Surface-assisted laser desorption ionization mass spectrometry techniques for application in forensics.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is an excellent analytical technique for the rapid and sensitive analysis of macromolecules (>700 Da), such as peptides, proteins, nucleic acids, and synthetic polymers. However, the detection of smaller organic molecules with masses below 700 Da using MALDI-MS is challenging due to the appearance of matrix adducts and matrix fragment peaks in the same spectral range. Recently, nanostructured substrates have been developed that facilitate matrix-free laser desorption ionization (LDI), contributing to an emerging analytical paradigm referred to as surface-assisted laser desorption ionization (SALDI) MS. Since SALDI enables the detection of small organic molecules, it is rapidly growing in popularity, including in the field of forensics. At the same time, SALDI also holds significant potential as a high throughput analytical tool in roadside, work place and athlete drug testing. In this review, we discuss recent advances in SALDI techniques such as desorption ionization on porous silicon (DIOS), nano-initiator mass spectrometry (NIMS) and nano assisted laser desorption ionization (NALDI™) and compare their strengths and weaknesses with particular focus on forensic applications. These include the detection of illicit drug molecules and their metabolites in biological matrices and small molecule detection from forensic samples including banknotes and fingerprints. Finally, the review highlights recent advances in mass spectrometry imaging (MSI) using SALDI techniques.

Silver Coating for High-Mass-Accuracy Imaging Mass Spectrometry of Fingerprints on Nanostructured Silicon.

Nanostructure imaging mass spectrometry (NIMS) using porous silicon (pSi) is a key technique for molecular imaging of exogenous and endogenous low molecular weight compounds from fingerprints. However, high-mass-accuracy NIMS can be difficult to achieve as time-of-flight (ToF) mass analyzers, which dominate the field, cannot sufficiently compensate for shifts in measured m/z values. Here, we show internal recalibration using a thin layer of silver (Ag) sputter-coated onto functionalized pSi substrates. NIMS peaks for several previously reported fingerprint components were selected and mass accuracy was compared to theoretical values. Mass accuracy was improved by more than an order of magnitude in several cases. This straightforward method should form part of the standard guidelines for NIMS studies for spatial characterization of small molecules.

Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing.

We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.

Properties of nucleic acid staining dyes used in gel electrophoresis.

Nucleic acid staining dyes are used for detecting nucleic acids in electrophoresis gels. Historically, the most common dye used for gel staining is ethidium bromide, however due to its toxicity and mutagenicity other dyes that are safer to the user and the environment are preferred. This Short Communication details the properties of dyes now available and their sensitivity for detection of DNA and their ability to permeate the cell membrane. It was found that GelRed™ was the most sensitive and safest dye to use with UV light excitation, and both GelGreen™ and Diamond™ Nucleic Acid Dye were sensitive and the safer dyes using blue light excitation.

Surface-assisted laser desorption/ionization mass spectrometry using ordered silicon nanopillar arrays.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is ideally suited for the high-throughput analysis of small molecules in bodily fluids (e.g. saliva, urine, and blood plasma). A key application for this technique is the testing of drug consumption in the context of workplace, roadside, athlete sports and anti-addictive drug compliance. Here, we show that vertically-aligned ordered silicon nanopillar (SiNP) arrays fabricated using nanosphere lithography followed by metal-assisted chemical etching (MACE) are suitable substrates for the SALDI-MS detection of methadone and small peptides. Porosity, length and diameter are fabrication parameters that we have explored here in order to optimize analytical performance. We demonstrate the quantitative analysis of methadone in MilliQ water down to 32 ng mL(-1). Finally, the capability of SiNP arrays to facilitate the detection of methadone in clinical samples is also demonstrated.

High-performance capillary electrophoretic separation of double-stranded oligonucleotides using a poly-(ethylpyrrolidine methacrylate-co-methylmethacrylate)-coated capillary.

Here we describe a capillary electrophoretic method for the separation of double-stranded oligonucleotides (ds-ODNs) ranging from 16-20 bp with 2 bp resolution using a low concentration of poly(ethylpyrrolidine methacrylate-co-methyl methacrylate) (PEPyM-co-PMMA) copolymer physically adsorbed to a capillary surface. Contrary to traditional DNA separations, we show that the ds-ODN with the highest molecular size eluted first and propose that this phenomena is due to a screening effect by the PEPyM-co-PMMA coating on the smaller ds-ODNs negative charge during elution. Key to the performance of this separation was a sample preparation time of less than 1 h and analysis time of 40 min. Repeatability of intraday migration time for the mixtures was typically < 1% relative standard deviation (n = 3). In addition, we demonstrate that the coating has an acceptable capillary lifetime of over 70 injections.

A fresh scientific look at transfer and persistence: From a materials science and tribology perspective.

Knowledge of the mechanisms governing transfer, persistence, and recovery of trace evidence, together with background prevalence in the population of interest, and other task relevant information, is key for the forensic interpretation and reconstruction of what happened at the activity level. Up to now, this informational "toolkit" has largely been developed through empirical forensic studies on specific trace materials such as glass, textile fibers, and soil. Combined with the identified systemic siloing between disciplines, while valuable, such research tends to be very material-dependent, introducing specific parameters and interpretations that may have actually impeded the recognition of underlying foundational factors applicable to most material types. In Australia, there has been a renewed interest in developing a discipline-independent framework for the interpretation and/or reconstruction of trace evidence to interpret specific circumstances in casework. In this paper, we present a discipline agnostic "way of thinking" that has been anchored in foundational science underpinning the trace evidence discipline. Physical and mechanical material properties such as material geometry and surface topography, strength, stiffness, and hardness collectively influence contact interactions through underlying friction, wear, and lubrication cause and effect mechanisms. We discuss how these fundamental factors and parameters stemming from materials science and tribology may be adopted and adapted by forensic practitioners and researchers to contribute to a better understanding of transfer, persistence, and recovery mechanisms irrespective of evidence discipline and material type. Examples are provided to demonstrate the practical significance to real-life casework and academic research.

Rapid drug detection in oral samples by porous silicon assisted laser desorption/ionization mass spectrometry.

The demand for analysis of oral fluid for illicit drugs has arisen with the increased adoption of roadside testing, particularly in countries where changes in legislation allow random roadside testing of drivers for the presence of a palette of illicit drugs such as methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA) and Delta9-tetrahydrocannabinol (THC). Oral samples are currently tested for such drugs at the roadside using an immunoassay-based commercial test kit. Positive roadside tests are sent for confirmatory laboratory analysis, traditionally by means of gas chromatography/mass spectrometry (GC/MS). We present here an alternative rapid analysis technique, porous silicon assisted laser desorption/ionization time-of-flight mass spectrometry (pSi LDI-MS), for the high-throughput analysis of oral fluids. This technique alleviates the need for sample derivatization, requires only sub-microliter sample volumes and allows fast analysis (of the order of seconds). In this study, the application of the technique is demonstrated with real samples from actual roadside testing. The analysis of oral samples resulted in detection of MA and MDMA with no extraction and analysis of THC after ethyl acetate extraction. We propose that, subject to miniaturization of a suitable mass spectrometer, this technique is well suited to underpin the deployment of oral fluid testing in the clinic, workplace and on the roadside.

Gunshot residue background on police officers: Considerations for secondary transfer in GSR evidence evaluation.

In order to have confidence in the integrity of a GSR test result, it is essential to understand the factors that may influence the presence of GSR particles on a person of interest's hands beyond the discharge of a firearm. One important consideration is the possibility that a person of interest (POI) may become contaminated with GSR through their interactions with firearms-carrying police officers during the process of apprehension or arrest. This situation has the potential to result in a false positive error, and potentially result in the mistaken inclusion of a POI in an investigation. Prior studies have indicated that receiving a firearm at the start of shift represents a significant contamination event, which may result in GSR persisting on the hands of police officers (Cook, 2016). Firearms-carrying police officers were sampled at various points throughout their shift to ascertain the extent of persistence of GSR on their hands, as a potential population that could then be transferred. A secondary transfer study was also conducted, with several 'mock arrest' scenarios used as a means of modelling the extent and dynamics of any secondary GSR transfer. When compared to the random population, it was found that police exhibit GSR contamination of their hands at a higher frequency. 7.9% of the officers sampled in this study returned at least one, 3-component particle characteristic of GSR. Further, 75% of the officers tested had at least one particle considered consistent with GSR present on their hands. However, the overall particle numbers were relatively low, with the average reported number of characteristic particles being less than five, and the greatest number reported being twelve. When considering the possibility of secondary transfer occurring during the process of arrest, it was further determined through secondary transfer experiments that the most probable transfer situation was fewer than 25% of the particles present on an officer's hands would be transferred to a POI as a result of a short 'mock arrest' contact. We conclude that although the possibility of GSR contamination through the process of arrest exists, and is worthy of consideration in the assessment of GSR evidence, it is not an overwhelming, or major concern.

Evaluation of the sub-surface morphology and composition of gunshot residue using focussed ion beam analysis.

Recent work in the forensic analysis of Gunshot residues (GSR) has suggested that the sub-surface or internal composition and morphology of these residues be explored. A particular area of interest is in heavy metal free, or non-toxic ammunition, which are becoming more frequently encountered in the marketplace. As the formulation of the primer compound changes the conditions of the firearm discharge, there is the possibility that different primer formulations may result in the formation of different GSR particles with distinct internal morphologies and compositions. To that end, the internal morphology and composition of GSR particles may provide additional information that could be useful in the investigation of firearms crime. This research investigated the internal morphology of GSR originating from a variety of different ammunition products. Both traditional three-component primed ammunition, and a selection of heavy metal free and non-toxic alternatives were considered. Particles were identified using SEM-EDS, before being cross-sectioned using a focussed ion beam (FIB) instrument. The FIB-sectioned particles were then re-acquired and mapped using SEM-EDS, to assess both internal morphology and composition. Particles observed in this study presented distinct morphological and compositional features at the sub-particle level that may provide an indication of the primer formulation from which they originated. That said, further investigation of a variety of samples should be undertaken to verify the consistency of these features, or any deviations that may be observed based on primer type. However, these results indicate that there may be promise in obtaining additional detail from sub-particle morphology and composition.

Methods for analysis of glass in glass-containing gunshot residue (gGSR) particles.

When lead, barium and antimony, or lead, barium, calcium, silicon and tin are found together in particles associated with a shooting investigation they are considered characteristic of gunshot residue (GSR). Antimony and tin are often absent from the primer of many low calibre rimfire ammunitions, which are the type most commonly used in Australia. Therefore, the likelihood of characteristic particles forming during the firing process of such rimfire ammunition is significantly less than the likelihood of these particles arising from higher calibre ammunition. The majority of rimfire ammunition examined in this research contains ground glass in the primer, which functions as a frictionator. These ammunitions produce a small number of gunshot residue particles containing glass coated with other primer components, which we refer to as glass-containing GSR (gGSR). If these particles are observed in an investigation, they have the potential to add a new dimension to gunshot residue analysis because they are not common in the environment. Furthermore, the composition of glass frictionator is stable during firing, which raises the possibility that chemical testing of the glass in gGSR may be used to identify the ammunition from which the residue was derived or to link deposits of GSR. This paper examines the application of scanning electron microscopy-energy dispersive X-ray spectrometry (SEM-EDS), focussed ion beam (FIB) techniques and time of flight-secondary ion mass spectrometry (ToF-SIMS) to the semi-quantitative analysis and comparisons of gGSR and frictionator extracted from unfired cartridges. SEM-EDS is effective for comparing gGSR with unfired frictionator, but the use of FIB to expose clean glass from the centre of gGSR followed by ToF-SIMS, or ToF-SIMS using ion sputtering to expose clean glass, offers more power for comparisons due to their capability for higher discrimination between frictionators from different sources.

Glass-containing gunshot residues and particles of industrial and occupational origins: Considerations for evaluating GSR traces.

In an ideal case, the value of traces would be determined numerically and presented through the use of likelihood ratios or verbal-equivalent scales. A problem in the evaluation of gunshot residue (GSR) evidence using these models is that in many shooting scenarios insufficient data exist to support a quantitative model of interpretation. The complex relationship that exists between ammunition composition and post-firing residues makes quantitative interpretation more difficult for GSR than for other traces such as glass. When evaluating the significance of traces in a quantitative model, the value of a trace is reduced as the number of random sources that could produce the trace increases. Previously published works have suggested that glass-containing GSR (gGSR), which is glass encrusted with lead (Pb) and barium (Ba) residues, are a new type of GSR not already classified under ASTM E1588 - 17. If random sources of particles resembling gGSR are rare, then gGSR may be valuable evidentiary traces. In order to potentially incorporate these particles into a future model, the general background prevalence of gGSR and specific sources capable of producing similar particles must be understood. Therefore, particles from fireworks, matches, and cartridge actuated nail guns were assessed on an individual basis and at a population level. These sources, known to produce particles resembling GSR, were assessed using backscattered electron - scanning electron microscopy - energy dispersive X-ray spectroscopy analysis (BSE-SEM-EDS) for the presence of glass-containing particles that resemble gGSR. In the experiments described in this article the nail gun produced particles compositionally indistinguishable from gGSR, due to the primer in the brand of nail gun cartridges used containing glass as the frictionator in addition to Pb and Ba compounds. In this study, no particles were located from fireworks or matches that were indistinguishable from gGSR, nor was any evidence observed or found in the literature that would suggest that such particles could be formed.

Analysis of elemental and isotopic variation in glass frictionators from 0.22 rimfire primers.

The majority of 0.22 calibre rimfire ammunition available in Australia, and overseas, tends to use glass powder rather than antimony sulfide frictionator in the primer. This glass can be the nucleus of a GSR particle, with other primer components condensing around and onto the glass structure. As the composition of glass frictionator remains largely unaltered during ammunition discharge [1] there is the possibility that frictionator composition could be used in GSR examinations to either correlate or discriminate between samples, thereby providing valuable information to an investigation. In this study, the composition of glass frictionator from a wide variety of ammunition was analysed by time-of-flight - secondary ion mass spectrometry (ToF-SIMS), sensitive high-resolution ion microprobe (SHRIMP) and scanning electron microscopy - energy dispersive X-ray spectrometry (SEM-EDS). Refractive index (RI) was measured using glass refractive index measurement (GRIM). Across the population of ammunition studied, it was found that the elemental and isotopic composition of frictionator varied. ToF-SIMS was able to discriminate 94.1% of brands in a pairwise comparison and SEM-EDS achieved a pairwise discrimination power of 79.4%. If SHRIMP was combined with the other two techniques, 95.6% of brands could be discriminated. Refractive index measurements supported the elemental data showing that there appeared, in most cases, to be only one population of glass within a cartridge. The results suggest that there is scope for frictionator analysis to contribute valuable, new capability to forensic GSR examinations.

An investigation into artefacts formed during gas chromatography/mass spectrometry analysis of firearms propellant that contains diphenylamine as the stabiliser.

In the course of providing assistance to legal counsel in a matter that involved the analysis of firearms propellant by gas chromatography/mass spectrometry it was noticed that phenoxazine was reported as a component of 0.22 calibre propellant that contained diphenylamine as the stabiliser. The research was conducted to find how phenoxazine was formed. The results showed that the compound was not phenoxazine but a diphenylamine derivative 4-(phenylimino) cyclohexa-2,5-dien-1-one that formed in the injection port of the gas chromatograph. In addition a second artefact was found to form in the ion source of the mass spectrometer. While the presence of the artefacts does not affect the ability to identify particles as propellant they may impact on comparison with source ammunition.

Patterns produced when soil is transferred to bras by placing and dragging actions: The application of digital photography and image processing to support visible observations.

A series of soil transference experiments (STEs) were undertaken to determine whether patterns identified in laboratory experiments could also be recognised at a simulated crime scene in the field. A clothed 55kg human rescue dummy dressed in a padded bra was either dragged or merely placed on a soil surface at sites with natural and anthropogenic soil types under both wet and dry soil conditions. Transfer patterns produced by dragging compared favourably with those of laboratory experiments. Twelve patterns were identified when a clothed human rescue dummy was dragged across the two soil types in the field. This expanded the original set of eight soil transfer patterns identified from dragging weighted fabric across soil samples in the laboratory. Soil transferred by placing the human rescue dummy resulted in a set of six transfer patterns that were different to those produced by dragging. By comparing trace soil patterns transferred to bras using each transfer method, it was revealed that certain transfer patterns on bras could indicate how the fabric had made contact with a soil surface. A photographic method was developed for crime scene examiners to capture this often subtle soil evidence before a body is transported or the clothing removed. This improved understanding of the dynamics of soil transference to bras and related clothing fabric may assist forensic investigators reconstruct the circumstances of a variety of forensic events.

Porous silicon mass spectrometry as an alternative confirmatory assay for compliance testing of methadone.

Porous silicon based surface-assisted laser desorption ionization mass spectrometry (pSi SALDI-MS) is an analytical technique well suited for high throughput analysis of low molecular weight compounds from biological samples. A potential application of this technology is the compliance monitoring of opioid addiction programmes, where methadone is used as a pharmacological treatment for drugs such as heroin. Here, we present the detection and quantification of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) from water and clinical samples (saliva, urine, and plasma) from opioid dependent participants using pSi SALDI-MS. A one-step solvent phase extraction using chloroform was developed for the detection of methadone from clinical samples for analysis by pSi SALDI-MS. Liquid chromatography-mass spectrometry (LC-MS) was used as a comparative technique for the quantification of methadone from clinical saliva and plasma samples. In all cases, we obtained a good correlation of pSi SALDI-MS and LC-MS results, suggesting that pSi SALDI-MS may be an alternative procedure for high-throughput screening and quantification for application in opioid compliance testing. Copyright © 2016 John Wiley & Sons, Ltd.

Quantifying gunshot residues in cases of suicide: Implications for evaluation of suicides and criminal shootings.

Gunshot residue (GSR) is a valuable form of forensic trace evidence in the investigation of firearms-related suicide and crime. In order to ensure that such evidence is given appropriate evidential weighting when it comes to case investigations, the dynamics of the deposition and distribution of GSR must be understood. As the activity of the subject after firing is a major factor in the retention of GSR, cases of suicide involving a firearm provide an opportunity to assess GSR distribution where post-shooting activity is not an issue, assuming the subject had not been moved. Seventy-one cases of suicide by gunshot in South Australia between 1998 and 2014 were examined to collect data on firearm type, calibre, and gunshot residue test results. Overall, 47% of cases were found to produce GSR results that were not of high probative value, even though it was known that the shooter had fired a firearm. Of particular interest were cases involving 0.22 rimfire ammunition, for which characteristic particles are not expected due to lack of antimony present in the primer. Despite this, 64% of these cases resulted in the detection of antimony-containing, three-component particles.

A study into the distribution of gunshot residue particles in the random population.

When considering the impact and value of gunshot residues (GSR) as forensic trace evidence, the likelihood of a suspect producing a positive GSR analysis result without having direct exposure to a firearm is a major consideration. Therefore, the random prevalence of GSR and 'GSR-like' residues in the wider population is a highly pertinent question when considering the probative value of such evidence. The random prevalence of GSR in two Australian jurisdictions - Victoria and South Australia - was assessed through the collection and analysis of GSR samples obtained from randomly selected members of the public. Volunteers were asked to declare any firearms use, hobbies or potential firearms exposure before allowing their hands to be sampled using aluminium GSR sample stubs coated in adhesive tape. A total of 289 samples, 120 from Victoria and 169 from South Australia were collected and analysed using scanning electron microscopy coupled with energy dispersive X-ray microanalysis (SEM-EDS). Across all samples, three 'characteristic' three-component Pb/Ba/Sb particles were detected from a single subject in South Australia, corresponding to an overall prevalence of 0.3%. Two-component 'consistent' particles were more prevalent, with Pb/Sb particles being the most frequently occurring, in 8% of samples, and in South Australia only. A number of samples, approximately 7%, showed populations of single element particles of Pb, Ba and Sb, which has the potential to generate a false positive for GSR if using a bulk analysis technique such as NAA or AAS. The prevalence of GSR or 'GSR like' particles in this study matches closely with similar surveys conducted in other jurisdictions. Such surveys are a useful foundation for the creation of a probabilistic method for the assessment of GSR evidence.

Soil transference patterns on bras: Image processing and laboratory dragging experiments.

In a recent Australian homicide, trace soil on the victim's clothing suggested she was initially attacked in her front yard and not the park where her body was buried. However the important issue that emerged during the trial was how soil was transferred to her clothing. This became the catalyst for designing a range of soil transference experiments (STEs) to study, recognise and classify soil patterns transferred onto fabric when a body is dragged across a soil surface. Soil deposits of interest in this murder were on the victim's bra and this paper reports the results of anthropogenic soil transfer to bra-cups and straps caused by dragging. Transfer patterns were recorded by digital photography and photomicroscopy. Eight soil transfer patterns on fabric, specific to dragging as the transfer method, appeared consistently throughout the STEs. The distinctive soil patterns were largely dependent on a wide range of soil features that were measured and identified for each soil tested using X-ray Diffraction and Non-Dispersive Infra-Red analysis. Digital photographs of soil transfer patterns on fabric were analysed using image processing software to provide a soil object-oriented classification of all soil objects with a diameter of 2 pixels and above transferred. Although soil transfer patterns were easily identifiable by naked-eye alone, image processing software provided objective numerical data to support this traditional (but subjective) interpretation. Image software soil colour analysis assigned a range of Munsell colours to identify and compare trace soil on fabric to other trace soil evidence from the same location; without requiring a spectrophotometer. Trace soil from the same location was identified by linking soils with similar dominant and sub-dominant Munsell colour peaks. Image processing numerical data on the quantity of soil transferred to fabric, enabled a relationship to be discovered between soil type, clay mineralogy (smectite), particle size and soil moisture content that would not have been possible otherwise. Soil type (e.g. Anthropogenic, gravelly sandy loam soil or Natural, organic-rich soil), clay mineralogy (smectite) and soil moisture content were the greatest influencing factors in all the dragging soil transference tests (both naked eye and measured properties) to explain the eight categories of soil transference patterns recorded. This study was intended to develop a method for dragging soil transference laboratory experiments and create a baseline of preliminary soil type/property knowledge. Results confirm the need to better understand soil behaviour and properties of clothing fabrics by further testing of a wider range of soil types and clay mineral properties.

Nanostructured Silicon-Based Fingerprint Dusting Powders for Enhanced Visualization and Detection by Mass Spectrometry.

Porous silicon microparticles (pSi MPs) functionalized with fluorescent dyes (lissamine and carboxy-5-fluorescein) and intrinsically luminescent pSi MPs were explored as novel fingerprint dusting powders. The versatility of luminescent pSi MPs is demonstrated through time-gated imaging of their long-lived (lifetime>28 µs) near-IR emission, and mass spectrometry analysis of fingerprints dusted with luminescent pSi MPs to provide further information on exogenous small molecules present in latent fingerprints.