Point-of-Care Diagnosis of Endometrial Cancer Using the Surgical Intelligent Knife (iKnife)-A Prospective Pilot Study of Diagnostic Accuracy.

Introduction: Delays in the diagnosis and treatment of endometrial cancer negatively impact patient survival. The aim of this study was to establish whether rapid evaporative ionisation mass spectrometry using the iKnife can accurately distinguish between normal and malignant endometrial biopsy tissue samples in real time, enabling point-of-care (POC) diagnoses. Methods: Pipelle biopsy samples were obtained from consecutive women needing biopsies for clinical reasons. A Waters G2-XS Xevo Q-Tof mass spectrometer was used in conjunction with a modified handheld diathermy (collectively called the 'iKnife'). Each tissue sample was processed with diathermy, and the resultant surgical aerosol containing ionic lipid species was then analysed, producing spectra. Principal component analyses and linear discriminant analyses were performed to determine variance in spectral signatures. Leave-one-patient-out cross-validation was used to test the diagnostic accuracy. Results: One hundred and fifty patients provided Pipelle biopsy samples (85 normal, 59 malignant, 4 hyperplasia and 2 insufficient), yielding 453 spectra. The iKnife differentiated between normal and malignant endometrial tissues on the basis of differential phospholipid spectra. Cross-validation revealed a diagnostic accuracy of 89% with sensitivity, specificity, positive predictive value and negative predictive value of 85%, 93%, 94% and 85%, respectively. Conclusions: This study is the first to use the iKnife to identify cancer in endometrial Pipelle biopsy samples. These results are highly encouraging and suggest that the iKnife could be used in the clinic to provide a POC diagnosis.

Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies.

Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the "double tap" method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a "second chance" at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.

Double-tap gene drive uses iterative genome targeting to help overcome resistance alleles.

Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.

Base Editors: Expanding the Types of DNA Damage Products Harnessed for Genome Editing.

Base editors are an innovative addition to the genome editing toolbox that introduced a new genome editing strategy to the field. Instead of using double-stranded DNA breaks, base editors use nucleobase modification chemistry to efficiently and precisely incorporate single nucleotide variants (SNVs) into the genome of living cells. Two classes of DNA base editors currently exist: deoxycytidine deamination-derived editors (CBEs, which facilitate C•G to T•A mutations) and deoxyadenosine deamination-derived base editors (ABEs, which facilitate A•T to G•C mutations). More recently, the development of mitochondrial base editors allowed the introduction of C•G to T•A mutations into mitochondrial DNA as well. Base editors show great potential as therapeutic agents and research tools, and extensive studies have been carried out to improve upon the original base editor constructs to aid researchers in a variety of disciplines. Despite their widespread use, there are few publications that focus on elucidating the biological pathways involved during the processing of base editor intermediates. Because base editors introduce unique types of DNA damage products (a U•G mismatch with a DNA backbone nick for CBEs, and an I•T mismatch with a DNA backbone nick for ABEs) to facilitate genome editing, a deep understanding of the DNA damage repair pathways that facilitate or impede base editing represents an important aspect for the further expansion and improvement of the technologies. Here, we first review canonical deoxyuridine, deoxyinosine, and single-stranded break repair. Then, we discuss how interactions among these different repair processes can lead to different base editing outcomes. Through this review, we hope to promote thoughtful discussions on the DNA repair mechanisms of base editing, as well as help researchers in the improvement of the current base editors and the development of new base editors.

Validation of Ultrasonic Harmonic Scalpel for Real-Time Tissue Identification Using Rapid Evaporative Ionization Mass Spectrometry.

In this study, we integrate rapid evaporative ionization mass spectrometry (REIMS) with the Harmonic scalpel, an advanced laparoscopic surgical instrument that utilizes ultrasound energy to dissect and coagulate tissues. It provides unparalleled manipulation capability to surgeons and has superseded traditional electrosurgical tools particularly in abdominal surgery, but is yet to be validated with REIMS. The REIMS platform coupled with the Harmonic device was shown to produce tissue-specific lipid profiles through the analysis of porcine tissues in both negative and positive ionization modes. Comparison with other methods of electrosurgical dissection, such as monopolar electrosurgery and CO2 laser, showed spectral differences in the profile dependent on the energy device used. The Harmonic device demonstrated major spectral differences in the phospholipid region of m/z 600-1000 compared with the monopolar electrosurgical and CO2 laser-generated spectra. Within the Harmonic REIMS spectra, high intensities of diglycerides and triglycerides were observed. In contrast, monopolar electrosurgical and laser spectra demonstrated high abundances of glycerophospholipids. The Harmonic scalpel was able to differentiate between the liver, muscle, colon, and small intestine, demonstrating 100% diagnostic accuracy. The validation of the Harmonic device-mass spectrometry combination will allow the platform to be used safely and robustly for real-time in vivo surgical tissue identification in a variety of clinical applications.

Endogenous aldehyde accumulation generates genotoxicity and exhaled biomarkers in esophageal adenocarcinoma.

Volatile aldehydes are enriched in esophageal adenocarcinoma (EAC) patients' breath and could improve early diagnosis, however the mechanisms of their production are unknown. Here, we show that weak aldehyde detoxification characterizes EAC, which is sufficient to cause endogenous aldehyde accumulation in vitro. Two aldehyde groups are significantly enriched in EAC biopsies and adjacent tissue: (i) short-chain alkanals, and (ii) medium-chain alkanals, including decanal. The short-chain alkanals form DNA-adducts, which demonstrates genotoxicity and confirms inadequate detoxification. Metformin, a putative aldehyde scavenger, reduces this toxicity. Tissue and breath concentrations of the medium-chain alkanal decanal are correlated, and increased decanal is linked to reduced ALDH3A2 expression, TP53 deletion, and adverse clinical features. Thus, we present a model for increased exhaled aldehydes based on endogenous accumulation from reduced detoxification, which also causes therapeutically actionable genotoxicity. These results support EAC early diagnosis trials using exhaled aldehyde analysis.

The intelligent knife (iKnife) and its intraoperative diagnostic advantage for the treatment of cervical disease.

Clearance of surgical margins in cervical cancer prevents the need for adjuvant chemoradiation and allows fertility preservation. In this study, we determined the capacity of the rapid evaporative ionization mass spectrometry (REIMS), also known as intelligent knife (iKnife), to discriminate between healthy, preinvasive, and invasive cervical tissue. Cervical tissue samples were collected from women with healthy, human papilloma virus (HPV) ± cervical intraepithelial neoplasia (CIN), or cervical cancer. A handheld diathermy device generated surgical aerosol, which was transferred into a mass spectrometer for subsequent chemical analysis. Combination of principal component and linear discriminant analysis and least absolute shrinkage and selection operator was employed to study the spectral differences between groups. Significance of discriminatory m/z features was tested using univariate statistics and tandem MS performed to elucidate the structure of the significant peaks allowing separation of the two classes. We analyzed 87 samples (normal = 16, HPV ± CIN = 50, cancer = 21 patients). The iKnife discriminated with 100% accuracy normal (100%) vs. HPV ± CIN (100%) vs. cancer (100%) when compared to histology as the gold standard. When comparing normal vs. cancer samples, the accuracy was 100% with a sensitivity of 100% (95% CI 83.9 to 100) and specificity 100% (79.4 to 100). Univariate analysis revealed significant MS peaks in the cancer-to-normal separation belonging to various classes of complex lipids. The iKnife discriminates healthy from premalignant and invasive cervical lesions with high accuracy and can improve oncological outcomes and fertility preservation of women treated surgically for cervical cancer. Larger in vivo research cohorts are required to validate these findings.

Global chemical effects of the microbiome include new bile-acid conjugations.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

Utilisation of Ambient Laser Desorption Ionisation Mass Spectrometry (ALDI-MS) Improves Lipid-Based Microbial Species Level Identification.

The accurate and timely identification of the causative organism of infection is important in ensuring the optimum treatment regimen is prescribed for a patient. Rapid evaporative ionisation mass spectrometry (REIMS), using electrical diathermy for the thermal disruption of a sample, has been shown to provide fast and accurate identification of microorganisms directly from culture. However, this method requires contact to be made between the REIMS probe and microbial biomass; resulting in the necessity to clean or replace the probes between analyses. Here, optimisation and utilisation of ambient laser desorption ionisation (ALDI) for improved speciation accuracy and analytical throughput is shown. Optimisation was completed on 15 isolates of Escherichia coli, showing 5 W in pulsatile mode produced the highest signal-to-noise ratio. These parameters were used in the analysis of 150 clinical isolates from ten microbial species, resulting in a speciation accuracy of 99.4% - higher than all previously reported REIMS modalities. Comparison of spectral data showed high levels of similarity between previously published electrical diathermy REIMS data. ALDI does not require contact to be made with the sample during analysis, meaning analytical throughput can be substantially improved, and further, increases the range of sample types which can be analysed in potential direct-from-sample pathogen detection.

Pragmatic and rapid analysis of carbonyl, oxidation and chlorination nucleoside-adducts in murine tissue by UPLC-ESI-MS/MS.

Nucleoside-adduct analysis by liquid chromatography mass spectrometry is a powerful tool in genotoxicity studies. Efforts to date have quantified an impressive array of DNA damage products, although methodological diversity suggests quantification is still a challenging task. For example, inadequate co-examination of normal nucleosides, cumbersome sample preparation and large DNA requirements were identified to be recurring issues. A six-minute ultra-performance liquid chromatography method is presented which adequately separates seven candidate nucleoside-adducts from the four unmodified nucleosides. The method was sensitive to 1 adduct per 108 normal bases with 20 µg DNA input for most targets. The method was shown to be accurate (81-119% across quintuplets of six tissue types) and precise (relative standard deviation 4-13%). The fast method time facilitated a second quantitation for normal nucleosides at an appropriate dilution, allowing DNA damage concentrations to be contextualised accurately sample-to-sample. From DNA samples, the analytical processing time was < 8 h, and 96 samples can easily be prepared in a day. The method was used to quantify carbonyl, chloro- and oxo- adducts in murine tissue samples.

The surgical intelligent knife distinguishes normal, borderline and malignant gynaecological tissues using rapid evaporative ionisation mass spectrometry (REIMS).

BACKGROUND: Survival from ovarian cancer (OC) is improved with surgery, but surgery can be complex and tumour identification, especially for borderline ovarian tumours (BOT), is challenging. The Rapid Evaporative Ionisation Mass Spectrometric (REIMS) technique reports tissue histology in real-time by analysing aerosolised tissue during electrosurgical dissection. METHODS: Aerosol produced during diathermy of tissues was sampled with the REIMS interface. Histological diagnosis and mass spectra featuring complex lipid species populated a reference database on which principal component, linear discriminant and leave-one-patient-out cross-validation analyses were performed. RESULTS: A total of 198 patients provided 335 tissue samples, yielding 3384 spectra. Cross-validated OC classification vs separate normal tissues was high (97·4% sensitivity, 100% specificity). BOT were readily distinguishable from OC (sensitivity 90.5%, specificity 89.7%). Validation with fresh tissue lead to excellent OC detection (100% accuracy). Histological agreement between iKnife and histopathologist was very good (kappa 0.84, P < 0.001, z = 3.3). Five predominantly phosphatidic acid (PA(36:2)) and phosphatidyl-ethanolamine (PE(34:2)) lipid species were identified as being significantly more abundant in OC compared to normal tissue or BOT (P < 0.001, q < 0.001). CONCLUSIONS: The REIMS iKnife distinguishes gynaecological tissues by analysing mass-spectrometry-derived lipidomes from tissue diathermy aerosols. Rapid intra-operative gynaecological tissue diagnosis may improve surgical care when histology is unknown, leading to personalised operations tailored to the individual.

Effect of Electrode Geometry on the Classification Performance of Rapid Evaporative Ionization Mass Spectrometric (REIMS) Bacterial Identification.

The recently developed automated, high-throughput monopolar REIMS platform is suited for the identification of clinically important microorganisms. Although already comparable to the previously reported bipolar forceps method, optimization of the geometry of monopolar electrodes, at the heart of the system, holds the most scope for further improvements to be made. For this, sharp tip and round shaped electrodes were optimized to maximize species-level classification accuracy. Following optimization of the distance between the sample contact point and tube inlet with the sharp tip electrodes, the overall cross-validation accuracy improved from 77% to 93% in negative and from 33% to 63% in positive ion detection modes, compared with the original 4 mm distance electrode. As an alternative geometry, round tube shaped electrodes were developed. Geometry optimization of these included hole size, number, and position, which were also required to prevent plate pick-up due to vacuum formation. Additional features, namely a metal "X"-shaped insert and a pin in the middle were included to increase the contact surface with a microbial biomass to maximize aerosol production. Following optimization, cross-validation scores showed improvement in classification accuracy from 77% to 93% in negative and from 33% to 91% in positive ion detection modes. Supervised models were also built, and after the leave 20% out cross-validation, the overall classification accuracy was 98.5% in negative and 99% in positive ion detection modes. This suggests that the new generation of monopolar REIMS electrodes could provide substantially improved species level identification accuracies in both polarity detection modes. Graphical abstract.

Determination of polycyclic aromatic hydrocarbons in infant formula using solid state urea clathrate formation with gas chromatography - tandem mass spectrometry.

An analytical method has been developed for the quantitative determination of the 16 EU priority polycyclic aromatic hydrocarbons (PAHs) in infant formula. Prior to analysis infant formula is subjected to sample preparation including the extraction of PAHs from the sample, transesterification of the triglyceride content of the extract to fatty acid methyl esters (FAMEs), and solid state urea clathrate formation to remove the FAMEs. Measurements were carried out with gas chromatography - tandem mass spectrometry. The developed method has been evaluated in terms of trueness, precision (repeatability), limit of detection (LOD), limit of quantification (LOQ), selectivity, sensitivity and linearity. Measurement range of 200-1500ngkg-1 was selected in accordance with the limit values set by the European Commission (835/2011/EC). LOD values were 75ngkg-1 for dibenzo(a,e)pyrene, dibenzo(a,i)pyrene and dibenzo(a,h)pyrene while 50ngkg-1 for the rest of the PAHs (benzo(c)fluorene, benzo(a)anthracene, cyclopenta(cd)pyrene, chrysene, 5-methylchrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(j)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perylene, dibenzo(a,l)pyrene), LOQ was 200ngkg-1 for all 16 PAHs. Trueness ranged between -29.7 and 29.8% while precision values were between 0.5 and 24.7% (RSD, n=3).

Rapid evaporative ionisation mass spectrometry of electrosurgical vapours for the identification of breast pathology: towards an intelligent knife for breast cancer surgery.

BACKGROUND: Re-operation for positive resection margins following breast-conserving surgery occurs frequently (average = 20-25%), is cost-inefficient, and leads to physical and psychological morbidity. Current margin assessment techniques are slow and labour intensive. Rapid evaporative ionisation mass spectrometry (REIMS) rapidly identifies dissected tissues by determination of tissue structural lipid profiles through on-line chemical analysis of electrosurgical aerosol toward real-time margin assessment. METHODS: Electrosurgical aerosol produced from ex-vivo and in-vivo breast samples was aspirated into a mass spectrometer (MS) using a monopolar hand-piece. Tissue identification results obtained by multivariate statistical analysis of MS data were validated by histopathology. Ex-vivo classification models were constructed from a mass spectral database of normal and tumour breast samples. Univariate and tandem MS analysis of significant peaks was conducted to identify biochemical differences between normal and cancerous tissues. An ex-vivo classification model was used in combination with bespoke recognition software, as an intelligent knife (iKnife), to predict the diagnosis for an ex-vivo validation set. Intraoperative REIMS data were acquired during breast surgery and time-synchronized to operative videos. RESULTS: A classification model using histologically validated spectral data acquired from 932 sampling points in normal tissue and 226 in tumour tissue provided 93.4% sensitivity and 94.9% specificity. Tandem MS identified 63 phospholipids and 6 triglyceride species responsible for 24 spectral differences between tissue types. iKnife recognition accuracy with 260 newly acquired fresh and frozen breast tissue specimens (normal n = 161, tumour n = 99) provided sensitivity of 90.9% and specificity of 98.8%. The ex-vivo and intra-operative method produced visually comparable high intensity spectra. iKnife interpretation of intra-operative electrosurgical vapours, including data acquisition and analysis was possible within a mean of 1.80 seconds (SD ±0.40). CONCLUSIONS: The REIMS method has been optimised for real-time iKnife analysis of heterogeneous breast tissues based on subtle changes in lipid metabolism, and the results suggest spectral analysis is both accurate and rapid. Proof-of-concept data demonstrate the iKnife method is capable of online intraoperative data collection and analysis. Further validation studies are required to determine the accuracy of intra-operative REIMS for oncological margin assessment.

In vitro and in silico characterization of fibrous scaffolds comprising alternate colistin sulfate-loaded and heat-treated polyvinyl alcohol nanofibrous sheets.

A multilayer mat for dispensing colistin sulfate through a body surface was prepared by electrospinning. The fabricated system comprised various polyvinyl alcohol fibrous layers prepared with or without the active ingredient. One of the electrospun layers contained water-soluble colistin sulfate and the other was prepared from the same polymer type and composition without the active drug and was finally heat-treated. The heat treatment modified the supramolecular structure and conferred the polymer nanofibre with the rate-controlling function. The microstructure of different layers was tracked by positron annihilation lifetime spectroscopy, and detailed morphological analysis of the fibre mats was performed using a scanning electron microscope. The drug-release profiles of various layer arrangements were studied in relation to their antimicrobial activity. The finite element method was applied to overcome the challenge of diffusion-controlled drug release from multilayer polymer scaffolds. The finite element method was first verified using analytical solutions for a simple arrangement (one drug-loaded swellable fibre and one rate-controlling nonswellable fibre) under perfect sink conditions and in a well-stirred finite volume. The effect of alternate layer arrangements on the drug-release profiles was also investigated to plan for controlled topical drug release from fibrous scaffolds. This design is expected to aid in increasing local effectiveness, thus reducing the systemic loading and the consequent side effects of colistin.

Combined cluster and discriminant analysis: An efficient chemometric approach in diesel fuel characterization.

Combined cluster and discriminant analysis (CCDA) as a chemometric tool in compound specific isotope analysis of diesel fuels was studied. The stable carbon isotope ratios (δ13C) of n-alkanes in diesel fuel can be used to characterize or differentiate diesels originating from different sources. We investigated 25 diesel fuel samples representing 20 different brands. The samples were collected from 25 different service stations in 11 European countries over a 2 year period. The n-alkane fraction of diesel fuels was separated using solid-state urea clathrate formation combined with silica gel fractionation. The stable carbon isotope ratios of C10-C24 n-alkanes were measured with gas chromatography-isotope ratio mass spectrometry (GC-IRMS) using perdeuterated n-alkanes as internal standards. Beside the 25 samples one additional diesel fuel was prepared and measured three times to get totally homogenous samples in order to test the performance of our analytical and statistical routine. Stable isotope ratio data were evaluated with hierarchical cluster analysis (HCA), principal component analysis (PCA) and CCDA. CCDA combines two multivariate data analysis methods hierarchical cluster analysis with linear discriminant analysis (LDA). The main idea behind CCDA is to compare the goodness of preconceived (based on the sample origins) and random groupings. In CCDA all the samples were compared pairwise. The results for the parallel sample preparations showed that the analytical procedure does not have any significant effect on the δ13C values of n-alkanes. The three parallels proved to be totally homogenous with CCDA. HCA and PCA can be useful tools when the examining of the relationship among several samples is in question. However, these two techniques cannot be always decisive on the origin of similar samples. The initial hypothesis that all diesel fuel samples are considered chemically unique was verified by CCDA. The main advantage of CCDA is that it gives an objective index number about the level of similarity among the investigated samples. Thus the application of CCDA supplemented by the traditionally used multivariate methods greatly improves the efficiency of statistical analysis in the CSIA of diesel fuel samples.

Rapid Evaporative Ionisation Mass Spectrometry (REIMS) Provides Accurate Direct from Culture Species Identification within the Genus Candida.

Members of the genus Candida, such as C. albicans and C. parapsilosis, are important human pathogens. Other members of this genus, previously believed to carry minimal disease risk, are increasingly recognised as important human pathogens, particularly because of variations in susceptibilities to widely used anti-fungal agents. Thus, rapid and accurate identification of clinical Candida isolates is fundamental in ensuring timely and effective treatments are delivered. Rapid Evaporative Ionisation Mass Spectrometry (REIMS) has previously been shown to provide a high-throughput platform for the rapid and accurate identification of bacterial and fungal isolates. In comparison to commercially available matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF), REIMS based methods require no preparative steps nor time-consuming cell extractions. Here, we report on the ability of REIMS-based analysis to rapidly and accurately identify 153 clinical Candida isolates to species level. Both handheld bipolar REIMS and high-throughput REIMS platforms showed high levels of species classification accuracy, with 96% and 100% of isolates classified correctly to species level respectively. In addition, significantly different (FDR corrected P value < 0.05) lipids within the 600 to 1000 m/z mass range were identified, which could act as species-specific biomarkers in complex microbial communities.

Determination of particulate phase polycyclic aromatic hydrocarbons and their nitrated and oxygenated derivatives using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry.

An analytical method has been developed for the quantitative determination of polycyclic aromatic hydrocarbons (PAHs) and their nitrated and oxygenated derivatives (nitro- and oxy-PAHs respectively) in particulate matter (PM) samples. The sample preparation procedure included only a simple and quick sonication-assisted extraction step, clean-up based on addition of water and centrifugation as well as pre-concentration under N2 stream. The determination of 16 PAHs and 4 oxy-PAHs was carried out by gas chromatography-mass spectrometry, while liquid chromatography-tandem mass spectrometry was used in the case of the 11 investigated nitro-PAHs. The optimized method was fully evaluated in terms of trueness, precision (repeatability), limit of detection (LOD), limit of quantification (LOQ), sensitivity and linearity. The LOQ values ranged at pgm-3 level for the investigated PAHs (42pgm-3), oxy-PAHs (either 42 or 83pgm-3) and nitro-PAHs (either 83 or 167pgm-3) as well. The developed method was applied for the quantitative determination of PAHs, nitro- and oxy-PAHs in urban PM2.5 (particles with aerodynamic diameter smaller than 2.5µm) samples (n=36) collected in Budapest, Hungary. Almost 100% of the PM2.5 samples contained the investigated PAHs and oxy-PAHs in detectable and quantifiable amounts; however, the concentration of the nitro-PAHs was generally lower than the corresponding LOD/LOQ values. According to our results, during the 3-year long sampling campaign the concentration of benzo(a)pyrene never exceeded the limit value (1ngm-3) set by the European Commission.

Automated High-Throughput Identification and Characterization of Clinically Important Bacteria and Fungi using Rapid Evaporative Ionization Mass Spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) has been shown to quickly and accurately speciate microorganisms based upon their species-specific lipid profile. Previous work by members of this group showed that the use of a hand-held bipolar probe allowed REIMS to analyze microbial cultures directly from culture plates without any prior preparation. However, this method of analysis would likely be unsuitable for a high-throughput clinical microbiology laboratory. Here, we report the creation of a customized platform that enables automated, high-throughput REIMS analysis that requires minimal user input and operation and is suitable for use in clinical microbiology laboratories. The ability of this high-throughput platform to speciate clinically important microorganisms was tested through the analysis of 375 different clinical isolates collected from distinct patient samples from 25 microbial species. After optimization of our data analysis approach, we achieved substantially similar results between the two REIMS approaches. For hand-held bipolar probe REIMS, a speciation accuracy of 96.3% was achieved, whereas for high-throughput REIMS, an accuracy of 93.9% was achieved. Thus, high-throughput REIMS offers an alternative mass spectrometry based method for the rapid and accurate identification of clinically important microorganisms in clinical laboratories without any preanalysis preparative steps.

Shotgun Lipidomic Profiling of the NCI60 Cell Line Panel Using Rapid Evaporative Ionization Mass Spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) was used for the rapid mass spectrometric profiling of cancer cell lines. Spectral reproducibility was assessed for three different cell lines, and the extent of interclass differences and intraclass variance was found to allow the identification of these cell lines based on the REIMS data. Subsequently, the NCI60 cell line panel was subjected to REIMS analysis, and the resulting data set was investigated for its distinction of individual cell lines and different tissue types of origin. Information content of REIMS spectral profiles of cell lines were found to be similar to those obtained from mammalian tissues although pronounced differences in relative lipid intensity were observed. Ultimately, REIMS was shown to detect changes in lipid content of cell lines due to mycoplasma infection. The data show that REIMS is an attractive means to study cell lines involving minimal sample preparation and analysis times in the range of seconds.