3-Methoxytyrosine as an indicator of dopaminergic manipulation in equine plasma.

The use of catechol-O-methyltransferase inhibitors may mask doping agents, primarily levodopa, administered to racehorses and prolong the stimulating effects of dopaminergic compounds such as dopamine. It is known that 3-methoxytyramine is a metabolite of dopamine and 3-methoxytyrosine is a metabolite of levodopa thus these compounds are proposed to be potential biomarkers of interest. Previous research established a urinary threshold of 4,000 ng/mL for 3-methoxytyramine to monitor misuse of dopaminergic agents. However, there is no equivalent biomarker in plasma. To address this deficiency a rapid protein precipitation method was developed and validated to isolate target compounds from 100 µL equine plasma. A liquid chromatography-high resolution accurate mass (LC-HRAM) method using an IMTAKT Intrada amino acid column provided quantitative analysis of 3-methoxytyrosine (3-MTyr) with lower limit of quantification of 5 ng/mL. Reference population profiling (n = 1129) investigated the expected basal concentrations for raceday samples from equine athletes and showed a right-skewed distribution (skewness = 2.39, kurtosis = 10.65) which resulted from large variation (RSD = 71%) within the data. Logarithmic transformation of the data provided a normal distribution (skewness = 0.26, kurtosis = 3.23) resulting in the proposal of a conservative threshold for plasma 3-MTyr of 1,000 ng/mL at a 99.995% confidence level. A 12-horse administration study of Stalevo® (800 mg L-DOPA, 200 mg carbidopa, 1600 mg entacapone) revealed elevated 3-MTyr concentrations for 24-hours post-administration.

Metabolomics in clinical and forensic toxicology, sports anti-doping and veterinary residues.

Metabolomics is a multidisciplinary field providing workflows for complementary approaches to conventional analytical determinations. It allows for the study of metabolically related groups of compounds or even the study of novel pathways within the biological system. The procedural stages of metabolomics; experimental design, sample preparation, analytical determinations, data processing and statistical analysis, compound identification and validation strategies are explored in this review. The selected approach will depend on the type of study being conducted. Experimental design influences the whole metabolomics workflow and thus needs to be properly assessed to ensure sufficient sample size, minimal introduced and biological variation and appropriate statistical power. Sample preparation needs to be simple, yet potentially global in order to detect as many compounds as possible. Analytical determinations need to be optimised either for the list of targeted compounds or a universal approach. Data processing and statistical analysis approaches vary widely and need to be better harmonised for review and interpretation. This includes validation strategies that are currently deficient in many presented workflows. Common compound identification approaches have been explored in this review. Metabolomics applications are discussed for clinical and forensic toxicology, human and equine sports anti-doping and veterinary residues.

Snapshot prediction of carbon productivity, carbon and protein content in a Southern Ocean diatom using FTIR spectroscopy.

Diatoms, an important group of phytoplankton, bloom annually in the Southern Ocean, covering thousands of square kilometers and dominating the region's phytoplankton communities. In their role as the major food source to marine grazers, diatoms supply carbon, nutrients and energy to the Southern Ocean food web. Prevailing environmental conditions influence diatom phenotypic traits (for example, photophysiology, macromolecular composition and morphology), which in turn affect the transfer of energy, carbon and nutrients to grazers and higher trophic levels, as well as oceanic biogeochemical cycles. The paucity of phenotypic data on Southern Ocean phytoplankton limits our understanding of the ecosystem and how it may respond to future environmental change. Here we used a novel approach to create a 'snapshot' of cell phenotype. Using mass spectrometry, we measured nitrogen (a proxy for protein), total carbon and carbon-13 enrichment (carbon productivity), then used this data to build spectroscopy-based predictive models. The models were used to provide phenotypic data for samples from a third sample set. Importantly, this approach enabled the first ever rate determination of carbon productivity from a single time point, circumventing the need for time-series measurements. This study showed that Chaetoceros simplex was less productive and had lower protein and carbon content during short-term periods of high salinity. Applying this new phenomics approach to natural phytoplankton samples could provide valuable insight into understanding phytoplankton productivity and function in the marine system.

Characterisation of Bone Beneficial Components from Australian Wallaby Bone.

Background: Osteoporosis is a condition in which the bones become brittle, increasing the risk of fractures. Complementary medicines have traditionally used animal bones for managing bone disorders, such as osteoporosis. This study aimed to discover new natural products for these types of conditions by determining mineral and protein content of bone extracts derived from the Australian wallaby. Methods: Inductively coupled plasma-mass spectrometry and Fourier transform infrared spectroscopic analysis were used for mineral tests, proteome analysis was using LC/MS/MS and the effects of wallaby bone extracts (WBE)s on calcium deposition and alkaline phosphatase activity were evaluated in osteogenic cells derived from adipose tissue-derived stem cells (ADSCs). Results: Concentrations of calcium and phosphorus were 26.21% and 14.72% in WBE respectively. Additionally, minerals found were wide in variety and high in concentration, while heavy metal concentrations of aluminium, iron, zinc and other elements were at safe levels for human consumption. Proteome analysis showed that extracts contained high amounts of bone remodelling proteins, such as osteomodulin, osteopontin and osteoglycin. Furthermore, in vitro evaluation of WBEs showed increased deposition of calcium in osteoblasts with enhanced alkaline phosphatase activity in differentiated adipose-derived stem cells. Conclusion: Our results demonstrate that wallaby bone extracts possess proteins and minerals beneficial for bone metabolism. WBEs may therefore be used for developing natural products for conditions such as osteoporosis and further investigation to understand biomolecular mechanism by which WBEs prevent osteoporosis is warranted.

Fingermark initial composition and aging using Fourier transform infrared microscopy (µ-FTIR).

This study investigated fingermark residues using Fourier transform infrared microscopy (µ-FTIR) in order to obtain fundamental information about the marks' initial composition and aging kinetics. This knowledge would be an asset for fundamental research on fingermarks, such as for dating purposes. Attenuated total reflection (ATR) and single-point reflection modes were tested on fresh fingermarks. ATR proved to be better suited and this mode was subsequently selected for further aging studies. Eccrine and sebaceous material was found in fresh and aged fingermarks and the spectral regions 1000-1850cm(-1) and 2700-3600cm(-1) were identified as the most informative. The impact of substrates (aluminium and glass slides) and storage conditions (storage in the light and in the dark) on fingermark aging was also studied. Chemometric analyses showed that fingermarks could be grouped according to their age regardless of the substrate when they were stored in an open box kept in an air-conditioned laboratory at around 20°C next to a window. On the contrary, when fingermarks were stored in the dark, only specimens deposited on the same substrate could be grouped by age. Thus, the substrate appeared to influence aging of fingermarks in the dark. Furthermore, PLS regression analyses were conducted in order to study the possibility of modelling fingermark aging for potential fingermark dating applications. The resulting models showed an overall precision of ±3 days and clearly demonstrated their capability to differentiate older fingermarks (20 and 34 days old) from newer ones (1, 3, 7 and 9 days old) regardless of the substrate and lighting conditions. These results are promising from a fingermark dating perspective. Further research is required to fully validate such models and assess their robustness and limitations in uncontrolled casework conditions.

Refining Fingermark Development using Diacetylene Copolymers on Difficult Surfaces(.).

In further work investigating the intriguing application of diacetylene copolymers in fingermark detection, methods were developed to control (inhibit or enhance) the diacetylene polymerization reaction in fingermarks treated with a mixture of the monomers 2,4-hexadiyne-1,6-bis-(phenylurethane) (HDDPU) and 2,4-hexadiyne-1,6-bis(p-chlorophenylurethane) (HDDCPU) in acetone solution. These methods included the use of a humidity chamber to reduce the amount of background development while promoting development on the fingermark, subjecting developed fingermarks to freezing temperatures and using a solvent to remove unreacted monomer in order to inhibit the polymerization reaction. Developed fingermarks were enhanced by conventional lighting (white light, filtered light) and fast Raman mapping, which was shown to be advantageous over FTIR imaging. This study also demonstrated the applicability of diacetylene copolymer solutions in the covert detection of fingermarks on difficult surfaces. Furthermore, fingermarks were successfully developed with good ridge detail on pig skin (used as a model for human skin, a notoriously difficult surface on which to develop fingermarks).

Phenotypic plasticity of southern ocean diatoms: key to success in the sea ice habitat?

Diatoms are the primary source of nutrition and energy for the Southern Ocean ecosystem. Microalgae, including diatoms, synthesise biological macromolecules such as lipids, proteins and carbohydrates for growth, reproduction and acclimation to prevailing environmental conditions. Here we show that three key species of Southern Ocean diatom (Fragilariopsis cylindrus, Chaetoceros simplex and Pseudo-nitzschia subcurvata) exhibited phenotypic plasticity in response to salinity and temperature regimes experienced during the seasonal formation and decay of sea ice. The degree of phenotypic plasticity, in terms of changes in macromolecular composition, was highly species-specific and consistent with each species' known distribution and abundance throughout sea ice, meltwater and pelagic habitats, suggesting that phenotypic plasticity may have been selected for by the extreme variability of the polar marine environment. We argue that changes in diatom macromolecular composition and shifts in species dominance in response to a changing climate have the potential to alter nutrient and energy fluxes throughout the Southern Ocean ecosystem.

Diacetylene copolymers for fingermark development.

In 1979, Miller and Patel showed that a solution containing two diacetylene monomers, 2,4-hexadiyne-1,6-bis(phenylurethane) (HDDPU) and 2,4-hexadiyne-1,6-bis(p-chlorophenylurethane) (HDDCPU) could be used to develop latent fingermarks on a non-porous surface. In the current work, the same mixture (HDDPU:HDDCPU=10:1, in acetone solution) was used to develop fingermarks on a wide variety of surfaces, both non-porous and porous, including paper. An airbrush system was optimized for the application of the reagent solution. Once the solution evaporates on a surface, the monomers co-crystallize in different ways, depending upon a number of factors, including the surface residue. "Active" co-crystallization leads (with heat or radiation) to the formation of purple polymer, while "inactive" crystallization results in a non-polymerizable white deposit. Fingermark contrast was achieved as a result of active co-crystallization (giving purple polymer) in either the ridges or the furrows, depending upon the surface and other factors. A general observation (supported by spot tests with linseed oil, salt and amino acid solutions) was that on paper, oily materials are more likely to lead to the formation of the purple polymer, while the presence of water inhibits polymerization. However, these observations are not consistent across all other substrates. It is hypothesized that water disrupts hydrogen bonding between diacetylene molecules, and thus prevents the topochemical polymerization of the diacetylenes, which occurs in the solid state between favourably aligned monomers. An interesting observation was the development of fingermarks deposited on paper that had already been treated with the diacetylene reagent.

Fingermark detection on non-porous and semi-porous surfaces using NaYF4:Er,Yb up-converter particles.

This article describes the first use of an anti-Stokes material, or up-converter, for the development of latent fingermarks on a range of non-porous surfaces. Anti-Stokes materials can absorb long-wavelength light and emit light at a shorter wavelength. This property is unusual in both natural and artificial materials and so fingermark detection techniques based on anti-Stokes luminescence are potentially sensitive and selective. Latent fingermarks on luminescent and non-luminescent substrates, including Australian polymer banknotes (a well-known 'difficult' surface), were developed with sodium yttrium tetrafluoride doped with erbium and ytterbium (NaYF(4):Er,Yb) by dry powder, wet powder, and cyanoacrylate staining techniques. This study illustrates the potential of up-converter phosphors for the detection of latent fingermarks.

Thermal development of latent fingermarks on porous surfaces--further observations and refinements.

In a further study of the thermal development of fingermarks on paper and similar surfaces, it is demonstrated that direct contact heating of the substrate using coated or ceramic surfaces at temperatures in excess of 230°C produces results superior to those obtained using hot air. Fingermarks can also be developed in this way on other cellulose-based substrates such as wood and cotton fabric, though ridge detail is difficult to obtain in the latter case. Fluorescence spectroscopy indicates that the phenomena observed during the thermal development of fingermarks can be reproduced simply by heating untreated white copy paper or filter paper, or these papers treated with solutions of sodium chloride or alanine. There is no evidence to suggest that the observed fluorescence of fingermarks heated on paper is due to a reaction of fingermark constituents on or with the paper. Instead, we maintain that the ridge contrast observed first as fluorescence, and later as brown charring, is simply an acceleration of the thermal degradation of the paper. Thermal degradation of cellulose, a major constituent of paper and wood, is known to give rise to a fluorescent product if sufficient oxygen is available [1-5]. However, the absence of atmospheric oxygen has only a slight effect on the thermal development of fingermarks, indicating that there is sufficient oxygen already present in paper to allow the formation of the fluorescent and charred products. In a depletion study comparing thermal development of fingermarks on paper with development using ninhydrin, the thermal technique was found to be as sensitive as ninhydrin for six out of seven donors. When thermal development was used in sequence with ninhydrin and DFO, it was found that only fingermarks that had been developed to the fluorescent stage (a few seconds of heating) could subsequently be developed with the other reagents. In the reverse sequence, no useful further development was noted for fingermarks that were treated thermally after having been developed with ninhydrin or DFO. Aged fingermarks, including marks from 1-year-old university examination papers were successfully developed using the thermal technique.

Four novel alkyl 2-cyanoacylate monomers and their use in latent fingermark detection by mid-infrared spectral imaging.

Four novel alkyl 2-cyanoacrylate monomers (alkyl=1-cyanoethyl, 2-cyanoethyl, trideuteromethyl and pentadeuteroethyl) have been tested for their ability to develop latent fingermarks that can then be visualized using mid-infrared spectral (chemical) imaging. Each of the four monomers was chosen for its potential to produce a strong, isolated infrared spectral band in its corresponding polymer (to provide spectral contrast against most backgrounds), as well as for its potential ability to be fumed onto fingermarks in the manner of conventional ethyl 2-cyanoacrylate (superglue). With the exception of the 2-cyanoethyl 2-cyanoacrylate, which had to be fumed under reduced pressure, all of the monomers were found to be sufficiently volatile to be fumed in a conventional fuming cabinet. All four monomers polymerized selectively on fingermark ridges on a variety of non-porous and semi-porous surfaces, leading to excellent development of the fingermarks. Unfortunately, although high quality mid-infrared spectral images of the fingermarks could be formed for all of the polymers at various frequencies, the new CN or CD stretching vibrations did not give rise to strong enough absorption intensities for good contrast on difficult backgrounds such as polymer banknotes. However, in the 1-cyanoethyl 2-cyanoacrylate polymer, the presence of the additional nitrile group had the unintended but desirable effect of shifting the strong CO absorption to higher frequencies, moving it away from interfering banknote absorptions. This enabled fingermark contrast to be achieved even against the intaglio printing on the banknotes.

Detection of illicit substances in fingerprints by infrared spectral imaging.

FTIR and Raman spectral imaging can be used to simultaneously image a latent fingerprint and detect exogenous substances deposited within it. These substances might include drugs of abuse or traces of explosives or gunshot residue. In this work, spectral searching algorithms were tested for their efficacy in finding targeted substances deposited within fingerprints. "Reverse" library searching, where a large number of possibly poor-quality spectra from a spectral image are searched against a small number of high-quality reference spectra, poses problems for common search algorithms as they are usually implemented. Out of a range of algorithms which included conventional Euclidean distance searching, the spectral angle mapper (SAM) and correlation algorithms gave the best results when used with second-derivative image and reference spectra. All methods tested gave poorer performances with first derivative and undifferentiated spectra. In a search against a caffeine reference, the SAM and correlation methods were able to correctly rank a set of 40 confirmed but poor-quality caffeine spectra at the top of a dataset which also contained 4,096 spectra from an image of an uncontaminated latent fingerprint. These methods also successfully and individually detected aspirin, diazepam and caffeine that had been deposited together in another fingerprint, and they did not indicate any of these substances as a match in a search for another substance which was known not to be present. The SAM was used to successfully locate explosive components in fingerprints deposited on silicon windows. The potential of other spectral searching algorithms used in the field of remote sensing is considered, and the applicability of the methods tested in this work to other modes of spectral imaging is discussed.

Classification of heterogeneous solids using infrared hyperspectral imaging.

Methods were explored for the classification of heterogeneous powder mixtures using Fourier transform infrared (FT-IR) hyperspectral image data. The images collected were non-congruent, meaning that samples of the same mixture do not have the same spatial arrangements of their components in their images. In order to classify such images on a one-image-per-object basis, dimension reduction was carried out so as to produce a score or feature vector for each image that preserved information about the heterogeneity of the sample. These feature vectors were then classified using discriminant analysis (DA) or soft independent modeling of class analogy (SIMCA). The most successful approach was the use of a median-interquartile range "super-spectrum" as the feature vector representing each image; using principal component analysis (PCA) DA classification, 87.5% of training samples were correctly classified using leave-one-out cross-validation, and 100% of a test set were correctly classified. This compares with 52.5% and 72%, respectively, when single-point spectra were used to classify the samples.

Elemental bio-imaging of melanoma in lymph node biopsies.

The spatial distribution of trace elements in human lymph nodes partially infiltrated by melanoma cells was determined by elemental bio-imaging. Imaging of (31)P within the nodal capsule and normal lymph node tissue showed a clear demarcation of the tumour boundary, with a significant decrease in relative (31)P concentration within the tumour. The location of the tumour boundary was confirmed by haematoxylin and eosin staining of serial sections and observation by light microscopy. Further enhancement of the tumour boundary was achieved by imaging the (31)P/(34)S ratio. (31)P/(66)Zn ratio images showed a decreasing ratio beyond the tumour boundary that extended into peritumour normal lymph node tissue.

Revisiting the thermal development of latent fingerprints on porous surfaces: new aspects and refinements.

Although the ability to develop latent fingerprints on paper using heat alone has been noted previously, it has been considered impractical for casework and inferior to other techniques. Here a new refinement of the technique is demonstrated for the high quality development of latent fingerprints on porous surfaces such as paper. Fingerprints deposited on various papers were developed by exposing them to hot air with a temperature in the vicinity of 300 degrees C, for periods of c. 10-20 sec. Several different heating methods were tested. The novel observation was made that after shorter heating times, fluorescent prints could be observed. These became visible after longer heating times, as noted by earlier workers, but with greatly improved contrast compared with their results. Prints from various donors (and aged prints) were developed with excellent ridge contrast. Direct heating methods (such as with a hot plate or press) produced inferior results. The refined technique, which is simple, safe and inexpensive compared with conventional methods, has great potential for use in forensic laboratories.

An examination of the sequence of intersecting lines using attenuated total reflectance-Fourier transform infrared spectral imaging.

In this study, the potential of attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectral imaging as a technique to determine the sequence of line crossings was examined. The technique was successful in determining the sequence of heterogeneous line intersections produced using ballpoint pens and laser printers. By imaging at characteristic frequencies, it was possible to form spectral images showing the spatial distribution of the materials. By examining the spectral images from the inks, it was possible to determine whether the ink was above or below the toner. In blind testing, ATR-FTIR spectral imaging results were directly compared to those obtained by eight experienced forensic document examiners using methods regularly employed in casework. ATR-FTIR spectral imaging was shown to achieve a 100% success rate in the blind tests, whereas some incorrect sequence determinations were made by the forensic document examiners when using traditional techniques. The technique was unable to image ink-jet printing, gel pens, roller ball pens, and felt-tip pens, and was also unable to determine the sequence of intersecting ballpoint pen lines.

The application of infrared chemical imaging to the detection and enhancement of latent fingerprints: method optimization and further findings.

Fourier transform infrared (FTIR) chemical imaging allows the collection of fingerprint images from backgrounds that have traditionally posed problems for conventional fingerprint detection methods. In this work, the suitability of this technique for the imaging of fingerprints on a wider range of difficult surfaces (including polymer banknotes, various types of paper, and aluminum drink cans) has been tested. For each new surface, a systematic methodology was employed to optimize settings such as spectral resolution, number of scans, and pixel aggregation in order to reduce collection time and file-size without compromising spatial resolution and the quality of the final fingerprint image. The imaging of cyanoacrylate-fumed fingerprints on polymer banknotes has been improved, with shorter collection times for larger image areas. One-month-old fingerprints on polymer banknotes have been successfully fumed and imaged. It was also found that FTIR chemical imaging gives high quality images of cyanoacrylate-fumed fingerprints on aluminum drink cans, regardless of the printed background. Although visible and UV light sources do not yield fingerprint images of the same quality on difficult, nonporous backgrounds, in many cases they can be used to locate a fingerprint prior to higher quality imaging by the FTIR technique. Attempts to acquire FTIR images of fingerprints on paper-based porous surfaces that had been treated with established reagents such as ninhydrin were all unsuccessful due to the swamping effect of the cellulose constituents of the paper.

Forensic analysis of bicomponent fibers using infrared chemical imaging.

The application of infrared chemical imaging to the analysis of bicomponent fibers was evaluated. Eleven nominally bicomponent fibers were examined either side-on or in cross-section. In six of the 11 samples, infrared chemical imaging was able to spatially resolve two spectroscopically distinct regions when the fibers were examined side-on. As well as yielding characteristic infrared spectra of each component, the technique also provided images that clearly illustrated the side-by-side configuration of these components in the fiber. In one case it was possible to prepare and image a cross-section of the fiber, but in general the preparation of fiber cross-sections proved very difficult. In five of the 11 samples, the infrared spectra could be used to identify the overall chemical composition of the fibers, according to a published classification scheme, but the fiber components could not be spatially resolved. Difficulties that are inherent to conventional "single-point" infrared spectroscopy, such as interference fringing and sloping baselines, particularly when analyzing acrylic type fibers, were also encountered in the infrared chemical image analysis of bicomponent fibers. A number of infrared sampling techniques were investigated to overcome these problems, and recommendations for the best sampling technique are given. Chemical imaging results were compared with those obtained using conventional fiber microscopy techniques.

Forensic applications of infrared chemical imaging: multi-layered paint chips.

This paper examines the potential of infrared chemical (hyperspectral) imaging as a technique for the forensic analysis of automotive paint chips in particular, and multicomponent (e.g., layered) samples in general. Improved sample preparation procedures for the infrared analysis of paint chips are detailed, with the recommendation that where mounting resins are chemically incompatible with the sample, it is better to mount and section the sample in a soft wax from which the sections can be removed and pressed into a KBr disk for transmission analysis. Infrared chemical images of multilayered paint chips have been successfully obtained, with the chief advantage over conventional infrared analysis being that thousands of infrared spectra are collected in a few minutes across the whole sample, at a spatial resolution of around 5 microm. As with conventional infrared spectroscopy, chemical species can be identified from their spectra, but the wealth of information available can be also extracted in a number of different ways that make multicomponent spectral (and hence chemical) comparisons between two samples easy to visualize and understand. In one approach, the infrared chemical images of two paint chips being compared side-by-side can be viewed as a "movie," in which each frame is an intensity map of the two samples at a given wavenumber (frequency) value. In another approach, the spectra (pixels) in the image files are classified into chemically similar groups, resulting in a "cluster" image that makes it possible to simultaneously compare all of the layers in two paint chips. These methods are applicable to other multicomponent samples, and also to other chemical imaging techniques.

The detection and enhancement of latent fingermarks using infrared chemical imaging.

The use of a new technique, Fourier transform infrared (FTIR) chemical imaging, has been demonstrated for the enhancement of latent fingermarks on a number of surfaces. Images of untreated fingermarks on glass backgrounds with excellent ridge detail were acquired using infrared chemical imaging. High quality fingermarks on glass backgrounds were also developed using ethyl cyanoacrylate (super glue) fuming and subsequent infrared chemical imaging. This new method allows the collection of images from backgrounds that traditionally pose problems for current fingermark detection methods. The background may, for example, be highly colored, have a complex pattern, or possess other pattern or image characteristics that make it difficult to separate fingermark ridges using traditional optical or luminescent visualization. One background that has proven to be a challenging surface for the development of latent fingermarks is the Australian polymer banknote. To demonstrate the power and applicability of infrared chemical imaging, fingermarks fumed with ethyl cyanoacrylate were successfully imaged from Australian polymer banknotes.