Shanlin Fu

The use of pre‐precursors to illicitly manufacture 3,4‐methylenedioxyphenyl‐2‐propanone (MDP2P) for the synthesis of 3,4‐methylenedioxymethylamphetamine (MDMA) has emerged in clandestine laboratories. Methyl 3‐(3,4‐methylenedioxyphenyl)‐2‐methyl glycidate (MMDMG) and ethyl 3‐(3,4‐methylenedioxyphenyl)‐2‐methyl glycidate (EMDMG) have recently been seized in clandestine laboratories in New South Wales, Australia. These glycidate esters can be converted to MDP2P via acid or base hydrolysis. This study investigated factors influencing MDP2P yield from MMDMG and EMDMG through both pathways. For acid hydrolysis, hydrochloric, sulphuric, phosphoric and acetic acids were examined at different reaction times and temperatures. Reactions at 100°C produced higher MDP2P yields than those at room temperature, and 18 h was sufficient for complete conversion. Hydrochloric and sulphuric acids gave the highest yields after 18 h at 100°C (79% and 78%, respectively), phosphoric acid gave moderate conversion (~30%), and acetic acid produced poor yields (< 10%). The conversion proceeded via two previously unreported diol‐ester intermediates. Base hydrolysis involved heating glycidate esters with sodium hydroxide in alcohol, forming sodium piperonyl methyl ketone (PMK) glycidate, followed by reaction with aqueous acid. Heating MMDMG in methanol or EMDMG in ethanol for 5 h produced sodium PMK glycidate. This intermediate was unstable in acidic aqueous solution and rapidly converted to MDP2P at room temperature. Yields obtained using hydrochloric, sulphuric and phosphoric acids (73%, 71% and 69%, respectively) were comparable to acid hydrolysis, whereas acetic acid produced lower yields (25%). This work may assist law enforcement agencies to predict illicit drug yields from pre‐precursors and support investigations into emerging illicit drug synthesis trends.

In recent years, a shift in the dynamics of the illicit drug market has been experienced, resulting in increasingly complex challenges for law-enforcement. In the past, the illicit drug market has ...

The infiltration of xylazine into the illicit drug supply, particularly as a fentanyl adulterant, has raised significant safety concerns due to its severe toxicological effects and the absence of an effective antidote for targeted therapeutic intervention. Despite the growing implications of xylazine in overdose cases, presumptive testing methods for its detection remain underdeveloped in forensic and public health settings. This study addresses the urgent need for reliable screening tools by evaluating the applicability of chemical spot tests, microcrystalline tests, commercially available xylazine immunoassay test strips, and other field-deployable drug test kits. A comprehensive panel of chemical spot test reagents was tested, and cobalt thiocyanate, Eosin Y (pH 7.0), and Liebermann, Mandelin, Mecke, Marquis, Scott, and Young reagents produced distinct and rapid color changes with xylazine. In parallel, different microcrystalline test reagents were evaluated, and the resulting microcrystal morphology was assessed by using light microscopy and scanning electron microscopy (SEM). Gold chloride reagent yielded immediate, reproducible, and well-defined microcrystals across different xylazine formulations, establishing it as the most effective reagent for microcrystalline testing. All xylazine test strips yielded clear positive test results when tested with xylazine. In contrast, many commercial and on-site screening tools displayed cross-reactivity or false-positive results with xylazine, raising concerns about its specificity and reliability. These findings highlight the need to develop and validate xylazine-specific presumptive screening tests to facilitate forensic investigations, support harm reduction efforts, and enable informed, safer decision-making by people who use drugs (PWUDs).

Altrenogest, also known as allyltrenbolone, is a synthetic form of progesterone used therapeutically to suppress unwanted symptoms of estrus in female horses. Altrenogest affects the system by decreasing levels of endogenous gonadotrophin and luteinizing and follicle-stimulating hormones, which in turn decreases estrogen and mimics the increase of progesterone production. This results in more manageable mares for training and competition alongside male horses while improving the workplace safety of riders and handlers. However, when altrenogest is administered, prohibited steroid impurities such as trendione, trenbolone, and epitrenbolone can be detected. It has been assumed that greater concentrations of these steroid impurities are present in injectable preparations and, therefore, pose a greater risk of causing anabolic effects when administered. For this reason, and due to the necessity of this therapeutic substance for the safety of thoroughbred racing participants, a metabolomic approach investigating the differentiation of two main administration routes was conducted. Liquid chromatography high-resolution mass spectrometry analysis of equine urine samples found five sulfated compounds, estrone sulfate, testosterone sulfate, 2-methoxyestradiol sulfate, pregnenolone sulfate, and cortisol sulfate, with the potential to differentiate between oral and intramuscularly administered altrenogest using a random forest classification model. The best model results, comparing two horses’ administration normalized peak area datasets, gave an AUC score of 0.965 with a confidence level of 95% (between 0.931 and 0.995). Identifications of these compounds were confirmed with assistance from the Shimadzu Insight Explore Assign feature, together with MS/MS spectrum and retention time matching of purchased and synthesized reference standards. This study proposes a new potential application for metabolomic multi-tool workflows and machine learning models in a forensic toxicological context.

Detecting the use of bisphosphonates (BPs) in equine athletes is of interest to regulators and laboratories due to the threat to welfare issues for the potential to provide analgesic effects and manipulating bone structure. The detection of BPs in biological matrices is challenging due to erratic biological elimination and inconsistent analytical recoveries. Therefore, complementary approaches are needed to provide evidence of their misuse in racehorses. BPs have two sub‐classes: nitrogenous and non‐nitrogenous. This study investigated plasma elimination following administration of one example from each sub‐class, together with changes in endogenous eicosanoid and corticosteroids. Zoledronic acid (ZA) and tiludronic acid (TA) were administered by IV infusion to 8 thoroughbred horses with an 11‐month washout period between each administration. Sample preparation for quantification of BPs by liquid chromatography–tandem mass spectrometry (LC–MS/MS) utilised a two‐step solid phase extraction (SPE) consisting of polymeric reversed‐phase followed by weak anion exchange prior to derivatisation using trimethyl orthoacetate. Endogenous biomarkers were analysed after protein precipitation and SPE with polymeric reversed‐phase prior to liquid chromatography–high resolution mass spectrometry (LC‐HRMS) using data independent acquisition. The LC–MS/MS analysis showed ZA was undetectable after 8 h post‐administration while TA was detected up to the final collection point of 28 days post‐administration. The LC‐HRMS analysis utilised targeted (i.e., prior inclusion list of compounds) approaches to monitor level changes of eicosanoid and corticosteroid biomarkers. Putative biomarkers were identified and now subject to validation for translation into routine sample analysis for improved retrospectivity to detecting BP misuse in equine plasma.

Accurate estimation of the postmortem interval (PMI) is crucial in forensic medico-legal investigations to understand case circumstances (e.g. narrowing down list of missing persons or include/exclude suspects). Due to the complex decomposition chemistry, estimation of PMI remains challenging and currently often relies on the subjective visual assessment of gross morphological/taphonomic changes of a body during decomposition or entomological data. The aim of the current study was to investigate the human decomposition process up to 3 months after death and propose novel time-dependent biomarkers (peptide ratios) for the estimation of decomposition time. An untargeted liquid chromatography tandem mass spectrometry–based bottom-up proteomics workflow (ion mobility separated) was utilized to analyse skeletal muscle, collected repeatedly from nine body donors decomposing in an open eucalypt woodland environment in Australia. Additionally, general analytical considerations for large-scale proteomics studies for PMI determination are raised and discussed. Multiple peptide ratios (human origin) were successfully proposed (subgroups < 200 accumulated degree days (ADD), < 655 ADD and < 1535 ADD) as a first step towards generalised, objective biochemical estimation of decomposition time. Furthermore, peptide ratios for donor-specific intrinsic factors (sex and body mass) were found. Search of peptide data against a bacterial database did not yield any results most likely due to the low abundance of bacterial proteins within the collected human biopsy samples. For comprehensive time-dependent modelling, increased donor number would be necessary along with targeted confirmation of proposed peptides. Overall, the presented results provide valuable information that aid in the understanding and estimation of the human decomposition processes. Graphical Abstract

The current approach to equine anti-doping is focused on the targeted detection of prohibited substances. However, as new substances are rapidly being developed, the need for complimentary methods for monitoring is crucial to ensure the integrity of the racing industry is upheld. Lipidomics is a growing field involved in the characterisation of lipids, their function and metabolism in a biological system. Different lipids have various biological effects throughout the equine system including platelet aggregation and inflammation. A certain class of lipids that are being reviewed are the eicosanoids (inflammatory markers). The use of eicosanoids as a complementary method for monitoring has become increasingly popular with various studies completed to highlight their potential. Studies including various corticosteroids, non-steroidal anti-inflammatories and cannabidiol have been reviewed to highlight the progress lipidomics has had in contributing to the equine anti-doping industry. This review has explored the techniques used to prepare and analyse samples for lipidomic investigations in addition to the statistical analysis and potential for lipidomics to be used for a longitudinal assessment in the equine anti-doping industry.