Time-of-flight Mass Spectrometry Research Articles

Identification of Southeast Asian Anopheles mosquito species with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a cross-correlation approach

BackgroundMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) is proposed for mosquito species identification. The absence of public repositories sharing mass spectra and open-source data analysis pipelines for fingerprint matching to mosquito species limits the widespread use of this technology. The objective of this study was to develop a free open-source data analysis pipeline for Anopheles species identification with MALDI–TOF MS.MethodsAnopheles mosquitoes were captured in 33 villages in Karen (Kayin) state in Myanmar. A subset of 403 specimens was selected for inclusion in either the reference or the test panel (270 and 133 specimens, respectively). Three hundred fifty-nine specimens could be identified with DNA barcodes and were assigned to 21 sensu stricto species and five sibling species pairs or complexes. A total of 3584 mass spectra of the head of these specimens identified with DNA barcoding were acquired and the similarity between mass spectra was quantified using a cross-correlation approach adapted from the published literature. A reference mass spectra database was created using all spectra of the PCR-identified specimens assigned to the reference panel. A simulation experiment was carried out by querying the reference database with the spectra of the test panel to evaluate the performance of species identification with MALDI–TOF MS at varying thresholds of the cross-correlation index for the algorithm to output an identification result and with varying numbers of technical replicates for the tested specimens, considering PCR identification results as the reference.ResultsWith one spot and a threshold value of −14 for the cross-correlation index on the log scale, the sensitivity was 0.99 [95% credible interval (CrI): 0.98–1.00], the predictive positive value was 0.99 (95% CrI: 0.98–0.99), and the accuracy was 0.98 (95% CrI: 0.97–0.99). It was not possible to directly estimate the sensitivity and negative predictive value because there was no true negative (i.e., queries of species not referenced in the database) in the assessment.ConclusionsThe cross-correlation approach can be used to match mass spectral fingerprints to predefined taxa. MALDI–TOF MS is a valuable tool for rapid, accurate, and affordable identification of Anopheles species.Graphical abstract

Photocatalytic production and biological activity of D-arabino-1,4-lactone from D-fructose

Lactones play crucial roles in various fields, such as pharmaceuticals, food, and materials science, due to their unique structures and diverse biological activities. However, certain lactones are difficult to obtain in large quantities from natural sources, necessitating their synthesis to study their properties and potential. In this study, we investigated the photocatalytic conversion of D-fructose, a biomass-derived and naturally abundant sugar, using a TiO2 photocatalyst under light irradiation in ambient conditions. The resulting products were identified using HPLC, LCMS, MALDI TOF MS, and 1H NMR. The results confirmed the successful production of D-arabino-1,4-lactone as a key product, along with the formation of other valuable compounds, including rare sugars such as erythrose and glyceraldehyde. Analysis of the reaction mechanism revealed that D-arabino-1,4-lactone can be directly produced by the α scission (C1-C2 position cleavage) of D-fructose. Furthermore, erythrose and glyceraldehyde, as rare sugars, can be produced from the decomposition of D-arabino-1,4-lactone, which means that D-arabino-1,4-lactone can be used as a source of rare sugars. Furthermore, to investigate the biological activity of D-arabino-1,4-lactone, it was administered to Bifidobacterium. The results showed that Bifidobacterium proliferated and produced more lactic acid than when cultured in a medium without D-arabino-1,4-lactone, suggesting that Bifidobacterium can utilize D-arabino-1,4-lactone.

Comparative metabolomic study of twelve Acacia species by UHPLC-q-tof-ESI-MS coupled with chemometrics in correlation with antibacterial activity.

Genus Acacia comprises around 1500 species. They are widely used to treat inflammation as well as bacterial and fungal infections as they are enriched in phytochemicals, especially phenolics. The aim of this study was to evaluate the antibacterial activity of leaves' methanolic extracts of twelve Acacia species growing in Egypt against Vibrio parahaemolyticus, Salmonella enterica, Listeria monocytogens, Klebsiella pnemoniae, Bacillus aquimaris, Bacillus subtilis, and Escherichia coli. These species are Acacia nilotica (wild and cultivated), Acacia seyal, Acacia auriculiformis, Acacia saligna, Acacia xanthophloea, Acacia tortilis subsp. raddiana (Gabal Elba and Aswan), Acacia tortilis, Acacia laeta (wild and cultivated), and Acacia albida. Furthermore, to study the metabolomic composition and variation among these species using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time of flight mass spectrometry (UHPLC-q-tof-ESI-MS) coupled with multivariate statistical analysis and correlate it to the antibacterial potential. Results showed that Acacia nilotica (AN) has superior antibacterial activity over the other species. In addition, it exhibited a distinct segregation in Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA). Full profiling of AN using UHPLC-ESI-q-tof-MS revealed 42 phenolics mainly catechins. It was further subjected to bio-guided fractionation and revealed the presence of methyl gallic acid, gallic acid, catechin gallate, and digallate isomers in its most bioactive fraction. These compounds were identical to the compounds annotated as VIPs and were responsible for the segregation of AN in both PCA and PLS-DA analyses. Hence, this study sheds light on the use of chemometrics as an early tool for the detection of bioactive compounds.

Cutaneous Prototheca wickerhamii infection identified with the matrix-assisted laser desorption/ionization time-of flight mass spectrometry after protein extraction protocol for filamentous fungi isolates from a facial discharge of a 66-year-old female patient in Korea: a case report

Prototheca wickerhamii is an achlorophyllic algae that rarely acts as an opportunistic pathogen in humans. We report the case of a 66-year-old female patient with a history of diabetes mellitus who presented with a pus-like discharge from face, facial redness, and swelling. The patient was admitted to the emergency room with worsening facial pain. Gram staining from pus-like discharge revealed yeast like features; however, an isolated colony on a blood agar plate was not identified using VITEK 2 (Biomerieux) or matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF) using VITEK®MS (Biomerieux). Lactophenol cotton blue staining of the colony revealed characteristic sporangia and endospore features. After performing the protein extraction procedure for filamentous fungi (mold) isolates using ethanol and formic acid, MALDI-TOF MS identified the colony as Prototheca wickerhamii, with 99.9% confidence. This case indicates that Prototheca spp. require specialized sample preparation steps, such as the ethanol/formic acid extraction procedure, for identification using MALDI-TOF MS.

Forced Degradation Study of an Anti-Diabetic Drug Imeglimin: Impurity Profiling and Structure Elucidation Using LC-Q-ToF-MS/MS and NMR.

The present study aims to establish structures of the degradation products of an anti-diabetic drug, Imeglimin (IMG) approved for the treatment of type 2 diabetes mellitus in the year 2021. Degradation pathways are proposed along with in silico toxicity assessments of the observed degradation products (DPs) of the drug. A reversed-phase high-performance liquid chromatography (RP-HPLC), equipped with a photodiode array detector, was used to separate the observed DPs with a Phenomenex Luna PFP (250 × 4.6 mm, 5 μm) column, using 10 mM ammonium formate (pH 4.5) and methanol as mobile phase. Liquid chromatography quadrupole time of flight mass spectrometry (LC-Q-ToF-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed for structural elucidation. Zeneth and Derek suites were used for in silico assessments. A total of four degradation products were observed, which were successfully separated on an RP-HPLC. The structural characterization of three of the four DPs was achieved using LC-Q-TOF-MS/MS by employing electro spray ionization as well as atmospheric pressure chemical ionization. Additionally, DP-3 was isolated using a preparative HPLC and was characterized by NMR. Computationally predicted structures were compared with the experimental observations. An HPLC method, capable of separating the Imeglimin and its four DPs, was developed and validated as per the ICH Q2(R1) guideline. Structure elucidation reveals a variety of products with metformin as one of the identified DPs along with a metabolite. The toxicity potential of DPs was assessed through docking studies.

Automatic identification of clinically important Aspergillus species by artificial intelligence-based image recognition: proof-of-concept study

ABSTRACT While morphological examination is the most widely used for Aspergillus identification in clinical laboratories, PCR–sequencing and MALDI–TOF MS are emerging technologies in more financially-competent laboratories. However, mycological expertise, molecular biologists and/or expensive equipment are needed for these. Recently, artificial intelligence (AI), especially image recognition, is being increasingly employed in medicine for fast and automated disease diagnosis. We explored the potential utility of AI in identifying Aspergillus species. In this proof-of-concept study, using 2813, 2814 and 1240 images from four clinically important Aspergillus species for training, validation and testing, respectively; the performances and accuracies of automatic Aspergillus identification using colonial images by three different convolutional neural networks were evaluated. Results demonstrated that ResNet-18 outperformed Inception-v3 and DenseNet-121 and is the best algorithm of choice because it made the fewest misidentifications (n = 8) and possessed the highest testing accuracy (99.35%). Images showing more unique morphological features were more accurately identified. AI-based image recognition using colonial images is a promising technology for Aspergillus identification. Given its short turn-around-time, minimal demand of expertise, low reagent/equipment costs and user-friendliness, it has the potential to serve as a routine laboratory diagnostic tool after the database is further expanded.

MALDI - TOF MS for the identification of obligate anaerobes and metronidazole susceptibility of anaerobic Gram negative bacilli

MALDI-TOF MS, though has facilitated rapid and accurate anaerobe identification, the problem of rising metronidazole resistance amongst the members of Bacteroides fragilis group is cause for concern. In this one-year study period,152 anaerobic Gram negative bacilli and 60 Gram positive anaerobes were isolated from 167 samples obtained from clinically suspected anaerobic infections. Bacteroides fragilis accounted for 56 % of the total anaerobic GNB and Peptoniphilus asacchrarolyticus was the most commonly isolated Gram positive cocci. All isolates were identified by the MALDI–TOF MS except one isolate each of Clostridium and Peptostreptococcus. E-test for members of Bacteroides fragilis group, demonstrated 26.8 % metronidazole resistance.

Pseudomonas serbiensis sp. nov. isolated from watermelon and muskmelon in Serbia.

Six Pseudomonas strains isolated from muskmelon and watermelon seedlings affected by stem rot and wilting in Serbia were reported as P. cichorii based on pathogenicity, LOPAT and cell wall fatty acid analyses. Recent bacterial isolates from cucurbit crops displaying P. cichorii-like symptoms in Alabama, USA, were identified as P. capsici, prompting polyphasic re-evaluation of the Serbian strains. All six strains were found to cause severe disease in watermelon and squash seedlings under greenhouse conditions. Strains KFB 138 and KFB 140 underwent whole-genome sequencing and were found to have the highest level of 16S rRNA similarity to P. lijiangensis LJ2T (both 99.87%). Phylogenies based on housekeeping genes and core-genome analysis placed both strains into phylogroup 11 of the Pseudomonas syringae species complex, with KFB 138 forming a lineage basal to all other phylogroup 11 members. In core-genome phylogeny, KFB 140 was placed into a clade alongside P. lijiangensis LJ2T. Average nucleotide identity based on blast (ANIb) identified KFB 140 as a member of P. lijiangensis (95.85%), though KFB 138 did not produce an ANIb value over 95% to any Pseudomonas type strain to which it was compared. Values for in silico DNA-DNA hybridization for both strains were below 70% to all reference strains tested, though KFB 140 was found to be most similar to P. lijiangensis (68.2%). KFB 138 and KFB 140 were further characterized using the online Type Genome Server, biochemical profiling with the Biolog Gen III MicroPlate system, matrix-assisted laser desorption/ionization time of flight mass spectrometry and imaged with transmission electron microscopy. From the results of the above analyses, we conclude that KFB 140 is a member of the species P. lijiangensis and that KFB 138 represents a novel Pseudomonas species, for which we propose the name Pseudomonas serbiensis (KFB 138T, NCPPB 4762=LMG 33366), named for its location of isolation.

Authenticity of cinnamon verum determined by MALDI TOF MS and machine learning

Authenticity of cinnamon verum determined by MALDI TOF MS and machine learning

The neurotoxic effect of Naja nubiae (Serpentes: Elapidae) venom from Sudan.

Neurotoxicity is a common feature of elapid snake envenomation. There are limited studies on the toxicity of Naja nubiae venom, the Nubian spitting cobra, from north-east Africa. We used the chick biventer cervicis nerve-muscle preparation to demonstrate the neurotoxic effect of N. nubiae venom and to compare it with the potent neurotoxic cobra Naja melanoleuca venom. Venoms were separated by successive reverse-phase high-performance liquid chromatography (RP-HPLC) runs and the molecular mass of the neurotoxins was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Both venoms caused time-dependent inhibition of nerve-mediated twitches with a t90 value of 22.2±1.9min and 12.9±1.2min for N. nubiae and N. melanoleuca venoms, respectively. Prior incubation of some commercial antivenom (EchiTab-Plus-ICP [Costa Rica], CSL, Parkville, Victoria, Australia) and snake venom antisera [India]) did not prevent the neurotoxic effect of N. nubiae venom. The chromatographic separation of N. nubiae and N. melanoleuca venoms followed by MALDI-TOF MS analysis revealed that short-chain α-neurotoxin accounted for 8.4% of N. nubiae and 14.8% of N. melanoleuca whole venoms. N. nubiae venom, which was previously known as cytotoxic venom, exhibits considerable in vitro neurotoxic effects on chick nerve-muscle preparations that may have consequences for antivenom development in north-east Africa.

Metabolite profiling in ten bread wheat (Triticum aestivum L.) genotypes in response to drought stress

Wheat is frequently constrained by extreme environmental conditions such as drought. Improving drought tolerance in wheat genotypes is crucial for ensuring food security, especially considering the challenges posed by climate change. To reveal the involvement of metabolites in drought response, ten diverse wheat genotypes were investigated under control and water scarcity conditions. The field experiments were set-up, using a 5 × 2 alpha lattice design, with two replicates per treatment, in the 2022 and 2023 growing seasons. Metabolites associated with drought tolerance were analysed using ultra-high performance liquid chromatography, coupled with a quadruple time of flight mass spectrometry (UHPLC-qTOF-MS). Multivariate statistical analysis (MVDA) tools, viz. principal component analysis (PCA) and the orthogonal projection to latent structures-discriminant analysis (OPLS-DA) loading scatter plot were used to identify the metabolites that are positively and negatively correlated to drought stress. Significant variation (p < 0.05) among genotypes was observed, with 58 metabolites annotated, including phenolic acids, carbohydrates, and fatty acids. The annotated compounds were linked to thirteen most significant pathways, with one carbon metabolism, cutin, suberin and wax synthesis and starch and sucrose metabolism being significantly affected by water stress, based on the KEGG pathway analysis. The two high-yielding wheat genotypes (LM48 and BW140) under drought stress displayed significant upregulation of key metabolites such as sinapoyl hydroxyagmatine, 7-oxostigmasterol, 1-O-caffeoyl-3-O-p-coumaroylglycerol, and 3-beta-3-lupanol, when compared to the non-stressed conditions. This study demonstrates the prospects of applied metabolomics for chemotaxonomic classification, phenotyping and selection in plant breeding, as well as potential use in crop improvement.

Evaluation of chelating agents based on pyridine-azacrown compounds H4PATA, PATAM, and H4PATPA for 68Ga and 177Lu

In this article, we present the synthesis and characterization of three macrocyclic chelators, H4PATA, PATAM, and H4PATPA, based on a pyridine-azacrown compound. Their complexation with 68Ga and 177Lu has been thoroughly investigated using MALDI TOF MS, 1H NMR spectroscopy, radiolabeling studies, and experiments in vitro with fetal bovine serum and a 1000-fold molar excess of H4EDTA. Our studies have shown that the chelators H4PATA and H4PATPA form complexes at room temperature with both radionuclides (RCY > 80 % and > 90 % for complexes with H4PATA and H4PATPA after 30 min, respectively). The chelator PATAM requires high temperature (95 °C) for complexation. In vitro stability assays in fetal bovine serum as well as H4EDTA-challenge revealed that transchelation occurs for all complexes with 68Ga. However, complexes of the ligands H4PATA and PATAM with 177Lu were found stable. Thus, taking into account the radiolabeling at room temperature and in vitro stability of the complex [177Lu]Lu·PATA, our investigations revealed the chelator H4PATA is a candidate for radiopharmaceutical use with 177Lu.

A Perspective of Multi-Reflecting TOF MS.

Time-of-flight mass spectrometry (TOF MS) excels in rapid and high-sensitivity analysis, making it a cornerstone of analytical chemistry. But as sample complexity explodes in omics studies, so does the need for higher resolving power to ensure accurate results. Traditional TOF instruments face a challenge: achieving high resolution often requires a very large instrument. To overcome this limitation, scientists developed alternative designs for TOF analyzers called multi-pass TOF analyzers (MPT). These MPT analyzers come in two main configurations: multi-turn (MTT) and multi-reflecting (MRT). Drawing on the authors' extensive experience, this review describes two decades of MPT advancements. It highlights the critical development of optimized analyzer designs, tracing the evolution towards mirror-based MRT instruments, generally providing superior resolution and spatial acceptance compared to MTT. While the manuscript attempts to overview MTT advances, it primarily focuses on MRT technology. Additionally, the review explores the role of orthogonal accelerators and trap pulse converters, comparing their efficiency and the dynamic range limits imposed by space charge effects. By comparing various MRT configurations and commercially available instruments, the review sets out to inform and empower researchers so they can make informed decisions about MRT mass spectrometers.

Multi-Domain Data Integration for Plasma Diagnostics in Semiconductor Manufacturing Using Tri-CycleGAN

The precise monitoring of chemical reactions in plasma-based processes is crucial for advanced semiconductor manufacturing. This study integrates three diagnostic techniques—Optical Emission Spectroscopy (OES), Quadrupole Mass Spectrometry (QMS), and Time-of-Flight Mass Spectrometry (ToF-MS)—into a reactive ion etcher (RIE) system to analyze CF4-based plasma. To synchronize and integrate data from these different domains, we developed a Tri-CycleGAN model that utilizes three interconnected CycleGANs for bi-directional data transformation between OES, QMS, and ToF-MS. This configuration enables accurate mapping of data across domains, effectively compensating for the blind spots of individual diagnostic techniques. The model incorporates self-attention mechanisms to address temporal misalignments and a direct loss function to preserve fine-grained features, further enhancing data accuracy. Experimental results show that the Tri-CycleGAN model achieves high consistency in reconstructing plasma measurement data under various conditions. The model’s ability to fuse multi-domain diagnostic data offers a robust solution for plasma monitoring, potentially improving precision, yield, and process control in semiconductor manufacturing. This work lays a foundation for future applications of machine learning-based diagnostic integration in complex plasma environments.

TOF-MS based characterization of polyglycerol polyricinoleate (PGPR) samples

Time-of-flight mass spectrometry (TOF-MS) was used to unravel the composition of commercial polyglycerol polyricinoleate (PGPR) samples, by identifying the various molecular species present. To cover the broad range of molecular weights for the present species, a combination of three ionisation conditions was used. Species exceeding the molecular weight of pentaglycerol hexaricinoleate were difficult to detect. Over 100 molecular species were observed and identified in the analysed samples, including free polyglycerols, ricinoleates, and PGPR-esters. Commercial PGPR samples were shown to be mainly composed of esterification products of di-, tri-, and tetraglycerol, while the esterification degree mainly varied from 1 to 5. The TOF-MS analysis was proven to be reproducible with a relative standard deviation (RSD) below 2.86% for three independent measurements on different days. The method proved to be very suitable to evaluate batch-to-batch variations and to compare the composition of different types of commercial PGPR’s. Moreover, this method can be applied to monitor the quality of PGPR products during the synthesis process. Furthermore, it can also provide fundamental knowledge for optimizing PGPR composition to improve its functionality.

Enhanced detection of Pythium insidiosum via lipid profiling with matrix-assisted laser desorption ionization time of flight mass spectrometry

Pythiosis is a severe disease in humans and animals globally, caused by the pathogenic oomycete Pythium insidiosum. Early and accurate detection is crucial for effective treatment, but traditional diagnostic methods have limitations. This study presents an alternative approach using Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) for lipid profiling to efficiently identify P. insidiosum. The study involved extracting microbial lipid components using optimized chloroform: methanol biphasic method and creating a lipid profile database with samples from 30 P. insidiosum isolates and 50 various fungi. The methodology was validated on 25 blinded samples for assay detection performance. Unique lipid profiles allowed species-specific identification with high efficiency: scores ≥ 2.682 indicated P. insidiosum, scores ≤ 2.512 suggested fungi, and scores in between pointed to other oomycetes. The assay demonstrated sensitivity, specificity, and accuracy of 100%, 80%, and 88%, respectively, for detecting P. insidiosum. The limited detection specificity was due to false positive samples from closely related Pythium species, which are not a significant clinical concern. The findings show that MALDI-TOF MS lipid profiling can efficiently identify P. insidiosum, offering significant advantages in sample preparation, stability, and reproducibility over protein profile-based methods. This study marks the first instance of lipid profiles being reported for P. insidiosum, paving the way for clinical use in improving accurate detection and facilitating timely treatment interventions.

Spatial distribution and temporal evolution of wall-stabilized DME/O2 premixed cool flames

The low-temperature oxidation of dimethyl ether (DME) was investigated in premixed wall-stabilized cool flames at two equivalence ratios (ϕ) of 0.2 and 0.5. Using a time-of-flight mass spectrometry (TOF-MS) coupled with gas chromatography (GC), the spatial distributions of major intermediate species, including DME, CH2O (formaldehyde), CO, CO2, and CH3OCHO (methyl formate), were quantified under well-controlled boundary conditions. Moreover, the temporal evolutions of multiple intermediate species in the wall-stabilized cool flame ignition process were measured via TOF-MS, while the wall temperature was gradually ramped up from 550 K to 730 K. Several kinetic models were examined herein to assess the estimated low-temperature reactivity of DME by comparing the one-dimensional axisymmetric simulation results with the experimental data. Wall-stabilized cool flame structures at equivalence ratios ϕ of 0.2 and 0.5 were quantitatively examined with the major intermediate species. It is found that the kinetic models reasonably predict the onset of the reaction zone near the wall. Among these models, Kurimoto et al.’s model gives better predictions for the distributions of CH2O and CO, which are characteristic species of cool flames. In addition, time-resolved measurements of the unsteady cool flames identified the negative temperature coefficient (NTC) turnover points for different species across various temperature regions. It is also found that the Kurimoto et al. model still indicates a slightly higher reactivity of DME in the low-temperature range, resulting in earlier DME consumption and a shift of NTC window to lower temperatures at ϕ = 0.2.

Decline of soil volatile organic compounds from a Mediterranean deciduous forest under a future drier climate

Biogenic volatile organic compounds (BVOCs) are crucial for ecosystem functioning, atmospheric chemistry and climate. While modulation of BVOC emissions from living vegetation with biotic and abiotic factors is well documented, how these factors drive soil BVOC emissions remain less understood, particularly in Mediterranean forests. To fill this gap, this pioneer study investigates whether BVOC fluxes from natural soil covered by litter (referred to as forest soil) vary under natural and amplified long-term water stress (35% annual rain exclusion over the past 10 years) in a deciduous oak Mediterranean forest (Quercus pubescens Willd.) compared to natural climate conditions. This aim has only been tackled in a single evergreen Mediterranean forest so far. Using proton transfer reaction time of flight mass spectrometer (PTR-ToF-MS) we also provide, for the first time, a detailed diurnal cycle of soil BVOCs in relation to air temperature, air humidity, and biotic factors including soil respiration and litter content in lignin, cellulose and hemicellulose. The main results revealed that forest soil represents a source of most BVOCs (e.g., acetaldehyde, acetone, acrolein, hexanol, monoterpenes) with maximum values at mid-day (42 μgC.m−2. h−1) in response to higher temperatures while it acts as a clear sink of isoprene. Total soil BVOC emission rates, together with soil respiration, decreased by 43% after a decade of partial rain restriction. These results will contribute to enhance further modeling of soil BVOC emissions under various climate scenarios both at regional and global scales.

Use of stable isotope combined with intact cell lipidomic by routine MALDI mass spectrometry analysis for rapid drug susceptibility assay in mycobacteria.

Rapid, accurate, and easy-to-perform diagnostic assays are required to address the current need for the diagnosis of resistant pathogens. That is particularly the case for mycobacteria, such as the human pathogen Mycobacterium tuberculosis, which requires up to 2weeks for the determination of the drug susceptibility profile using the conventional broth microdilution method. To address this challenge, we investigated the incorporation of deuterium, the stable isotope of hydrogen, into lipids as a read out of the drug susceptibility profile. Deuterium is incorporated into newly synthesized proteins or lipids in place of hydrogen as bacterial cells grow, increasing the mass of the macromolecules, which can then be observed via matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). As proof-of-concept, we used the non-pathogenic Mycobacterium smegmatis mc2155 strain, which is susceptible to the aminoglycoside antibiotic kanamycin, and M.smegmatis mc2155 containing the empty vector pVV16, which is kanamycin-resistant. Bacteria were incubated in a culture medium containing 50% of deuterium oxide (D2O) and either 1 or 2 times the minimal inhibitory concentration (MIC50) of kanamycin. Lipids were then analyzed using the MBT lipid Xtract matrix combined with routine MALDI mass spectrometry in the positive ion mode to evaluate the changes in the lipid profile. Using this approach, we were able to distinguish susceptible from resistant bacteria in less than 5h, a process that would take 72 h using the conventional broth microdilution method. We therefore propose a solution for the rapid determination of drug susceptibility profiles using a phenotypic assay combining D2O stable isotope labelling and lipid analysis by routine MALDI mass spectrometry.

Changing composition of street drug nyaope and its implications in South Africa

Nyaope is a Novel Psychoacve Substance (NPS) that connues to bring major challenges to the health and social well-being of users and their communies in South Africa. The acve ingredients that make up this cocktail drug Nyaope were first idenfied in 2015 using Time-of- flight mass spectrometry (TOF-MS) in the samples collected from the surrounding areas of the capital city Pretoria, and they were found to be variable across the samples. This variability had posed challenges to the treatment and rehabilitaon programmes. The current study was conducted in 2020, and included significantly a larger number of Nyaope samples collected from two provinces: Gauteng and Kwa Zulu-Natal of South Africa. The analycal method applied was also an improved model of TOF-MS analyser with a larger library of various drugs and their metabolites for matching the ingredients. Strikingly, many non-addicve drugs that were found in the previous samples were not found in this study. The new trend of formulaon has become more addicve and compact with major opiates and a few selected opioids, and with paracetamol and caffeine in different combinaons. This study reports differences in the physical appearance and the chemical composion of street drug Nyaope across the 2 provinces of South Africa: Gauteng and KwaZulu Natal (KZN), and also compared with the previous findings of 2015 in the samples collected in Gauteng province. The drug appears to be widely consumed and has serious health consequences as per previous studies, and therefore, understanding the chemical substances used in the drug will be helpful in both treatment and rehabilitaon efforts.