A New Strategy for Efficient Retrospective Data Analyses for Designer Benzodiazepines in Large LC-HRMS Datasets.

The expanding and dynamic market of new psychoactive substances (NPSs) poses challenges for laboratories worldwide. The retrospective data analysis (RDA) of previously analyzed samples for new targets can be used to investigate analytes missed in the first data analysis. However, RDA has historically been unsuitable for routine evaluation because reprocessing and reevaluating large numbers of forensic samples are highly work- and time-consuming. In this project, we developed an efficient and scalable retrospective data analysis workflow that can easily be tailored and optimized for groups of NPSs. The objectives of the study were to establish a retrospective data analysis workflow for benzodiazepines in whole blood samples and apply it on previously analyzed driving-under-the-influence-of-drugs (DUID) cases. The RDA workflow was based on a training set of hits in ultrahigh-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UHPLC-QTOF-MS) data files, corresponding to common benzodiazepines that also had been analyzed with a complementary UHPLC-tandem mass spectrometry (MS/MS) method. Quantitative results in the training set were used as the true condition to evaluate whether a hit in the UHPLC-QTOF-MS data file was true or false positive. The training set was used to evaluate and set filters. The RDA was used to extract information from 47 DBZDs in 13,514 UHPLC-QTOF-MS data files from DUID cases analyzed from 2014 to 2020, with filters on the retention time window, count level, and mass error. Sixteen designer and uncommon benzodiazepines (DBZDs) were detected, where 47 identifications had been confirmed by using complementary methods when the case was open (confirmed positive finding), and 43 targets were not reported when the case was open (tentative positive finding). The most common tentative and confirmed findings were etizolam (n = 26), phenazepam (n = 13), lorazepam (n = 9), and flualprazolam (n = 8). This method efficiently found DBZDs in previously acquired UHPLC-QTOF-MS data files, with only nine false-positive hits. When the standard of an emerging DBZD becomes available, all previously acquired DUID data files can be screened in less than 1 min. Being able to perform a fast and accurate retrospective data analysis across previously acquired data files is a major technological advancement in monitoring NPS abuse.

Identification of the synthetic cannabinoid-type new psychoactive substance, CH-PIACA, in seized material.

Synthetic cannabinoids (SCs) remain the largest class of new psychoactive substances (NPS), and while the number of NPS that are reported to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) for the first time each year declines, the number of newly reported SCs still exceeds other NPS classes. This decline can be seen as a result of legislative changes by different jurisdictions which have sometimes transitioned to a more generalized approach when controlling substances by defining common structural scaffolds rather than explicit structures. While the consequences of such legislative changes have been expected over the years, the introduction of so-called "class-wide" bans puts further pressure on clandestine laboratories to synthesize compounds which are out of the scope of the legislation, and thus, these compounds are initially harder to detect and/or identify in the absence of analytical data. Recently, a SC with an indole-3-acetamide core-linker scaffold, AD-18 (i.e., ADB-FUBIATA or ADB-FUBIACA), was reported for the first time in China in 2021. Here, an additional cannabinoid with the indole-3-acetamide scaffold, N-cyclohexyl-2-(1-pentyl-1H-indol-3-yl)acetamide (CH-PIACA), is reported which was identified for the first time in a seized material in Denmark. Structural characterization was performed using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-high-resolution mass spectrometry (LC-HRMS), and nuclear magnetic resonance (NMR) spectroscopy.

Postmortem Brain-Blood Ratios of Codeine, Fentanyl, Oxycodone and Tramadol.

The analgesics, codeine, fentanyl, oxycodone and tramadol, frequently occur in postmortem cases and determining their role in the cause of death can be challenging. However, postmortem blood is susceptible to redistribution and may not be available in cases of severe blood loss, putrefaction or burns. Brain tissue may serve as a viable supplement to blood or on its own, as it is resistant to postmortem redistribution and often available as a sample matrix when blood is not available. We present brain and blood concentrations and brain-blood ratios of the four analgesics from 210 autopsy cases. The cases were classified according to the presumed cause of death: A: The compound was believed to have solely caused a fatal intoxication. B: The compound was assumed to have contributed to a fatal outcome in combination with other drugs, alcohol or disease. C: The compound was not regarded as being related to the cause of death. Blood and brain samples were prepared by automatic solid phase extraction and quantified by liquid chromatography-mass spectrometry. The squared correlation coefficients between concentrations in brain tissue and blood ranged 0.45-0.91. The median brain-blood ratios were codeine 1.8 (range 0.47-4.6), fentanyl 2.1 (range 0.29-16), oxycodone 1.8 (range 0.11-6.0) and tramadol 1.8 (range 0.047-6.8). A significantly higher brain-blood ratio of codeine was observed in cases where heroin had been administered, although there was a wide overlap. Intravenous and transdermal fentanyl administration could not be distinguished based on the blood or brain concentration or the brain-blood ratio. The results of this study may benefit the toxicological investigation in postmortem cases where one of the four analgesics are suspected of having contributed to or caused a fatal intoxication.

Brain-blood ratio of morphine in heroin and morphine autopsy cases.

Brain tissue is a useful supplement to blood in postmortem investigations, but reference concentrations are scarce for many opioids. Heroin cases may be difficult to distinguish from morphine cases as heroin and its metabolites are rapidly degraded. We present concentrations from brain and blood and brain-blood ratios of 98 cases where morphine was quantified. These cases were grouped according to the cause of death: A: The compound was solely assumed to have caused a fatal intoxication. B: The compound presumably contributed to a fatal outcome in combination with other drugs, alcohol or disease. C: The compound was not regarded to be related to the cause of death. The cases were further classified as heroin cases if 6-acetyl-morphine or noscapine were detected. The analyses were carried out using solid-phase extraction or protein precipitation followed by ultra high-performance liquid chromatography coupled to mass spectrometry. The average brain-blood ratios of morphine were 1.2 and 1.8 for 69 morphine and 29 heroin cases, respectively. Differences in the brain-blood ratios were found for cases where heroin and morphine were involved in the cause of death, either in combination or on its own (P<0.001 and P=0.004, respectively). However, the overlap between morphine and heroin cases precludes the use of the brain-blood ratio to distinguish heroin from morphine intake. Morphine-6-glucuronide and 6-acetyl-morphine were quantified in brain and blood in a subset of the samples, yielding median brain-blood ratios of 5.1 and 8.3, respectively. The brain concentrations may aid the toxicological investigation in cases where heroin or morphine intoxications are suspected, but blood is not available.

Postmortem Brain-Blood Ratios of Amphetamine, Cocaine, Ephedrine, MDMA and Methylphenidate.

Brain tissue may serve as a useful supplement to blood in postmortem investigations. However, reference concentrations for central stimulant drugs are scarce in brain tissue. This study involves some frequently used stimulants: amphetamine, cocaine, ephedrine, MDMA and methylphenidate. We present concentrations from brain and blood and brain-blood ratios of the analytes from autopsies. The cases were grouped according to the cause of death: A: The compound solely caused a fatal intoxication. B: The compound contributed to a fatal outcome in combination with other drugs, alcohol or disease. C: The compound was not related to the cause of death. Analyses were carried out using solid-phase extraction and ultra high-performance liquid chromatography. Paired brain and femoral blood concentrations from 133 cases were analysed. Positive correlations were observed for all analytes with correlation coefficients ranging from 0.58 to 0.95. The following median brain-blood ratios were obtained: cocaine 2.0 (range 0.20-7.0), amphetamine 3.2 (range 1.5-4.5), ephedrine 2.3 (range 1.1-6.2), MDMA 3.9 (range 0.92-5.1) and methylphenidate 2.4 (0.92-4.6). The concentrations in femoral blood generally agreed with the literature for all compounds. The metabolite of cocaine, benzoylecgonine, was also quantified in brain and blood from 60 cases, and the median brain-blood ratio was 0.66 with 10-90 percentiles of 0.39-1.27. The results of this study can aid the toxicological investigation in determining the cause of death.

Reference Brain and Blood Concentrations of Olanzapine in Postmortem Cases.

The antipsychotic drug olanzapine may be subject to postmortem redistribution. This complicates the toxicological evaluation in postmortem cases and a supplementary analysis of brain tissue may be an advantage. We report reference brain and blood concentrations of olanzapine from 40 forensic autopsy cases. Each case was assigned to one of three groups according to the cause of death: (A) fatal intoxication by olanzapine alone; (B) fatal intoxication by olanzapine in combination with other drugs and (C) olanzapine was not related to the cause of death. Quantification of olanzapine in brain and blood was performed by ultra-performance liquid chromatography with tandem mass spectrometry using a validated method. A linear correlation between concentrations in blood and brain from 40 cases was found with a correlation coefficient of 0.87. The median brain:blood ratio was 2.5 (10-90%: 1.2-5.8, range: 0.72-10.4). For the A cases (n = 2), the concentrations in brain (Br) and femoral blood (FB) were: Br: 2.1-3.6 mg/kg, FB: 0.99-1.2 mg/kg; for the B cases (n = 17) the 10-90% were: Br: 0.27-1.0 mg/kg (range: 0.13-1.3 mg/kg) FB: 0.11-0.57 mg/kg (range: 0.096-0.65 mg/kg) and the 10-90% of the C cases (n = 21): Br: 0.05-0.49 mg/kg (range: 0.040-0.87 mg/kg) FB: 0.02-0.14 mg/kg (range: 0.008-0.15 mg/kg). These results can serve as reference concentrations for the interpretation of postmortem forensic cases.

Validation of a fully automated solid-phase extraction and ultra-high-performance liquid chromatography-tandem mass spectrometry method for quantification of 30 pharmaceuticals and metabolites in post-mortem blood and brain samples.

In this study, we present the validation of an analytical method capable of quantifying 30 commonly encountered pharmaceuticals and metabolites in whole blood and brain tissue from forensic cases. Solid-phase extraction was performed by a fully automated robotic system, thereby minimising manual labour and human error while increasing sample throughput, robustness, and traceability. The method was validated in blood in terms of selectivity, linear range, matrix effect, extraction recovery, process efficiency, carry-over, stability, precision, and accuracy. Deuterated analogues of each analyte were used as internal standards, which corrected adequately for any inter-individual variability in matrix effects on analyte accuracy and precision. The lower limit of quantification (LLOQ) spanned from 0.0008 to 0.010 mg/kg, depending on the analyte, while the upper LOQ ranged between 0.40 and 2.0 mg/kg. Thus, the linear range covered both therapeutic and toxic levels. The method showed acceptable accuracy and precision, with accuracies ranging from 80 to 118% and precision below 19% for the majority of the analytes. Linear range, matrix effect, extraction recovery, process efficiency, precision, and accuracy were also tested in brain homogenate and the results agreed with those from blood. An additional finding was that the analyte concentrations in brain samples could be quantified by calibration curves obtained from spiked blood samples with acceptable precision and accuracy when using deuterated analogues of each analyte as internal standards. This method has been successfully implemented as a routine analysis procedure for quantification of pharmaceuticals in both blood and brain tissue since 2015.

Reference Brain/Blood Concentrations of Citalopram, Duloxetine, Mirtazapine and Sertraline.

Postmortem blood samples may not accurately reflect antemortem drug concentrations, as the levels of some drugs increase due to postmortem redistribution (PMR). The brain has been suggested as an alternative sampling site. The anatomically secluded site of the brain limits redistribution and prolongs the detection window, thereby enabling sampling from deceased individuals where blood is no longer suitable for analysis. We report concentrations in brain tissue and blood from 91 cases for the four antidepressants citalopram, duloxetine, mirtazapine and sertraline. The cases were classified according to their role in the cause of death, as follows: (A) concentrations where the drug was the sole cause of fatal intoxication; (B) concentrations where the drug contributed to a fatal outcome; and (C) concentrations where the drug was not related to the cause of death. The analytical method was successfully validated in brain tissue in terms of linearity, process efficiency, precision and accuracy. Quantification of analytes was performed by ultra-performance liquid chromatography with tandem mass spectrometry. Correlations between blood and brain concentrations were achieved with R2-values between 0.67 and 0.91. The following median brain-blood ratios were obtained: 3.71 for citalopram (range: 1.4-5.9), 11.0 for duloxetine (range: 5.0-21.6), 1.53 for mirtazapine (range: 1.02-4.71) and 7.38 for sertraline (range: 3.2-14.2). The S/R ratio of racemic citalopram was the same in brain (0.80) and blood (0.85), whereas the median citalopram/N-desmethylcitalopram ratio was higher in brain (9.1) than blood (4.1). The results of this study may serve as reference concentrations in brain for forensic cases.