MALDI Research Articles

Delay in the diagnosis of Brucella abortus bacteremia in a nonendemic country: a case report

BackgroundIt is challenging to diagnose brucellosis in nonendemic regions because it is a nonspecific febrile disease. The accurate identification of Brucella spp. in clinical microbiology laboratories (CMLs) continues to pose difficulties. Most reports of misidentification are for B. melitensis, and we report a rare case of misidentified B. abortus.Case presentationA 67-year-old man visited an outpatient clinic complaining of fatigue, fever, and weight loss. The patient had a history of slaughtering cows with brucellosis one year prior, and his Brucella antibody tests were negative twice. After blood culture, the administration of doxycycline and rifampin was initiated. The patient was hospitalized due to a positive blood culture. Gram-negative coccobacilli were detected in aerobic blood culture bottles, but the CML's lack of experience with Brucella prevented appropriate further testing. Inaccurate identification results were obtained for a GN ID card of VITEK 2 (bioMérieux, USA) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) using a MALDI Biotyper (Bruker, Germany). The strain showed 100.0% identity with Brucella spp. according to 16S rRNA sequencing. MALDI–TOF MS peaks were reanalyzed using the CDC MicrobeNet database to determine Brucella spp. (score value: 2.023). The patient was discharged after nine days of hospitalization and improved after maintaining only doxycycline for six weeks. The isolate was also identified as Brucella abortus by genomic evidence.ConclusionAutomated identification instruments and MALDI–TOF MS are widely used to identify bacteria in CMLs, but there are limitations in accurately identifying Brucella spp. It is important for CMLs to be aware of the possibility of brucellosis through communication with clinicians. Performing an analysis with an additional well-curated MALDI–TOF MS database such as Bruker security-relevant (SR) database or CDC MicrobeNet database is helpful for quickly identifying the genus Brucella.

DNA Walker-Driven Mass Nanotag Assembly System for Simultaneously Profiling Dual Markers of Oxidative Stress at Different Cellular Locations.

Simultaneous profiling of redox-regulated markers at different cellular sublocations is of great significance for unraveling the upstream and downstream molecular mechanisms of oxidative stress in living cells. Herein, by synchronizing dual target-triggered DNA machineries in one nanoentity, we engineered a DNA walker-driven mass nanotag (MNT) assembly system (w-MNT-AS) that can be sequentially activated by oxidative stress-associated mucin 1 (MUC1) and apurinic/apyrimidinic endonuclease 1 (APE1) from plasma membrane to cytoplasm and induce recycled assembly of MNTs for multiplex detection of the two markers by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In the working cascade, the sensing process governs the separate activation of w-MNT-AS by MUC1 and APE1 in diverse locations, while the assembly process contributes to the parallel amplification of the ion signal of the characteristic mass tags. In this manner, the differences between MCF-7, HeLa, HepG2, and L02 cells in membrane MUC1 expression and cytoplasmic APE1 activation were fully characterized. Furthermore, the oxidative stress level and dynamics caused by exogenous H2O2, doxorubicin, and simvastatin were comprehensively demonstrated by tracking the fate of the two markers across different cellular locations. The proposed w-MNT-AS coupled MS method provides an effective route to probe multiple functional molecules that lie at different locations while participating in the same cellular event, facilitating the mechanistic studies on cellular response to oxidative stress and other disease-related cellular processes.

Biological Activity and Phytochemical Characteristics of Star Anise (Illicium verum) Essential Oil and Its Anti-Salmonella Activity on Sous Vide Pumpkin Model.

Illicium verum, commonly known as star anise, represents one of the notable botanical species and is recognized for its rich reservoir of diverse bioactive compounds. Beyond its culinary application as a spice, this plant has been extensively utilized in traditional medicine. Given the contemporary emphasis on incorporating natural resources into food production, particularly essential oils, to enhance sensory attributes and extend shelf life, our study seeks to elucidate the chemical composition and evaluate the antibacterial (in vitro, in situ) and insecticidal properties of Illicium verum essential oil (IVEO). Also, microbiological analyses of pumpkin sous vide treated with IVEO after inoculation of Salmonella enterica were evaluated after 1 and 7 days of study. GC/MS analysis revealed a significantly high amount of (E)-anethole (88.4%) in the investigated EO. The disc diffusion method shows that the antibacterial activity of the IVEO ranged from 5.33 (Streptococcus constellatus) to 10.33 mm (Citrobacter freundii). The lowest minimal inhibition concentration was found against E. coli and the minimum biofilm inhibition concertation was found against S. enterica. In the vapor phase, the best antimicrobial activity was found against E. coli in the pears model and against S. sonei in the beetroot model. The application of the sous vide method in combination with IVEO application decreased the number of microbial counts and eliminated the growth of S. enterica. The most isolated microbiota identified from the sous vide pumpkin were Bacillus amyloliquefaciens, B. cereus, B. licheniformis, and Ralstonia picketii. Modifications to the protein composition of biofilm-forming bacteria S. enterica were suggested by the MALDI TOF MS instigations. The IVEO showed insecticidal potential against Harmonia axyridis. Thanks to the properties of IVEO, our results suggest it can be used in the food industry as a natural supplement to extend the shelf life of foods and as a natural insecticide.

Implementation of the EUCAST rapid antimicrobial susceptibility test (RAST) for carbapenemase/ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates, and its effect on mortality.

With the rise in antimicrobial resistance, there is a growing demand for rapid antimicrobial susceptibility testing (RAST). In this study, we applied the EUCAST RAST method to ESBL/carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolates without using advanced identification systems and analysed the effect of this method on mortality rates Also the clinical impact of this method on patients infected with these bacteria and its effect on mortality rates were investigated. RAST was used for clinical blood cultures containing carbapenemase/ESBL-producing E. coli and K. pneumoniae without advanced identification systems (e.g. MALDI TOF), with preliminary identification by simple diagnostic tests (predicted RAST, or p-RAST), and its categorical agreement was investigated. The impact of the method on mortality was analysed by comparing the clinical data of patients whose blood cultures were subject to p-RAST (p-RAST group, n = 49) and those who were not subject to p-RAST (non-RAST group, n = 145). p-RAST results were analysed based on 539 antibiotic-bacteria combinations. Total error rates at 4, 6 and 8 h of incubation were 2.9%, 3.9% and 3.8%, respectively. In the p-RAST group, patients who did not receive appropriate antibiotics (29/45, 59.1%) were switched to appropriate treatment within 8 h at the latest. In contrast, in the non-RAST group, treatment of patients who received inappropriate antibiotics (79/145, 54.5%) could be changed after at least 24 h. Mortality rates were lower in the p-RAST group than in the non-RAST group (28.6% versus 51.7%, P = 0.005). p-RAST can be used safely in hospital laboratories with high rates of antimicrobial resistance and can reduce mortality rates by shortening the transition time to appropriate treatment.

Clinical Evaluation of VITEK MS PRIME with PICKME Pen for Bacteria and Yeasts, and RUO Database for Filamentous Fungi.

The VITEK MS PRIME (bioMérieux, Marcy-l'Étoile, France), a newly developed matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system, alongside the VITEK PICKME pen (PICKME), offers easy sample preparation for bacteria and yeasts. The VITEK MS PRIME also offers two software platforms for filamentous fungi: the IVD database and the RUO database. Our study evaluated its identification agreement on 320 clinical isolates of bacteria and yeasts, comparing PICKME and traditional wooden toothpick sampling techniques against MicroIDSys Elite (ASTA) results. Additionally, we assessed the IVD (v3.2) and SARAMIS (v4.16) RUO databases on 289 filamentous fungi against molecular sequencing. The concordance rates for species-level identification of bacteria and yeasts were about 89.4% (286/320) between the PICKME and wooden toothpick, and about 83.4-85.3% between the VITEK MS PRIME and ASTA MicroIDSys Elite. Retesting with PICKME improved concordance to 91.9%. For filamentous fungi, species-level identification reached 71.3% with the IVD database and 85.8% with RUO, which significantly enhanced basidiomycetes' identification from 35.3% to 100%. Some strains in the IVD database, like Aspergillus versicolor, Exophiala xenobiotica, and Nannizzia gypsea, failed to be identified. The VITEK MS PRIME with PICKME offers reliable and efficient microorganism identification. For filamentous fungi, combined use of the RUO database can be beneficial, especially for basidiomycetes.

Penetration Profile of Terbinafine Compared to Amorolfine in Mycotic Human Toenails Quantified by Matrix-Assisted Laser Desorption Ionization-Fourier Transform Ion Cyclotron Resonance Imaging.

Amorolfine 5% lacquer is an established topical treatment for fungal infection of the nails. The success of topical therapy for onychomycosis depends on whether the permeated drug concentration in the deep nail bed is retained above the effective antifungal minimum inhibitory concentration (MIC). We compared the penetration profile of amorolfine and a new topical formula of terbinafine in human mycotic toenails using matrix-assisted laser desorption ionization mass spectrometry imaging-Fourier transform ion cyclotron resonance (MALDI-FTICR) imaging. Amorolfine 5% lacquer and terbinafine 7.8% lacquer were applied to mycotic nails (n = 17); nail sections were prepared, and MALDI-FTICR analysis was performed. Based on the MICs of amorolfine and terbinafine needed to kill 90% (MIC90) of Trichophyton rubrum, the fold differences between the MIC90 and the antifungal concentrations in the nails (the multiplicity of the MIC90) were calculated overall and for the keratin-unbound fractions. Both amorolfine and terbinafine penetrated the entire thickness of the nail. The mean concentration across the entire nail section 3h following terbinafine treatment was 1414μg/g of tissue (equivalent to 4.9mM) compared with 780μg/g (2.5mM) following amorolfine treatment (not significantly different; p = 0.878). The median multiplicity of the MIC90 was significantly higher in amorolfine- than terbinafine-treated nails overall (191 vs. 48; p = 0.010) and for the keratin-unbound fractions only (7.4 vs. 0.8; p = 0.002). In this ex vivo study, MALDI-FTICR demonstrated that, although amorolfine 5% and terbinafine 7.8% had similar distribution profiles, both penetrating from the surface to the nail bed, the concentration of amorolfine in the nail was significantly higher than that of terbinafine relative to their respective MIC90 values. Clinical studies are required to determine whether these effects translate to a clinical difference in treatment success.

Development of a defined mixed starter culture for the improvement of Tej, Ethiopian honey wine.

Ethiopian honey wine, Tej, is the most popular traditionally fermented alcoholic beverage in Ethiopia. Owing to the spontaneous fermentation process, the final product is neither predictable nor consistent in quality. Attempts have not been made before to solve this problem. Thus, the aim of this study was to develop a potential mixed starter culture of yeast and lactic acid bacteria isolated from Tej samples for the production of Tej with consistent quality. One hundred seventy-seven lactic acid bacteria and 194 yeasts were isolated from 30 Tej samples collected from southwest Ethiopia. After sequentially testing the isolates towards physiological stress tolerance and desired metabolic products, 10 lactic acid bacteria and 10 yeast isolates were screened. Later, four lactic acid bacteria and four yeast isolates were found to be compatible in co-culture tests. Finally, the combination of lactic acid bacteria and yeast isolates was formulated using the design of expert version 7.0.0 software, and six formulates (F #1-6) were designed. Controlled Tej fermentation was performed under laboratory conditions using six lactic acid bacteria-yeast starter culture formulations. The sensory attributes, in terms of color, flavor, odor, turbidity, and overall acceptance analysis scored 4.8/5.0 (F #2) and 4.7/5.0 (F #6), with mean significant variations (p < 0.05) among the other formulates. These two formulates were considered the best-mixed starter cultures compared to the control and other formulates. Matrix-assisted laser desorption ionization-time of flight analysis revealed that the lactic acid bacteria starters (AAUL7 and AAUL10) belonged to Lactobacillus paracasei. While the yeast starters (AAUY2 and AAUY8) belonged to Saccharomyces cerevisiae. These mixed lactic acid bacteria-yeast starter cultures could be used as the best starter culture for the fermentation of Ethiopian honey wine, Tej, with consistent quality.

Penetration Profiles of Four Topical Antifungals in Mycotic Human Toenails Quantified by Matrix-Assisted Laser Desorption Ionization-Fourier Transform Ion Cyclotron Resonance Imaging.

Onychomycosis is a fungal infection of the nails that can be challenging to treat. Here, matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) imaging was applied to the quantitative analysis of the penetration profile of the antifungal compound, amorolfine, in human mycotic toenails. The amorolfine profile was compared with those of three other antifungals, ciclopirox, naftifine, and tioconazole. Antifungal compounds (amorolfine 5% lacquer, ciclopirox 8% lacquer, naftifine 1% solution, and tioconazole 28% solution) were applied to mycotic nails (n = 42). Nail sections were prepared, and MALDI-FTICR analysis was performed on the sections at a spatial resolution of 70 μm to compare the distribution profiles. Based on the minimum inhibitory concentrations of the four test compounds needed to kill 90% (MIC90) of the fungal organism, Trichophyton rubrum, the fold differences between the MIC90 and the antifungal concentrations in the nails (termed the multiplicity of the MIC90) were calculated for each. The penetration profiles indicated higher concentrations of amorolfine and ciclopirox in the deeper layers of the nails 3h after treatment, compared with naftifine and tioconazole. The mean concentrations across the entire nail sections at 3h were significantly different among the four antifungals: amorolfine, 2.46mM; ciclopirox, 0.95mM; naftifine, 0.63mM; and tioconazole, 1.36mM (p = 0.016; n = 8 per compound). The median multiplicity of the MIC90 at 3h was 191-fold for amorolfine, tenfold for ciclopirox, 52-fold for naftifine, and 208-fold for tioconazole. In this study, MALDI-FTICR was successfully applied to the quantitative analysis of antifungal distribution in human mycotic nails. The findings suggest that amorolfine penetrates deeper layers of the nail and accumulates at concentrations far exceeding the MIC needed to exert antimycotic activity.

Enhancing pneumococcal bacteraemia diagnosis: A comparative assessment of culture-independent assays (MALDI–TOF–MS Sepsityper® module and a lateral flow inmunochromatography test)

IntroductionPneumococcal bacteraemia is a major contributor to global morbidity and mortality. Traditional culture-based methods lack sensitivity and are time-consuming. This study aimed to assess the effectiveness of two culture-independent assays, the MALDI–TOF–MS Sepsityper® module and the lateral flow inmunochromatography test (LFICT) with the Standard F® Streptococcus pneumoniae, directly from positive blood culture (BC) bottles. MethodsA prospective study was conducted from December 2021 to July 2022. For all BC positives for S. pneumoniae a double centrifugation protocol was implemented. The resulting pellet was subsequently processed using both techniques. ResultsThe LFICT showed exceptional performance with 100% sensitivity and specificity, outperforming the MALDI–TOF–MS Sepsityper® module, which achieved 85.2% sensitivity and 100% specificity. Nevertheless, the combination of these assays offers a robust and comprehensive approach to diagnosis. ConclusionsThe simultaneous use of both techniques offers a promising alternative that can be integrated into routine practices directly from BC samples.

Mass Spectrometry-Based Analyses of Carbon Nanodots: Structural Elucidation.

Carbon nanodots (CNDs) are nanomaterials with ubiquitous applications in health for diagnosis and treatments. The key to enhancing the applications of carbon nanodots in various fields lies on how deep its structure is understood. Here, we review the mass spectroscopy (MS) techniques employed for carbon nanodot analysis. We aimed to revive the use of MS to support the structural elucidation of carbon nanodots. General techniques used in nanomaterials characterization include laser desorption/ionization (LDI), matrix-assisted LDI (MALDI), inductively coupled plasma (ICP), and electrospray ionization (ESI) MS. For CNDs characterization, LDI-MS, MALDI-MS, and ESI-MS were employed. The techniques required further instrumentations of time-of-flight (TOF), for MALDI, and TOF, quadrupole (Q), and tandem (MS/MS) for ESI. LDI-MS could be applied to prove the surface and core structural composition of carbon nanodots. Meanwhile, MALDI-MS was used to elucidate the surface structures of CNDs. Finally, ESI-MS could provide significant insight into the carbon nanodots' structural composition and bonding patterns. In summary, MS could be combined with other techniques to unambiguously elucidate the structure of carbon nanodots.

Detection accuracy and clinical applications of DP-TOF mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently used in clinical microbiology laboratories. This study aimed to determine whether dual-polarity time-of-flight mass spectrometry (DP-TOF MS) could be applied to clinical nucleotide detection. This prospective study included 40 healthy individuals and 110 patients diagnosed with cardiovascular diseases. We used DP-TOF MS and Sanger sequencing to evaluate 17 loci across 11 genes associated with cardiovascular drug responses. In addition, we used DP-TOF MS to test 998 retrospectively collected clinical DNA samples with known results. A, T, and G nucleotide detection by DP-TOF MS and Sanger sequencing revealed 100% concordance, whereas the C nucleotide concordance was 99.86%. Genotyping based on the results of the two methods showed 99.96% concordance. Regarding clinical applications, DP-TOF MS yielded a 99.91% concordance rate for known loci. The minimum detection limit for DNA was 0.4 ng; the inter-assay and intra-assay precision rates were both 100%. Anti-interference analysis showed that aerosol contamination greater than 1013 copies/µL in the laboratory environment could influence the results of DP-TOF MS. The DP-TOF MS platform displayed good detection performance, as demonstrated by its 99.96% concordance rate with Sanger sequencing. Thus, it may be applied to clinical nucleotide detection.

Detection of KPC directly from positive blood cultures by MALDI-TOF: From research to the clinical microbiology laboratory routine

Bloodstream infections (BSI) caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) are a subject of major clinical concern, mainly those associated with carbapenemase-producing isolates. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed to detect specific β-lactamases, including KPC. We aimed to detect KPC enzyme directly from positive blood cultures using MALDI-TOF MS. Overall, 146 clinical Gram-negative bacilli (46 CR-GNB) recovered from consecutive blood cultures were evaluated. Proteins were extracted using formic acid, isopropyl alcohol, and water and spotted onto a steel target plate using the double-layer sinapinic acid method. The relative ions intensity ≥120 arbitrary units (a.u.) of a peak close to 28,700 m/z indicated the presence of KPC. The results were compared to HRM-qPCR methodology. This specific peak was observed in 11/14 blood bottles with blaKPC positive isolates (78.6% sensitivity), with 3 false-positive results (97.7% specificity). Analysis from colonies reached identical sensitivity (78.6%), but higher specificity (100%). The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid. It's excellent specificity indicates that positive results are consistently associated with the presence of a KPC producer in positive blood culture.

Identifying organisms directly from BacT/Alert FAN plus blood culture bottles using Vitek MS in a state-wide laboratory network

Identifying organisms directly from positive blood culture bottles using matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has many advantages to patients, clinical services, and laboratories. However, few published methods have demonstrated good performance using the current BioMérieux culture bottles and MALDI-TOF system: BacT/Alert FAN plus and Vitek MS. The effect of transporting bottles on test performance has not been assessed for any direct-from-bottle MS method. In this study, 802 positive blood culture bottles were analysed including 234 requiring inter-laboratory transport, using a method involving protein extraction with formic acid and acetonitrile. Correct identification rates were high for Staphylococcus aureus (58/58 of new diagnostic samples), Enterococcus faecalis (27/27), Gram-negative bacilli (160/176, 90.1%), and coagulase-negative Staphylococcus species (108/132, 81.8%). Three false identifications were made, none with clinical significance. For Gram-positive cocci in pairs or chains, more correct identifications were made from bottles analysed immediately compared to transported bottles (67% vs 44%, p=0.016), and longer transport time was associated with slightly lower probability of correct identification (OR 0.984 per additional hour, p=0.040). Transportation was not associated with a difference for other organism types. This technique is a vastly more cost-effective alternative to molecular techniques for rapid identification of bacteraemia isolates, and performance is minimally affected by inter-laboratory transport of bottles at ambient temperature.

Low molar mass polycarbonate diols from degradation of terpolymers obtained by epoxide/o-phthalaldehyde/CO2 copolymerization

In this manuscript, we describe the synthesis of low molar mass polycarbonate diols by acid cleavage of labile acetal linkages included in high molar mass poly(ether-co-carbonate-co-acetal) terpolymers. These copolymers were prepared by terpolymerization of propylene oxide (PO) with o-phthalaldehyde (OPA) and carbon dioxide (CO2), using triethyl borane (TEB) as activator and an onium salt as initiator. The advantage of this strategy of synthesis of poly(propylene carbonate) diols (PPC-diols) lies in the minute amounts of TEB and initiator required. Moreover, OPA could be isolated through post-hydrolysis of the terpolymers and recycled for subsequent use. We also demonstrated that this strategy works for the synthesis of low molar mass poly(ethylene carbonate) diols (PEC-diols). The structural integrity of the terpolymers before and after acid treatment, the characterization of low molar mass PPC-diols, and recycling process of OPA were carried out by 1H NMR spectroscopy, gel permeation chromatography, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) MS.

Monitoring of enteropathogenic Gram-negative bacteria in wastewater treatment plants: a multimethod approach

The wastewater treatment processes are associated with the emission of microbial aerosols, including enteropathogenic bacteria. Their presence in this work environment poses a real threat to the health of employees, both through the possibility of direct inhalation of the contaminated air and indirectly through the pollution of all types of surfaces with such bioaerosol particles. This study aimed to investigate the prevalence of enteropathogenic bacteria in the air, on surfaces, and in wastewater samples collected in four wastewater treatment plants (WWTPs). The effectiveness of conventional culture-biochemical, as well as spectrometric and molecular methods for the rapid detection of enteropathogenic bacteria at workstations related to particular stages of wastewater processing, was also evaluated. Bioaerosol, surface swab, and influent and effluent samples were collected from wastewater plants employing mechanical–biological treatment technologies. The air samples were collected using MAS-100 NT impactor placed at a height of 1.5 m above the floor or ground, simulating aspiration from the human breathing zone. Surface samples were collected with sterile swabs from different surfaces (valves, handles, handrails, and coveyor belts) at workplaces. The raw influent and treated effluent wastewater samples were aseptically collected using sterile bottles. The identification of bacterial entheropathogens was simultaneously conducted using a culture-based method supplemented with biochemical (API) tests, mass-spectrometry (MALDI TOF MS), and molecular (multiplex real-time PCR) methods. This study confirmed the common presence of bacterial pathogens (including enteropathogenic and enterotoxigenic Escherichia coli, Salmonella spp., Campylobacter spp., and Yersinia enterocolitica) in all air, surface, and wastewater samples at studied workplaces. Higher concentrations of enteropathogenic bacteria were observed in the air and on surfaces at workplaces where treatment processes were not hermetized. The results of this study underline that identification of enteropathogenic bacteria in WWTPs is of great importance for the correct risk assessment at workplaces. From the analytical point of view, the control of enteropathogenic bacterial air and surface pollution using rapid multiplex-PCR method should be routinely performed as a part of hygienic quality assessment in WWTPs.

Quantitative MALDI-MS and Imaging of Fungicide Pyrimethanil in Strawberries with 2-Nitrophloroglucinol as an Effective Matrix.

This work explores the use of 2-nitrophloroglucinol (2-NPG) as a matrix for quantitative analysis of the fungicide Pyrimethanil (PYM) in strawberries using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and imaging. 2-NPG was selected for PYM analysis for optimum sensitivity and precision compared to common matrices α-cyano-4-hydroxylcinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). PYM-sprayed strawberries were frozen 0, 1, 3, and 4 days after treatment and sectioned for MALDI imaging. The remaining part of each strawberry was processed using quick easy cheap effective rugged and safe (QuEChERS) extraction and analyzed by MALDI-MS and ultraperformance liquid chromatography multireaction-monitoring (UPLC-MRM). MALDI-MS showed comparable performance to UPLC-MRM in calibration, LOD/LOQ, matrix effect, and recovery, with the benefit of fast analysis. The MALDI imaging results demonstrated that PYM progressively penetrated the interior of the strawberry over time and the PYM concentration on tissue measured by MALDI imaging correlated linearly with MALDI-MS and UPLC-MRM measurements and accounts for 79% MALDI-MS and 85% UPLC-MRM values on average. Additionally, quartz crystal microbalance (QCM) was introduced as a new approach to determine strawberry tissue mass per area for MALDI imaging absolute quantitation with sensitive, direct, and localized measurements. This work demonstrates the first example of absolute quantitative MALDI imaging of pesticides in a heterogeneous plant tissue. The novel use of the 2-NPG matrix in quantitative MALDI-MS and imaging could be applied to other analytes, and the new QCM tissue mass per area method is potentially useful for quantitative MALDI imaging of heterogeneous tissues in general.

Incorporation of Three Different Optical Trains into the IR-MALDESI Mass Spectrometry Imaging Platform to Characterize Artemisia annua.

Artemisinin is the leading medication for the treatment of malaria and is only produced naturally in Artemisia annua. The localization of artemisinin in both the glandular and non-glandular trichomes of the plant makes it an ideal candidate for mass spectrometry imaging (MSI) as a model system for method development. Infrared matrix-assisted laser desorption electrospray ionization MSI (IR-MALDESI-MSI) has the capability to detect hundreds to thousands of analytes simultaneously, providing abundance information in conjunction with species localization throughout a sample. The development of several new optical trains and their application to the IR-MALDESI-MSI platform has improved data quality in previous proof-of-concept experiments but has not yet been applied to analysis of native biological samples, especially the MSI analysis of plants. This study aimed to develop a workflow and optimize MSI parameters, specifically the laser optical train, for the analysis of Artemisia annua with the NextGen IR-MALDESI platform coupled to an Orbitrap Exploris 240 mass spectrometer. Two laser optics were compared to the conventional set up, of which include a Schwarzschild-like reflective objective and a diffractive optical element (DOE). These optics, respectively, enhance the spatial resolution of imaging experiments or create a square spot shape for top-hat imaging. Ultimately, we incorporated and characterized three different optical trains into our analysis of Artemisia annua to study metabolites in the artemisinin pathway. These improvements in our workflow, resulted in high spatial resolution and improved ion abundance from previous work, which will allow us to address many different questions in plant biology beyond this model system.

Development of benzimidazole derivatives as efficient matrices for the analysis of acidic small-molecule compounds using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in negative ion mode.

With the development of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) in spatial localisation omics research on small molecules, the detection sensitivity of the matrix must increase. However, the types of matrices suitable for detecting acidic small molecules in (-) MALDI-MS mode are very limited and are either not sensitive enough or difficult to obtain. More than 10 commercially available benzimidazole and benzothiazole derivatives were selected as MALDI matrices in negative ion mode. MALDI-MS analysis was performed on 38 acidic small molecules and mouse serum, and the matrix effects were compared with those of the common commercial matrices 9-aminoacridine (9AA), 1,5-naphthalenediamine (DAN) and 3-aminoquinoline (3AQ). Moreover, the proton affinity (PA) of the selected potential matrix was calculated, and the relationships among the compound structure, PA value and matrix effect were discussed. In (-) MALDI-MS mode, a higher PA value generally indicates a better matrix effect. Amino-substituted 2-phenyl-1H-benzo[d]imidazole derivatives had well-defined matrix effects on all analytes and were generally superior to the commonly used matrices 9AA, DAN and 3AQ. Among them, 2-(4-(dimethylamino-phenyl)-1H-benzo[d]imidazole-5-amine (E-4) has the best sensitivity and versatility for detecting different analytes and has the best ability to detect fatty acids in mouse serum; moreover, the limit of detection (LOD) of some analytes can reach as low as ng/L. Compared to 9AA, DAN and 3AQ, matrix E-4 is more effective at detecting low-molecular-weight acidic compounds in (-) MALDI-MS mode, with higher sensitivity and better versatility. In addition, there is a clear correlation between compound structure, PA and matrix effects, which provides a basis for designing more efficient matrices.

Elucidating Gender-Specific Distribution of Imipramine, Chloroquine, and Their Metabolites in Mice Kidney Tissues through AP-MALDI-MSI.

Knowledge of gender-specific drug distributions in different organs are of great importance for personalized medicine and reducing toxicity. However, such drug distributions have not been well studied. In this study, we investigated potential differences in the distribution of imipramine and chloroquine, as well as their metabolites, between male and female kidneys. Kidneys were collected from mice treated with imipramine or chloroquine and then subjected to atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry imaging (AP-MALDI-MSI). We observed differential distributions of the drugs and their metabolites between male and female kidneys. Imipramine showed prominent distributions in the cortex and medulla in male and female kidneys, respectively. Desipramine, one of the metabolites of imipramine, showed significantly higher (*** p < 0.001) distributions in the medulla of the male kidney compared to that of the female kidney. Chloroquine and its metabolites were accumulated in the pelvis of both male and female kidneys. Interestingly, they showed a characteristic distribution in the medulla of the female kidney, while almost no distributions were observed in the same areas of the male kidney. For the first time, our study revealed that the distributions of imipramine, chloroquine, and their metabolites were different in male and female kidneys.

Argon nonthermal plasma etching of poly(L-lactic acid) films: Tunning the local surface degradation and hydrolytic degradation rate

Plasma technology is widely used in the industry for the surface modification of polymeric materials, improving the adhesion of paints in polymeric packaging and cellular adhesion on polymers' surfaces without using hazardous chemicals. Plasma etching is a fast way to modify polymers, creating new nanostructure surfaces and adding chemical groups for new applications like antimicrobial surfaces. Poly(L-lactic acid) (PLLA) is a biodegradable polymer with exciting properties for biomedical, tissue engineering, food packaging, and other applications. In many cases, these products must undergo plasma surface treatments to meet the technical requirements of their function. Herein, we evaluated the effects of surface modification of PLLA using Argon nonthermal plasma etching. The results from Gel Permeation Chromatography (GPC), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC), X-ray Excited Photoelectron Spectroscopy (XPS), and Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF-MS) show that increasing the plasma exposure time causes an enhancement in bulk and local surface degradation, crystallization kinetics and susceptibility to hydrolytic degradation for PLLA. Atomic Force Microscopy (AFM) show that the PLLA films present the highest surface roughness after 60 s of plasma etching times. Our findings suggest that plasma etching can be a suitable tool to adjust the polymer degradation rate to design intracorporeal devices with controlled degradation kinetics.