Profiling ephedrine/pseudoephedrine and methamphetamine synthesised from benzaldehyde, nitroethane and dimethyl carbonate.

In previous work, a novel pathway for the synthesis of ephedrine/pseudoephedrine and methamphetamine using the precursors benzaldehyde, nitroethane and dimethyl carbonate was investigated, and an impurity profile presented. This paper presents chiral and stable isotope ratios of ephedrine/pseudoephedrine and methamphetamine synthesised by this pathway. Based on the chiral profile and the more negative δ13C (avg. -33.2‰) and more positive δ2H values, it is possible to distinguish ephedrine/pseudoephedrine and methamphetamine prepared from this pathway from those of "fully synthetic", "semi-synthetic" or "natural" origin. The more positive δ2H values of methamphetamine from this pathway allowed for differentiation from methamphetamine produced from phenyl-2-propanone. It was noted, however, that the use of stable isotope profiling would likely be limited when a benzaldehyde source having a negative δ2H value was used as a precursor. Furthermore, the stable isotope values alone could not be used to differentiate from methamphetamine prepared by the Akabori-Momotani reaction, highlighting the need for combination with impurity profiling.

Once upon a twine: The Donna Steele murder investigation and an improved methodology for the comparison of synthetic twine.

In 2017 Cooktown resident Donna Steele was murdered and the case remained unsolved for more than a year. The forensic evidence from the investigation included two lengths of synthetic twine and a new protocol for the comparison of twine was developed to enhance the potential value of any evidence. The method was developed using 23 samples of similar twine collected across Australia. Traditional methods of physical and microscopic comparisons and polymer analysis by infrared spectroscopy were retained. Micro-spectrophotometry was used as an objective assessment of colour and was able to identify five groupings within the background samples. Measurements of hydrogen and carbon stable isotopic composition provided further delineation of the background samples. Combining traditional methods with micro-spectrophotometry and stable isotope measurements, the two case samples were found to be distinct from the background population and were indistinguishable when compared to each other.

Fingerprinting eukaryotic metabolism across the animal kingdom using position-specific isotope analysis (PSIA) 13C/12C measurements.

Despite differences in their overall metabolism, eukaryotes share a common mitochondrial biochemistry. We investigated how this fundamental biochemistry supports overall metabolism using a high-resolution carbon isotope approach, position-specific isotope analysis. We measured carbon isotope 13C/12C cycling in animals, focusing on amino acids that are formed in mitochondrial reactions and are most metabolically active. Carboxyl isotope determinations for amino acids showed strong signals related to common biochemical pathways. Contrasting isotope patterns were measured for metabolism associated with major life history patterns, including growth and reproduction. Turnover of proteins and lipids as well as gluoconeogensis dynamics could be estimated for these metabolic life histories. The high-resolution isotomics measurements fingerprinted metabolism and metabolic strategies across the eukaryotic animal kingdom, yielding results for humans, ungulates, whales, and diverse fish and invertebrates in a nearshore marine food web.

Stable isotope characterisation of 3,4-methylenedioxyphenyl-2-propanone and 3,4-methylenedioxyamphetamine prepared from piperonal.

RATIONALE: Stable isotopic ratios can provide information for illicit drug profiling. The research presented here investigated the variations in stable isotopic ratios of hydrogen (δ2 H), carbon (δ13 C), nitrogen (δ15 N) and oxygen (δ18 O) during the synthesis of MDP2P (3,4-methylenedioxyphenyl-2-propanone) and MDA (3,4-methylenedioxyamphetamine) prepared via the 'nitrostyrene' route. METHODS: Samples of MDA and MDP2P were synthesised from two isotopically characterised starting materials, piperonal and nitroethane. The isotopic compositions of the nitrostyrene intermediate (3,4-methylenedioxyphenyl-2-nitropropene, MDP2NP) and products MDP2P and MDA were also measured by isotope ratio mass spectrometry. RESULTS: A significantly negative change occurred to δ2 H values during the production of MDP2NP, MDP2P and MDA, indicating a mechanism that favours inclusion or retention of 1 H over 2 H. This suggests that the δ2 H compositions of MDA/MDP2P prepared from piperonal will not provide information on the synthetic history. Minimal changes were observed in δ13 C composition during the synthesis of MDP2NP, MDP2P and MDA, and minimal δ15 N compositional changes occurred in MDP2NP and MDA. Progressing from piperonal to MDP2NP, a minimal change occurred to δ18 O composition. A variable change to δ18 O was observed from MDP2NP with one sample becoming more positive in δ18 O composition and two samples becoming more negative. Progressing from MDP2NP to MDA, a significant negative change occurred to δ18 O composition. CONCLUSIONS: The changes to stable isotopic ratios observed during the preparation of MDA and MDP2P from piperonal may prove useful when attempting to compare batch-to-batch variations between seizures and provide information with tactical intelligence applications.

Feeding Sugars to Stingless Bees: Identifying the Origin of Trehalulose-Rich Honey Composition.

The beneficial disaccharide, trehalulose, is a feature of stingless bee honey, while not dominant in any other foods. By experimentally feeding sugar solutions to confined colonies of the Australian stingless bee Tetragonula carbonaria, the origin of trehalulose has now been established. Complete conversion of fed sucrose was observed, by analysis of the honey, forming trehalulose (64-72%) with lesser erlose (18-23%), fructose (9-12%), and minor glucose detected. Remarkably, feeding solutions of glucose/fructose (1:1) mixtures did not result in trehalulose/erlose formation. Hence, stingless bees with natural access to floral nectar high in sucrose will produce honey high in trehalulose, with its associated beneficial properties. Any temptation to artificially increase trehalulose content by feeding sucrose to stingless bees would produce "fake" honey lacking key natural phytochemicals available to the foraging bee. The sucrose-fed fake and natural honey were however readily distinguished via isotope ratio mass spectrometry δ13C values, to combat such potential indirect adulteration.

Food Matrix Reference Materials for Hydrogen, Carbon, Nitrogen, Oxygen, and Sulfur Stable Isotope-Ratio Measurements: Collagens, Flours, Honeys, and Vegetable Oils.

An international project developed, quality-tested, and measured isotope-delta values of 10 new food matrix reference materials (RMs) for hydrogen, carbon, nitrogen, oxygen, and sulfur stable isotope-ratio measurements to support food authenticity testing and food provenance verification. These new RMs, USGS82 to USGS91, will enable users to normalize measurements of samples to isotope-delta scales. The RMs include (i) two honeys from Canada and tropical Vietnam, (ii) two flours from C3 (rice) and C4 (millet) plants, (iii) four vegetable oils from C3 (olive, peanut) and C4 (corn) plants, and (iv) two collagen powders from marine fish and terrestrial mammal origins. An errors-in-variables regression model included the uncertainty associated with the measured and assigned values of the RMs, and it was applied centrally to normalize results and obtain consensus values and measurement uncertainties. Utilization of these new RMs should facilitate mutual compatibility of stable isotope data if accepted normalization procedures are applied and documented.

Stable carbon isotope diagnostics of mammalian metabolism, a high-resolution isotomics approach using amino acid carboxyl groups.

The carbon isotopic compositions of amino acids are increasingly measured to characterize diets and metabolic response to diets. We report a new high-resolution system to measure the stable carbon isotopic composition of carboxyl atoms within amino acids. The automated system used HPLC to separate amino acids followed by addition of ninhydrin for decarboxylation and transfer of the evolved CO2 to a stable isotope ratio mass spectrometer for δ13CCARBOXYL measurement. The ninhydrin reaction was conducted at acidic pH (1.5) and elevated temperature (160 oC) giving yields close to 100% for most common amino acids. Eight mammalian keratin samples from herbivores (kudu and caribou), omnivores (humans) and carnivores (bowhead and humpback zooplanktivorous whales) were analysed with this new system. The data provide an initial calibration of reference materials to be used in studies of this type and is the first report of carboxyl carbon isotope distributions in mammals. Results showed widespread 13C enrichments in both essential and non-essential amino acid carboxyl groups, likely linked to decarboxylation of amino acids during normal metabolism. Analyses of non-essential amino acid isotope profiles showed (1) consistent and general taxon-level metabolic differences between the herbivore, human and whale samples, (2) marked differences among individual humans, ruminants and whales (3) evidence for gluconeogenesis in the wildlife samples, and (4) extensive 13C enrichment likely associated with fasting in the humpback whale sample. Future mammalian research related to the metabolism of growth, reproduction, aging and disease may benefit from using this technique. Values obtained for internationally available samples USGS42 and USGS43 (Tibetan and Indian human hair) provide a first characterization of reference materials for δ13CCARBOXYL profiles.

Position-specific 13 C/12 C analysis of amino acid carboxyl groups - automated flow-injection analysis based on reaction with ninhydrin.

RATIONALE: The fundamental level of stable isotopic knowledge lies at specific atomic positions within molecules but existing methods of analysis require lengthy off-line preparation to reveal this information. An automated position-specific isotope analysis (PSIA) method is presented to determine the stable carbon isotopic compositions of the carboxyl groups of amino acids (δ13 CCARBOXYL values). This automation makes PSIA measurements easier and routine. METHODS: An existing high-performance liquid chromatography (HPLC) gas handling interface/stable isotope ratio mass spectrometry system was modified by the addition of a post-column derivatisation unit between the HPLC system and the interface. The post-column reaction was optimised to yield CO2 from the carboxyl groups of amino acids by reaction with ninhydrin. RESULTS: The methodology described produced δ13 CCARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences (Δ13 CCARBOXYL values) between amino acids over a 1-year period. First estimates are presented for the δ13 CCARBOXYL values of a number of internationally available amino acid reference materials. CONCLUSIONS: The PSIA methodology described provides a further dimension to the stable isotopic characterisation of amino acids at a more detailed level than the bulk or averaged whole-molecule level. When combined with on-line chromatographic separation or off-line fraction collection of protein hydrolysates the technique will offer an automated and routine way to study position-specific carboxyl carbon isotope information for amino acids, enabling more refined isotopic studies of carbon uptake and metabolism.

A comparison of plastic cable ties based on physical, chemical and stable isotopic measurements.

Plastic cable ties can be utilised in a range of serious criminal activities and a comparison of cable ties, or fragments, may form part of the physical evidence presented to a Court of law. This research assessed the potential value of evidence based on the analysis of plastic cable ties. Twenty packets of black coloured plastic cable ties (nominally 200mm×4.8mm) were purchased in pack sizes ranging from 25 to 100 individual cable ties (Brisbane, Australia, March 2015). Representative samples from each packet were visually examined, compared and tested to determine their physical dimensions, chemical compositions and stable isotopic compositions (δ2H, δ13C and δ15N). All of the individual cable ties from a given packet were found to be indistinguishable with respect to appearance, physical, chemical and isotopic measurements (within-batch variability). Individual cable ties were also found to be isotopically homogeneous with respect to hydrogen, carbon and nitrogen. All of the cable ties analysed were found to have very similar chemical compositions and to be manufactured predominantly from nylon 6,6. The elemental compositions of composite samples, prepared from each packet, were found to be highly variable and, as such, were of very limited value. Cable ties from ten of the twenty packets were uniquely characterised by physical appearance (between-batch variability). Physical measurements such as the width, thickness and tooth-count of the grip section did not provide additional discrimination. Cable ties from nineteen of the twenty packets were uniquely characterised by isotopic composition, based on δ2H and δ15N measurements. Samples from two packets of Crescent brand cable ties were found to be indistinguishable with respect to all of the tests applied in this study. These two packets were inadvertently purchased from the same retailer and had the same barcode and batch number. It was considered a reasonable assumption that these two packets originated from the same manufacturing batch. The authors reason that a likelihood ratio (that might be presented to a Court of law) can be derived from this type of discrete data based on a calculation of the possible combinations of distinguishable objects (unordered sampling with replacement) in a convenience sample collected from the background population. In this example, a database of 19 distinguishable objects can yield a likelihood ratio as high as 210, with a verbal equivalent of "moderately strong support" for a proposition that two cable ties have the same isotopic composition because they originate from the same batch rather than by random chance.

Prawn biomonitors of nutrient and trace metal pollution along Asia-Pacific coastlines.

To assess coastal ecosystem status and pollution baselines, prawns were collected from the commercial catches of eight Asia-Pacific countries (Australia, Bangladesh, Indonesia, Myanmar, Philippines, Pakistan, Sri Lanka and Thailand). Samples collected from 21 sites along regional coastlines were analysed for trace metal and stable isotopic compositions of H, C, N, O and S. A combination of simple averaging and multivariate analyses was used to evaluate the data. Sites could be assigned to easily recognise polluted and unpolluted groups based on the prawn results. Some filter-feeding clams were also collected and analysed together with the benthic-feeding prawns, and the prawns generally had lower trace metal burdens. Climate change effects were not strongly evident at this time, but altered ocean circulation and watershed run-off patterns accompanying future climate change are expected to change chemical patterns recorded by prawns along these and other coastlines. Stable isotopes, especially (15)N, can help to distinguish between relatively polluted and unpolluted sites.

Isotopic and Elemental Composition of Roasted Coffee as a Guide to Authenticity and Origin.

This study presents the stable isotopic and elemental compositions of single-origin, roasted coffees available to retail consumers. The δ(13)C, δ(15)N, and δ(18)O compositions were in agreement with those previously reported for green coffee beans. The δ(15)N composition was seen to be related to organic cultivation, reflected in both δ(2)H and δ(18)O compositions. The δ(13)C composition of extracted caffeine differed little from that of the bulk coffee. Stepwise discriminant analysis with jackknife tests, using isotopic and elemental data, provided up to 77% correct classification of regions of production. Samples from Africa and India were readily classified. The wide range in both isotopic and elemental compositions of samples from other regions, specifically Central/South America, resulted in poor discrimination between or within these regions. Simpler X-Y and geo-spatial plots of the isotopic data provided effective visual means to distinguish between coffees from different regions.

Isotopic profiling of seized benzylpiperazine and trifluoromethylphenylpiperazine tablets using δ13C and δ15N stable isotopes.

This paper demonstrates the use of isotopic analysis of 23 benzylpiperazine (BZP) and trifluoromethylphenylpiperazine (TFMPP) containing tablets seized on two independent occasions by the Northern Territory (NT) Police, Australia. Isolation (High Performance Liquid Chromatography (HPLC)) of BZP and TFMPP followed by Isotope Ratio Mass Spectrometry (IRMS) (carbon and nitrogen stable isotopes) analysis was performed. Results are presented for δ13C and δ15N values of the respective piperazine analogues. The isotopic data and statistical analysis suggest a common source of manufacture for the BZP samples but suggest different sources for the TFMPP isolated from the corresponding BZP containing tablets investigated. The use of IRMS in this case study demonstrated the ability to obtain information regarding the BZP/TFMPP sources unattainable via conventional chemical analysis.

Precursor discrimination of designer drug benzylpiperazine using δ13C and δ15N stable isotopes.

Advances in analytical technology and emerging techniques have resulted in the increased exploitation of chemical and isotopic profiling for source linkage/discrimination of illicit drugs for forensic purposes. Although not routinely used for illicit drug investigations, such information has been obtained and its application demonstrated through the use of isotope ratio mass spectrometry (IRMS). There is a solid platform of research available relating to the isotopic analysis of methylenedioxymethamphetamine (MDMA) and methamphetamine (MA), however with the recently flourishing designer drug market it was of interest to examine the isotopic profiles of the popular 'party drug' benzylpiperazine hydrochloride (BZP·HCl). A preliminary analysis of δ13C and δ15N isotopic ratios in BZP·HCl products and corresponding synthetic intermediates (piperazine·HCl) synthesized in-house from three different precursor suppliers was conducted using IRMS. Analysis of the δ13C and δ15N isotopic data indicated that discrimination and correct grouping of all the intermediates and some of the product samples examined in this study were achievable.

Stable isotope and chemical compositions of European and Australasian ciders as a guide to authenticity.

This paper presents a data set derived from the analysis of bottled and canned ciders that may be used for comparison with suspected counterfeit or substitute products. Isotopic analysis of the solid residues from ciders (predominantly sugar) provided a means to determine the addition of C4 plant sugars. The added sugars were found to comprise cane sugar, high-fructose corn syrup, glucose, or combinations. The majority of ciders from Australia and New Zealand were found to contain significant amounts of added sugar, which provided a limited means to distinguish these ciders from European ciders. The hydrogen and oxygen isotopic compositions of the whole ciders (predominantly water) were shown to be controlled by two factors, the water available to the parent plant and evaporation. Analysis of data derived from both isotopic and chemical analysis of ciders provided a means to discriminate between regions and countries of manufacture.

The role of isotope ratio mass spectrometry as a tool for the comparison of physical evidence.

This paper considers how likelihood ratios can be derived for a combination of physical, chemical and isotopic measurements. Likelihood ratios were formulated based on the characteristics of a small convenience sample of 20 duct tapes. The propositions considered were: The physical and isotopic characteristics of ten rolls of duct tape were shown to be consistent throughout each roll. The width and thickness of the tapes and the density of the scrim fibres provided equivalent information and the combined physical characteristics provided a basis upon which to discriminate between many of the samples. Scatter-plots and confidence ellipses provided a convenient method to group the isotopic composition of the tape backing material and provided a basis to discriminate between samples which were physically indistinguishable. Considering both the physical and isotopic characteristics it was possible, at best, to ascertain that the evidence provided moderately strong support for the proposition that two samples of tape were derived from the same batch (LR=400). Kernel density estimates were used to model the distribution of isotopic compositions of the backing material. Using this technique it was possible to estimate objectively the probability that a sample with given characteristics could be drawn, at random, from the background population and to calculate a likelihood ratio based on the propositions above. The strength of evidence which could be presented by either model was ultimately limited by the size of the background sample.

Laparoscopic removal of abdominal cerclage at 19 weeks' gestation.

We discuss laparoscopic removal of an abdominal cerclage in a 39-year-old woman, gravida 4, para 0, abortus 3, who presented at 19 weeks' gestation with ruptured membranes. This patient had a failed previous vaginal cerclage. An abdominal cerclage was performed at the time of abdominal myomectomy. A subsequent pregnancy was diagnosed, with ruptured membranes at 19 weeks' gestation, and the patient opted for pregnancy termination. After laparoscopic removal of the cerclage, cervical laminaria were placed, and the patient underwent an uncomplicated dilation and curettage procedure the following day.