Professor Melanie Bailey

Immunoglobulin gene heterogeneity reflects the diversity and focus of the humoral immune response towards different infections, enabling inference of B cell development processes. Detailed compositional and lineage analysis of long read IGH repertoire sequencing, combining examples of pandemic, epidemic and endemic viral infections with control and vaccination samples, demonstrates general responses including increased use of IGHV4-39 in both Zaire Ebolavirus (EBOV) and COVID-19 patient cohorts. We also show unique characteristics absent in Respiratory Syncytial Virus or yellow fever vaccine samples: EBOV survivors show unprecedented high levels of class switching events while COVID-19 repertoires from acute disease appear underdeveloped. Despite the high levels of clonal expansion in COVID-19 IgG1 repertoires there is a striking lack of evidence of germinal centre mutation and selection. Given the differences in COVID-19 morbidity and mortality with age, it is also pertinent that we find significant differences in repertoire characteristics between young and old patients. Our data supports the hypothesis that a primary viral challenge can result in a strong but immature humoral response where failures in selection of the repertoire risk off-target effects.

Standard drug testing is regularly carried out using urine, blood or oral fluid. However, fingerprints present a good alternative, as the sample collection is non-invasive, rapid and safe. Herein, we describe the application of two different testing methods for the detection of cocaine in fingerprint samples.

We report the first demonstration of a microfluidics-based approach to measure lipids in single living cells using widely available liquid chromatography mass spectrometry (LC-MS) instrumentation. The method enables the rapid sorting of live cells into liquid chambers formed on standard Petri dishes and their subsequent dispensing into vials for analysis using LC-MS. This approach facilitates automated sampling, data acquisition, and analysis and carries the additional advantage of chromatographic separation, aimed at reducing matrix effects present in shotgun lipidomics approaches. We demonstrate that our method detects comparable numbers of features at around 200 lipids in populations of single cells versus established live single-cell capillary sampling methods and with greater throughput, albeit with the loss of spatial resolution. We also show the importance of optimization steps in addressing challenges from lipid contamination, especially in blanks, and demonstrate a 75% increase in the number of lipids identified. This work opens up a novel, accessible, and high-throughput way to obtain single-cell lipid profiles and also serves as an important validation of single-cell lipidomics through the use of different sampling methods.

Mass spectrometry is a method of identifying molecules within a sample, based on a characteristic mass to charge ratio. Over the last decades, it has become possible to use mass spectrometry to obtain high resolution molecular images of surfaces. In this chapter, we will show how mass spectrometry techniques can be used to obtain high quality images of fingerprints, determine their placement compared with other traces (for example overlapping fingerprints or inks) and determine their chemical make-up for offender profiling purposes.

Secondary ion mass spectrometry (SIMS) is a technique that can be used to provide high resolution images of elements and molecules in 3D, and it has been widely used for material characterisation, particularly of inorganic materials. Recent developments in SIMS instrumentation are now enabling the analysis of organic materials, and there is, therefore, considerable scope for exploitation in forensic science. In this chapter, we describe the principles of operation of SIMS and outline the progress that has been made towards its application in forensic science.

The COVID-19 pandemic has led to an unprecedented demand for testing - for diagnosis and prognosis - as well as for investigation into the impact of the disease on the host metabolism. Sebum sampling has the potential to support both needs by looking at what the virus does to us, rather than looking for the virus itself. In this pilot study, sebum samples were collected from 67 hospitalised patients (30 COVID-19 positive and 37 COVID-19 negative) by gauze swab. Lipidomics analysis was carried out using liquid chromatography mass spectrometry, identifying 998 reproducible features. Univariate and multivariate statistical analyses were applied to the resulting feature set. Lipid levels were depressed in COVID-19 positive participants, indicative of dyslipidemia; p-values of 0·022 and 0·015 were obtained for triglycerides and ceramides respectively, with effect sizes of 0·44 and 0·57. Partial Least Squares-Discriminant Analysis showed separation of COVID-19 positive and negative participants with sensitivity of 57% and specificity of 68%, improving to 79% and 83% respectively when controlled for confounding comorbidities. COVID-19 dysregulates many areas of metabolism; in this work we show that the skin lipidome can be added to the list. Given that samples can be provided quickly and painlessly, we conclude that sebum is worthy of future consideration for clinical sampling. The authors acknowledge funding from the EPSRC Impact Acceleration Account for sample collection and processing, as well as EPSRC Fellowship Funding EP/R031118/1, the University of Surrey and BBSRC BB/T002212/1. Mass Spectrometry was funded under EP/P001440/1.

A fingerprint offers a convenient, noninvasive sampling matrix for monitoring therapeutic drug use. However, a barrier to widespread adoption of fingerprint sampling is the fact that the sample volume is uncontrolled. Fingerprint samples (n = 140) were collected from patients receiving the antibiotic isoniazid as part of their treatment, as well as from a drug-naive control group (n = 50). The fingerprint samples were analyzed for isoniazid (INH) and acetylisoniazid (AcINH), using liquid chromatography high-resolution mass spectrometry. The data set was analyzed retrospectively for metabolites known to be present in eccrine sweat. INH or AcINH was detected in 89% of the fingerprints collected from patients and in 0% of the fingerprints collected from the control group. Metabolites lysine, ornithine, pyroglutamic acid, and taurine were concurrently detected alongside INH/AcINH and were used to determine whether the fingerprint sample was sufficient for testing. Given a sufficient sample volume, the fingerprint test for INH use has sensitivity, specificity, and accuracy of 100%. Normalization to taurine was found to reduce intradonor variability. Fingerprints are a novel and noninvasive approach to monitor INH therapy. Metabolites can be used as internal markers to demonstrate a sufficient sample volume for testing and reduce intradonor variability.

Here we show a new and significant application area for mass spectrometry imaging. The potential for fingerprints to reveal drug use has been widely reported, with potential applications in forensics and workplace drug testing. However, one unsolved issue is the inability to distinguish between drug administration and contamination by contact. Previous work using bulk mass spectrometry analysis has shown that this distinction can only be definitively made if the hands are washed prior to sample collection. Here, we illustrate how three mass spectrometry imaging approaches, desorption electrospray ionisation (DESI), matrix assisted laser desorption ionisation (MALDI) and time of flight secondary ion mass spectrometry (ToF-SIMS) can be used to visualise fingerprints at different pixel sizes, ranging from the whole fingerprint down to the pore structure. We show how each of these magnification scales can be used to distinguish between cocaine use and contact. We also demonstrate the first application of water cluster SIMS to a fingerprint sample, which was the sole method tested here that was capable of detecting excreted drug metabolites in fingerprints, while providing spatial resolution sufficient to resolve individual pore structure. We show that after administration of cocaine, lipids and salts in the fingerprint ridges spatially correlate with the cocaine metabolite, benzoylecgonine. In contrast after contact, we have observed that cocaine and its metabolite show a poor spatial correlation with the flow of the ridges.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been shown to enhance fingermark recovery compared to standard processes used by police forces, but there is no data to show how generally applicable the improvement is. Additionally, ToF-SIMS can be run in either positive or negative ion mode (or both), and there is no data on which mode of operation is most effective at revealing fingerprints. This study aims to fill these gaps by using ToF-SIMS to image fingerprints deposited on two common exhibit-type surfaces (polyethylene and stainless steel) using 10 donors and ageing fingerprints in either ambient, rainwater, or underground for 1 and 5 months. In all, 120 fingerprints were imaged using ToF-SIMS, and each was run in positive and negative modes. A fingerprint expert compared the fingerprint ridge detail produced by the standard process to the ToF-SIMS images. In over 50% of the samples, ToF-SIMS was shown to improve fingerprint ridge detail visualised by the respective standard process for all surfaces tested. In over 90% of the samples, the ridge detail produced by ToF-SIMS was equivalent to standard development across all different ageing and exposure conditions. The data shows that there is a benefit to running the ToF-SIMS in both positive and negative modes, even if no ridge detail was seen in one mode.

Description This dataset of participant, field blank and quality control liquid-chromatography-mass spectrometry .raw files supports the following article: Changes to the sebum lipidome upon COVID-19 infection observed via rapid sampling from the skin - EClinicalMedicine (thelancet.com) Background The COVID-19 pandemic has led to an unprecedented demand for testing - for diagnosis and prognosis - as well as for investigation into the impact of the disease on the host metabolism. Sebum sampling has the potential to support both needs by looking at what the virus does to us, rather than looking for the virus itself. Methods and attached dataset description In this pilot study, sebum samples were collected from 67 hospitalised patients (30 COVID-19 positive and 37 COVID-19 negative) by gauze swab. Lipidomics analysis was carried out using liquid chromatography mass spectrometry, identifying 998 reproducible features. Univariate and multivariate statistical analyses were applied to the resulting feature set. The dataset uploaded here represents .raw liquid chromatography-mass spectrometry files for participants (triplicate injections), field blanks and pooled quality control standards, as well as the output peak:area matrix. Findings Lipid levels were depressed in COVID-19 positive participants, indicative of dyslipidemia; p-values of 0·022 and 0·015 were obtained for triglycerides and ceramides respectively, with effect sizes of 0·44 and 0·57. Partial Least Squares-Discriminant Analysis showed separation of COVID-19 positive and negative participants with sensitivity of 57% and specificity of 68%, improving to 79% and 83% respectively when controlled for confounding comorbidities. Interpretation COVID-19 dysregulates many areas of metabolism; in this work we show that the skin lipidome can be added to the list. Given that samples can be provided quickly and painlessly, we conclude that sebum is worthy of future consideration for clinical sampling.

Treatments for COVID-19 infections have improved dramatically since the beginning of the pandemic, and glucocorticoids have been a key tool in improving mortality rates. The UK’s National Institute for Health and Care Excellence guidance is for treatment to be targeted only at those requiring oxygen supplementation, however, and the interactions between glucocorticoids and COVID-19 are not completely understood. In this work, a multi-omic analysis of 98 inpatient-recruited participants was performed by quantitative metabolomics (using targeted liquid chromatography-mass spectrometry) and data-independent acquisition proteomics. Both ‘omics datasets were analysed for statistically significant features and pathways differentiating participants whose treatment regimens did or did not include glucocorticoids. Metabolomic differences in glucocorticoid-treated patients included the modulation of cortisol and bile acid concentrations in serum, but no alleviation of serum dyslipidemia or increased amino acid concentrations (including tyrosine and arginine) in the glucocorticoid-treated cohort relative to the untreated cohort. Proteomic pathway analysis indicated neutrophil and platelet degranulation as influenced by glucocorticoid treatment. These results are in keeping with the key role of platelet-associated pathways and neutrophils in COVID-19 pathogenesis and provide opportunity for further understanding of glucocorticoid action. The findings also, however, highlight that glucocorticoids are not fully effective across the wide range of ‘omics dysregulation caused by COVID-19 infections.

Abstract The majority of metabolomics studies to date have utilised blood serum or plasma, biofluids that do not necessarily address the full range of patient pathologies. Here, correlations between serum metabolites, salivary metabolites and sebum lipids are studied for the first time. 83 COVID-19 positive and negative hospitalised participants provided blood serum alongside saliva and sebum samples for analysis by liquid chromatography mass spectrometry. Widespread alterations to serum-sebum lipid relationships were observed in COVID-19 positive participants versus negative controls. There was also a marked correlation between sebum lipids and the immunostimulatory hormone dehydroepiandrosterone sulphate in the COVID-19 positive cohort. The biofluids analysed herein were also compared in terms of their ability to differentiate COVID-19 positive participants from controls; serum performed best by multivariate analysis (sensitivity and specificity of 0.97), with the dominant changes in triglyceride and bile acid levels, concordant with other studies identifying dyslipidemia as a hallmark of COVID-19 infection. Sebum performed well (sensitivity 0.92; specificity 0.84), with saliva performing worst (sensitivity 0.78; specificity 0.83). These findings show that alterations to skin lipid profiles coincide with dyslipidaemia in serum. The work also signposts the potential for integrated biofluid analyses to provide insight into the whole-body atlas of pathophysiological conditions.

The effect of COVID-19 infection on the human metabolome has been widely reported, but to date all such studies have focused on a single wave of infection. COVID-19 has generated numerous waves of disease with different clinical presentations, and therefore it is pertinent to explore whether metabolic disturbance changes accordingly, to gain a better understanding of its impact on host metabolism and enable better treatments. This work used a targeted metabolomics platform (Biocrates Life Sciences) to analyze the serum of 164 hospitalized patients, 123 with confirmed positive COVID-19 RT-PCR tests and 41 providing negative tests, across two waves of infection. Seven COVID-19-positive patients also provided longitudinal samples 2–7 months after infection. Changes to metabolites and lipids between positive and negative patients were found to be dependent on collection wave. A machine learning model identified six metabolites that were robust in diagnosing positive patients across both waves of infection: TG (22:1_32:5), TG (18:0_36:3), glutamic acid (Glu), glycolithocholic acid (GLCA), aspartic acid (Asp) and methionine sulfoxide (Met-SO), with an accuracy of 91%. Although some metabolites (TG (18:0_36:3) and Asp) returned to normal after infection, glutamic acid was still dysregulated in the longitudinal samples. This work demonstrates, for the first time, that metabolic dysregulation has partially changed over the course of the pandemic, reflecting changes in variants, clinical presentation and treatment regimes. It also shows that some metabolic changes are robust across waves, and these can differentiate COVID-19-positive individuals from controls in a hospital setting. This research also supports the hypothesis that some metabolic pathways are disrupted several months after COVID-19 infection.

In this work, we demonstrate the development and first application of nanocapillary sampling followed by analytical flow liquid chromatography–mass spectrometry for single-cell lipidomics. Around 260 lipids were tentatively identified in a single cell, demonstrating remarkable sensitivity. Human pancreatic ductal adenocarcinoma cells (PANC-1) treated with the chemotherapeutic drug gemcitabine can be distinguished from controls solely on the basis of their single-cell lipid profiles. Notably, the relative abundance of LPC(0:0/16:0) was significantly affected in gemcitabine-treated cells, in agreement with previous work in bulk. This work serves as a proof of concept that live cells can be sampled selectively and then characterized using automated and widely available analytical workflows, providing biologically relevant outputs.

Single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) offers an attractive option for rapidly measuring trace metal heterogeneity at the single cell level. Chemical fixation has been previously applied to mammalian cells prior to sample introduction so that they can be resuspended in a solution suitable for SC-ICP-MS. However, the effect of fixation on the elemental composition of suspended cells is unknown, and robust methodologies are urgently needed so that the community can measure the effects of intracellular pathogens on elemental composition of their host cells. We demonstrate that different fixatives impact measured cell elemental composition. We have compared suspensions treated using different fixatives (methanol 60-100% in H2O and 4% paraformaldehyde in phosphate-buffered saline solution), and the number of distinguishable single cell events, keeping a constant particle number concentration. Significantly more single cell events (n = 3, P ≤ 0.05) were observed for Ca and Mg when cells were fixed in 4% paraformaldehyde than for the methanol-based fixatives, confirming the hypothesis that methanol fixatives cause leaching of these elements from the cells. The impact of fixation on Mn and Zn was less pronounced. Microbial and viral infection of eukaryotic cells can have profound effects on their elemental composition, but chemical fixation is necessary to render infected cells safe before analysis. We have successfully applied our methodology to a macrophage model of tuberculosis demonstrating utility in understanding metal homeostasis during microbial infection of mammalian cells.Single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) offers an attractive option for rapidly measuring trace metal heterogeneity at the single cell level. Chemical fixation has been previously applied to mammalian cells prior to sample introduction so that they can be resuspended in a solution suitable for SC-ICP-MS. However, the effect of fixation on the elemental composition of suspended cells is unknown, and robust methodologies are urgently needed so that the community can measure the effects of intracellular pathogens on elemental composition of their host cells. We demonstrate that different fixatives impact measured cell elemental composition. We have compared suspensions treated using different fixatives (methanol 60-100% in H2O and 4% paraformaldehyde in phosphate-buffered saline solution), and the number of distinguishable single cell events, keeping a constant particle number concentration. Significantly more single cell events (n = 3, P ≤ 0.05) were observed for Ca and Mg when cells were fixed in 4% paraformaldehyde than for the methanol-based fixatives, confirming the hypothesis that methanol fixatives cause leaching of these elements from the cells. The impact of fixation on Mn and Zn was less pronounced. Microbial and viral infection of eukaryotic cells can have profound effects on their elemental composition, but chemical fixation is necessary to render infected cells safe before analysis. We have successfully applied our methodology to a macrophage model of tuberculosis demonstrating utility in understanding metal homeostasis during microbial infection of mammalian cells.

The colocation of elemental species with host biomolecules such as lipids and metabolites may shed new light on the dysregulation of metabolic pathways and how these affect disease pathogeneses. Alkali metals have been the subject of extensive research, are implicated in various neurodegenerative and infectious diseases and are known to disrupt lipid metabolism. Desorption electrospray ionisation (DESI) is a widely used approach for molecular imaging, but previous work has shown that DESI delocalises ions such as potassium (K) and chlorine (Cl), precluding the subsequent elemental analysis of the same section of tissue. The solvent typically used for the DESI electrospray is a combination of methanol and water. Here we show that a novel solvent system, (50:50 (%v/v) MeOH:EtOH) does not delocalise elemental species and thus enables elemental mapping to be performed on the same tissue section post-DESI. Benchmarking the MeOH:EtOH electrospray solvent against the widely used MeOH:H2O electrospray solvent revealed that the MeOH:EtOH solvent yielded increased signal-to-noise ratios for selected lipids. The developed multimodal imaging workflow was applied to a lung tissue section containing a tuberculosis granuloma, showcasing its applicability to elementally rich samples displaying defined structural information.

Elemental imaging is widely used for imaging cells and tissues but rarely in combination with organic mass spectrometry, which can be used to profile lipids and measure drug concentrations. Here, we demonstrate how elemental imaging and a new method for spatially resolved lipidomics (DAPNe-LC-MS, based on capillary microsampling and liquid chromatography mass spectrometry) can be used in combination to probe the relationship between metals, drugs, and lipids in discrete areas of tissues. This new method for spatial lipidomics, reported here for the first time, has been applied to rabbit lung tissues containing a lesion (caseous granuloma) caused by tuberculosis infection. We demonstrate how elemental imaging with spatially resolved lipidomics can be used to probe the association between ion accumulation and lipid profiles and verify local drug distribution.

The radical ring-opening polymerization (RROP) of thionolactones provides access to thioester backbone-functional copolymers but has, to date, only been demonstrated on acrylic copolymers. Herein, the thionolactone dibenzo[c,e]oxepane-5-thione (DOT) was subjected to azobisisobutyronitrile (A1BN)-initiated free-radical homopolymerization, which produced a thioester-functional homopolymer with a glass-transition temperature of 95 degrees C and the ability to degrade exclusively into predetermined small molecules. However, the homopolymerization was impractically slow and precluded the introduction of functionality. Conversely, the reversible addition-fragmentation chain-transfer (RAFT)-mediated copolymerization of DOT with N-methylmaleimide (MeMI), N-phenylmaleimide (PhMI), and N-2,3,4,5,6-pentafluorophenylmaleimide (PFPMI) rapidly produced well-defined copolymers with the tendency to form alternating sequences increasing in the order MeMI

Metabolic flux is the final output of cellular regulation and has been extensively studied for carbon but much less is known about nitrogen, which is another important building block for living organisms. For the tuberculosis pathogen, this is particularly important in informing the development of effective drugs targeting the pathogen's metabolism. Here we performed (CN)-C-13-N-15 dual isotopic labeling of Mycobacterium bovis BCG steady state cultures, quantified intracellular carbon and nitrogen fluxes and inferred reaction bidirectionalities. This was achieved by model scope extension and refinement, implemented in a multi-atom transition model, within the statistical framework of Bayesian model averaging (BMA). Using BMA-based (CN)-C-13-N-15-metabolic flux analysis, we jointly resolve carbon and nitrogen fluxes quantitatively. We provide the first nitrogen flux distributions for amino acid and nucleotide biosynthesis in mycobacteria and establish glutamate as the central node for nitrogen metabolism. We improved resolution of the notoriously elusive anaplerotic node in central carbon metabolism and revealed possible operation modes. Our study provides a powerful and statistically rigorous platform to simultaneously infer carbon and nitrogen metabolism in any biological system.

Identification of features with high levels of confidence in liquid chromatography-mass spectrometry (LC-MS) lipidomics research is an essential part of biomarker discovery, but existing software platforms can give inconsistent results, even from identical spectral data. This poses a clear challenge for reproducibility in biomarker identification. In this work, we illustrate the reproducibility gap for two open-access lipidomics platforms, MS DIAL and Lipostar, finding just 14.0% identification agreement when analyzing identical LC-MS spectra using default settings. Whilst the software platforms performed more consistently using fragmentation data, agreement was still only 36.1% for MS2 spectra. This highlights the critical importance of validation across positive and negative LC-MS modes, as well as the manual curation of spectra and lipidomics software outputs, in order to reduce identification errors caused by closely related lipids and co-elution issues. This curation process can be supplemented by data-driven outlier detection in assessing spectral outputs, which is demonstrated here using a novel machine learning approach based on support vector machine regression combined with leave-one-out cross-validation. These steps are essential to reduce the frequency of false positive identifications and close the reproducibility gap, including between software platforms, which, for downstream users such as bioinformaticians and clinicians, can be an underappreciated source of biomarker identification errors.

•Ensuring adherence and sufficient exposure to anti-tuberculosis medications is essential for successful treatment.•Finger sweat offers a non-invasive alternative to monitor patients.•Isoniazid can be detected in finger sweat for 1–6 h following controlled administration of the drug.•Normalisation to creatinine can be used to account for differences in sample volume and was shown to increase correlation to serum. Insufficient exposure and poor compliance with anti-tuberculosis (TB) medications are risk factors for treatment failure and the development of drug resistance. Measurement of drugs in biological samples, such as blood and saliva, can be used to assess adherence and make dose adjustments by therapeutic drug monitoring (TDM). Finger sweat testing is a convenient and non-invasive method to monitor patients. To assess the feasibility of finger sweat testing for medication adherence and as a semi-quantitative tool for TDM analysis. Ten patients provided finger sweat, blood and saliva samples following a controlled dose of isoniazid. Samples were analysed by liquid chromatography-mass spectrometry. Isoniazid can be detected in finger sweat 1–6 h following administration at typically prescribed dosages. The normalisation of isoniazid to creatinine increases the correlation between finger sweat and serum isoniazid concentration and provides a means to account for inconsistent sample volumes. We describe the time-course measurement of isoniazid (or drug-to-creatinine ratio) in finger sweat compared to the pharmacokinetic profile in blood for the first time. This technique, adaptable for other drugs, could reduce the burden on clinics and improve patient experience. [Display omitted]

RATIONALE: Paper spray offers a rapid screening test without the need for sample preparation. The incomplete extraction of paper spray allows for further testing using more robust, selective and sensitive techniques such as liquid chromatography mass spectrometry (LC-MS). Here we develop a two-step process of paper spray followed by LC-MS to (1) rapidly screen a large number of samples and (2) confirm any disputed results. This demonstrates the applicability for testing medication adherence from a fingerprint. METHODS: Following paper spray analysis, drugs of abuse samples were analysed using LC-MS. All analyses were completed using a Q Exactive™ Plus Orbitrap™ mass spectrometer. This two-step procedure was applied to fingerprints collected from patients on a maintained dose of the antipsychotic drug quetiapine. RESULTS: The extraction efficiency of paper spray for two drugs of abuse and metabolites was found to be between 15-35% (analyte dependent). For short acquisition times, the extraction efficiency was found to vary between replicates by less than 30%, enabling subsequent analysis by LC-MS. This two-step process was then applied to fingerprints collected from two patients taking the antipsychotic drug quetiapine, which demonstrates how a negative screening result from paper spray can be resolved using LC-MS. CONCLUSIONS: We have shown for the first time the sequential analysis of the same sample using paper spray and LC-MS, as well as the detection of an antipsychotic drug from a fingerprint. We propose that this workflow may also be applied to any type of sample compatible with paper spray, and will be especially convenient where only one sample is available for analysis.

Direct analyte probed nanoextraction (DAPNe) is a technique that allows extraction of drug and endogenous compounds from a discrete location on a tissue sample using a nano capillary filled with solvent. Samples can be extracted from a spot diameters as low as 6 µm. Studies previously undertaken by our group have shown that the technique can provide good precision (5%) for analysing drug molecules in 150 µm diameter areas of homogenised tissue, provided an internal standard is sprayed on to the tissue prior to analysis. However, without an isotopically labelled standard, the repeatability is poor, even after normalisation to and the spot area or matrix compounds. By application to tissue homogenates spiked with drug compounds, we can demonstrate that it is possible to significantly improve the repeatability of the technique by incorporating a liquid chromatography separation step. Liquid chromatography is a technique for separating compounds prior to mass spectrometry (LC-MS) which enables separation of isomeric compounds that cannot be discriminated using mass spectrometry alone, as well as reducing matrix interferences. Conventionally, LC-MS is carried out on bulk or homogenised samples, which means analysis is essentially an average of the sample and does not take into account discrete areas. This work opens a new opportunity for spatially resolved liquid chromatography mass spectrometry with precision better than 20%.

ollection of finger sweat is explored here as a rapid and convenient way of monitoring patient adherence to antipsychotic drugs. Finger sweat samples (n = 426) collected from patients receiving treatment with clozapine, quetiapine and olanzapine were analysed by liquid chromatography mass spectrometry, including a subgroup of patients with paired plasma samples. Finger sweat samples were also analysed from a negative control group and patients who had handled antipsychotic medication only. The finger sweat test (based on the detection of parent drug in one donated sample) was 100% effective in monitoring adherence within commonly prescribed dosing ranges. In comparison to participants who handled the medication only, the test could distinguish between contact and administration through monitoring of the drug metabolite, or the level of parent drug. Additionally, in a subgroup of patients prescribed clozapine, a statistically significant correlation was observed between the mass of parent drug in finger sweat and plasma concentration. The finger sweat technology shows promise as a dignified, noninvasive method to monitor treatment adherence in patients taking antipsychotics.

The substances deposited from the fingertip onto a surface during contact between them represent a highly complex range of chemicals that can be exploited in a variety of ways in a forensic investigation. An overview is given of the multitude of chemicals that have been detected in fingermarks, including those occurring in endogenous sweat, metabolites of ingested substances, and exogenous substances picked up on the fingertip. Changes in chemistry that may occur between deposition of the fingermark and its subsequent forensic analysis are discussed, with particular reference to the ways in which these changes have been considered as a means of dating fingermarks. The ways in which fingermark enhancement reagents utilise the different chemicals present to reveal ridge is reviewed, together with how different classes of chemical can be sequentially targeted to optimise the number of fingermarks recovered. A field of increasing interest is the use of advanced analytical techniques incorporating mass spectrometry and imaging capability to simultaneously obtain additional contextual information about the donor of the mark whilst visualising the fingermark ridge pattern. Examples are given of how such information can be applied in forensic investigations. It is concluded that an extensive ‘tool kit’ of fingermark enhancement processes is already available to utilise the different chemicals present, and the advances that can be made in this field using conventional approaches are limited. There is, instead, significant potential to utilise analytical techniques to forensically exploit the chemical information within fingermarks but there are also significant barriers to their implementation in this way.

This work describes the development of a new approach to measure drug levels and lipid fingerprints in single living mammalian cells. Nanocapillary sampling is an approach that enables the selection and isolation of single living cells under microscope observation. Here, live single cell nanocapillary sampling is coupled to liquid chromatography for the first time. This allows molecular species to be separated prior to ionisation and improves measurement precision of drug analytes. The efficiency of transferring analytes from the sampling capillary into a vial was optimised in this work. The analysis was carried out using standard flow liquid chromatography coupled to widely available mass spectrometry instrumentation, highlighting opportunities for widespread adoption. The method was applied to 30 living cells, revealing cell-to-cell heterogeneity in the uptake of different antibiotics. Using this system, we detected 14-158 lipid features per single cell, revealing the association between bedaquiline uptake and lipid fingerprints.

There are many technical challenges in the fabrication of devices from novel materials. The characterization of these materials is critical in the development of efficient photovoltaic systems. We show how the application of recent advances in MeV IBA, providing the self-consistent treatment of RBS (Rutherford backscattering) and PIXE (particle induced X-ray emission) spectra, makes a new set of powerful complementary depth profiling techniques available for all thin film technologies, including the chalcopyrite compound semiconductors. We will give and discuss a detailed analysis of a CuInAl metallic precursor film, showing how similar methods are also applicable to other films of interest.

Background The COVID-19 pandemic is likely to represent an ongoing global health issue given the potential for new variants, vaccine escape and the low likelihood of eliminating all reservoirs of the disease. Whilst diagnostic testing has progressed at a fast pace, the metabolic drivers of outcomes-and whether markers can be found in different biofluids-are not well understood. Recent research has shown that serum metabolomics has potential for prognosis of disease progression. In a hospital setting, collection of saliva samples is more convenient for both staff and patients, and therefore offers an alternative sampling matrix to serum. Methods Saliva samples were collected from hospitalised patients with clinical suspicion of COVID-19, alongside clinical metadata. COVID-19 diagnosis was confirmed using RT-PCR testing, and COVID-19 severity was classified using clinical descriptors (respiratory rate, peripheral oxygen saturation score and C-reactive protein levels). Metabolites were extracted and analysed using high resolution liquid chromatography-mass spectrometry, and the resulting peak area matrix was analysed using multivariate techniques. Results Positive percent agreement of 1.00 between a partial least squares-discriminant analysis metabolomics model employing a panel of 6 features (5 of which were amino acids, one that could be identified by formula only) and the clinical diagnosis of COVID-19 severity was achieved. The negative percent agreement with the clinical severity diagnosis was also 1.00, leading to an area under receiver operating characteristics curve of 1.00 for the panel of features identified. Conclusions In this exploratory work, we found that saliva metabolomics and in particular amino acids can be capable of separating high severity COVID-19 patients from low severity COVID-19 patients. This expands the atlas of COVID-19 metabolic dysregulation and could in future offer the basis of a quick and non-invasive means of sampling patients, intended to supplement existing clinical tests, with the goal of offering timely treatment to patients with potentially poor outcomes.

Inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify the total amount of trace elements in retina from adult male Sprague-Dawley rats (n = 6). Concentration of trace elements within individual retinal areas in frozen sections of the fellow eye was established with the use of two methodologies: (1) particle-induced X-ray emission (PIXE) in combination with 3D depth profiling with Rutherford backscattering spectrometry (RBS) and (2) synchrotron X-ray fluorescence (SXRF) microscopy. The most abundant metal in the retina was zinc, followed by iron and copper. Nickel, manganese, chromium, cobalt, selenium and cadmium were present in very small amounts. The PIXE and SXRF analysis yielded a non-homogenous pattern distribution of metals in the retina. Relatively high levels of zinc were found in the inner part of the photoreceptor inner segments (RIS)/outer limiting membrane (OLM), inner nuclear layer and plexiform layers. Iron was found to accumulate in the retinal pigment epithelium/choroid layer and RIS/OLM. Copper in turn, was localised primarily in the RIS/OLM and plexiform layers. The trace elements iron, copper, and zinc exist in different amounts and locations in the rat retina.

The processes routinely used by police forces to visualise fingermarks in casework may not provide sufficient ridge pattern quality to aid an investigation. Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS) has been proposed as a technique to enhance fingermark recovery. The technique is currently designated a Category C process in the Fingermark Visualisation Manual (FVM) as it shows potential for effective fingermark visualisation but has not yet been fully evaluated. Here the sensitivity of ToF-SIMS on three common exhibit-type surfaces - paper, polyethylene and stainless-steel was compared to standard processes. An adapted Home Office grading scale was used to evaluate the efficacy of fingerprint development by ToF-SIMS and to provide a framework for comparison with standard processes. ToF-SIMS was shown to visualise more fingerprints than the respective standard process, for all surfaces tested. In addition, ToF-SIMS was applied after the standard processes and successfully enhanced the fingerprint detail, even when the standard process failed to visualise ridge detail. This demonstrates the benefit for incorporating it into current operational fingermark development workflows. Multivariate analysis (MVA), using simsMVA, was additionally explored as a method to simplify the data analysis and image generation process.

The suite of techniques which are available with the small accelerators used for MeV ion beam analysis (IBA) range from broad beams, microbeams or external beams using the various particle and photon spectrometries (including RBS, EBS, ERD, STIM, PIXE, PIGE, NRA and their variants), to tomography and secondary particle spectrometries like MeV-SIMS. These can potentially yield almost everything there is to know about the 3-D elemental composition of types of samples that have always been hard to analyse, given the sensitivity and the spacial resolution of the techniques used. Molecular and chemical information is available in principle with, respectively, MeV-SIMS and high resolution PIXE. However, these techniques separately give only partial information – the secret of “Total IBA” is to find synergies between techniques used simultaneously which efficiently give extra information. We here review how far “Total IBA” can be considered already a reality, and what further needs to be done to realise its full potential.

Context. The Tarantula Nebula (a.k.a. 30Dor) is a spectacular star-forming region in the LargeMagellanic Cloud (LMC), seen through gas in the Galactic disc and halo. Diffuse interstellar bands (DIBs) offer a unique probe of the diffuse, cool-warm gas in these regions. Aims. The aim is to use DIBs as diagnostics of the local interstellar conditions, whilst at the same time deriving properties of the yet-unknown carriers of these enigmatic spectral features. Methods. Spectra of over 800 early-type stars from the Very Large Telescope Flames Tarantula Survey (VFTS) were analysed. Maps were created, separately, for the Galactic and LMC absorption in the DIBs at 4428 and 6614 Å and - in a smaller region near the central cluster R136 - neutral sodium (the Na iD doublet); we also measured the DIBs at 5780 and 5797 Å. Results. The maps show strong 4428 and 6614 Å DIBs in the quiescent cloud complex to the south of 30 Dor but weak absorption in the harsher environments to the north (bubbles) and near the OB associations. The Na maps show at least five kinematic components in the LMC and a shell-like structure surrounding R136, and small-scale structure in the Milky Way. The strengths of the 4428, 5780, 5797 and 6614 Å DIBs are correlated, also with Na absorption and visual extinction. The strong 4428 Å DIB is present already at low Na column density but the 6614, 5780 and 5797 Å DIBs start to be detectable at subsequently larger Na column densities. Conclusions. The carriers of the 4428, 6614, 5780 and 5797 Å DIBs are increasingly prone to removal from irradiated gas. The relative strength of the 5780 and 5797 Å DIBs clearly confirm the Tarantula Nebula as well as Galactic high-latitude gas to represent a harsh radiation environment. The resilience of the 4428 Å DIB suggests its carrier is large, compact and neutral. Structure is detected in the distribution of cool-warm gas on scales between one and >100 pc in the LMC and as little as 0.01 pc in the Sun's vicinity. Stellar winds from the central cluster R136 have created an expanding shell; some infalling gas is also detected, reminiscent of a galactic "fountain". © ESO 2013.

The CdS window layer in thin film solar cells is frequently grown by chemical bath deposition (CBD). Deposited films are typically less than 100 nm thick and the inability to identify the exact start of the deposition can make CBD an imprecise process. This paper describes the construction and testing of a simple optical fibre sensor that detects the start of the deposition process and also allows for its mechanism to be studied. The in situ optical fibre monitoring technique utilises the change in optical reflectance off the glass/deposited film/precursor solution interfaces at an operating wavelength of 1550 nm. A theoretical expression for the reflection of light from the interface is discussed and compared with experimental results. The monitoring technique shows the presence of two different deposition mechanisms. This result is confirmed by film densities calculated by Rutherford backscattering spectrometry and an optical model for ellipsometry measurements which indicates that the deposited CdS films consist of a double layer structure with a porous layer on top of a dense under layer. The application of the theoretical expression is optimised by assuming the refractive index of the CdS layer to be 2.02. The ellipsometry model shows that the refractive index of the CdS deposited is 2.14 for a two layer model of the film that included a porous upper layer through the effective medium approximation.

The metallome has been involved in the pathological investigation into ocular tissue for decades; however, as technologies advance, more information can be ascertained from individual tissue sections that were not previously possible. Herein, a demonstration of complementary techniques has been utilized to describe the distribution and concentrations of essential metals in both wildtype (WT) and rhodopsin (Rho−/−) ocular tissues. The multimodal approach described is an example of complementary datasets that can be produced when employing a multifaceted analytical approach. Heterogenous distributions of copper and zinc were observable within both WT and Rho−/− tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), and the distributions of further trace elements notoriously problematic for ICP-MS analysis (phosphorous, Sulfur, chlorine, potassium, calcium, iron, and aluminum) were analysed by particle-induced X-ray emission (PIXE).

Liquid Extraction Surface Analysis (LESA) is a new, high throughput tool for ambient mass spectrometry. A solvent droplet is deposited from a pipette tip onto a surface and maintains contact with both the surface and the pipette tip for a few seconds before being re-aspirated. The technique is particularly suited to the analysis of trace materials on surfaces due to its high sensitivity and low volume of sample removal. In this work, we assess the suitability of LESA for obtaining detailed chemical profiles of fingerprints, oral fluid and urine, which may be used in future for rapid medical diagnostics or metabolomics studies. We further show how LESA can be used to detect illicit drugs and their metabolites in urine, oral fluid and fingerprints. This makes LESA a potentially useful tool in the growing field of fingerprint chemical analysis, which is relevant not only to forensics but also to medical diagnostics. Finally, we show how LESA can be used to detect the explosive material RDX in contaminated artificial fingermarks.

We report the development and validation of an untargeted single-cell lipidomics method based on microflow chromatography coupled to a data-dependent mass spectrometry method for fragmentation-based identification of lipids. Given the absence of single-cell lipid standards, we show how the methodology should be optimized and validated using a dilute cell extract. The methodology is applied to dilute pancreatic cancer and macrophage cell extracts and standards to demonstrate the sensitivity requirements for confident assignment of lipids and classification of the cell type at the single-cell level. The method is then coupled to a system that can provide automated sampling of live, single cells into capillaries under microscope observation. This workflow retains the spatial information and morphology of cells during sampling and highlights the heterogeneity in lipid profiles observed at the single-cell level. The workflow is applied to show changes in single-cell lipid profiles as a response to oxidative stress, coinciding with expanded lipid droplets. This demonstrates that the workflow is sufficiently sensitive to observing changes in lipid profiles in response to a biological stimulus. Understanding how lipids vary in single cells will inform future research into a multitude of biological processes as lipids play important roles in structural, biophysical, energy storage, and signaling functions.

The rapid emergence of antibiotic resistant bacterial pathogens constitutes a critical problem in healthcare and requires the development of novel treatments. Potential strategies include the exploitation of microbial social interactions based on public goods, which are produced at a fitness cost by cooperative microorganisms, but can be exploited by cheaters that do not produce these goods. Cheater invasion has been proposed as a 'Trojan horse' approach to infiltrate pathogen populations with strains deploying built-in weaknesses (e.g., sensitiveness to antibiotics). However, previous attempts have been often unsuccessful because population invasion by cheaters was prevented by various mechanisms including the presence of spatial structure (e.g., growth in biofilms), which limits the diffusion and exploitation of public goods. Here we followed an alternative approach and examined whether the manipulation of public good uptake and not its production could result in potential 'Trojan horses' suitable for population invasion. We focused on the siderophore pyoverdine produced by the human pathogen Pseudomonas aeruginosa MPAO1 and manipulated its uptake by deleting and/or overexpressing the pyoverdine primary (FpvA) and secondary (FpvB) receptors. We found that receptor synthesis feeds back on pyoverdine production and uptake rates, which led to strains with altered pyoverdine-associated costs and benefits. Moreover, we found that the receptor FpvB was advantageous under iron-limited conditions but revealed hidden costs in the presence of an antibiotic stressor (gentamicin). As a consequence, FpvB mutants became the fittest strain under gentamicin exposure, displacing the wildtype in liquid cultures, and in biofilms and during infections of the wax moth larvae Galleria mellonella, which both represent structured environments. Our findings reveal that an evolutionary trade-off associated with the costs and benefits of a versatile pyoverdine uptake strategy can be harnessed for devising a Trojan-horse candidate for medical interventions.

The finding that drugs and metabolites can be detected from fingerprints is of potential relevance to forensic science and as well as toxicology and clinical testing. However, discriminating between dermal contact and ingestion of drugs has never been verified experimentally. The inability to interpret the result of finding a drug or metabolite in a fingerprint has prevented widespread adoption of fingerprints in drug testing and limits the probative value of detecting drugs in fingermarks. A commonly held belief is that the detection of metabolites of drugs of abuse in fingerprints can be used to confirm a drug has been ingested. However, we show here that cocaine and its primary metabolite, benzoylecgonine, can be detected in fingerprints of non-drug users after contact with cocaine. Additionally, cocaine was found to persist above environmental levels for up to 48 hours after contact. Therefore the detection of cocaine and benzoylecgonine (BZE) in fingermarks can be forensically significant, but do not demonstrate that a person has ingested the substance. In contrast, the data here shows that a drug test from a fingerprint (where hands can be washed prior to donating a sample) CAN distinguish between contact and ingestion of cocaine. If hands were washed prior to giving a fingerprint, BZE was detected only after the administration of cocaine. Therefore BZE can be used to distinguish cocaine contact from cocaine ingestion, provided donors wash their hands prior to sampling. A test based on the detection of BZE in at least one of two donated fingerprint samples has accuracy 95%, sensitivity 90% and specificity of 100% (n = 86).

Imaging and analyzing gunshot residue (GSR) particles using the scanning electron microscope equipped with an energy dispersive X-ray spectrometer (SEM-EDS) is a standard technique that can provide important forensic evidence, but the discrimination power of this technique is limited due to low sensitivity to trace elements and difficulties in obtaining quantitative results from small particles. A new, faster method using a scanning proton microbeam and Particle Induced X-ray Emission (μ-PIXE), together with Elastic Backscattering Spectrometry (EBS) is presented for the non-destructive, quantitative analysis of the elemental composition of single GSR particles. In this study, the GSR particles were all Pb, Ba, Sb. The precision of the method is assessed. The grouping behaviour of different makes of ammunition is determined using multivariate analysis. The protocol correctly groups the cartridges studied here, with a confidence >99%, irrespective of the firearm or population of particles selected.

Gunshot Residue (GSR) produced by the discharge of a firearm often provides very useful information in criminal investigations in cases involving the use of firearms. Scanning Electron Microscopy equipped with an Energy Dispersive X-ray Spectrometer (SEM-EDS) is typically used worldwide to visualize micrometric particles constituting GSR and to analyse their elemental composition. The 2017 ASTM Standard guide for gunshot residue analysis by scanning electron microscopy/energy dispersive X-ray spectroscopy specifies that “Particles classified as characteristic of GSR will have one of the following elemental compositions: Lead, antimony, barium; Lead, barium, calcium, silicon, tin”. For the first time, the presence of an additional element, such as Sn, plays a key role in ASTM particle classification. It is known that some ammunitions, used for pistols, revolvers and rifles, contain tin foil discs for sealing the primer mixture into the cup, resulting in GSR particles containing Sn. The authors faced some cases in which Sn was unexpectedly found in GSR particles from a 0.22 Long Rifle derringer and from some 12 gauge shotguns. No tin foil discs are used in rimfire ammunitions and there is no published evidence of tin foil discs in shotshell ammunitions. Following a “case by case” approach, experimental research has been carried out to explain how Sn can be present in GSR particles when the last discharged cartridge also does not contain any Sn either in components and in the explosive charges. Moreover, the use of Particle Induced X-ray Emission (PIXE) showed the capability to overcome overlap ambiguity of Sb and Sn peaks in the X-ray spectra, being a possible key issue in real shooting cases if Sn quantities are below the lower limit of SEM detection, especially when Sb is also present.

Metabolic flux is the final output of cellular regulation and has been extensively studied for carbon but much less is known about nitrogen, which is another important building block for living organisms. For the tuberculosis pathogen, this is particularly important in informing the development of effective drugs targeting the pathogen's metabolism. Here we performed 13C15N dual isotopic labeling of Mycobacterium bovis BCG steady state cultures, quantified intracellular carbon and nitrogen fluxes and inferred reaction bidirectionalities. This was achieved by model scope extension and refinement, implemented in a multi-atom transition model, within the statistical framework of Bayesian model averaging (BMA). Using BMA-based 13C15N-metabolic flux analysis, we jointly resolve carbon and nitrogen fluxes quantitatively. We provide the first nitrogen flux distributions for amino acid and nucleotide biosynthesis in mycobacteria and establish glutamate as the central node for nitrogen metabolism. We improved resolution of the notoriously elusive anaplerotic node in central carbon metabolism and revealed possible operation modes. Our study provides a powerful and statistically rigorous platform to simultaneously infer carbon and nitrogen metabolism in any biological system.

The use of a low aerosol dispersion ablation chamber within a LA-ICP-MS set up allows for high-resolution, high-speed imaging of the distribution of elements within a sample. Here we show how this enhanced capability creates new analytical problems and solutions. We report the distribution of platinum at the cellular level in non-small cell lung cancer (NSCLC) explant models after treatment with clinically relevant doses of cisplatin. This revealed for the first time a correlation between the platinum signal and the presence of carbon deposits within lung tissue. We show how complementary ion beam analysis techniques, particle induced X-ray emission (PIXE) and elastic backscattering spectrometry (EBS) can be used to explore potential matrix effects in LA-ICP-MS data. For these samples, it was confirmed that the enhancement was unlikely to have resulted from a matrix effect alone. Thus, the presence of carbon deposits within tissue has potential implications for the effective distribution of the cisplatin drug.

The first analytical intercomparison of fingerprint residue using equivalent samples of latent fingerprint residue and characterized by a suite of relevant techniques is presented. This work has never been undertaken, presumably due to the perishable nature of fingerprint residue, the lack of fingerprint standards, and the intradonor variability, which impacts sample reproducibility. For the first time, time-of-flight secondary ion mass spectrometry, high-energy secondary ion mass spectrometry, and X-ray photoelectron spectroscopy are used to target endogenous compounds in fingerprints and a method is presented for establishing their relative abundance in fingerprint residue. Comparison of the newer techniques with the more established gas chromatography/mass spectrometry and attenuated total reflection Fourier transform infrared spectroscopic imaging shows good agreement between the methods, with each method detecting repeatable differences between the donors, with the exception of matrix-assisted laser desorption ionization, for which quantitative analysis has not yet been established. We further comment on the sensitivity, selectivity, and practicability of each of the methods for use in future police casework or academic research.

Darwin glass is an impact glass resulting from the melting of local rocks during the meteorite impact that formed the 1.2 km diameter Darwin Crater in western Tasmania. These glass samples have small spheroidal inclusions, typically a few tens of microns in diameter, that are of great interest to the geologists. We have analysed one such inclusion in detail with proton microbeam ion beam analysis (IBA). A highly heterogeneous composition is observed, both laterally and in depth, by using self-consistent fitting of photon emission and particle backscattering spectra. With various proton energies near 2 MeV we excite the C-12(p,p)C-12 resonance at 1734 keV at various depths, and thus we can probe both the C concentration, and also the energy straggling of the proton beam as a function of depth which gives information on the sample structure. This inclusion has an average composition of (C, O, Si) = (28, 56, 16) mol% with S, K, Ca, Ti and Fe as minor elements and Cr, Mn, Ni, Cu, Zn and Br as trace elements. This composition includes, at specific points, an elemental depth profile and a density variation with depth consistent with discrete quartz crystals a few microns in size. (c) 2009 Elsevier B.V. All rights reserved.

The global COVID-19 pandemic resulted in widespread harms but also rapid advances in vaccine development, diagnostic testing, and treatment. As the disease moves to endemic status, the need to identify characteristic biomarkers of the disease for diagnostics or therapeutics has lessened, but lessons can still be learned to inform biomarker research in dealing with future pathogens. In this work, we test five sets of research-derived biomarkers against an independent targeted and quantitative Liquid Chromatography-Mass Spectrometry metabolomics dataset to evaluate how robustly these proposed panels would distinguish between COVID-19-positive and negative patients in a hospital setting. We further evaluate a crowdsourced panel comprising the COVID-19 metabolomics biomarkers most commonly mentioned in the literature between 2020 and 2023. The best-performing panel in the independent dataset-measured by F1 score (0.76) and AUROC (0.77)-included nine biomarkers: lactic acid, glutamate, aspartate, phenylalanine, & beta;-alanine, ornithine, arachidonic acid, choline, and hypoxanthine. Panels comprising fewer metabolites performed less well, showing weaker statistical significance in the independent cohort than originally reported in their respective discovery studies. Whilst the studies reviewed here were small and may be subject to confounders, it is desirable that biomarker panels be resilient across cohorts if they are to find use in the clinic, highlighting the importance of assessing the robustness and reproducibility of metabolomics analyses in independent populations.

Single cell metabolomics is a rapidly advancing field of bio-analytical chemistry which aims to observe cellular biology with the greatest detail possible. Mass spectrometry imaging and selective cell sampling (e.g. using nanocapillaries) are two common approaches within the field. Recent achievements such as observation of cell-cell interactions, lipids determining cell states and rapid phenotypic identification demonstrate the efficacy of these approaches and the momentum of the field. However, single cell metabolomics can only continue with the same impetus if the universal challenges to the field are met, such as the lack of strategies for standardisation and quantification, and lack of specificity/sensitivity. Mass spectrometry imaging and selective cell sampling come with unique advantages and challenges which, in many cases are complementary to each other. We propose here that the challenges specific to each approach could be ameliorated with collaboration between the two communities driving these approaches.

A new protocol using time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been developed to identify the deposition order of a fingerprint overlapping an ink line on paper. By taking line scans of fragment ions characteristic of the ink molecules (m/z 358.2 and 372.2) where the fingerprint and ink overlap and by calculating the normalised standard deviation of the intensity variation across the line scan, it is possible to determine whether or not a fingerprint is above ink on a paper substrate. The protocol adopted works for a selection of fingerprints from four donors tested here and for a fingerprint that was aged for six months; for one donor, the very faint fingerprints could not be visualized using either standard procedures (ninhydrin development) or SIMS and therefore the protocol correctly gives an inconclusive result.

Metals have a fundamental role in microbiology, and accurate methods are needed for their identification and quantification. The inability to assess cellular heterogeneity is considered an impediment to the successful treatment of different diseases. Unlike bulk approaches, single-cell analysis allows elemental heterogeneity across genetically identical populations to be related to specific biological events and to the effectiveness of drugs. Single particle-inductively coupled plasma-mass spectrometry (SP-ICP-MS) can analyse single cells in suspension and measure this heterogeneity. Here we explore advances in instrumental design, compare mass analysers and discuss key parameters requiring optimisation. This review has identified that the effect of pre-treatment of cell suspensions and cell fixation approaches require further study and novel validation methods are needed as using bulk measurements is unsatisfactory. SP-ICP-MS has the advantage that a large number of cells can be analysed; however, it does not provide spatial information. Techniques based on laser ablation (LA) enable elemental mapping at the single-cell level, such as laser-induced breakdown spectroscopy (LIBS) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The sensitivity of commercial LIBS instruments restricts its use for sub-tissue applications; however, the capacity to analyse endogenous bulk components paired with developments in nano-LIBS technology shows great potential for cellular research. LA-ICP-MS offers high sensitivity for the direct analysis of single cells, but standardisation requires further development. The hyphenation of these trace elemental analysis techniques and their coupling with multi-omic technologies for single-cell analysis have enormous potential in answering fundamental biological questions.

Determination of the deposition order of different writing tools is very important for the forensic investigation of questioned documents. Here we present a novel application of two ion beam analysis (IBA) techniques: secondary ion mass spectrometry using MeV ions (MeV-SIMS) and particle induced X-ray emission (PIXE) to determine the deposition order of intersecting lines made of ballpoint pen ink, inkjet printer ink, and laser printer toners. MeV-SIMS is an emerging mass spectrometry technique where incident heavy MeV ions are used to desorb secondary molecular ions from the uppermost layers of an organic sample. In contrast, PIXE provides information about sample elemental composition through characteristic X-ray spectra coming from greater depth. In the case of PIXE, the information depth depends on incident ion energy, sample matrix and self-absorption of X-rays on the way out from the sample to the X-ray detector. The measurements were carried out using a heavy ion microprobe at the Ruđer Bošković Institute. Principal component analysis (PCA) was employed for image processing of the data. We will demonstrate that MeV-SIMS alone was successful to determine the deposition order of all intersections not involving inkjet printer ink. The fact that PIXE yields information from deeper layers was crucial to resolve cases where inkjet printer ink was included due to its adherence and penetration properties. This is the first time the different information depths of PIXE and MeV-SIMS have been exploited for a practical application. The use of both techniques, MeV-SIMS and PIXE, allowed the correct determination of deposition order for four out of six pairs of samples.

The use of a low aerosol dispersion ablation chamber within a LA-ICP-MS set up allows for high-resolution, high-speed imaging of the distribution of elements within a sample. Here we show how this enhanced capability creates new analytical problems and solutions. We report the distribution of platinum at the cellular level in non-small cell lung cancer (NSCLC) explant models after treatment with clinically relevant doses of cisplatin. This revealed for the first time a correlation between the platinum signal and the presence of carbon deposits within lung tissue. We show how complementary ion beam analysis techniques, particle induced X-ray emission (PIXE) and elastic backscattering spectrometry (EBS) can be used to explore potential matrix effects in LA-ICP-MS data. For these samples, it was confirmed that the enhancement was unlikely to have resulted from a matrix effect alone. Thus, the presence of carbon deposits within tissue has potential implications for the effective distribution of the cisplatin drug.

BACKGROUND: Recent publications have explored the possibility of using fingerprints to confirm drug use, but none has yet dealt with environmental contamination from fingertips. Here we explored the possibility of establishing an environmental cutoff for drug testing from a single fingerprint. METHODS: Fingerprint samples (n=100) were collected from the hands of 50 nondrug users before and after handwashing to establish separate environmental cutoff values and testing protocols for cocaine, benzoylecgonine, heroin, and 6-monoacetylmorphine. The cutoff was challenged by testing the fingerprints of drug-free volunteers after shaking hands with drug users. Fingerprints from patients who testified to taking cocaine (n = 32) and heroin (n = 24) were also collected and analyzed. RESULTS: A different cutoff value needed to be applied, depending on whether the fingerprints were collected as presented or after handwashing. Applying these cutoffs gave a 0%false-positive rate from the drug-free volunteers. After application of the cutoff, the detection rate (compared to patient testimony) for washed hands of patients was 87.5% for cocaine use and 100% for heroin use. CONCLUSIONS: Fingerprints show enhanced levels of cocaine, heroin, and their respective metabolites in patients who testified to taking the substances, compared with the population of naı¨ve drug users surveyed, and a cutoff (decision level) can be established. The cutoff is robust enough to account for small increases in analyte observed after secondary transfer.

The use of synthetic stimulants, including designer cathinones, remains a significant concern worldwide. Thus, the detection and identification of synthetic cathinones in biological matrices is of paramount importance for clinical and forensic laboratories. In this study, distribution of mephedrone and its metabolites was investigated in fingerprints. Following a controlled human mephedrone administration (100 mg nasally insufflated), two mass spectrometry-based methods for fingerprint analysis have been evaluated. The samples deposited on triangular pieces of chromatography paper were directly analysed under ambient conditions by paper spray-mass spectrometry (PS-MS) while those deposited on glass cover slips were extracted and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method was 5–6 times more sensitive than PS-MS but required sample preparation and longer analysis time. Mephedrone was detected in 62% and in 38% of all post-administration samples analysed by LC-MS/MS and PS-MS, respectively. Nor-mephedrone was the only metabolite detected in 3.8% of all samples analysed by LC-MS/MS. A large inter- and intra-subject variation was observed for mephedrone which may be due to several factors, such as the applied finger pressure, angle and duration of contact with the deposition surface and inability to control the ‘amount’ of collected fingerprint deposits. Until these limitations are addressed, we suggest that the sole use of fingerprints can be a useful diagnostic tool in qualitative rather than quantitative analysis, and requires a confirmatory analysis in a different biological matrix.

Gunshot Residue (GSR) is residual material from the discharge of a firearm, which frequently provides crucial information in criminal investigations. Changes in ammunition manufacturing are gradually phasing out the heavy metals on which current forensic GSR analysis is based, and the latest Heavy Metal Free (HMF) primers urgently demand new forensic solutions. Proton scanning microbeam Ion Beam Analysis (IBA), in conjunction with the Scanning Electron Microscope equipped with an Energy Dispersive X-ray Spectrometer (SEM-EDS), can be introduced into forensic analysis to solve both new and old problems, with a procedure entirely commensurate with current forensic practice. Six cartridges producing GSR particles known to be interesting in casework by both experience and the literature were selected for this study. A standard procedure to relocate the same particles previously analysed by SEM-EDS, based on both secondary electron (SE) and X-ray imaging was developed and tested. Elemental Particle Induced X-ray Emission (PIXE) mapping of the emitted X-rays allowed relocation in a scan of 10μm×10μm of even a 1μm GSR particle. The comparison between spectra from the same particle obtained by SEM-EDS and IBA-PIXE showed that the latter is much more sensitive at mid-high energies. Results that are very interesting in a forensic context were obtained with particles from a cartridge containing mercury fulminate in the primer. Particle-induced gamma-ray emission (PIGE) maps of a particles from HMF cartridges allowed identification of Boron and Sodium in particles from hands using the B(p,αγ)Be, B(p,pγ)B and Na(p,pγ)Na reactions, which is extraordinary in a forensic context. The capability for quantitative analysis of elements within individual particles by IBA was also demonstrated, giving the opportunity to begin a new chapter in the research on GSR particles. The integrated procedure that was developed, which makes use of all the IBA signals, has unprecedented characterisation and discrimination power for GSR samples. © 2013 Elsevier Ireland Ltd.

Characterizing proton beam damage in biological materials is of interest to enable the integration of proton microprobe elemental mapping techniques with other imaging modalities. It is also of relevance to obtain a deeper understanding of mechanical damage to lipids in tissues during proton beam cancer therapy. We have developed a novel strategy to characterize proton beam damage to lipids in biological tissues based on mass spectrometry imaging. This methodology is applied to characterize changes to lipids in tissues ex vivo, irradiated under different conditions designed to mitigate beam damage. This work shows that performing proton beam irradiation at ambient pressure, as well as including the application of an organic matrix prior to irradiation, can reduce damage to lipids in tissues. We also discovered that, irrespective of proton beam irradiation, placing a sample in a vacuum prior to desorption electrospray ionization imaging can enhance lipid signals, a conclusion that may be of future benefit to the mass spectrometry imaging community.

The independent verification in a forensics context of quartz grain morphological typing by scanning electron microscopy was demonstrated using particle-induced X-ray emission (PIXE) and particle-induced γ-ray emission (PIGE). Surface texture analysis by electron microscopy and high-sensitivity trace element mapping by PIXE and PIGE are independent analytical techniques for identifying the provenance of quartz in sediment samples in forensic investigations. Trace element profiling of the quartz grain matrix separately from the quartz grain inclusions served to differentiate grains of different provenance and indeed went some way toward discriminating between different quartz grain types identified in a single sample of one known forensic provenance. These results confirm the feasibility of independently verifying the provenance of critical samples from forensic cases.

Elemental and molecular imaging play a crucial role in understanding disease pathogenesis. To accurately correlate elemental and molecular markers, it is desirable to perform sequential elemental and molecular imaging on a single tissue section. However, very little is known about the impact of performing these measurements in sequence. In this work, we highlight some of the challenges and successes associated with performing elemental mapping in sequence with mass spectrometry imaging. Specifically, the feasibility of molecular mapping using the mass spectrometry imaging (MSI) techniques matrix assisted laser desorption ionisation (MALDI) and desorption electrospray ionisation (DESI) in sequence with the elemental mapping technique particle induced X-ray emission (PIXE) is explored. Challenges for integration include substrate compatibility, as well as delocalisation and spectral changes. We demonstrate that whilst sequential imaging comes with some compromises, sequential DESI-PIXE imaging is sufficient to correlate sulphur, iron and lipid markers in a single tissue section at the 50-micrometre scale.

Paper spray mass spectrometry is a rapid and sensitive tool for explosives detection but has so far only been demonstrated using high resolution mass spectrometry, which bears too high a cost for many practical applications. Here we explore the potential for paper spray to be implemented in field applications with portable mass spectrometry. This involved (a) replacing the paper substrate with a swabbing material (which we call “swab spray”) for compatibility with standard collection materials; (b) collection of explosives from surfaces; (c) an exploration of interferences within a ± 0.5 m/z window; and (d) demonstration of the use of high-field assisted waveform ion mobility spectrometer (FAIMS) for enhanced selectivity. We show that paper and Nomex® are viable collection materials, with Nomex providing cleaner spectra and therefore greater potential for integration with portable mass spectrometers. We show that sensitive detection using swab spray will require a mass spectrometer with a mass resolving power of 4000 or more. We show that by coupling the swab spray ionisation source with FAIMS, it is possible to reduce background interferences, thereby facilitating the use of a low resolving power (e.g. quadrupole) mass spectrometer.

Background The global COVID-19 pandemic has led to extensive development in many fields, including the diagnosis of COVID-19 infection by mass spectrometry. The aim of this systematic review and meta-analysis was to assess the accuracy of mass spectrometry diagnostic tests developed so far, across a wide range of biological matrices, and additionally to assess risks of bias and applicability in studies published to date. Method 23 retrospective observational cohort studies were included in the systematic review using the PRISMA-DTA framework, with a total of 2 858 COVID-19 positive participants and 2 544 controls. Risks of bias and applicability were assessed via a QUADAS-2 questionnaire. A meta-analysis was also performed focusing on sensitivity, specificity, diagnostic accuracy and Youden’s Index, in addition to assessing heterogeneity. Findings Sensitivity averaged 0.87 in the studies reviewed herein (interquartile range 0.81 - 0.96) and specificity 0.88 (interquartile range 0.82 - 0.98), with an area under the receiver operating characteristic summary curve of 0.93. By subgroup, the best diagnostic results were achieved by viral proteomic analyses of nasopharyngeal swabs and metabolomic analyses of plasma and serum. The performance of other sampling matrices (breath, sebum, saliva) was less good, indicating that these protocols are currently insufficiently mature for clinical application. Conclusions This systematic review and meta-analysis demonstrates the potential for mass spectrometry and ‘omics in achieving accurate test results for COVID-19 diagnosis, but also highlights the need for further work to optimize and harmonize practice across laboratories before these methods can be translated to clinical applications.

Rapid and comprehensive genetic sequencing has shed light on the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and allowed timely implementation of PCR tests to determine the presence of viral RNA. PCR tests for SARS-CoV-2 are some way from being reliably qualitative and will never indicate how the disease might progress in an individual. As COVID-19 becomes endemic, there is a concomitant need for accurate serological assays to detect antibodies to SARS-CoV-2 antigens and ultimately tests for prognostic markers to target treatment options.1,2 With this considerable genetic insight, and the emerging structural information, comes associated questions regarding the molecular descriptors that contribute to disease progression, especially when we consider spread across different populations. The power of mass spectrometry to generate rapid, precise, and reproducible diagnostic information that complements genomic information and accelerates our understanding of the disease, is now becoming a reality.

Fingerprints have been proposed as a promising new matrix for drug testing. In previous work it has been shown that a fingerprint can be used to distinguish between drug users and non-users. Herein, we look at the possibility of using a fingerprint to distinguish between dermal contact and administration of heroin. Fingerprint samples were collected from (a) 10 patients attending a drug rehabilitation clinic (b) 50 non-drug users (c) participants who touched 2 mg street heroin, before and after various hand cleaning procedures. Oral fluid was also taken from the patients. All samples were analysed using a liquid chromatography – high resolution mass spectrometry (LC-HRMS) method validated in previous work for heroin and 6-AM. The HRMS data was analysed retrospectively for morphine, codeine, 6-acetylcodeine and noscapine. Heroin and 6-AM were detected in all fingerprint samples produced from contact with heroin, even after handwashing. In contrast, morphine, acetylcodeine and noscapine were successfully removed after handwashing. In patient samples, the detection of morphine, noscapine and acetylcodeine (alongside heroin and 6-AM) gave a closer agreement to patient testimony on whether they had recently used heroin use than the detection of heroin and 6-AM alone. This research highlights the importance of washing hands prior to donating a fingerprint sample to distinguish recent contact with heroin from heroin use.

Surface mass spectrometry methods can be difficult to use effectively with low cost, portable mass spectrometers. This is because commercially available portable (single quadrupole) mass spectrometers lack the mass resolution to confidently differentiate between analyte and background signals. Additionally, current surface analysis methods provide no facility for chromatographic separation and therefore are vulnerable to ion suppression. Here we present a new analytical method where analytes are extracted from a sample using a solvent flushed across the surface under high pressure, separated using a chromatography column and then analysed using a portable mass spectrometer. The use of chromatography reduces ion suppression effects and this, used in combination with in-source fragmentation, increases selectivity, thereby allowing high sensitivity to be achieved with a portable and affordable quadrupole mass spectrometer. We demonstrate the efficacy of the method for the quantitative detection of cocaine and benzoylecgonine in urine and oral fluid. The method gives relative standard deviations below 15% (with one exception), and R2 values above 0.998. The limits of detection for these analytes in oral fluid and urine are

Surface analysis techniques have rapidly evolved in the last decade. Some of these are already routinely used in forensics, such as for the detection of gunshot residue or for glass analysis. Some surface analysis approaches are attractive for their portability to the crime scene. Others can be very helpful in forensic laboratories owing to their high spatial resolution, analyte coverage, speed, and specificity. Despite this, many proposed applications of the techniques have not yet led to operational deployment. Here, we explore the application of these techniques to the most important traces commonly found in forensic casework. We highlight where there is potential to add value and outline the progress that is needed to achieve operational deployment. We consider within the scope of this review surface mass spectrometry, surface spectroscopy, and surface X-ray spectrometry. We show how these tools show great promise for the analysis of fingerprints, hair, drugs, explosives, and microtraces. Expected final online publication date for the Volume 15 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

In this publication we work towards providing fast, sensitive and selective analysis of explosive compounds collected on swabs using paper spray mass spectrometry. We have (a) increased the size of the paper spray substrate to 1.6×2.1 cm for compatibility with current practise in swabbing for explosive material; (b) developed a method for determining a successful extraction of analyte from the substrate to reduce false negative events; and (c) expanded the range of analytes that can be detected using paper spray to include the peroxide explosive HMTD, as well as nitroglycerine (NG), picric acid (PA) and tetryl. We report the development of a 30 s method for the simultaneous detection of 7 different explosive materials using PSMS with detection limits below 25 pg, as well as detection of HMTD at 2500 pg, showing an improvement on previously published work.

The constantly growing field of the true one cell analysis provides important information on the direct chemical composition of various cells and cellular compartments. Since the heterogeneity of individual cells has been established, more researchers are interested in the chemical differences between individual cells and that is the only analysis of the one cell can determine. This results in new technologies and methods being reported regularly. This review highlights the common techniques of micro- and nanomanipulation, Raman spectroscopy, microsocopy, and mass spectrometric imaging as they pertain to the true one cell chemical analysis.

Direct analyte-probed nano-extraction (DAPNe) is a method of extracting material from a microscale region of a sample and provides the opportunity for detailed mass spectrometry analysis of extracted analytes from a small area. The technique has been shown to provide enhanced sensitivity compared with bulk analysis by selectively removing analytes from their matrix and has been applied for selective analysis of single cells and even single organelles. However, the quantitative capabilities of the technique are yet to be fully evaluated. In this study, various normalisation techniques were investigated in order to improve the quantitative capabilities of the technique. Two methods of internal standard incorporation were applied to test substrates, which were designed to replicate biological sample matrices. Additionally, normalisation to the extraction spot area and matrix compounds were investigated for suitability in situations when an internal standard is not available. The variability observed can be significantly reduced by using a sprayed internal standard, and in some cases, by normalising to the extracted area.

BACKGROUND: Paper spray mass spectrometry6 is a technique that has recently emerged and has shown excellent analytical sensitivity to a number of drugs in blood. As an alternative to blood, fingerprints have been shown to provide a noninvasive and traceable sampling matrix. Our goal was to validate the use of fingerprint samples to detect cocaine use. METHODS: Samples were collected on triangular pieces (168 mm2) of washed Whatman Grade I chromatography paper. Following application of internal standard, spray solvent and a voltage were applied to the paper before mass spectrometry detection. A fingerprint visualization step was incorporated into the analysis procedure by addition of silver nitrate solution and exposing the sample to ultraviolet light. RESULTS: Limits of detection for cocaine, benzoylecgonine, and methylecgonine were 1, 2, and 31 ng/mL respectively, with relative standard deviations of less than 33%. No matrix effects were observed. Analysis of 239 fingerprint samples yielded a 99% true-positive rate and a 2.5% false-positive rate, based on the detection of cocaine, benzoylecgonine, or methylecgonine with use of a single fingerprint. CONCLUSIONS: The method offers a qualitative and noninvasive screening test for cocaine use. The analysis method developed is rapid (4 min/sample) and requires no sample preparation.