between the power law coefficient and dilution factor. The intensity distribution of matrix peaks was found to spread (unevenly) on a broad range, fitting well the Weibull distribution function with all matrices and extracts. The quantitative approach demonstrated here gives a measure of the actual capabilities and limitations of LC-MS in the analysis of nontargets in complex matrices. It may be used to estimate and compare the complexity of matrices and predict the typical detection limits of unknowns.

Integrating Effect-Directed Analysis and Chemically Indicative Mass Spectral Fragmentation to Screen for Toxic Organophosphorus Compounds.

Analytical chemists are often challenged to screen for bioactive compounds in complex matrices, sometimes without a priori knowledge of the exact compound of interest. Therefore, "flagging" techniques, highlighting common characteristics of bioactive compounds, are highly sought after. In this work, we demonstrate a double flagging method, where unknown organophosphorus acetylcholinesterase inhibitors are "flagged" out of a complex matrix by the presence of organophosphorus-indicative ions as well as their acetylcholinesterase inhibition. This is accomplished by flagging the LC chromatographic retention time of phosphorus-indicative ions using accurate mass high-energy in-source CID products, and the retention time of acetylcholinesterase inhibiting compounds using a parallel microfractionation-based bioassay. We successfully apply this method to screen VX, VM, and RVX nerve agents as well as methomyl, a carbamate pesticide, out of soil and whole blood samples at low µM to sub-µM concentrations. This methodology can be easily extended to diverse chemical families and biological activities of interest.

Placing CF2 in the Center: Major Physicochemical Changes Upon a Minor Structural Alteration in Gem-Difunctional Compounds.

Fluorine atoms play an important role in all branches of chemistry and accordingly, it is very important to study their unique and varied effects systematically, in particular, the structure-physicochemical properties relationship. The present study describes exceptional physicochemical effects resulting from a H/F exchange at the methylene bridge of gem-difunctional compounds. The Δlog P(CF2-CH2) values, that is, the change in lipophilicity, observed for the CH2 /CF2 replacement in various α,α-phenoxy- and thiophenoxy-esters/amides, diketones, benzodioxoles and more, fall in the range of 0.6-1.4 units, which for most cases, is far above the values expected for such a replacement. Moreover, for compounds holding more than one such gem-difunctional moiety, the effect is nearly additive, so one can switch from a hydrophilic compound to a lipophilic one in a limited number of H/F exchanges. DFT studies of some of these systems revealed that polarity, conformational preference as well as charge distributions are strongly affected by such hydrogen to fluorine atom substitution. The pronounced effects described, are a result of the interplay between changes in polarity, H-bond basicity and molecular volume, which were obtained with a very low 'cost' in terms of molecular weight or steric effects and may have a great potential for implementation in various fields of chemical sciences.

Software-Assisted Automated Detection and Identification of "Unknown" Analogues: Implementation on V-Type Nerve Agents.

V-type nerve agents are among the most toxic organophosphorus chemical warfare agents, and they are under strict regulation and supervision by the OPCW (Organization for the Prohibition of Chemical Weapons). The V-type class of materials refers to a potentially large number of analogues and isomers. In order to expose instances of unfulfillment of the OPCW treaty, it is essential to have the ability to detect and identify "unknown" analogues of this family, even in the absence of an analytical standard. This work demonstrates a new automated tool for the detection and identification of V-type analogues, using high-resolution-accurate-mass LC-MS analysis, followed by "Compound Discoverer" software data processing. This software, originally developed for metabolism and metabolomics screening, is used here to automatically detect various V-type analogues by picking peaks and comparing them to "in-silico" calculated modifications made on a predefined basic V-backbone structure (according to the OPCW definitions for V-type agents). Subsequently, a complete structural elucidation for the proposed molecular formula is obtained by MS/MS data analysis of the suspected component, for both the V-type analogue (using ESI(+) analysis) as well as its hydrolysis product (using ESI(-) analysis) for a better elucidation of the phosphonate "head" structure. This method was found to be useful for the detection and identification of several "unknown" analogues, at low ng/mL levels in soil extracts.

Studying Lipophilicity Trends of Phosphorus Compounds by 31P-NMR Spectroscopy: A Powerful Tool for the Design of P-Containing Drugs.

Systematically studying the lipophilicity of phosphorus compounds is of great importance for many chemical and biological fields and particularly for medicinal chemistry. Here, we report on the study of trends in the lipophilicity of a wide set of phosphorus compounds relevant to drug design including phosphates, thiophosphates, phosphonates, thiophosphonates, bis-phosphonates, and phosphine chalcogenides. This was enabled by the development of a straightforward log P determination method for phosphorus compounds based on 31P-NMR spectroscopy. The log P values measured ranged between -3.2 and 3.6, and the trends observed were interpreted using a DFT study of the dipole moments and by H-bond basicity (pKHB) measurements of selected compounds. Clear signal separation in 31P-NMR spectroscopy grants the method high tolerability to impurities. Moreover, the wide range of chemical shifts for the phosphorus nucleus (250 to -250 ppm) enables a direct simultaneous log P determination of phosphorus compound mixtures in a single shake-flask experiment and 31P-NMR analysis.

A multiple-method comparative study using GC-MS, AMDIS and in-house-built software for the detection and identification of "unknown" volatile organic compounds in breath.

The human respiratory system is a highly complex matrix that exhales many volatile organic compounds (VOCs). Breath-exhaled VOCs are often "unknowns" and possess low concentrations, which make their analysis, peak digging and data processing challenging. We report a new methodology, applied in a proof-of-concept experiment, for the detection of VOCs in breath. For this purpose, we developed and compared four complementary analysis methods based on solid-phase microextraction and thermal desorption (TD) tubes with two GC-mass spectrometer (MS) methods. Using eight model compounds, we obtained an LOD range of 0.02-20 ng/ml. We found that in breath analysis, sampling the exhausted air from Tedlar bags is better when TD tubes are used, not only because of the preconcentration but also due to the stability of analytes in the TD tubes. Data processing (peak picking) was based on two data retrieval approaches with an in-house script written for comparison and differentiation between two populations: sick and healthy. We found it best to use "raw" AMDIS deconvolution data (.ELU) rather than its NIST (.FIN) identification data for comparison between samples. A successful demonstration of this method was conducted in a pilot study (n = 21) that took place in a closed hospital ward (Covid-19 ward) with the discovery of four potential markers. These preliminary findings, at the molecular level, demonstrate the capabilities of our method and can be applied in larger and more comprehensive experiments in the omics world.

Structural elucidation of amino amide-type local anesthetic drugs and their main metabolites in urine by LC-MS after derivatization and its application for differentiation between positional isomers of prilocaine.

The demand for clinical toxicology analytical methods for identifying drugs of abuse and medicinal drugs is steadily increasing. Structural elucidation of amino amide-type local anesthetic drugs and their main metabolites by GC-EI-MS and LC-ESI-MS/MS is of great analytical challenge. These compounds exhibit only/mostly fragments/product ions representing the amine-containing residue, while the aromatic amide moiety remains unidentified. This task becomes even more complicated when discrimination between positional isomers of such compounds is required. Here, we report the development of a derivatization procedure for the differentiation and structural elucidation of a mixture of local anesthetic drugs and their metabolites that possess tertiary and secondary amines in water and urine. A method based on two sequential "in-vial" instantaneous derivatization processes at ambient temperature followed by LC-ESI-MS/MS analysis was developed. 2,2,2-Trichloro-1,1-dimethylethyl chloroformate (TCDMECF) was utilized to selectively convert the secondary amines into their carbamate derivatives, followed by hydrogen peroxide addition to produce the corresponding tertiary amine oxides. The resulting derivatives exhibited rich fragmentation patterns, enabling improved structural elucidation of the original compounds. The developed method was successfully applied to the differentiation and structural elucidation of prilocaine and its four positional isomers, which all possess similar GC and LC retention times and four of them exhibit almost identical EI-MS and ESI-MS/MS spectra, enabling their structural elucidation in a single LC-ESI-MS/MS analysis. The developed technique is fast and simple and enables discrimination between isomers based on different diagnostic ions/fragmentation patterns.

Diameter-selective dispersion of carbon nanotubes by ß-lactoglobulin whey protein.

The ß-lactoglobulin (ß-LG) protein was discovered to be an efficient and selective dispersant for carbon nanotubes (CTNs) with certain diameters. A dispersion process of CTNs by the ß-LG was studied, focusing on the relationships between the surface curvature of the CNT and the ß-LG's efficiency in dispersing them, using cryogenic-transmission electron microscopy (cryo-TEM) and optical spectroscopy. Plausible binding sites of the ß-LG, responsible for the interaction of the protein with CNTs of various diameters (surface curvatures) were also investigated and were found to be in good agreement with corresponding docking calculations.

Enhanced bioavailability of polyaromatic hydrocarbons in the form of mucin complexes.

Increasing exposure of biological systems to large amounts of polycyclic aromatic hydrocarbons is of great public concern. Organisms have an array of biological defense mechanisms, and it is believed that mucosal gel (which covers the respiratory system, the gastrointestinal tract, etc.) provides an effective chemical shield against a range of toxic materials. However, in this work, we demonstrate, for the first time, that, upon complexation of polyaromatic hydrocarbons with mucins, enhanced bioavailability and, therefore, toxicity are obtained. This work was aimed to demonstrate how complexation of various highly hydrophobic polycyclic aromatic hydrocarbons with representative mucin glycoprotein could lead to the formation of previously undescribed materials, which exhibit increased toxicity versus pristine polycyclic aromatic hydrocarbons. In the present work, we show that a representative mucin glycoprotein, bovine submaxillary mucin, has impressive and unprecedented capabilities of binding and solubilizing water-insoluble materials in physiological solution. The complexes formed between the mucin and a series of polycyclic aromatic hydrocarbons were comprehensively characterized, and their toxicity was evaluated by both in vivo and in vitro assays. In addition, the bioavailability and membrane-penetration capabilities were tested using an internalization assay. Our results provide, for the first time, evidence of an unknown route by which hydrophobic materials may achieve higher bioavailability, penetrating some of the biological defense systems, in the form of water-soluble complexes with mucosal proteins.

Mucin complexes of nanomaterials: first biochemical encounter.

In recent years, the exposure of biological systems to various nanomaterials has become an issue of great public concern. Although living organisms have arrays of biological defense mechanisms against exposure to exogenous compounds, the biochemical mechanisms allowing various nanomaterials to enter the body are not well understood. A unique example of a typical mucosal glycoprotein capable of binding and solubilizing nanomaterials in physiological solution is provided, suggesting a possible route for entry into biological systems.

Synthesis and evaluation of a pseudocyclic tristhiourea-based anion host.

A novel methodology for the evaluation of receptor arrangement in structurally flexible anion chemosensors was developed and applied to map the binding site of a new pseudocyclic tristhiourea chemosensor (6). The syntheses of 6 and related macrocyclic chemosensor 10 (a model of the folded monomeric structure of 6) are reported. Both chemosensors were evaluated by titration with a variety of structurally different anions in CH3Cl and DMSO, showing a common preference for F-, CH3CO2-, and H2PO4-. However, within this group of anions, the binding patterns of the chemosensors differed, indicating dissimilarity in the arrangement of the binding sites of 6 and 10.