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.

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 cosubstrates, 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. KEYWORDSin vitro metabolism, new psychoactive substances NPS, synthetic cannabinoids

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.

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.

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