Chris Kostakis

Increases in use of novel synthetic stimulant are not directly linked to decreased use of 3,4-methylenedioxy-N-methylamphetamine (MDMA)

A decline in 3,4-methylenedioxy-N-methylamphetamine (MDMA) use in Adelaide, Australia from 2009 to 2010 was confirmed by us previously. Reports suggested that the shortage in MDMA supply was associated with an increased prevalence of other synthetic stimulants, but quantitative measurements were unavailable. To obtain objective data on the community use of synthetic stimulants, we collected wastewater samples from multiple treatment plants in Adelaide, Australia from 2009 to 2011 and analysed them using solid-phase extraction/liquid chromatography/tandem mass spectrometry (SPE-LC-MS/MS), targeting MDMA and some of the most reported synthetic cathinones and piperazines. Data were temporally compared. MDMA and six other synthetic stimulants were detected and quantified in wastewater samples. While MDMA level decreased markedly from 2009 to 2010 and remained low in 2011, localized increased use of mephedrone, methylone, methylenedioxypyrovalerone (MDPV), benzylpiperazine (BZP), 3-trifluoromethylphenylpiperazine (TFMPP), but not methcathinone, was observed in 2010 and 2011. This suggested that the decline in MDMA use was associated with an increase in the use of a number of other synthetic stimulants. However, the lag time from the decrease in MDMA to the increase in use of a number of these stimulants, together with the highly regionalized use of all synthetic stimulants except methcathinone indicates that there was no direct population wide substitution in response to the reduction in MDMA.

Towards finding a population biomarker for wastewater epidemiology studies

Wastewater analysis has the potential to provide objective information on community drug use. Introducing a population biomarker (PB) in the sample analysis may significantly reduce errors in the back-calculation associated with population estimation and wastewater volume measurement. A number of potential PBs have been suggested but no systematic evaluation has been conducted so far. This study evaluated the eligibility of the previously suggested PB candidates (creatinine, cholesterol, coprostanol and cotinine) as well as three new ones (cortisol, androstenedione and 5-hydroxyindoleacetic acid (5-HIAA)) using five criteria. We assessed the quantification method, affinity to particulate matter and stability of candidates in wastewater, as well as the constancy of inter-day excretion and correlation between excretion and census population. All PB candidates were quantifiable in wastewater. Cholesterol and coprostanol were eliminated from further consideration due to affinity to particulate matters in the wastewater. Creatinine, cortisol and androstenedione were disqualified for stability reasons. On a population scale, both cotinine and 5-HIAA were excreted (RSD=8.01 ± 1.13% and 10.20 ± 0.89%, respectively) at a constant rate and concentrations of each correlated well with the census population (r=0.9809 and 0.9442, respectively). Overall, both cotinine and 5-HIAA are eligible PBs, but the neurotransmitter metabolite 5-HIAA may be more suitable for international comparisons.

Evaluation of pre‐analysis loss of dependent drugs in wastewater: stability and binding assessments

Wastewater analysis has the potential to provide objective and timely data on population drug consumption, but some crucial factors such as pre-analysis drug loss during sample storage and filtration could affect the accuracy and reliability of the method, and these uncertainties have yet to be fully assessed. This study was designed to evaluate analyte stability in wastewater stored under different conditions with the aim of optimizing the sample storage procedures for future studies. It also investigated whether there is significant analyte loss during filtration before sample extraction and storage after that. The studied substances and metabolites were: cotinine, cocaine and its metabolite benzoylecgonine, phenethylamines amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), opioids including codeine, methadone, 6-monoacetylmorphine (MAM) and morphine. In most situations, storing samples at 4 °C is sufficient to stabilize analytes for at least 2 weeks, and refrigeration is unnecessary during sample transportation within 3 days. However, additional measures need to be taken if unstable analytes such as cocaine and MAM are to be analyzed. No significant analyte loss was observed in the filtration process or in reconstituted extract stored at 4 °C or -20 °C for 2 weeks. By choosing stable analytes and proper storage conditions, wastewater analysis has the potential to provide accurate data for estimation of community drug use.

Diabetic Ketoacidosis – A possible Complicating Factor in Deaths associated with Drug Overdose

Two cases are described where diabetic ketoacidosis was found in conjunction with significant levels of prescription drugs. Case 1: A 45-year-old woman with a history of insulin-dependent diabetes mellitus was found to have Armanni-Ebstein nephropathy characteristic of hyperglycaemia with a vitreous humour glucose level of 63.1 mmol/L and a β-hydroxybutyrate level of 14.25 mmol/L. Ancillary toxicological evaluation revealed a lethal level of sertraline (2.5 mg/L), with an elevated level of methadone (0.23 mg/L). Death was due to diabetic ketoacidosis complicating mixed drug toxicity. Case 2: A 27-year-old man with a history of insulin-dependent diabetes mellitus was found to have Armanni-Ebstein nephropathy with a vitreous humour glucose level of 51.7 mmol/L and a β-hydroxybutyrate level of 18.6 mmol/L. Ancillary toxicological evaluation revealed a potentially lethal level of methadone of 0.39 mg/L. Death was attributed to diabetic ketoacidosis complicating methadone toxicity. These cases demonstrate a situation where drug toxicity led to diabetic ketoacidosis resulting in death most likely from a combination of factors. Measuring vitreous humour glucose and β-hydroxybutyrate levels is important in individuals with histories, or scene evidence, of insulin-dependent diabetes mellitus, in addition to toxicological screening when there is evidence of possible drug taking. It appears that drug intoxication in both cases had impaired the ability to administer insulin, resulting in the development over time of diabetic ketoacidotic states. Lethal mechanisms were, therefore, more complex than simple drug toxicity or diabetic ketoacidosis in isolation.

Circumstances of Death and Diagnostic Difficulties in Brushfire Fatalities

Abstract:  The deaths of 10 bushfire (brushfire) victims (aged 2–59 years; M/F 1:1) from the files of Forensic Science SA in Adelaide, South Australia, over an 8‐year period (January 2002 to December 2009) are reported. Nine of the victims were found in or near motor vehicles. Death was attributed to incineration (N = 5), trauma from bushfire‐related vehicle crashes (N = 2), inhalation of products of combustion with hyperthermia (N = 1), inhalation of products of combustion (N = 1), and undetermined (N = 1). Death scenes covered large areas and involved many victims. Loss of infrastructure and closure of local roads owing to debris limited access and made the finding of bodies difficult. Bodies in such fires may be exposed to the damaging effects of weather and animal predation. Heat damage hindered pathological assessment with resultant delays in identification. Assessment of antemortem injuries and determination of causes of death were also complicated by the condition of some of the bodies.

The presence of licit and illicit drugs in police stations and their implications for workplace drug testing

The presence of licit and illicit drug residues on surfaces was studied in 10 police stations and a central drug evidence store in New South Wales, Australia, with the results compared to similar surfaces in four public buildings (to establish a community baseline). The results of almost 850 workplace surface swabs were also compared to the outcome of drug analysis in urine and hair samples volunteered by police officers. Surfaces were swabbed with alcohol and the swabs were extracted and analysed by LC-MS/MS. Low level concentrations of the more commonly used drugs were detected at four public sites and one restricted access police office facility. Surface swabs taken in 10 city and country police stations yielded positive results for a broader suite of drugs than at background sites however 75-93% of the positive drug results detected in police stations were below 40ng, which is only slightly greater than the largest background result measured in the current study. This study indicates that contamination issues are more likely to be focussed in higher risk areas in police stations, such as counters and balances in charge areas, and surfaces within drug safes although front reception counters also returned surface contamination. All 64 urine samples collected in this study were negative, while only 2 of the 11 hair samples collected from donors resulted in trace concentrations for cocaine, but not its metabolite benzoylecgonine. Positive hair samples were only obtained from police donors in very high risk jobs, indicating that the exposure risk is low. Minor changes to the materials used as work surfaces, and some procedural changes in police stations and large evidence stores are suggested to decrease the likelihood of drugs contaminating work surfaces, thereby reducing the potential exposure of police officers to drugs in the workplace.

Quantification of licit and illicit drugs on typical police station work surfaces using LC-MS/MS

Licit and illicit drug use is widespread in the community and as a result, drug residues can be transferred onto handles and work surfaces in shared places. Police officers are more likely than members of the public to encounter drug residues while performing their work duties. As a result, sampling and analysis methodology must be developed to assess their work environments to determine which drug residues are present, at what concentration, and how long they may persist on the work surface to attempt to determine whether the residues pose a risk. The following reports a method for determining residues on work surfaces using cotton swabs, solvent extraction and analysis with LC-MS/MS. LC column type, swab extraction time, solvent composition, and analyte suppression were investigated. The reported method is simple, allows high throughput at low cost, simultaneously analyses for 23 licit and illicit drugs and metabolites, and has the scope for inclusion of additional analytes. Additionally, the method could be adapted easily to suit other organic chemicals, such as pesticides. The optimised method was used to investigate the persistence of 23 drugs and metabolites on five different surface types commonly found in police stations, under both dark and illuminated incubation conditions. The results demonstrated that different drugs within a given class can have dramatically different rates of loss, and general predictions cannot be made for other drugs in the same class. Illuminated incubation conditions generally accelerated the loss of drugs on surfaces, either by enhanced volatilisation, photocatalysis, or a combination of both. Only drugs such as amphetamine, methamphetamine and ketamine deviated from this trend because their disappearance from all surfaces under both incubation conditions was so rapid that no real difference was observed.

Supported liquid extraction (SLE) for the analysis of methylamphetamine, methylenedioxymethylamphetamine and delta-9-tetrahydrocannabinol in oral fluid and blood of drivers

Since 2006, the South Australian Government has been conducting roadside oral fluid testing of drivers for the illicit drugs methylamphetamine (MA), methylenedioxymethylamphetamine (MDMA) and Δ(9)-tetrahydrocannabinol (THC) using the Securetec Drugwipe II Twin and Alere DDS 805 AP saliva collection kit. Forensic Science South Australia carries out the confirmatory analysis by LC/MS for the positive test results of oral fluid roadside testing along with the pre-screened ELISA positive road traffic accident blood samples. The number of blood and oral fluid samples received in the laboratory has been steadily increasing during this time, and over 10,000 samples were received in 2014. The proportion of positive results from these samples has also been increasing over the decade of driver drug testing, and this data is presented. A simple and efficient method has been developed for the analysis of the three drugs using Biotage Isolute(®) SLE+ 96-well plates. Sample preparation included 1:1 dilution with a dilute ammonia solution for buffered oral fluids (1:3 dilution for blood samples), and addition of deuterated internal standards. Samples were loaded onto the phase, left to absorb for 5min then eluted with methyl t-butyl ether (MTBE). The samples were evaporated and reconstituted in methanol. LC/MS analysis was performed on an AB Sciex 5500 Q-Trap in positive ion mode, monitoring 3 transitions for each analyte. Separation was achieved on a Restek Ultrabiphenyl 50×2.1mm column with a gradient system of acetonitrile/0.1% formic acid over 5min. Method validation and recoveries were carried out on drug free ante mortem blood and DDS buffer solution provided by Alere, Australia. Recoveries above 80% were achieved for MA and MDMA at a concentration of 25ng/mL, whilst recoveries of greater than 65% were achieved for THC at 4.5ng/mL. Accuracy and precision were acceptable down to the LLOQ for all three analytes (5, 5 and 1ng/mL for MA, MDMA and THC, respectively). Mean matrix effects were 1.0, 0.97 and 0.78 in DDS buffer and 0.96, 0.96 and 0.62 in blood for MA, MDMA and THC, respectively. Linearity was achieved up to 1250ng/mL for MA and MDMA, and 112ng/mL for THC (r(2)>0.999 for all analytes). The method is designed for easy transfer to an automated liquid handling platform.

Work place drug testing of police officers after THC exposure during large volume cannabis seizures

Police officers responsible for the seizure and removal of illegally grown cannabis plants from indoor and outdoor growing operations face the prospect of THC exposure while performing their work duties. As a result, a study investigating the amount of THC on hands and uniforms of officers during raids on cannabis growing houses (CGHs) and forest cannabis plantations (FCPs) and in the air at these sites was conducted. Swabs of gloves/hands, chests, and heads/necks were collected and analysed for THC. Results of hand swabs indicated that officers removing plants from FCPs were exposed to THC concentrations up to 20 times those involved in raids at CGHs, which was mainly associated with the number and size of plants seized. Air samples collected inside cannabis houses showed no detectable THC. Air samples collected inside the cargo area of the storage trucks used during FCP raids indicated that THC can be volatilised when lush plants are compressed by other seized plants loaded on top of them in the truck over a period of several days, allowing composting of plants at the bottom of the load to commence. The elevated temperature and humidity inside the truck may assist the decarboxylation of THCA to THC, as well as increasing the rate of volatilisation of THC. More than 100 urine samples were collected from officers in raids on both CGHs and FCPs and all tested negative for THC. Removal of cannabis plants by officers often resulted in cuts, abrasions and ruptured blisters on exposed skin surfaces, particularly at FCPs. The results in this study suggest that even when small areas of damaged skin are directly exposed to THC by contact transfer, the likelihood of showing a positive THC urine test is low.

A Validated Method for the Screening of 320 Forensically Significant Compounds in Blood by LC/QTOF, with Simultaneous Quantification of Selected Compounds

A broad drug screening method for toxicologically significant drugs and metabolites in whole blood using liquid chromatography time-of-flight mass spectrometry (LC/QTOF) was developed and comprehensively validated. The method qualitatively screens for 320 compounds while simultaneously quantifying 39. Compounds were extracted from the blood using alkaline liquid/liquid extraction and chromatographic separation was achieved in 12 min. The QTOF was operated using positive mode electrospray ionization using data dependent acquisition. Qualitative validation was performed for all 320 compounds, and included selectivity, recovery, limit of detection, matrix effects, carryover and extract stability. The limits of detection were in the low to sub ng/mL range for the majority of compounds. Full quantitative validation was performed for 39 compounds and accuracy and precision were within 15 and 18%, respectively. The qualitative data processing method uses an in-house retention time, accurate mass and MSMS spectral database, which can be easily updated with new compounds of interest as they emerge onto the market, without affecting method performance. The use of a non-targeted data acquisition method coupled with targeted data processing has proven to be a highly versatile, efficient and robust approach to screening, well suited to meet the needs of the modern toxicology laboratory involved in systematic toxicological analysis.