Time- and Site-Dependent Postmortem Redistribution of Antidepressants and Neuroleptics in Blood and Alternative Matrices.

Postmortem redistribution (PMR) leads to challenges in postmortem case interpretation. Particularly antidepressants and neuroleptics are expected to undergo PMR based on their physico-chemical properties. For the current study, time- and site-dependent PMR of 20 antidepressants and neuroleptics were investigated in humans (authentic cases); five of which are discussed in detail (citalopram, mirtazapine, quetiapine, risperidone and venlafaxine) along with two metabolites (9-OH-risperidone and O-desmethylvenlafaxine). Blood [femoral (pB) and heart blood (HB)] and tissue biopsy samples (lung, kidney, liver, spleen, thigh muscle and adipose tissue) were collected upon admission to the institute utilizing a computed tomography-guided sample collection workflow (t1). Approximately 24 h later (t2; mean 23 ± 9.3 h), samples from the same body regions were collected manually. Liquid chromatography-tandem mass spectrometry was used for quantification. Most antidepressants and neuroleptics showed significant time-dependent concentration changes indicating the occurrence of PMR. For the first time, two phases of redistribution in pB for quetiapine were proposed (concentration decreases in the early postmortem phase, followed by concentration increases) and contrasting existing literature, both concentration increases and decreases in pB overtime were observed for risperidone and 9-OH-risperidone. Venlafaxine and its metabolite only showed minimal concentration changes, while citalopram exhibited a trend for concentration increases and mirtazapine for concentration decreases in pB overtime. Based on time-dependent tissue data, passive diffusion processes along the muscle-to-pB, liver-to-HB and lung-to-HB concentration gradients could be proposed along with bacterial degradation. Overall, no case interpretation had to be adjusted, which suggests that PMR changes of antidepressants and neuroleptics do not seem to be relevant for forensic case interpretation within the 24 h period that was investigated. However, limitations of the current study (e.g., temperature-controlled storage of the bodies) could have led to an underestimation of occurring postmortem changes, hence, interpretation of postmortem results should always be conducted with care, considering PMR phenomena and inter-individual variability.

Postmortem metabolomics: Correlating time-dependent concentration changes of xenobiotic and endogenous compounds.

Postmortem redistribution (PMR) describes the artificial postmortem concentration changes of xenobiotics that may pose major challenges in forensic toxicology. Only a few studies have systematically investigated time-dependent postmortem drug concentration changes so far and the a posteriori estimation of the occurrence of PMR is not yet possible. In this context, the general concept that postmortem biochemical changes in blood might parallel drug redistribution mechanisms seems promising. Thus, the current study investigated the possible correlations between time-dependent postmortem concentration changes of xenobiotic and endogenous compounds; exemplified for authentic morphine (n = 19) and methadone (n = 11) cases. Peripheral blood samples at two time-points postmortem were analyzed for morphine and methadone concentrations and an (un)targeted postmortem metabolomics approach was utilized to combine targeted quantitative analysis of 56 endogenous analytes and untargeted screening for endogenous compounds (characterizing 1174 features); liquid and gas chromatography-mass spectrometry was used respectively. Individual statistically significant correlations between morphine/methadone and endogenous compounds/features could be determined. Hence, the general applicability of the proposed concept could successfully be confirmed. To verify the reproducibility and robustness of the correlating behavior, a larger dataset must be analyzed next. Once a marker/set of markers is found (e.g. robust correlation with specific xenobiotic or xenobiotic class), these could be used as surrogates to further study the time-dependent PMR in a broader variety of cases (e.g. independent of a xenobiotic drug present). A crucial next step will also be the attempt to create a statistical model that allows a posteriori estimation of PMR occurrence of xenobiotics to assist forensic toxicologists in postmortem case interpretation.

Liquid chromatography-tandem mass spectrometry screening method using information-dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine.

The total number of synthetic cannabinoids (SCs) - a group of new psychoactive substances (NPS) - is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and finally included to the MS/MS spectra in-house library. Sample preparation was performed using a cheap-and-easy salting-out liquid-liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05 ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.

Fatal poisoning involving cyclopropylfentanyl - Investigation of time-dependent postmortem redistribution.

A growing number of fatal overdoses involving opioid drugs, in particular involving fentanyl and its analogues, pose an immense threat to public health. Postmortem casework of forensic toxicologists in such cases is challenging, as data on pharmacodynamic and pharmacokinetic properties as well as reference values for acute toxicities and data on potential postmortem redistribution (PMR) mechanisms often do not exist. A fatal case involving cyclopropylfentanyl was investigated at the Zurich Institute of Forensic Medicine and the Zurich Forensic Science Institute; an unknown powder found at the scene was reliably identified as cyclopropylfentanyl by gas chromatography-infrared spectroscopy (GC-IR). Femoral blood samples were collected at two time points after death; 11h postmortem (t1) and during the medico-legal autopsy 29h after death (t2). At the autopsy, additional samples from the heart blood, urine and gastric content were collected. Cyclopropylfentanyl was quantified using a validated liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. Femoral blood concentration of cyclopropylfentanyl at autopsy was 19.8ng/mL (t1=15.7ng/mL; heart blood concentration at autopsy=52.4ng/mL). In the light of the current literature and under the exclusion that no other morphological findings could explain the cause of death, contribution of cyclopropylfentanyl to death was proposed (polydrug use). Significant postmortem concentration increases of cyclopropylfentanyl in femoral blood during 18h after the first sampling were observed, thus indicating a relevant potential to undergo PMR. A central-to-peripheral blood concentration ratio of 2.6 supports this. Consequently, the current case suggests that postmortem cyclopropylfentanyl concentration should always be interpreted with care.

Identification of urinary metabolites of the synthetic cannabinoid 5F-CUMYL-P7AICA in human casework.

Synthetic cannabinoids belong to one of the largest groups of new psychoactive substances (NPS) and are challenging regarding analytical detection because of the often complete metabolic degradation of the parent compounds. 5F-CUMYL-P7AICA contains a 7-azaindole moiety and appeared first on the market in 2015. In the frame of abstinence control cases, possible 5F-CUMYL-P7AICA metabolites were detected in three urine samples. The samples were reanalyzed using liquid-chromatography-high resolution mass spectrometry (LC-HRMS) and human phase I and II metabolites were identified. Major in vivo biotransformation steps of 5F-CUMYL-P7AICA in humans were oxidative defluorination followed by carboxylation, and monohydroxylation followed by sulfation and glucuronidation. Evaluation of the metabolites as marker for 5F-CUMYL-P7AICA consumption revealed the defluorinated and carboxylated metabolite to be important for sensitive detection. For higher specificity, additional monitoring of a monohydroxylated metabolite is recommended. Comparison with previously published in vitro metabolism data revealed good accordance of the results, with exception of a of the dihydroxylated metabolites from the in vitro assays, which were not detected in the in vivo samples.

Time-Dependent Postmortem Redistribution of Opioids in Blood and Alternative Matrices.

Forensic postmortem case interpretation can be challenging, in particular due to postmortem redistribution (PMR) phenomena. Recent studies have shown that computed tomography (CT)-guided collection of biopsy samples using a robotic arm (virtobot) provides a valuable tool for systematic studies on time-dependent PMR. Utilizing this strategy, several cases involving opioid use such as methadone, fentanyl, tramadol, codeine, oxycodone and hydrocodone were evaluated for time-dependent concentration changes and potential redistribution mechanisms. Upon admission to the institute (t1), blood (femoral and right ventricle heart blood) and tissue biopsy samples (lung, kidney, liver, spleen, thigh muscle and adipose tissue) were collected utilizing CT-guided biopsy. Approximately 24 h later (t2; mean 28 ± 15 h), during the autopsy, samples from the same body regions were collected manually and in addition brain tissue, gastric content, urine and left ventricle heart blood. Analysis was conducted with liquid chromatography tandem mass spectrometry. Significant time-dependent methadone concentration increases in femoral blood (pB) indicate the occurrence of PMR, however, ultimately not relevant for forensic interpretation. The main metabolite of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), showed a less significant trend for PMR. Redistribution by passive diffusion along the muscle-to-pB concentration gradient seems likely for methadone, but not for EDDP. Results for fentanyl suggest extensive PMR. Other opioids such as tramadol, codeine, hydrocodone and oxycodone showed no consistent trend for significant PMR. Overall, CT-guided biopsy sampling proved to be a valuable tool for the investigation of PMR mechanisms.

In vitro metabolism of the synthetic cannabinoids CUMYL-PINACA, 5F-CUMYL-PINACA, CUMYL-4CN-BINACA, 5F-CUMYL-P7AICA and CUMYL-4CN-B7AICA.

Synthetic cannabinoid consumption trends underlie fast changes and provide several challenges to clinical and forensic toxicologists. Due to their extensive metabolism, parent compounds are hardly detectable in urine. Therefore, knowledge of the metabolism of synthetic cannabinoids is essential to allow their detection in biological matrices. The aim of the present study was the elucidation of the metabolism of CUMYL-PINACA, 5F-CUMYL-PINACA, CUMYL-4CN-BINACA, 5F-CUMYL-P7AICA, and CUMYL-4CN-B7AICA with a focus on the analytical and interpretational differentiation of the compounds. Microsomal assay mixtures containing co-substrates, 10 µg/mL substrate and 1 mg/mL pooled human liver microsomes were incubated for 1 hour at 37°C. Investigation of the metabolites was performed on a Thermo Fischer Ultimate 3000 UHPLC system coupled to a Sciex 6600 QTOF System. Hydroxylation was observed to be a major biotransformation step for all 5 cumyl-derivatives, followed by dihydroxylation. For CUMYL-PINACA, a major metabolic pathway was hydroxylation at the pentyl moiety, followed by a second hydroxylation at that pentyl moiety or oxidation to ketone. A major metabolic pathway for the compounds containing a nitrile function was nitrile hydrolysis followed by carboxylation and further hydroxylation. For the fluorinated compounds, oxidative defluorination and carboxylation were abundant metabolic steps. Some of the metabolic transformations lead to structurally identical metabolites, which should not be used as marker for the intake of a particular parent compound. In addition, several constitutional isomers containing either an indazole or azaindole core structure were detected, which should be differentiated by retention time rather than by their mass spectra alone.

Structural characterization of the new synthetic cannabinoids CUMYL-PINACA, 5F-CUMYL-PINACA, CUMYL-4CN-BINACA, 5F-CUMYL-P7AICA and CUMYL-4CN-B7AICA.

Synthetic cannabinoids are a group of new psychoactive compounds (NPS) that act as agonists at the cannabinoid receptor. First reported in 2008, they currently represent one of the largest groups of NPS that are monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). Five samples (4 from the European RESPONSE project and one from daily casework) containing different synthetic cannabinoids were analyzed by a complex of analytical methods including gas chromatography-electron ionization mass spectrometry (GC-EI-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS), infrared spectroscopy (IR) and nuclear magnetic resonance spectroscopy (NMR). Five new synthetic cannabinoids containing a cumyl moiety as a linked group were identified: CUMYL-PINACA, 5F-CUMYL-PINACA, CUMYL-4CN-BINACA, 5F-CUMYL-P7AICA, CUMYL-4CN-B7AICA. 5F-CUMYL-PINACA and 5F-CUMYL-P7AICA as well as CUMYL-4CN-BINACA and CUMYL-4CN-B7AICA are constitutional isomers and only differ in the position of a nitrogen atom. The article contains all analytical data for a proper identification and differentiation of the five cumyl compounds.

Postmortem distribution and redistribution of MDAI and 2-MAPB in blood and alternative matrices.

Intoxication cases involving new psychoactive substances (NPS) provide several challenges for forensic toxicologists as data on pharmacodynamic and pharmacokinetic properties are lacking, especially on potency and toxicity. Furthermore, reference values and information on postmortem redistribution (PMR) do not exist so far for most NPS. A fatal case involving the amphetamine-derivatives MDAI (5,6-methylenedioxy-2-aminoindane) and 2-MAPB (1-(benzofuran-2-yl)-N-methylpropan-2-amine) was investigated at the Zurich Institute of Forensic Medicine. At admission at the institute approx. 11h after death (first time point, t1), femoral and heart blood (right ventricle) was collected using computed tomography (CT)-guided biopsy sampling. At autopsy (t2), samples from the same body regions as well as various tissue samples were collected manually. In addition, an antemortem blood sample collected 6h before death was available. MDAI and 2-MAPB were quantified using a validated LC-MS/MS method. A significant concentration decrease between the antemortem and the first peripheral postmortem blood sample was observed, which most probably can be explained by remaining metabolism and excretion within the last 6h prior to death. No significant concentration change was observed between the two postmortem heart blood and peripheral blood samples. Accordingly, MDAI and 2-MAPB did not seem to undergo relevant postmortem redistribution in peripheral and heart blood in the presented case. This is the first study on postmortem redistribution of the new psychoactive substances MDAI and 2-MAPB. However, more studies covering more cases are necessary to generate universal statements on the PMR with these two NPSs.

Studies on the metabolism of the fentanyl-derived designer drug butyrfentanyl in human in vitro liver preparations and authentic human samples using liquid chromatography-high resolution mass spectrometry (LC-HRMS).

Increasing numbers of new psychoactive substances (NPS) among them fentanyl derivatives has been reported by the European monitoring centre for drugs and drug addiction (EMCDDA). Butyrfentanyl is a new fentanyl derivative whose potency ratio was found to be seven compared to morphine and 0.13 compared to fentanyl. Several case reports on butyrfentanyl intoxications have been described. Little is known about its pharmacokinetic properties including its metabolism. However, knowledge of metabolism is essential for analytical detection in clinical and forensic toxicology. Therefore, in vitro and in vivo phase I and phase II metabolites of butyrfentanyl were elucidated combining liquid chromatography with a qTOF high resolution mass spectrometer. Human liver microsomes and recombinant cytochrome P450 enzymes (CYP) were used for in vitro assays. Authentic blood and urine samples from a fatal intoxication case were available for in vivo comparison. Butyrfentanyl was shown to undergo extensive metabolism. Six pathways could be postulated with hydroxylation and N-dealkylation being the major ones in vitro. In vivo, hydroxylation of the butanamide side chain followed by subsequent oxidation to the carboxylic acid represented the major metabolic step in the authentic case. Initial screening experiments with the most relevant CYPs indicated that mainly CYP2D6 and 3A4 were involved in the primary metabolic steps. Altered CYP2D6 and CYP3A4 status might cause a different metabolite pattern, making the inclusion of metabolites of different pathways recommendable when applying targeted screening procedures in clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd.

Time-dependent postmortem redistribution of morphine and its metabolites in blood and alternative matrices-application of CT-guided biopsy sampling.

Interpretation of postmortem morphine concentrations in forensic toxicology provides several pitfalls such as missing information on tolerance, analyte stability, or postmortem redistribution (PMR). Recently, it had been shown that computed tomography (CT)-guided collection of biopsies using a robotic arm (virtobot) provides a valuable strategy for systematic studies on time-dependent PMR. Using this technique, time-dependent PMR of morphine and its metabolites was investigated in 12 cases. At admission to the institute (t1), femoral and heart blood (right ventricle) as well as biopsies from the right lung, the right kidney, liver, spleen, and muscle tissue were collected. At autopsy approximately 24 h later (t2), samples from the same body regions were collected again. Additionally, gastric contents, urine, brain tissue, and heart blood from the left ventricle was collected. Morphine, normorphine, hydromorphone, morphine-3-glucuronide, morphine-6-glucuronide, and morphine-sulfate were quantified with LC-MS/MS. In femoral blood, significant increase of morphine concentrations was observed, although ultimately not relevant for forensic interpretation. In the alternative matrices, increases as well as decreases were observed without a clear trend. The morphine metabolites did not exhibit relevant concentration changes. Investigation of underlying redistribution mechanisms indicated that concentration change (i.e., increase) of morphine in femoral blood rather resulted from diffusion processes than from release of morphine from its conjugates. Concentration changes in heart blood might have been caused by redistribution from lung tissue or gastric content. This study also proved that CT-guided collection of biopsies using a virtobot arm is an invaluable tool for future studies on PMR redistribution of other substance groups.

Time-dependent postmortem redistribution of butyrfentanyl and its metabolites in blood and alternative matrices in a case of butyrfentanyl intoxication.

A fatal case of butyrfentanyl poisoning was investigated at the Zurich Institute of Forensic Medicine. At admission at the institute approx. 9h after death (first time point, t1), femoral and heart blood (right ventricle) was collected, as well as samples from the lung, liver, kidney, spleen, muscle and adipose tissue using computed tomography (CT)-guided biopsy sampling. At autopsy (t2), samples from the same body regions were collected manually. Additionally, urine, heart blood (left ventricle), gastric content, brain samples and hair were collected. Butyrfentanyl concentrations and relative concentrations of the metabolites carboxy-, hydroxy-, nor-, and desbutyrfentanyl were determined by LC-MS/MS and LC-QTOF. At t1, butyrfentanyl concentrations were 66ng/mL in femoral blood, 39ng/mL in heart blood, 110ng/g in muscle, 57ng/g in liver, 160ng/g in kidney, 3100ng/g in lung, 590ng/g in spleen and 550ng/g in adipose tissue. At t2, butyrfentanyl concentration in urine was 1100ng/mL, in gastric content 2000ng/mL, in hair 11,000pg/mg and brain concentrations ranged between 200-340ng/g. Carboxy- and hydroxybutyrfentanyl were identified as most abundant metabolites. Comparison of t1 and t2 showed a concentration increase of butyrfentanyl in femoral blood of 120%, in heart blood of 55% and a decrease in lung of 30% within 19h. No clear concentration changes could be observed in the other matrices. Postmortem concentration changes were also observed for the metabolites. In conclusion, butyrfentanyl seems to be prone to postmortem redistribution processes and concentrations in forensic death cases should be interpreted with caution.

Development of CT-guided biopsy sampling for time-dependent postmortem redistribution investigations in blood and alternative matrices--proof of concept and application on two cases.

The postmortem redistribution (PMR) phenomenon complicates interpretation in forensic toxicology. Human data on time-dependent PMR are rare and only exist for blood so far. A new method for investigation of time-dependent PMR in blood as well as in alternative body fluids and tissues was developed and evaluated using automated biopsy sampling. At admission of the bodies, introducer needles were placed in liver, lung, kidney, muscle, spleen, adipose tissue, heart, femoral vein, and lumbar spine using a robotic arm guided by a computed tomography scanner (CT). Needle placement accuracy was analyzed and found to be acceptable for the study purpose. Tissue biopsies and small volume body fluid samples were collected in triplicate through the introducer needles. At autopsy (around 24 h after admission), samples from the same body regions were collected. After mastering of the technical challenges, two authentic cases were analyzed as a proof of concept. Drug concentrations of venlafaxine, O-desmethylvenlafaxine, bromazepam, flupentixol, paroxetine, and lorazepam were determined by LC-MS/MS, and the percentage concentration changes between the two time points were calculated. Concentration changes were observed with both increases and decreases depending on analyte and matrix. While venlafaxine, flupentixol, paroxetine, and lorazepam generally showed changes above 30% and more, O-desmethylvenlafaxine and bromazepam did not undergo extensive PMR. The presented study shows that CT-controlled biopsy collection provides a valuable tool for systematic time-dependent PMR investigation, demanding only minimal sample amount and causing minimal damage to the body.

Development and validation of a dynamic range-extended LC-MS/MS multi-analyte method for 11 different postmortem matrices for redistribution studies applying solvent calibration and additional (13)C isotope monitoring.

Postmortem redistribution (PMR) is one of numerous problems in postmortem toxicology making correct interpretation of measured drug concentrations difficult or even impossible. Time-dependent PMR in peripheral blood and especially in tissue samples is still under-explored. For further investigation, an easy applicable method for the simultaneous quantitation of over 80 forensically relevant compounds in 11 different postmortem matrices should be developed and validated overcoming the challenges of high inter-matrix and intra-matrix concentration variances. Biopsy samples (20 mg) or body fluids (20 µL) were spiked with an analyte mix and deuterated internal standards, extracted by liquid-liquid extraction, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). For highest applicability, an easy solvent calibration was used. Furthermore, time-consuming dilution of high concentration samples showing detector saturation was circumvented by two overlapping calibration curves using (12)C isotope monitoring for low concentrations and (13)C isotopes for high concentration, respectively. The method was validated according to international guidelines with modifications. Matrix effects and extraction efficiency were strongly matrix and analyte dependent. In general, brain and adipose tissue produced the highest matrix effects, whereas cerebrospinal fluid showed the least matrix effects. Accuracy and precision results were rather matrix independent with some exceptions. Despite using an external solvent calibration, the accuracy requirements were fulfilled for 66 to 81 % of the 83 analytes. Depending on the matrix, 75-93 % of the analytes showed intra-day precisions at <20 %. (12)C and (13)C calibrations gave comparable results and proved to be a useful tool in expanding the dynamic range.

Comparison of conventional liquid chromatography-tandem mass spectrometry versus microflow liquid chromatography-tandem mass spectrometry within the framework of full method validation for simultaneous quantification of 40 antidepressants and neuroleptics in whole blood.

Microflow liquid chromatography (MFLC) coupled to mass spectrometry (MS) is claimed to improve analysis throughput, reduce matrix effects and lower mobile phase consumption. This statement was checked within the framework of method validation of a multi-analyte procedure in clinical and forensic toxicology employing MFLC-MS/MS and conventional LC-MS/MS. 200 µL whole blood were spiked with 50 µL internal standard mixture and extracted by protein precipitation. The concentrated extract was separated into two vials. One was analyzed using a Thermo Fisher Ultimate liquid chromatography system coupled to an ABSciex 5500 QTrap mass spectrometer (LC-MS/MS) and one by an ABSciex Eksigent Microflow LC system coupled to an ABSciex 4500 linear ion trap quadrupole MS (MFLC-MS/MS). Both methods were fully validated and compared in terms of selectivity, stability, limits, calibration model, recovery (RE), matrix effects (ME), bias, imprecision and beta tolerance interval for 40 antidepressants and neuroleptics including 9 metabolites. Both methods had comparable LODs, LOQs and calibration models with some exceptions. The MFLC system showed slightly higher coefficients of variation (CVs) in the RE experiments. ME were reproducible in both systems but with lower CVs in the conventional LC system. Acceptance criteria for imprecision and bias were fulfilled for 32 analytes on the LC and for 28 analytes on the MFLC system. Beta tolerance intervals indicated better reproducibility in terms of narrower intervals for the conventional LC system. The advantages of the MFLC system were low mobile phase consumption, short run time, and better peak separation. The systems were comparable in terms of peak interference, LOD, ME, bias and imprecision. The advantages of the conventional LC system were more data points per peak, linear calibration models, stable retention times and better beta tolerance intervals. Due to higher robustness, the conventional LC system was finally chosen for routine application in forensic toxicology.