A method for the sensitive targeted screening of synthetic cannabinoids and opioids in whole blood by LC-QTOF-MS with simultaneous suspect screening using HighResNPS.com.

A sensitive method for the qualitative screening of synthetic cannabinoids and opioids in whole blood was developed and validated using alkaline liquid-liquid extraction (LLE) and liquid chromatography-time-of-flight mass spectrometry (LC-QTOF-MS). Estimated limits of detection for validated compounds ranged from 0.03 to 0.29 µg/L (median, 0.04 µg/L) for the 27 opioids and from 0.04 to 0.5 µg/L (median, 0.07 µg/L) for the 23 synthetic cannabinoids. Data processing occurred in two stages; first, a targeted screen was performed using an in-house database containing retention times, accurate masses and MS-MS spectra for 79 cannabinoids and 53 opioids. Suspect screening was then performed using a database downloaded from the crowd sourced NPS data website HighResNPS.com which contains mass, consensus MS-MS data and laboratory-specific predicted retention times for a far greater number of compounds. The method was applied to 61 forensic cases where synthetic cannabinoid or opioid screening was requested by the client or their use was suspected due to case information. CUMYL-PEGACLONE was detected in two cases and etodesnitazine, 5 F-MDMB-PICA, 4-cyano-CUMYL-BUTINACA and carfentanil were detected in one case each. These compounds were within the targeted scope of the method but were also detected through the suspect screening workflow. The method forms a solid base for expansion as more compounds emerge onto the illicit drug market.

Characteristics of analytically confirmed gamma-hydroxybutyrate (GHB) positive patients in the emergency department: presentation, poly-drug use, disposition and impact on intensive care resource utilisation.

BACKGROUND: Gamma-hydroxybutyrate is a potent central nervous system depressant with a narrow recreational dose window and analytical detection time. We describe data relating to intoxicated patients presenting to emergency departments across metropolitan Adelaide who tested positive for gamma-hydroxybutyrate. This work was part of the Emergency Department Admission Blood Psychoactive Testing study. METHODS: Over a 15-month period, patients presenting to four metropolitan emergency departments with symptoms of drug intoxication were enrolled in the study. The methodology involved the collection of demographic and clinical data and a de-identified blood sample which underwent comprehensive toxicological analysis. Gamma-hydroxybutyrate was determined using an acid-catalysed cyclisation followed by liquid-liquid extraction and gas chromatography-mass spectrometry. Data relating to samples positive for gamma-hydroxybutyrate were examined. RESULTS AND DISCUSSION: A total of 1120 patients were enrolled between March 2019 and May 2020, 309 of whom were positive for gamma-hydroxybutyrate (27.6%). Of these, 256 (83%) were also positive for metamfetamine (methamphetamine). The most common clinical observation in gamma-hydroxybutyrate-positive patients was central nervous system depression (89%). There was a significant relationship between gamma-hydroxybutyrate status and sex; although males outnumbered females in absolute terms, a higher proportion of females (32%) tested positive for gamma-hydroxybutyrate than males (25%, P = 0.0155). Blood gamma-hydroxybutyrate concentrations ranged from 10 to 651 mg/L (0.096-6.2 mmol/L) and increasing gamma-hydroxybutyrate concentration correlated with severe toxicity. The presence of gamma-hydroxybutyrate had a significant impact on the patient discharge destination: the majority (69.2%) of gamma-hydroxybutyrate-positive patients were managed and discharged from the emergency department or their attached short stay wards. A significantly higher proportion of gamma-hydroxybutyrate-positive patients were admitted to the intensive care unit (28.2%) compared with gamma-hydroxybutyrate-negative patients (12.7%, chi-squared = 36.85, P <0 .001). Gamma-hydroxybutyrate positive cases accounted for 45.8% of all study-related intensive care unit admissions. CONCLUSIONS: Gamma-hydroxybutyrate is commonly detected in illicit drug-related emergency department presentations and is detected disproportionately in the patient cohort who require intensive care unit level care.

Establishing the protocols for the South Australian Emergency Department Admission Blood Psychoactive Testing (EDABPT) programme for drug surveillance.

OBJECTIVE: ED presentations because of illicit use of psychotropic drugs and pharmaceuticals result in significant medical harm and resource consumption. Patient assessment is complicated by the regular emergence of new psychoactive substances, difficulties associated with their identification and a lack of information about their effects. Here we report the protocol for the Emergency Department Admission Blood Psychoactive Testing (EDABPT) programme, an observational study utilising clinical data capture and definitive drug identification to assess the medical impact and patterns of illicit drug use in the community, and their geographic and temporal fluctuations. The study provides data to an early warning system targeting an improved public health response to emerging drugs of concern. METHODS: Enrolment of adult patients presenting with suspected illicit drug use occurs at four major EDs in a single urban setting. Clinical and demographic data are collected by treating clinicians. Blood samples are collected at presentation and frozen on site prior to transport to a specialised forensic facility for comprehensive toxicological screening. RESULTS: Results are fed back to clinicians and disseminated more broadly via an existing local early warning system. Targeted warnings and public health releases are instigated where heightened risk or harm is identified. CONCLUSION: The study pairs city-wide patient enrolment with analytically confirmed toxicology results to allow broad sampling and identification of illicit drugs causing medical harm. It provides a mechanism for the identification of new agents as they emerge in the community, delivers a relevant and reliable source of information for public health agencies and clinicians and supplements existing local early warning mechanisms.

Identification of a thermal degradation product of CUMYL-PEGACLONE and its detection in biological samples.

The structural diversity of synthetic cannabinoids makes it a challenging task to have a comprehensive screening method for this class of drugs. The difficulty is increased by the fact that some synthetic cannabinoids undergo thermal decomposition during common routes of administration, such as smoking or vaping. CUMYL-PEGACLONE is a relatively new synthetic cannabinoid which has a structural variant from most other synthetic cannabinoids: a γ-carbolinone core. To investigate its thermal stability, CUMYL-PEGACLONE was heated in an oven at temperatures ranging from 200 to 350o C, and a major thermal degradation product, N-pentyl-γ-carbolinone, was subsequently identified. Unlike some other synthetic cannabinoids, the thermal degradation product of CUMYL-PEGACLONE is not one of its known metabolites, nor were any known metabolites detected during the thermal stability experiments. The degradation product was formed in significant amounts at temperatures above 250°C, and has been detected (along with CUMYL-PEGACLONE) in case samples, including post-mortem blood and urine, and residue found at a scene.

A Fatality Involving Furanylfentanyl and MMMP, with Presumptive Identification of Three MMMP Metabolites in Urine.

The prevalence of new psychoactive substances (NPS) on the illicit drug market continues to grow, with new analogs being routinely synthesized. Routes of administration for these compounds are also diversifying, and recent research has shown an increase in the incorporation of NPS into vaping liquids. Among the most commonly encountered NPS are the cathinone and fentanyl analogs. Fentanyl analogs in particular have been implicated in a significant number of deaths, usually in combination with other prescription and illicit drugs. We report the case of a 44-year-old male with a history of polysubstance abuse found deceased at his home address. Items located within the vicinity of the deceased were found to contain furanylfentanyl and 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MMMP also known as MTMP, MMTMP, Irgacure 907 and Caccure 907). Both of these compounds were detected in the post-mortem peripheral blood of the deceased: furanylfentanyl at 1.6 ng/mL and MMMP at 6.7 ng/mL. MMMP is an unrestricted, commercially available photo-initiator used in the printing and polymer industry, which structurally can be classed as a highly modified cathinone. Although MMMP has been found previously in drug seizures, this is the first fatality in which MMMP has been detected. A number of other prescription and illicit drugs were also detected in the blood. MMMP was not detected in the post-mortem urine; however three metabolites, beta-hydroxy-MMMP, beta-hydroxy-MMMP-sulfoxide and beta-hydroxy-MMMP-sulfone, were presumptively identified. The significance of MMMP to the cause of death is uncertain as its pharmacological and toxicological profile is unclear.

A Case Study Involving U-47700, Diclazepam and Flubromazepam-Application of Retrospective Analysis of HRMS Data.

The number of new psychoactive substances (NPS) available is constantly increasing, making it difficult for toxicology laboratories to keep screening methods up to date. Full scan high-resolution mass spectrometry (HRMS) is a versatile technique which allows for progressive updating of spectral databases to increase the scope of screening. It also allows for retrospective screening of data-specifically, reprocessing of data files using an updated spectral database without the need for re-extraction or reanalysis.The coronial case reported here illustrates the application of retrospective processing of HRMS data in the detection of emerging NPS. A 28-year-old male with a history of illicit drug use was found deceased at home. Initial routine screening of the post-mortem peripheral blood identified only methylamphetamine, amphetamine and trace amounts of lorazepam. A compound with an accurate mass and isotope ratio consistent with the opioid AH-7921 was also detected in the liquid chromatography (LC)-HRMS screen; however; the retention time and mass spectrum did not match the library. Further investigation confirmed the compound to be U-47700, another opioid and structural isomer of AH-7921. Several months later, after additional NPS had been added to the in-house HRMS database, retrospective screening of the HRMS data was performed, revealing the presence of designer benzodiazepines, diclazepam and flubromazepam as well as the psychedelic drug 2,5-dimethoxy-4-chloroamphetamine (DOC). Quantitative analysis gave the following results in peripheral post-mortem blood: U-47700 (330 µg/L), diclazepam (70 µg/L), flubromazepam (10 µg/L), methylamphetamine (290 µg/L) and amphetamine (150 µg/L) (DOC not quantitated). These substances, along with lorazepam and etizolam, were also confirmed in the post-mortem urine and an investigation into blood and urinary metabolites was carried out. All analyses were performed using the same LC-quadrupole-time of flight method. The cause of death was aspiration (of gastric content into airways and lungs) due to mixed drug toxicity.

The Increasing Use and Abuse of Tapentadol and Its Incorporation Into a Validated Quantitative Method.

Tapentadol is a centrally acting synthetic analgesic which is prescribed for the treatment of a range of chronic pain conditions. Its use in treating various pain conditions is increasing and, as with other opioids, it has the potential to be abused. We describe a three-stage incorporation of tapentadol into validated screening and quantitative methods through: (i) addition of retention time/mass spectral data to a database, (ii) qualitative validation and (iii) quantitative validation. This represents an efficient and flexible approach to the incorporation of new compounds of interest to existing screening methods. Tapentadol was analyzed in blood and serum samples using alkaline liquid-liquid extraction with identification and quantitation by liquid chromatography/time-of-flight mass spectrometry. In a series of six post-mortem cases where tapentadol was detected but was not a primary causative factor in death, blood concentrations ranged from 0.01 to 1.0 mg/L. In two cases where tapentadol was a significant contributor to death, post-mortem blood concentrations were 1.7 and 3.9 mg/L. In one of these fatal cases, ante-mortem blood and serum were also analyzed. The tapentadol concentration in the post-mortem blood was 30% higher than in the ante-mortem blood after a post-mortem interval of 13 days, indicating some potential for post-mortem redistribution. The measured ante-mortem blood:serum ratio was 1.7, and is the first such ratio to be reported. Other drugs were detected in almost all cases, with the majority being prescription analgesics, sedatives and antidepressants. The number of cases in which tapentadol has been detected has increased in recent years, highlighting the importance of screening for this drug in forensic toxicological laboratories.

Air Quality Inside Police Drug Safes and Drug Storage Areas.

Storage of drug-based evidence inside sealed safes may allow chemical vapors to accumulate, creating concerns of drug exposure by inhalation, or the possibility of cross-contamination of drug evidence. Air samples were taken from inside eight drug safes and one small storage room at nine city and country police stations, as well as a large centralized drug evidence storage vault, in New South Wales (NSW), Australia. Sorbent tubes containing charcoal were used to determine whether any drug residues could be detected in the air, and to identify the types of chemicals present. Carbon traps were extracted and analyzed by LC-MS-MS for a suite of 22 licit and illicit drug residues and 2 metabolites. Carbon traps and SPME fibers were also analyzed by GC-MS for general volatile organic compound (VOC) residues. No detectable drug residues, either as airborne dust or vapor, were found in the safes, the storage room or the large central repository vault. No drugs were detected in any of the 34 urine samples collected at 8 of the 10 sampling locations, while only one of the five hair samples was positive for cocaine (9 pg/mg) provided by police exhibit officers at 3 of the 10 sampling locations. VOC analysis identified a variety of solvents associated with drug manufacture, plasticisers, personal care products and volatiles associated with plants such as cannabis. The results indicate that strong chemical odours emanating from drug safes are unlikely to be drug residues due to low volatility of drugs, and are more likely VOCs associated with their manufacture or from plant growing operations. Consideration should be given to the quality of air flow in rooms in which safes are housed and the use of air filtering inside safes to reduce the likelihood of VOC accumulation, and therefore the risk of human exposure.

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.

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.

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.

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.

An alternative approach for assessment of liquid chromatography-mass spectrometry matrix effects using auto-sampler programmed co-injection.

We report the use of auto-sampler programmable functions to co-inject analyte standard solution and matrix extract to assess ion enhancement and suppression (matrix effects) in LC-MS. This is effectively an automated post-extraction addition (APEA) procedure, emulating the manual post-extraction addition (PEA) approach widely adopted for assessment of matrix effects. To verify that APEA was comparable to the conventional PEA approach, matrix effects were determined using both methods for a selection of 31 illicit and pharmaceutical drugs in 10 different human urine extracts. Matrix effects measured using APEA were statistically indistinguishable from manual PEA methodology for 27 of the 31 drugs. Of the four drugs that showed significant differences using the two methods, three differed by less than 2 %, which is within the expected accuracy limits required for matrix effect determinations. The remaining analyte, trimeprazine, was found to degrade in the spiked PEA matrix extract, accounting for the difference between matrix effects measured by the PEA and APEA approaches. APEA enables a single matrix extract to be assessed at multiple analyte concentrations, resulting in a considerable reduction in sample preparation time. In addition, APEA can reduce the quantity of analyte-free sample matrix required for matrix effect assessment, which is an important consideration in certain analytical and bioanalytical fields. This work shows that APEA may be considered as an acceptable alternative to PEA for the assessment of matrix effects in LC-MS method validation and may be applicable to a variety of matrices such as environmental samples.

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.

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.

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.

An audit of the toxicology findings in 555 medico-legal autopsies finds manner of death changed in 5 cases.

An audit of toxicological analysis in Coronial autopsies performed at Forensic Science South Australia was conducted on the cases of three pathologists. Toxicological analysis had been performed in 555 (68 %) from a total of 815 autopsies. It was found that the proffered manner of death was changed from the provisional report (provided immediately after the post-mortem examination) in five cases (just under 1 %) as a consequence of the toxicological findings. This is a limited study as it is retrospective, not all cases had toxicological analysis and the findings are constrained by the range of the substances that could be detected. Nonetheless, the audit supports the application of toxicological analysis in medico-legal death investigation and suggests that an inclusive policy should be adopted.