High-resolution Tandem Mass Spectrometry Research Articles

Identification of Granatane Alkaloids from Duboisia myoporoides (Solanaceae) using Molecular Networking and Semisynthesis.

The Solanaceae plant family contains at least 98 genera and over 2700 species. The Duboisia genus stands out for its ability to produce pyridine and tropane alkaloids, which are relatively poorly characterized at the phytochemical level. In this study, we analyzed dried leaves of Duboisia spp. using supercritical CO2 extraction and ultra-high-pressure liquid chromatography coupled to high-resolution tandem mass spectrometry, followed by feature-based molecular networking. Thirty-one known tropane alkaloids were putatively annotated, and the identity of six (atropine, scopolamine, anisodamine, aposcopolamine, apoatropine, and noratropine) were identified using reference standards. Two new granatane alkaloids connected in the molecular network were highlighted from Duboisia myoporoides, and their α-granatane tropate and α-granatane isovalerate structures were unambiguously established by semisynthesis.

Empowering the rapid authentication of the botanical origin of monofloral honey by coated blade spray mass spectrometry (CBS-MS)

In the quest for a rapid and cost-effective tool for determining the botanical origin of monofloral honey, the analytical capabilities of coated blade spray mass spectrometry (CBS-MS) were investigated. To this aim, the chemical profiles of 64 honey samples from seven different botanical origins (acacia, dandelion, chestnut, rhododendron, citrus, sunflower, and linden) were captured by the absorbent of the coated blades and then analyzed by mass spectrometry. An exploratory analysis was performed by principal component analysis (PCA) to generate a graphical representation of the CBS-MS data that allows the discovery of patterns, outliers, or relations between types of honey in an unsupervised manner. Additionally, the performances of four different classification algorithms (least absolute shrinkage and selection operator (LASSO), random forest (RF), and neural network (NNET) partial least squares discriminant analysis (PLS-DA)) were built up and compared. The performances of the four classifiers were verified by a 50 times-repeated, 5-fold-cross-validation and permutation test. Although all classifiers performed well, the RF showed significantly higher performances in cross-validation (with area under the curve (AUC) of 0.99, overall accuracy 0.94, Kappa 0.93, sensitivity 0.94, and specificity 0.99). Moreover, the permutation tests showed the models were not overfitted. Finally, to determine the molecular identities of the ions that most contribute to the classification, extracts from the same honey samples were prepared, analyzed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS), and the most significant features were annotated. This proof-of-principle work warrants a future large-scale study to validate and challenge this CBS-MS-based method with a greater number of honeys from different years and geographical origins.

UPLC-LTQ-Orbitrap Study on Rat Urinary Metabolites of 5-Methoxy-Alpha-Methyltryptamine.

5-Methoxy-α-Methyltryptamine (5-MeO-AMT) is a new psychoactive substance which is abused due to its hallucinogenic and euphoric effects. This study aimed to study the metabolic characteristics of 5-MeO-AMT. Five rats were given intraperitoneal injection at a dose of 50 mg/kg of 5-MeO-AMT, and their urine was subsequently collected at different times within 7 days. Ultra-high performance liquid chromatography-- tandem high-resolution mass spectrometry (UPLC-LTQ-Orbitrap) was used to detect the precise molecular weight and fragment ions of 5-MeO-AMT and its possible metabolites in the urine sample extracted with benzene-ethyl acetate. Three metabolites, including OH-5-MeO-AMT, α-Me-5-HT, and N-Acetyl-5-MeO-AMT were identified in rats' urine. The major metabolic pathways involved O-demethylation, hydroxylation of indole ring, and Acetylation on aliphatic amines. The results of this study are an important reference for the identification and screening of toxicants of 5-MeO-AMT.

Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) β-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG β-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Universal Identification of Pathogenic Viruses by liquid chromatography coupled to tandem mass spectrometry Proteotyping

Accurate and rapid identification of viruses is crucial for an effective medical diagnosis when dealing with infections. Conventional methods, including DNA amplification techniques or lateral-flow assays, are constrained to a specific set of targets to search for. In this study, we introduce a novel tandem mass spectrometry proteotyping-based method that offers a universal approach for the identification of pathogenic viruses and other components, eliminating the need for a priori knowledge of the sample composition. Our protocol relies on a time and cost-efficient peptide sample preparation, followed by an analysis with liquid chromatography coupled to high-resolution tandem mass spectrometry. As a proof of concept, we first assessed our method on publicly available shotgun proteomics datasets obtained from virus preparations and fecal samples of infected individuals. Successful virus identification was achieved with 53 public datasets, spanning 23 distinct viral species. Furthermore, we illustrated the method's capability to discriminate closely related viruses within the same sample, using alphaviruses as an example. The clinical applicability of our method was demonstrated by the accurate detection of the vaccinia virus in spiked saliva, a matrix of paramount clinical significance due to its non-invasive and easily obtainable nature. This innovative approach represents a significant advancement in pathogen detection and paves the way for enhanced diagnostic capabilities.

Enhancing drug analysis through advanced mass spectrometry techniques and quantitative modeling

The integration of advanced mass spectrometry (MS) techniques with quantitative modeling has revolutionized the field of drug analysis, offering unprecedented accuracy, sensitivity, and specificity in the detection and quantification of pharmaceutical compounds and their metabolites. High-Resolution Mass Spectrometry (HRMS) and Tandem Mass Spectrometry (MS/MS) have emerged as pivotal tools, capable of distinguishing isobaric species and providing detailed structural information, respectively. Ambient ionization techniques such as Desorption Electrospray Ionization (DESI) and Direct Analysis in Real Time (DART) have further simplified sample preparation, enabling rapid, direct analyses under ambient conditions. This review discusses the significant strides made in enhancing drug analysis through these innovations, alongside the application of calibration curves and mathematical models for quantitation and pharmacokinetic modeling. Additionally, it addresses the challenges inherent in quantitative MS, including matrix effects, analyte stability, and the need for improved sensitivity and specificity. Through a comprehensive overview, this article underscores the transformative impact of these advancements on drug analysis, paving the way for more accurate and efficient pharmaceutical research and diagnostics.

Hearing loss and its association with the proteome of perilymph, cerebrospinal fluid, and tumor tissue in patients with vestibular schwannoma

This study examined the association between hearing loss in sporadic vestibular schwannoma patients and the proteome of perilymph (PL), cerebrospinal fluid (CSF), and vestibular schwannoma. Intraoperative sampling of PL and of CSF, and biopsy of vestibular schwannoma tissue, was performed in 32, 32, and 20 patients with vestibular schwannoma, respectively. Perilymph and CSF in three patients with meningioma and normal hearing were also sampled. The proteomes were identified by liquid chromatography coupled to high-resolution tandem mass spectrometry. Preoperative hearing function of the patients was evaluated with pure tone audiometry, with mean values at frequencies of 500, 1000, 2000, and 4000 Hz (PTA4) in the tumor-affected ear used to delineate three hearing groups. Analysis of the PL samples revealed significant upregulation of complement factor H-related protein 2 (CFHR2) in patients with severe to profound hearing loss after false discovery rate correction. Pathway analysis of biofunctions revealed higher activation scores in the severe/profound hearing loss group of leukocyte migration, viral infection, and migration of cells in PL. Upregulation of CFHR2 and activation of these pathways indicate chronic inflammation in the cochlea of vestibular schwannoma patients with severe to profound hearing loss compared with patients with normal hearing or mild hearing loss.

Cytotoxicity of Physalis minima Linn (Solanaceae) fruit against HCT116 and HT29 colorectal cancer cell lines

The pantropical Physalis minima are traditionally used for the prevention and treatment of various illnesses, diseases, and cancers. While most earlier studies on the species have focused on the phytochemistry of the leaf and stem extracts, recent studies have indicated that its fruit may contain bioactive compounds of medical interest. In this study, we investigated the cytotoxicity of extracts from the fruit of P. minima against colorectal cancer cell lines and revealed its phytochemical profile via high-resolution tandem mass spectrometry analysis. Following a 24-h treatment with the fruit extract, cytoplasm shrinkage and nucleus condensation were observed in the colorectal cancer cell lines HCT116 and HT29, indicating the induction of programmed cell death. Phytochemically, 71 putative metabolites were identified. Some of these metabolites have been reported to inhibit cancers to varying degrees, further supporting the correlation of the putative metabolites with the cytotoxicity against colorectal cancer cells demonstrated in this study.

Post-metabolism impurity profiling of carfentanil, remifentanil, sufentanil, and benzylfentanyl

Carfentanil, remifentanil, and sufentanil are potent fentanyl analogues that are regularly mixed with illicit drugs causing many overdose deaths. Chemical impurity profiling of these drugs is a well-established technique for linking evidence found at a crime scene to other seized samples. The current study aims to expand the application of impurity profiling to metabolized samples to find synthesis specific markers. This is particularly relevant when the drug has been consumed, and no intact material is present at a crime scene. Carfentanil, remifentanil, and sufentanil were synthesized according to the Ugi or 7-step method and benzylfentanyl was produced using the Siegfried method. After in-vitro metabolism with human liver microsomes, the samples were analyzed by gas chromatography-mass spectrometry (GC–MS) and liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS). Characteristic markers were found by applying a match criterion approach and principal component analysis (PCA). The precursors 4-ANBP, aniline, and N-phenylacetamide and several metabolites were identified in post-metabolism samples, indicating that specific synthesis information is retained after in-vitro metabolism. The detected levels were in line with concentrations reported in case work. In addition, LDA was applied to maximize discrimination between synthesis methods and to establish likelihood ratios (LRs). Calibrated LR values were in the range of 0.083 to 16 with very low false positive and false negative error rates. In conclusion, the presented work demonstrates the possibility of combining chemical profiling and retrospective biomarker analysis to obtain information about the synthesis method, which could be useful for forensic reconstructions and attribution investigations.

Metaproteomic analysis of King Ghezo tomb wall (Abomey, Benin) confirms 19th century voodoo sacrifices.

The palace of King Ghezo in Abomey, capital of the ancient kingdom of Dahomey (present-day Benin), houses two sacred huts which are specific funerary structures. It is claimed that the binder in their walls is made of human blood. In the study presented here, we conceived an original strategy to analyze the proteins present on minute amounts of the cladding sampled from the inner facade of the cenotaph wall and establish their origin. The extracted proteins were proteolyzed and the resulting peptides were characterized by high-resolution tandem mass spectrometry. Over 6397 distinct molecular entities were identified using cascading searches. Starting from without a priori searches of an extended generic database, the peptide repertoire was narrowed down to the most representative organisms-identified by means of taxon-specific peptides. A wide diversity of bacteria, fungi, plants, and animals were detected through the available protein material. This inventory was used to archaeologically reconstruct the voodoo rituals of consecration and maintenance of vitality. Several indicators attested to the presence of traces of human and poultry blood in the material taken. This study shows the essential advantages of paleoproteomics and metaproteomics for the study of ancient residues from archaeological excavations or historical monuments.

A novel strategy for detoxification of deoxynivalenol via modification of both toxic groups

Deoxynivalenol (DON) is the most abundant mycotoxin in cereal crops and derived foods and is of great concern in agriculture. Bioremediation strategies have long been sought to minimize the impact of mycotoxin contamination, but few direct and effective enzyme-catalyzed detoxification methods are currently available. In this study, we established a multi-enzymatic cascade reaction and successfully achieved detoxification at double sites: glutathionylation for the C-12,13 epoxide group and epimerization for the C-3 hydroxyl group. This yielded novel derivatives of DON, 3-epi-DON-13-glutathione (3-epi-DON-13-GSH) as well as its by-product, 3-keto-DON-13-GSH, for which precise structures were validated via liquid chromatography–high-resolution tandem mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Both cell viability and DNA synthesis assays demonstrated dramatically decreased cytotoxicity of the double-site modified product 3-epi-DON-13-GSH. These findings provide a promising and urgently needed novel method for addressing the problem of DON contamination in agricultural and industrial settings.

UHPLC-HRMS-based Multiomics to Explore the Potential Mechanisms and Biomarkers for Colorectal Cancer

BackgroundUnderstanding the metabolic changes in colorectal cancer (CRC) and exploring potential diagnostic biomarkers is crucial for elucidating its pathogenesis and reducing mortality. Cancer cells are typically derived from cancer tissues and can be easily obtained and cultured. Systematic studies on CRC cells at different stages are still lacking. Additionally, there is a need to validate our previous findings from human serum.MethodsUltrahigh-performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS)-based metabolomics and lipidomics were employed to comprehensively measure metabolites and lipids in CRC cells at four different stages and serum samples from normal control (NR) and CRC subjects. Univariate and multivariate statistical analyses were applied to select the differential metabolites and lipids between groups. Biomarkers with good diagnostic efficacy for CRC that existed in both cells and serum were screened by the receiver operating characteristic curve (ROC) analysis. Furthermore, potential biomarkers were validated using metabolite standards.ResultsMetabolite and lipid profiles differed significantly among CRC cells at stages A, B, C, and D. Dysregulation of glycerophospholipid (GPL), fatty acid (FA), and amino acid (AA) metabolism played a crucial role in the CRC progression, particularly GPL metabolism dominated by phosphatidylcholine (PC). A total of 46 differential metabolites and 29 differential lipids common to the four stages of CRC cells were discovered. Eight metabolites showed the same trends in CRC cells and serum from CRC patients compared to the control groups. Among them, palmitoylcarnitine and sphingosine could serve as potential biomarkers with the values of area under the curve (AUC) more than 0.80 in the serum and cells. Their panel exhibited excellent performance in discriminating CRC cells at different stages from normal cells (AUC = 1.00).ConclusionsTo our knowledge, this is the first research to attempt to validate the results of metabolism studies of serum from CRC patients using cell models. The metabolic disorders of PC, FA, and AA were closely related to the tumorigenesis of CRC, with PC being the more critical factor. The panel composed of palmitoylcarnitine and sphingosine may act as a potential biomarker for the diagnosis of CRC, aiding in its prevention.

How Wastewater Reflects Human Metabolism─Suspect Screening of Pharmaceutical Metabolites in Wastewater Influent.

Pharmaceuticals and their human metabolites are contaminants of emerging concern in the aquatic environment. Most monitoring studies focus on a limited set of parent compounds and even fewer metabolites. However, more than 50% of the most consumed pharmaceuticals are excreted in higher amounts as metabolites than as parents, as confirmed by a literature analysis within this study. Hence, we applied a wide-scope suspect screening approach to identify human pharmaceutical metabolites in wastewater influent from three Swiss treatment plants. Based on consumption amounts and human metabolism data, a suspect list comprising 268 parent compounds and over 1500 metabolites was compiled. Online solid phase extraction combined with liquid chromatography coupled to high-resolution tandem mass spectrometry was used to analyze the samples. Data processing, annotation, and structure elucidation were achieved with various tools, including molecular networking as well as SIRIUS/CSI:FingerID and MetFrag for MS2 spectra rationalization. We confirmed 37 metabolites with reference standards and 16 by human liver S9 incubation experiments. More than 25 metabolites were detected for the first time in influent wastewater. Semiquantification with MS2Quant showed that metabolite to parent concentration ratios were generally lower compared to literature expectations, probably due to further metabolite transformation in the sewer system or limitations in the metabolite detection. Nonetheless, metabolites pose a large fraction to the total pharmaceutical contribution in wastewater, highlighting the need for metabolite inclusion in chemical risk assessment.

Mono- to tetra-alkyl ether cardiolipins in a mesophilic, sulfate-reducing bacterium identified by UHPLC-HRMSn: a novel class of membrane lipids.

The composition of membrane lipids varies in a number of ways as adjustment to growth conditions. Variations in head group composition and carbon skeleton and degree of unsaturation of glycerol-bound acyl or alkyl chains results in a high structural complexity of the lipidome of bacterial cells. We studied the lipidome of the mesophilic, sulfate-reducing bacterium, Desulfatibacillum alkenivorans strain PF2803T by ultra-high-pressure liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMSn). This anaerobic bacterium has been previously shown to produce high amounts of mono-and di-alkyl glycerol ethers as core membrane lipids. Our analyses revealed that these core lipids occur with phosphatidylethanomamine (PE) and phosphatidylglycerol (PG) head groups, representing each approximately one third of the phospholipids. The third class was a novel group of phospholipids, i.e., cardiolipins (CDLs) containing one (monoether/triester) to four (tetraether) ether-linked saturated straight-chain or methyl-branched alkyl chains. Tetraether CDLs have been shown to occur in archaea (with isoprenoid alkyl chains) but have not been previously reported in the bacterial Domain. Structurally related CDLs with one or two alkyl/acyl chains missing, so-called monolyso-and dilyso-CDLs, were also observed. The potential biosynthetic pathway of these novel CDLs was investigated by examining the genome of D. alkenivorans. Three CDL synthases were identified; one catalyzes the condensation of two PGs, the other two are probably involved in the condensation of a PE with a PG. A heterologous gene expression experiment showed the in vivo production of dialkylglycerols upon anaerobic expression of the glycerol ester reductase enzyme of D. alkenivorans in E. coli. Reduction of the ester bonds probably occurs first at the sn-1 and subsequently at the sn-2 position after the formation of PEs and PGs.

Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

Alpibectir: Early Qualitative and Quantitative Metabolic Profiling from a First-Time-in-Human Study by Combining 19F-NMR (Nuclear Magnetic Resonance), 1H-NMR, and High-Resolution Mass Spectrometric Analyses.

Alpibectir (also known as BVL-GSK098 and GSK3729098) is a new chemical entity (NCE) with a novel mechanism for the treatment of tuberculosis. The disposition of alpibectir was determined in subjects from a first-time-in-human trial after a single oral dose of 40 mg and after 7 days repeat dosing at 30 mg. Here we present a combined approach of 19F-NMR (nuclear magnetic resonance), 1H-NMR, and high-resolution mass spectrometry (HRMS) to confidently determine the human metabolic fate of alpibectir. Utilizing multiple sites of fluorination in the molecule, it was possible to fractionate human urine and plasma to confidently detect and quantify the metabolite responses using 19F-NMR. Qualitative detection and structural characterization of F-containing NMR fractions were performed using complementary high-resolution ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analyses to further add confidence to the metabolite responses in these fractions. Subsequent 1H-NMR then provided unequivocal standard-free structural confirmation for key metabolites, which would not be possible with conventional radioactivity detection and LC-MS/MS techniques. Alpibectir was shown to undergo extensive hydrolysis of the central amide moiety, where the resultant N-dealkylated amine and trifluorobutyric acid products were detected initially by unbiased 19F-NMR detection along with major downstream biotransformations to form a carbamoyl glucuronide conjugate and trifluoroacetic acid, respectively. Parallel UHPLC-MS/MS analyses provided confirmatory or additional structural characterization only where relevant. These concerted data allowed for the qualitative metabolic profile and quantitative determination of drug-related material (DRM) in urine and plasma, along with the percentage of dose excreted in urine, to be reported in a comprehensive, efficient, and data-led manner. SIGNIFICANCE STATEMENT: Combining the selectivity of 19F-NMR (nuclear magnetic resonance) for unfractionated samples as first-intent, data-led sample fractionation prior to 19F-NMR and structure-rich 1H-NMR detection, along with the sensitivity of high-resolution ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), a novel alternative for time-efficient detection and quantification of drug-related material (DRM) in human without use of radiolabeled drug is reported. This allowed more complete data rationalization of human metabolism, permitting early risk assessment and progression of the development of antitubercular agent, alpibectir.

Biotransformation of Ginsenoside Rb1 to Ginsenoside Rd and 7 Rare Ginsenosides Using Irpex lacteus with HPLC-HRMS/MS Identification.

The biotransformation of ginsenosides using microorganisms represents a promising and ecofriendly approach for the production of rare ginsenosides. The present study reports on the biotransformation of ginsenoside Rb1 using the fungus Irpex lacteus, resulting in the production of ginsenoside Rd and seven rare ginsenosides with novel structures. Employing high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry, the identities of the transformation products were rapidly determined. Two sets of isomers with molecular weights of 980.56 and 962.55 were discovered among the seven rare ginsenosides, which were generated through the isomerization of the olefin chain in the protopanaxadiol (PPD)-type ginsenoside skeleton. Each isomer exhibited characteristic fragment ions and neutral loss patterns in their tandem mass spectra, providing evidence of their unique structures. Time-course experiments demonstrated that the transformation reaction reached equilibrium after 14 days, with Rb1 initially generating Rd and compound 5, followed by the formation of other rare ginsenosides. The biotransformation process catalyzed by I. lacteus was found to involve not only the typical deglycosylation reaction at the C-20 position but also hydroxylation at the C-22 and C-23 positions, as well as hydrogenation, transfer, and cyclization of the double bond at the C-24(25) position. These enzymatic capabilities extend to the structural modification of other PPD-type ginsenosides such as Rc and Rd, revealing the potential of I. lacteus for the production of a wider range of rare ginsenosides. The transformation activities observed in I. lacteus are unprecedented among fungal biotransformations of ginsenosides. This study highlights the application of a medicinal fungi-based biotransformation strategy for the generation of rare ginsenosides with enhanced structural diversity, thereby expanding the variety of bioactive compounds derived from ginseng.

In Vivo and In Vitro Metabolic Fate and Urinary Detectability of Five Deschloroketamine Derivatives Studied by Means of Hyphenated Mass Spectrometry.

Ketamine derivatives such as deschloroketamine and deschloro-N-ethyl-ketamine show dissociative and psychoactive properties and their abuse as new psychoactive substances (NPSs) has been reported. Though some information is available on the biotransformation of dissociative NPSs, data on deschloro-N-cyclopropyl-ketamine deschloro-N-isopropyl-ketamine and deschloro-N-propyl-ketamine concerning their biotransformation and, thus, urinary detectability are not available. The aims of the presented work were to study the in vivo phase I and II metabolism; in vitro phase I metabolism, using pooled human liver microsomes (pHLMs); and detectability, within a standard urine screening approach (SUSA), of five deschloroketamine derivatives. Metabolism studies were conducted by collecting urine samples from male Wistar rats over a period of 24 h after their administration at 2 mg/kg body weight. The samples were analyzed using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) and gas chromatography-mass spectrometry (GC-MS). The compounds were mainly metabolized by N-dealkylation, hydroxylation, multiple oxidations, and combinations of these metabolic reactions, as well as glucuronidation and N-acetylation. In total, 29 phase I and 10 phase II metabolites were detected. For the LC-HRMS/MS SUSA, compound-specific metabolites were identified, and suitable screening targets could be recommended and confirmed in pHLMs for all derivatives except for deschloro-N-cyclopropyl-ketamine. Using the GC-MS-based SUSA approach, only non-specific acetylated N-dealkylation metabolites could be detected.

Plasma lipidome differences in patients with and without significant carotid plaque

BackgroundAtherosclerosis is a major cause of ischemic stroke, and early detection of advanced atherosclerosis in the carotid artery is important for reducing morbidity and mortality. What is even more important is not only detection of atherosclerosis but early determination whether the patients are at high risk of an event with adverse effects as the size of the plaque does not necessarily reflect its potential to trigger such events. AimWe studied whether plasma lipidomics profile can be used as a diagnostic tool for stratification of stable or unstable plaques without the need of removing the carotid plaque. MethodsThis study used liquid chromatography high-resolution tandem mass spectrometry lipidomics to characterize lipid profiles in patients' plasma and found that patients with significant and complicated (vulnerable) atherosclerotic plaque had distinct lipid profiles compared to those with insignificant plaques. ResultsThe lipid classes that were most predictive of vulnerable plaque were lysophosphoethanolamines, fatty acyl esters of hydroxy fatty acids, free fatty acids, plasmalogens, and triacylglycerols. Most of these compounds were found decreased in plasma of patients with unstable plaques which enabled sufficient performance of a statistical model used for patient stratification. ConclusionsPlasma lipidomes measured by liquid chromatography-mass spectrometry show differences in patients with stable and unstable carotid plaques, therefore these compounds could potentially be used as biomarkers for unstable plaque in future clinical diagnosis.

Sequence confirmation of synthetic DNA exceeding 100 nucleotides using restriction enzyme mediated digestion combined with high-resolution tandem mass spectrometry

Oligonucleotides have emerged as important therapeutic options for inherited diseases. In recent years, RNA therapeutics, especially mRNA, have been pushed to the market. Analytical methods for these molecules have been published extensively in the last few years. Notably, mass spectrometry has proven as a state-of-the-art quality control method. For RNA based therapeutics, numerous methods are available, while DNA therapeutics lack of suitable MS-based methods when it comes to molecules exceeding approximately 60 nucleotides. We present a method which combines the use of common restriction enzymes and short enzyme-directing oligonucleotides to generate DNA digestion products with the advantages of high-resolution tandem mass spectrometry. The instrumentation includes ion pair reverse phase chromatography coupled to a time-of-flight mass spectrometer with a collision induced dissociation (CID) for sequence analysis. Utilizing this approach, we increased the sequence coverage from 23.3% for a direct CID-MS/MS experiment of a 100 nucleotide DNA molecule to 100% sequence coverage using the restriction enzyme mediated approach presented in this work. This approach is suitable for research and development and quality control purposes in a regulated environment, which makes it a versatile tool for drug development.