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.

Recent trends in barbiturate detection in medicolegal deaths.

A study was undertaken of 51 cases where barbiturates were detected in post-mortem blood samples from 2000 to 2019 at Forensic Science South Australia, Adelaide, Australia. The cause of death was drug toxicity in only 27 (53%) (M:F = 19:8; age range 19-74yrs, mean 46yrs). In 17 cases, barbiturate toxicity was the primary cause of death, 14 due to pentobarbitone and 3 to phenobarbitone. All were suicides. Barbiturates were obtained by online purchase from overseas sources in 9 cases (33%), and through veterinary practice in 2 cases (7%). Drug toxicity deaths where barbiturates were detected rose from 1 in 2000-2004 to 11 in 2015-2019, and those where deaths were primarily due to barbiturate toxicity rose from 1 in 2000-2004 to 9 in 2015-2019. However, the mere detection of barbiturates in post mortem samples did not equate with illicit use, as 23 of the deaths (45%) were due to natural causes in individuals prescribed barbiturates for epilepsy. The usefulness of examining subset populations separate from accrued national data is also demonstrated in the significantly younger age of decedents in South Australia dying from deliberately administered barbiturates (46 yrs) compared to the national average of 57.9 yrs. The reasons for this difference will require further investigation as this may impact upon local suicide prevention strategies.

Delivering harm reduction to the community and frontline medical practitioners through the South Australian Drug Early Warning System (SADEWS).

Australia does not have a formal drug early warning system. A coordinated program of fixed or event-based drug-checking is expensive and provides harm reduction information to atargeted user group. The South Australian Drug Early Warning System (SADEWS) is an informal inter-agency collaboration which rapidly and confidentially exchanges contemporary,evidence-based information about drug seizures, usage trends and clinical outcomes associated with drug use in South Australia. Information is sourced from policing, forensic analysis,waste-water analysis, medical research, clinical data and directly from people using drugs. SADEWS exchanges information relating to new drug emergences and clusters of adverseoutcomes following drug use, amongst members via secure digital platforms. The diverse but complimentary expertise of members allows a comprehensive assessment of changes tothe baseline risk associated with drug use and, where a potential community harm is identified, enables the timely delivery of warnings through formal mechanisms existing withinmember agencies. It is expected that these warnings contribute to significantly reduced medical consequences associated with community drug use through decreased drug overdosefatalities and hospital presentations rates, contributing to reduced healthcare costs. Importantly, this drug early warning system is politically risk-free, is achieved simply and without external funding or significant administrative overheads.

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.

Characteristics of drowning deaths in an inner city river.

A study was undertaken of all drowning deaths that occurred over a 30-year period from 1988 to 2017 in the urban section of the River Torrens, Adelaide, South Australia, an augmented waterway that runs through the central business district. Autopsy records from Forensic Science South Australia (FSSA) were reviewed. There were 34 drownings (0-5 cases/yr) with 28 males and 6 females (M;F = 4.6:1), with an age range for males of 18-76yrs (mean 42.0; SD 18.0) and for females of 20-84yrs (mean 69.3; SD 24.5). There were 15 (44%) accidents, 11 (32%) suicides, 1 (3%) homicide and 7 (21%) undetermined. Of the 22 cases during or after 1994 with complete toxicology reports, 10 (45%) had a blood alcohol concentration (BAC) of greater than 0.05% (g/100 mL) with an illicit substance detected in 4 (18%) cases: (MDMA (3,4-methylenedioxymethamphetamine), methylamphetamine and THC (delta-9-tetrahydrocannabinol) acid). The presence of various therapeutic drugs was also detected in 10 cases (45%) including temazepam, fluoxetine, diazepam, olanzapine, amitriptyline, carbamazepine, codeine, citalopram and valproate. Although the numbers of cases were not high, the urban portion of the River Torrens had a much higher number of drowning events per kilometre compared to other inland waterways in South Australia such as the Murray River. This is most likely due to the vulnerability that exists for intoxicated individuals in the city from falls into the water and to the availability of the river as a means of suicide to members of the adjacent urban population.

The complexities associated with new psychoactive substances in influent wastewater: The case of 4-ethylmethcathinone.

Consumption of new psychoactive substances (NPS) is an international problem for health, policing, forensic, and analytical laboratories. The transience of these substances in the community, combined with continual slight structural changes to evade legislation makes the elucidation of NPS an analytical challenge. This is amplified in a matrix as complex as wastewater. For that reason, suspect and non-target methodologies, employing high resolution mass spectrometry are the most appropriate current tool to facilitate the identification of new and existing compounds. In the current work, a qualitative screening method of influent wastewater using liquid chromatography-high resolution mass spectrometry showed a strong signal at m/z 192.1382 - identical to that of two NPS standards that were in our method (pentedrone and 4-methylethcathinone), and with identical fragment ions, but the retention times did not match. This work shows the methodology followed to identify this compound, highlighting the challenges of the identifying "new" compounds in influent wastewater.

HighResNPS.com: An Online Crowd-Sourced HR-MS Database for Suspect and Non-targeted Screening of New Psychoactive Substances.

The number of new psychoactive substances (NPS) is constantly increasing. However, although the number might be large, most NPS have a low prevalence of use, so keeping screening libraries updated with the relevant analytical targets becomes a challenge. One way to ensure sufficient screening coverage is to use shared high resolution-mass spectrometry (HR-MS) databases, such as HighResNPS.com: a free, online, spreadsheet-format, crowd-sourced HR-MS database for NPS screening. The aims of this study were (i) to present the database to the scientific community and (ii) to verify that the HighResNPS database can be utilized in suspect screening workflows for LC-HR-MS instruments and software from four different instrument vendors. A sample was spiked with 10 NPS, and participating laboratories then analyzed the sample with their respective HR-MS vendor platforms and the HighResNPS database. The HighResNPS data were obtained via a spreadsheet converted to fit the import specifications of the different vendor platforms. Suspect screening was performed using LC-HR-MS vendor platforms from Thermo Fisher, Waters, Bruker and Agilent. All 10 NPS were identified in at least three workflows used for the four different vendor platforms. Multiple users have submitted data to HighResNPS for the same NPS, which resulted in multiple true-positive identifications for these NPS. Suspect screening with LC-HR-MS can be based on diagnostic fragment ions reported by users of different vendor platforms and can support NPS identification in biological samples and/or seizure analyses when no reference standard is available in-house. The present work clearly demonstrates that HighResNPS data is compatible with instruments and screening software from at least four different vendor platforms. The database can thus serve as a useful add-on in LC-HR-MS screening workflows.

Investigating the appearance of new psychoactive substances in South Australia using wastewater and forensic data.

New psychoactive substances (NPS) have increased in use and popularity worldwide. Wastewater analysis has been successfully applied to evaluate illicit drugs use within a population. However, for NPS, such an approach may be limited due to low doses of NPS combined with their ever-changing composition and usage. The dynamic nature of the NPS market means use may be opportunistic, infrequent, and with few users. Hence, the use of complementary information sources is recommended to improve the knowledge on NPS consumption. The aim of this study was to investigate the changing landscape of NPS use on a community scale by combining wastewater analysis and forensic toxicology. Forensic analysis provided specific information on NPS prevalence in post-mortem blood samples in Adelaide, South Australia over five years, while wastewater analysis showed community use over the same period. A qualitative liquid chromatography--high resolution mass spectrometry method was initially used to screen the wastewater samples. A total of 24 NPS were found: 6 in wastewater only, 13 in forensic post-mortem toxicology samples only, and 5 in both. As these results showed the presence of NPS, a targeted method was subsequently employed to quantify levels of these NPS in wastewater. Temporal trends were found in wastewater with distinct tendencies for synthetic cathinones visible over the period studied.

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.

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.

Increasing the linear dynamic range in LC-MS: is it valid to use a less abundant isotopologue?

Liquid chromatography-mass spectrometry (LC-MS) has quickly become the analytical method of choice in forensic toxicology laboratories due to its ability to detect a very wide range of compounds in a single analysis. One of the major limitations of LC-MS however, is a relatively limited linear dynamic range for quantitation. A new approach to combating this problem is to use the [+1 M + H]+ isotope mass peak for quantitation, thereby reducing saturation at the detector and extending the linear range. This is particularly useful in full-scan applications, such as quadrupole-time-of-flight (QTOF) mass spectrometry, where the isotopic mass peaks are acquired as a matter of course. Due to the variation in abundance of naturally occurring isotopes for common elements, especially 13 C, this technique has the potential to lead to additional quantitative error. Through a review of published isotope ratio mass spectrometry data, we have assessed this potential for error and found that it is likely to be less than 2% and unlikely to be more than 4%, although this may not apply to compounds containing high numbers of nitrogen or sulphur atoms. This additional potential error must be considered before using this technique as it may not be appropriate for all applications. We have deemed it fit for purpose for our application and demonstrate the applicability of this technique to a quantitative LC-TOF method. © 2017 Commonwealth of Australia. Drug Testing and Analysis © 2017 John Wiley & Sons, Ltd.

Porous silicon mass spectrometry as an alternative confirmatory assay for compliance testing of methadone.

Porous silicon based surface-assisted laser desorption ionization mass spectrometry (pSi SALDI-MS) is an analytical technique well suited for high throughput analysis of low molecular weight compounds from biological samples. A potential application of this technology is the compliance monitoring of opioid addiction programmes, where methadone is used as a pharmacological treatment for drugs such as heroin. Here, we present the detection and quantification of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) from water and clinical samples (saliva, urine, and plasma) from opioid dependent participants using pSi SALDI-MS. A one-step solvent phase extraction using chloroform was developed for the detection of methadone from clinical samples for analysis by pSi SALDI-MS. Liquid chromatography-mass spectrometry (LC-MS) was used as a comparative technique for the quantification of methadone from clinical saliva and plasma samples. In all cases, we obtained a good correlation of pSi SALDI-MS and LC-MS results, suggesting that pSi SALDI-MS may be an alternative procedure for high-throughput screening and quantification for application in opioid compliance testing. Copyright © 2016 John Wiley & Sons, Ltd.

Blunt Craniofacial Trauma as a Manifestation of Excited Delirium Caused by New Psychoactive Substances.

The body of a 19-year-old male was found apparently concealed underneath bushes with recent head and facial trauma, and multiple superficial abrasions. Subsequently, it was discovered that the decedent had been running into objects and buildings following the ingestion the evening before of what was thought to be lysergic acid diethylamide (LSD). Blood staining of a nearby wall close to where the body was lying was in keeping with the described behavior. Toxicology revealed 3,4-methylenedioxymethamphetamine (Ecstasy), in addition to two only recently available drugs 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine, (25B-NBOMe), and 1-(3,4-methylenedioxyphenyl)-2-(1-pyrrolidinyl)-1-butanone, (MDPBP). At autopsy, the skull was fractured with cerebral swelling, contusions, and subarachnoid hemorrhage. Death was due to blunt cranial trauma against a background of mixed drug toxicity. The case demonstrates a rare cause of death in a drug-induced acute delirium, as well as highlighting two new designer street drugs that may result in significant aberrant behavior.

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.

Bacterial degradation of risperidone and paliperidone in decomposing blood.

The stability of two benzisoxazole antipsychotics was determined in vitro in decomposing porcine blood inoculated with bacteria, utilizing a high-performance liquid chromatography with ultraviolet and fluorescence detection method for drug quantitation. Stability experiments for risperidone and paliperidone were conducted at 7, 20 and 37°C for 4 days using sterile and bacterially inoculated porcine blood. The drugs were stable in sterile blood at each temperature and in inoculated blood at 7°C, but degraded significantly in inoculated blood at 20 and 37°C. Complete loss occurred within 2 days when incubated at 37°C. The benzisoxazole-cleaved degradation products for both drugs were identified as 2-hydroxybenzoyl-risperidone and 2-hydroxybenzoyl-paliperidone utilizing liquid chromatography quadrupole-time-of-flight mass spectrometry and accurate mass measurements. The degradation products have been found in postmortem case studies, including one case where risperidone and paliperidone were not detected, indicating complete conversion can occur in situ.

Stability of serotonin-selective antidepressants in sterile and decomposing liver tissue.

It is well established that bacteria are capable of degrading selected drugs during decomposition. The aim of this study was to investigate the stability of several serotonin-selective reuptake inhibitor antidepressants and venlafaxine during putrefaction in porcine liver macerate inoculated with porcine cecal contents rich in bacteria. Blank liver matrices, sterile liver macerates, and sterile aqueous controls were included with the experiment performed for 57 days at 20°C under anaerobic conditions. A liquid chromatography/mass spectrometry method was developed for quantitative determination of the drugs investigated in both sterile and decomposed liver matrices. The method was found to encounter matrix effects not detected during the validation stage. Citalopram, paroxetine, sertraline, venlafaxine, and fluoxetine were found to be stable under the experimental conditions; however, fluvoxamine was found to be decreased by c. 50% over 57 days in bacterially inoculated liver macerate. This study suggests that fluvoxamine concentrations in cases with evidence of decomposition/putrefaction should be interpreted with extra caution.