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A highly discussed step in hair sample preparation for forensic analytics is the applied decontamination. The here presented investigations aim to gain insight and give recommendations on how to conduct this decontamination for the analysis of cocaine consumption in hair. Key insights were gained from the investigation of cocaine consumer hair, which was artificially contaminated in a humid atmosphere with 13C6 labelled cocaine and from cocaine powder contaminated hair. Several decontamination protocols were investigated, whereby the usage of a decontamination protocol consisting of multiple short repetitive washes allowed to visualize the wash out of (13C6-) cocaine. Multiple methanol washes proved to be an efficient and simple decontamination approach. Our findings showed that decontamination protocols can successfully wash out recent cocaine contaminations. They were observed to be rather quickly washed out, whereas cocaine from consumption or "older" cocaine contaminations were shown to eliminate both at a constant rate (from inner hair compartments). Thus, the usage of decontamination protocols to differentiate between consumption and contamination was shown to be limited. As contamination can happen any time at any level, only the application of elaborated decision trees, based on cocaine metabolite ratios and thresholds, can provide the distinction between consumption and contamination. Thus, the authors highly recommend the usage of such tools on all hair samples analyzed for cocaine consumption., (© The Author(s) 2020. Published by Oxford University Press.)

The number of new psychoactive substances (NPS) on the illicit drug market increases fast, posing a need to urgently understand their toxicity and behavioural effects. However, with currently available rodent models, NPS assessment is limited to a few substances per year. Therefore, zebrafish (Danio rerio) embryos and larvae have been suggested as an alternative model that would require less time and resources to perform an initial assessment and could help to prioritize substances for subsequent evaluation in rodents. To validate this model, more information on the concordance of zebrafish larvae and mammalian responses to specific classes of NPS is needed. Here, we studied toxicity and behavioural effects of opioids in zebrafish early life stages. Synthetic opioids are a class of NPS that are often used in pain medication but also frequently abused, having caused multiple intoxications and fatalities recently. Our data shows that fentanyl derivatives were the most toxic among the tested opioids, with toxicity in the zebrafish embryo toxicity test decreasing in the following order: butyrfentanyl>3-methylfentanyl>fentanyl>tramadol> O-desmethyltramadol>morphine. Similar to rodents, tramadol as well as fentanyl and its derivatives led to hypoactive behaviour in zebrafish larvae, with 3-methylfentanyl being the most potent. Physico-chemical properties-based predictions of chemicals' uptake into zebrafish embryos and larvae correlated well with the effects observed. Further, the biotransformation pattern of butyrfentanyl in zebrafish larvae was reminiscent of that in humans. Comparison of toxicity and behavioural responses to opioids in zebrafish and rodents supports zebrafish as a suitable alternative model for rapidly testing synthetic opioids., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Urine sample manipulation including substitution, dilution, and chemical adulteration is a continuing challenge for workplace drug testing, abstinence control, and doping control laboratories. The simultaneous detection of sample manipulation and prohibited drugs within one single analytical measurement would be highly advantageous. Machine learning algorithms are able to learn from existing datasets and predict outcomes of new data, which are unknown to the model., Methods: Authentic human urine samples were treated with pyridinium chlorochromate, potassium nitrite, hydrogen peroxide, iodine, sodium hypochlorite, and water as control. In total, 702 samples, measured with liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, were used. After retention time alignment within Progenesis QI, an artificial neural network was trained with 500 samples, each featuring 33,448 values. The feature importance was analyzed with the local interpretable model-agnostic explanations approach., Results: Following 10-fold cross-validation, the mean sensitivity, specificity, positive predictive value, and negative predictive value was 88.9, 92.0, 91.9, and 89.2%, respectively. A diverse test set (n=202) containing treated and untreated urine samples could be correctly classified with an accuracy of 95.4%. In addition, 14 important features and four potential biomarkers were extracted., Conclusions: With interpretable retention time aligned liquid chromatography high-resolution mass spectrometry data, a reliable machine learning model could be established that rapidly uncovers chemical urine manipulation. The incorporation of our model into routine clinical or forensic analysis allows simultaneous LC-MS analysis and sample integrity testing in one run, thus revolutionizing this field of drug testing., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

In forensic toxicology, gamma-hydroxybutyrate (GHB) still represents one of the most challenging drugs of abuse in terms of analytical detection and interpretation. Given its rapid elimination, the detection window of GHB in common matrices is short (maximum 12 h in urine). Additionally, the differentiation from naturally occurring endogenous GHB, is challenging. Thus, novel biomarkers to extend the detection window of GHB are urgently needed. The present study aimed at searching new potential biomarkers of GHB use by means of mass spectrometry (MS) metabolomic profiling in serum (up to 16.5 h) and urine samples (up to 8 h after intake) collected during a placebo-controlled crossover study in healthy men. MS data acquired by different analytical methods (reversed phase and hydrophilic interaction liquid chromatography; positive and negative electrospray ionization each) were filtered for significantly changed features applying univariate and mixed-effect model statistics. Complementary to a former study, conjugates of GHB with glycine, glutamate, taurine, carnitine and pentose (ribose) were identified in urine, with particularly GHB-pentose being promising for longer detection. None of the conjugates were detectable in serum. Therein, mainly energy metabolic substrates were identified, which may be useful for more detailed interpretation of underlying pathways but are too unspecific as biomarkers.

Endogenous steroid hormones and endocannabinoids (ECs) are important regulators in the stress response of the human body. For the measurement of chronic stress, hair analysis has been established as method of choice for long-term and retrospective determination of endogenous stress markers. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of five steroid hormones (cortisone, cortisol, androstenedione, testosterone, progesterone) and four endocannabinoids (anandamide, palmitoylethanolamide, 2-arachidonylglycerol, oleoylethanolamide) in hair was developed and validated. The hair samples were extracted with methanol and cleaned up with a fully automated supported liquid extraction (SLE) before analysis. Special attention was paid to the difficulties accompanying the quantification of endogenous analytes in hair. Five different strategies for endogenous compound quantification in hair (surrogate analyte, standard addition, background correction, stripped matrix and solvent calibration) were tested and compared. As a result, the approach of the surrogate analyte was used for the quantification of steroid hormones whereas background correction was used for endocannabinoids. The measurement of 58 samples from healthy young adults allowed insights into endocannabinoid ranges in hair and the correlation to steroid hormones. No significant differences in steroid and EC concentration levels of male and female in hair were found, except for testosterone (p < 0.001) and androstenedione (p < 0.0001). Cortisol to cortisone and testosterone to androstenedione concentrations were significantly and positively correlated. There were significant intercorrelations between endocannabinoids., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hair analysis has become an integral part in forensic toxicological laboratories for e.g. assessment of drug or alcohol abstinence. However, hair samples can be manipulated by cosmetic treatments, altering drug concentrations which eventually leads to false negative hair test results. In particular oxidative bleaching of hair samples under alkaline conditions significantly affects incorporated drug concentrations. To date, current techniques to detect cosmetic hair adulterations bear limitations such as the implementation of cut-off values or the requirement of specialized instrumentations. As a new approach, untargeted hair metabolomics analysis was applied to detect altered, endogenous biomolecules that could be used as biomarkers for oxidative cosmetic hair treatments. For this, genuine hair samples were treated in vitro with 9% hydrogen peroxide (H2O2) for 30 minutes. Untreated and treated hair samples were analyzed using liquid-chromatography high-resolution time-of-flight mass spectrometry. In total, 69 metabolites could be identified as significantly altered after hair bleaching. The majority of metabolites decreased after bleaching, yet totally degraded metabolites were most promising as suitable biomarkers. The formation of biomarker ratios of metabolites decreasing and increasing in concentrations improved the discrimination of untreated and treated hair samples. With the results of this study, the high variety of identified biomarkers now offers the possibility to include single biomarkers or biomarker selections into routine screening methods for improved data interpretation of hair test results.

Untargeted metabolomic studies are used for large-scale analysis of endogenous compounds. Due to exceptional long detection windows of incorporated substances in hair, analysis of hair samples for retrospective monitoring of metabolome changes has recently been introduced. However, information on the general behavior of metabolites in hair samples is scarce, hampering correct data interpretation so far. The presented study aimed to investigate endogenous metabolites depending on hair color and along the hair strand and to propose recommendations for best practice in hair metabolomic studies. A metabolite selection was analyzed using untargeted data acquisition in genuine hair samples from different hair colors and after segmentation in 3 cm segments. Significant differences in metabolites among hair colors and segments were found. In conclusion, consideration of hair color and hair segments is necessary for hair metabolomic studies and, subsequently, recommendations for best practice in hair metabolomic studies were proposed.

Metabolomics studies that aim to qualitatively and quantitatively characterize the entirety of small endogenous biomolecules in an organism are widely conducted in the clinical setting. They also become more and more popular in the field of forensics (toxicology), e.g., to assist in postmortem investigations by objective postmortem interval estimation. However, other issues in postmortem toxicology, such as the phenomenon of (time-dependent) postmortem redistribution, have not yet been tackled by metabolomics studies. Hence, the aim of the current study was to develop an (un)targeted gas chromatography-high-resolution mass spectrometry-based method for endogenous metabolites as a tool for large-scale (un)targeted human postmortem metabolomics investigations (e.g., to objectively assess PMR) with thorough analytical evaluation of this method to ensure fitness-to-purpose in terms of reliability and robustness. This was achieved by using a targeted metabolite subset (n = 56) and a targeted processing workflow. Evaluation experiments have shown that using an artificial matrix (revised simulated body fluid (rSBF) + 5% bovine serum albumin (BSA)) for calibration purposes, all parameters lay within the scope of the method (sensitivity, selectivity, calibration model, accuracy, precision, processed sample stability, and extraction efficiency). When applying this method to large-scale studies, samples should be run in randomized order if analysis time is expected to exceed 18-24 h and potential biomarkers that are found with this method should be verified by a specialized, targeted method (e.g., by using standard addition in authentic matrix for quantification purposes). Overall, the current method can be successfully used for conduction of time-dependent postmortem metabolomics investigations. Graphical abstract.

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., (© 2020 John Wiley & Sons, Ltd.)

Psychoactive stimulants are a popular drug class which are used recreationally. Over the last decade, large numbers of new psychoactive substances (NPS) have entered the drug market and these pose a worldwide problem to human health. Metabolomics approaches are useful tools for simultaneous detection of endogenous metabolites affected by drug use. They allow identification of pathways or characteristic metabolites, which might support the understanding of pharmacological actions or act as indirect biomarkers of consumption behavior or analytical detectability. Herein, we performed a comparative metabolic profiling of three psychoactive stimulant drugs 3,4-methylenedioxymethamphetamine (MDMA), amphetamine and the NPS mephedrone by liquid chromatography-high resolution mass spectrometry (LC-HRMS) in order to identify common pathways or compounds. Plasma samples were obtained from controlled administration studies to humans. Various metabolites were identified as increased or decreased based on drug intake, mainly belonging to energy metabolism, steroid biosynthesis and amino acids. Linoleic acid and pregnenolone-sulfate changed similarly in response to intake of all drugs. Overall, mephedrone produced a profile more similar to that of amphetamine than MDMA in terms of affected energy metabolism. These data can provide the basis for further in-depth targeted metabolome studies on pharmacological actions and search for biomarkers of drug use.

Drug testing in hair is a controversial subject of discussion. Claims that decontamination protocols could generate false-positive samples, by washing contamination in hair, have unsettled many toxicologists. At least for zolpidem (known for showing only minor contamination), it could be shown that differentiation of the drug incorporated via the bloodstream from contamination was possible. The current work addresses cocaine and methadone, known for their high concentrations and contamination issues. Longitudinally and cross-sectioned samples of drug-soaked hair, consumer hair and cocaine powder contaminated hair were investigated using time of flight-secondary ion mass spectrometry (ToF-SIMS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). In addition, the resulting wash solutions were investigated using LC-MS/MS. Differentiation of contamination from incorporation was possible for soaked and consumer hair samples. Therefore, contamination could be localized in the superficial compartments of hair and could be removed using strong wash protocols. In the case of powder contaminated hair samples, a small amount of cocaine remained in the inner structures even after the application of the strongest wash protocols. However, taking into consideration the differences in their behavior during decontamination steps compared to both soaked and authentic hair samples, the validity of this contamination protocol (rubbing cocaine powder into hair) must be questioned. Furthermore, when using cut-off values and metabolite ratios (from routine hair analysis), the differentiation of incorporation from contamination was possible also for all our experimental samples in this study. Inclusion of metabolites and application of cut-off values are therefore a must in routine hair analysis.

Liquid chromatography coupled to high-resolution mass spectrometry (HRMS) enables data independent acquisition (DIA) and untargeted screening. However, to avoid the handling of the resulting large dataset, most laboratories in that field still use targeted screening methods, which offer good sensitivity and specificity but are limited to known compounds. The promising field of machine learning offers new possibilities such as artificial neural networks that can be trained to classify large amounts of data. In this proof of concept study, we exemplify such a machine learning approach for raw HRMS-DIA data files. We evaluated a machine learning model using training, validation, and test sets of solvent and whole blood samples containing drugs (of abuse) common in forensic toxicology. For that purpose, different platforms were used. With a feedforward neural network model architecture, a category prediction (blank sample vs. drug containing sample) was aimed for. With the applied machine learning approaches, the sensitivity and specificity, of the validation and test set, for the prediction of sample classes were in a suitable range for an actual use in a (routine) laboratory (e.g. workplace drug testing). In conclusion, this proof of concept study clearly demonstrated the huge potential of machine learning in the analysis of HRMS-DIA data., (© 2020 John Wiley & Sons, Ltd.)

Background: There is no universally accepted age cut-off for defining young strokes., Aims: We aimed to determine, based on the profile of young stroke patients in our regional centre, an appropriate age cut-off for young strokes., Methods: A retrospective analysis of all ischaemic stroke patients admitted to our centre from 2015 to 2017. We identified 391 ischaemic stroke patients; 30 patients between the ages of ≤50, 40 between 51-60 inclusive and 321 ≥ 61 years of age. We collected data on demographic profiles, risk factors and stroke classification using the Trial of Org 10 172 in Acute Stroke Treatment criteria., Results: We found significant differences between the ≤50 and ≥61 age groups for most of the risk factors and similarities between the 51-60 inclusive and ≥ 61 age groups. At least one of the six risk factors assessed in the study was present in 86.7% of the youngest group, 97.5% of the intermediate age group and 97.2% in the oldest group. In terms of the mechanisms of stroke, the youngest and oldest age groups in our study differed in the prevalence of cryptogenic, cardioembolic and other causes of stroke. The middle and older age groups had similar mechanisms of stroke., Conclusions: The prevalence of vascular risk factors and mechanisms of stroke likewise differed significantly across age groups. This study suggests that 50 years is an appropriate age cut-off for defining young strokes and reinforces the importance of primary prevention in all age groups., (© 2019 Royal Australasian College of Physicians.)

Benzodiazepines (BDZ) and z-drugs belong to the most frequently prescribed medicines worldwide used for anxiety, epilepsy and sleeping disorders. Due to their pharmacology, they have a high potential for misuse. Hair analysis is being performed for the retrospective monitoring of drug exposure. However, there is a lack of reference values to obtain indication of BDZ/z-drug misuse. Further, there is no consensus on BDZ/z-drug cut-off concentrations above which a hair sample is reported as positive. The objective of the present study was to retrospectively evaluate BDZ/z-drug levels in hair for better interpretation of hair testing results. For this purpose, 4,630 authentic samples (head/body hair) from a heterogeneous cohort in Switzerland tested for the presence of 20 BDZ/BDZ metabolites and three z-drugs by liquid chromatography-tandem mass spectrometry were included. Drug concentrations in hair were statistically evaluated by box-plots in 1,726 positive samples. Further, metabolite-to-parent drug ratios were determined. Zolpidem, diazepam, nordazepam, oxazepam, lorazepam, midazolam, alprazolam, and bromazepam were among the most frequently detected drugs. Generally, drug concentration ranges varied strongly between the lower limit of quantification and 30,000 pg/mg hair. Sixteen BDZ/BDZ metabolites and zopiclone (z-drug) displayed a median below 50 pg/mg which is recommended as cut-off or minimum requirement for the limit of detection (LOD) by institutions. In case of ten drugs even the 75 th percentile was below 50 pg/mg. Therefore, we strongly recommend to reconsider whether the use of an equal BDZ/z-drug cut-off is reasonable and whether minimum requirements for LODs should be lowered. The statistical evaluation of BDZ/Z-drug hair concentrations by box-plots can help in the development of analytical methods for hair samples and in the interpretation of BDZ/z-drug hair levels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Importance: Intravenous thrombolysis with tenecteplase improves reperfusion prior to endovascular thrombectomy for ischemic stroke compared with alteplase., Objective: To determine whether 0.40 mg/kg of tenecteplase safely improves reperfusion before endovascular thrombectomy vs 0.25 mg/kg of tenecteplase in patients with large vessel occlusion ischemic stroke., Design, Setting, and Participants: Randomized clinical trial at 27 hospitals in Australia and 1 in New Zealand using open-label treatment and blinded assessment of radiological and clinical outcomes. Patients were enrolled from December 2017 to July 2019 with follow-up until October 2019. Adult patients (N = 300) with ischemic stroke due to occlusion of the intracranial internal carotid, \basilar, or middle cerebral artery were included less than 4.5 hours after symptom onset using standard intravenous thrombolysis eligibility criteria., Interventions: Open-label tenecteplase at 0.40 mg/kg (maximum, 40 mg; n = 150) or 0.25 mg/kg (maximum, 25 mg; n = 150) given as a bolus before endovascular thrombectomy., Main Outcomes and Measures: The primary outcome was reperfusion of greater than 50% of the involved ischemic territory prior to thrombectomy, assessed by consensus of 2 blinded neuroradiologists. Prespecified secondary outcomes were level of disability at day 90 (modified Rankin Scale [mRS] score; range, 0-6); mRS score of 0 to 1 (freedom from disability) or no change from baseline at 90 days; mRS score of 0 to 2 (functional independence) or no change from baseline at 90 days; substantial neurological improvement at 3 days; symptomatic intracranial hemorrhage within 36 hours; and all-cause death., Results: All 300 patients who were randomized (mean age, 72.7 years; 141 [47%] women) completed the trial. The number of participants with greater than 50% reperfusion of the previously occluded vascular territory was 29 of 150 (19.3%) in the 0.40 mg/kg group vs 29 of 150 (19.3%) in the 0.25 mg/kg group (unadjusted risk difference, 0.0% [95% CI, -8.9% to -8.9%]; adjusted risk ratio, 1.03 [95% CI, 0.66-1.61]; P = .89). Among the 6 secondary outcomes, there were no significant differences in any of the 4 functional outcomes between the 0.40 mg/kg and 0.25 mg/kg groups nor in all-cause deaths (26 [17%] vs 22 [15%]; unadjusted risk difference, 2.7% [95% CI, -5.6% to 11.0%]) or symptomatic intracranial hemorrhage (7 [4.7%] vs 2 [1.3%]; unadjusted risk difference, 3.3% [95% CI, -0.5% to 7.2%])., Conclusions and Relevance: Among patients with large vessel occlusion ischemic stroke, a dose of 0.40 mg/kg, compared with 0.25 mg/kg, of tenecteplase did not significantly improve cerebral reperfusion prior to endovascular thrombectomy. The findings suggest that the 0.40-mg/kg dose of tenecteplase does not confer an advantage over the 0.25-mg/kg dose in patients with large vessel occlusion ischemic stroke in whom endovascular thrombectomy is planned., Trial Registration: ClinicalTrials.gov Identifier: NCT03340493.

Hair analysis has become a valuable tool in forensic toxicology to assess drug or alcohol abstinence. Yet, hair adulteration by cosmetic products presents a major challenge for forensic hair analysis. Oxidative treatments, e.g. bleaching, may lead to analyte loss and thereby to false negative results. Currently, the eumelanin degradation product 1H-pyrrole-2,3,5-tricarboxylic acid (PTCA) serves as a marker for oxidative hair treatment, but requires the definition of cut-off values. To investigate further eumelanin degradation products as markers for oxidative hair treatment, hair samples with and without in vitro bleaching (hydrogen peroxide (H 2 O 2 ) concentrations 1.9% up to 12%; incubation times 15 min, 30 min, 60 min) were analyzed by liquid chromatography coupled to high-resolution time of flight mass spectrometry (HPLC-HRMS). The distribution of eumelanin degradation products along the hair shaft was investigated for routine applicability after segmentation of cosmetically untreated hair samples and authentically treated hair samples. The signals of the eumelanin degradation products PTCA, 1H-pyrrole-2,3,4-tricarboxylic acid (isoPTCA), and 1H-pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) were found to be significantly elevated after in vitro bleaching already with low H 2 O 2 concentrations and after short incubation times. In contrast to PTCA and isoPTCA, PTeCA was not detectable in cosmetically untreated segments up to 12 cm from hair root and was only formed through the oxidation process. The results of the study show that the detection of PTeCA within the proximal 3 to 6 cm segment can be applied to reliably detect hair adulteration attempts through hair bleaching., (© 2019 John Wiley & Sons, Ltd.)

Target-controlled infusion (TCI) is based on pharmacokinetic models designed to achieve a desired drug level in the blood. TCI's predictive accuracy of plasma propofol levels at the end of surgery with major blood loss has not been well established. This prospective observational study included adult patients (BMI 20-35 kg/m 2 ) undergoing surgery with expected blood loss ≥ 1500 mL. The study was conducted with the Schnider TCI propofol model (Alaris PK Infusion Pump, CareFusion, Switzerland). Propofol levels were assessed in steady-state at the end of anaesthesia induction (T initial ) and before the end of surgery (T final ). Predicted propofol levels (C TCI ) were compared to measured levels (C blood ). Twenty-one patients were included. The median estimated blood loss was 1600 mL (IQR 1000-2300), and the median fluid balance at T final was + 3200 mL (IQR 2320-4715). Heart rate, mean arterial blood pressure, and blood lactate did not differ significantly between T initial and T final . The median bispectral index (0-100) was 50 (IQR 42-54) and 49 (IQR 42-56) at the two respective time points. At T initial , median C TCI was 2.2 µmol/L (IQR 2-2.45) and C blood was 2.0 µmol/L (bias 0.3 µmol/L, limits of agreement - 1.1 to 1.3, p = 0.33). C TCI and C blood at T final were 2.0 µmol/L (IQR 1.6-2.2) and 1 µmol/L (IQR 0.8-1.4), respectively (bias 0.6 µmol/L, limits of agreement - 0.89 to 1.4, p < 0.0001). Propofol TCI allows clinically unproblematic conduct of general anaesthesia. In cases of major blood loss, the probability of propofol TCI overestimating plasma levels increases.Trial registration German Clinical Trials Register (DRKS; DRKS00009312).

Steroid hormone analysis is widely used in health- and stress-related research to get insights into various diseases and the adaption to stress. Hair analysis has been used as a tool for the long-term monitoring of these steroid hormones. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous identification and quantification of seven steroid hormones (cortisone, cortisol, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, progesterone) in hair. Cortisol, cortisone, androstenedione, testosterone and progesterone were detected and quantified in authentic hair samples of different individuals. Significantly higher concentrations for body hair were found for cortisone and testosterone compared to scalp hair. Furthermore, weak correlations for the majority of steroids between scalp and body hair indicate that body hair is not really suitable as alternative when scalp hair is not available. The influence of hair pigmentation was analyzed by comparing pigmented to non-pigmented hair of grey-haired individuals. The results showed no differences for cortisol, cortisone, androstenedione, testosterone and progesterone concentrations (p > 0.05) implying that hair pigmentation has not a strong effect on steroid hormone concentrations. Correlations between hair and nail steroid levels were also studied. Higher concentrations of cortisol and cortisone in hair were found compared to nails (p < 0.0001). No significant correlation for cortisone, cortisol, androstenedione, testosterone and progesterone concentrations were found between hair and nails. These results demonstrate that matrix-dependent value ranges for hair and nail steroid levels should be established and applied for interpretation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Gamma-hydroxybutyrate (GHB; or sodium oxybate) is an endogenous GHB-/gamma-aminobutyric acid B receptor agonist. It is approved for application in narcolepsy and has been proposed for the potential treatment of Alzheimer's disease, Parkinson's disease, fibromyalgia, and depression, all of which involve neuro-immunological processes. Tryptophan catabolites (TRYCATs), the cortisol-awakening response (CAR), and brain-derived neurotrophic factor (BDNF) have been suggested as peripheral biomarkers of neuropsychiatric disorders. GHB has been shown to induce a delayed reduction of T helper and natural killer cell counts and alter basal cortisol levels, but GHB's effects on TRYCATs, CAR, and BDNF are unknown., Methods: Therefore, TRYCAT and BDNF serum levels, as well as CAR and the affective state (Positive and Negative Affect Schedule [PANAS]) were measured in the morning after a single nocturnal dose of GHB (50 mg/kg body weight) in 20 healthy male volunteers in a placebo-controlled, balanced, randomized, double-blind, cross-over design., Results: In the morning after nocturnal GHB administration, the TRYCATs indolelactic acid, kynurenine, kynurenic acid, 3-hydroxykynurenine, and quinolinic acid; the 3-hydroxykynurenine to kynurenic acid ratio; and the CAR were significantly reduced (P < 0.05-0.001, Benjamini-Hochberg corrected). The quinolinic acid to kynurenic acid ratio was reduced by trend. Serotonin, tryptophan, and BDNF levels, as well as PANAS scores in the morning, remained unchanged after a nocturnal GHB challenge., Conclusions: GHB has post-acute effects on peripheral biomarkers of neuropsychiatric disorders, which might be a model to explain some of its therapeutic effects in disorders involving neuro-immunological pathologies. This study was registered at ClinicalTrials.gov as NCT02342366., (© The Author(s) 2019. Published by Oxford University Press on behalf of CINP.)

Gamma-hydroxybutyrate (GHB) is an endogenous GHB/GABA B receptor agonist, which has demonstrated potency in consolidating sleep and reducing excessive daytime sleepiness in narcolepsy. Little is known whether GHB's efficacy reflects the promotion of physiological sleep mechanisms and no study has investigated its sleep consolidating effects under low sleep pressure. GHB (50 mg/kg p.o.) and placebo were administered in 20 young male volunteers at 2:30 a.m., the time when GHB is typically given in narcolepsy, in a randomized, double-blinded, crossover manner. Drug effects on sleep architecture and electroencephalographic (EEG) sleep spectra were analyzed. In addition, current source density (CSD) analysis was employed to identify the effects of GHB on the brain electrical sources of neuronal oscillations. Moreover, lagged-phase synchronization (LPS) analysis was applied to quantify the functional connectivity among sleep-relevant brain regions. GHB prolonged slow-wave sleep (stage N3) at the cost of rapid eye movement (REM) sleep. Furthermore, it enhanced delta-theta (0.5-8 Hz) activity in NREM and REM sleep, while reducing activity in the spindle frequency range (13-15 Hz) in sleep stage N2. The increase in delta power predominated in medial prefrontal cortex, parahippocampal and fusiform gyri, and posterior cingulate cortex. Theta power was particularly increased in the prefrontal cortex and both temporal poles. Moreover, the brain areas that showed increased theta power after GHB also exhibited increased lagged-phase synchronization among each other. Our study in healthy men revealed distinct similarities between GHB-augmented sleep and physiologically augmented sleep as seen in recovery sleep after prolonged wakefulness. The promotion of the sleep neurophysiological mechanisms by GHB may thus provide a rationale for GHB-induced sleep and waking quality in neuropsychiatric disorders beyond narcolepsy.

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., (© 2019 John Wiley & Sons, Ltd.)

Given that external contamination must be considered in hair analysis, there is still a demand for reliable tools to differentiate between incorporation of drugs into the hair as a result of drug consumption and of the hair shaft by external contamination. With the aim of establishing alternative discrimination parameters, some of the hydroxy metabolites of cocaine i.e., para- and meta-hydroxycocaine and para- and meta-hydroxybenzoylecgonine were measured together with cocaine, benzoylecgonine, cocaethylene, and norcocaine in five seized street cocaine samples and in hair samples from different cohorts: cohort 1 (in vivo external contamination study, n = 28), cohort 2 (individuals with self-reported cocaine use, n = 92), and cohort 3 (individuals with suspected cocaine use or contamination, n = 198). Statistical evaluation of the data of cohort 1 and 2 using ROC curves yielded metabolic ratios indicating cocaine use. Based on these results, a decision workflow was established for the discrimination between cocaine use and external contamination. The power of this approach was finally statistically validated across the different cohorts., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Although there is an established association between cancer and stroke, the role of malignancy as a causative agent or comorbidity is not always clear. Moreover, there are no established guidelines on the acute treatment of cancer-associated stroke or optimal anticoagulation. This case report illustrates the significance of these practice gaps., Case Presentation: A 62-year-old Caucasian woman presented to our institute with acute neurological deficits and was found to have an occluded left middle cerebral artery on a computed tomographic angiogram. She was administered intravenous alteplase and underwent unsuccessful endovascular clot retrieval. Besides smoking and her age, she had no cerebrovascular risk factors, and the results of baseline investigations for the cause of stroke were negative. Subsequent computed tomography of the chest, abdomen, and pelvis showed metastatic malignancy, and in the context of a significantly elevated serum cancer antigen 19-9, we suspected a pancreatic primary cancer. A transthoracic echocardiogram demonstrated mitral regurgitation but no visible vegetation. The patient died of her illness. We made a diagnosis of cancer-associated stroke, specifically a likely case of nonbacterial thrombotic endocarditis., Conclusions: This case highlights the importance of having a high threshold of suspicion for malignancy as a cause of stroke.

The interest in metabolomic studies has rapidly increased over the past few years. Changes of endogenous compounds are typically detected in plasma or urine. However, the use of hair allows for long-term monitoring of metabolomic changes and has recently started being applied to metabolomic studies. Within the proposed study, we aimed for a systematical investigation of different pre-analytical parameters on detected metabolites from different chemical classes in hair. For this purpose, three different parameters were varied: (1) multi-step decontamination (dichloromethane (DCM), acetone, H 2 O, acetone; H 2 O, acetone, DCM, acetone; and H 2 O, methanol/acetone), (2) homogenization (pulverization vs. cutting into snippets), and (3) extraction (acetonitrile (ACN)/buffer pH 4 vs. ACN/H 2 O vs. ACN/buffer pH 8.5). To include as many metabolites as possible, samples were analyzed by high-resolution time of flight mass spectrometry coupled to liquid chromatography (HPLC-HRMS) and additionally by gas chromatography high-resolution mass spectrometry (GC-HRMS) followed by untargeted-like data processing, respectively. The application of different decontamination procedures yielded similar results, although pointing to a trend towards increased washing-out effects if protic solvents were used as a first washing step. Pulverization of hair samples was favorable in terms of detected and tentatively identified metabolites. Evaluation of extraction solvents showed differences in extraction yield for the majority of investigated metabolites, yet, a prediction of metabolite extraction according to their pK a values was not possible. Overall, successive decontamination with DCM, acetone, H 2 O, and acetone; homogenization by pulverization; and extraction with ACN/H 2 O produced reliable results. Graphical abstract.

Gamma-hydroxybutyrate (GHB) is a short-chain fatty acid that occurs naturally in the mammalian brain and is prescribed as a medication against narcolepsy or used as a drug of abuse. Particularly, its use as a knock-out drug in cases of drug-facilitated crimes is of major importance in forensic toxicology. Because of its rapid metabolism and resulting narrow detection windows (<12 hours in urine), detection of GHB remains challenging. Thus, there is an urgent call for new markers to improve the reliable detection of GHB use. In the framework of a randomized, placebo-controlled, crossover study in 20 healthy male volunteers, urine samples obtained 4.5 hours post-administration were submitted to untargeted mass spectrometry [MS, quadrupole time of flight (QTOF)] analysis to identify possible new markers of GHB intake. MS data from four different analytical methods (reversed phase and hydrophilic interaction liquid chromatography; positive and negative electrospray ionization) were filtered for significantly changed features applying univariate and multivariate statistics. From the resulting 42 compounds of interest, 8 were finally identified including conjugates of GHB with carnitine, glutamate, and glycine as well as the endogenous compounds glycolate and succinylcarnitine. While GHB conjugates were only detectable in the GHB, but not in the placebo group, glycolate and succinylcarnitine were present in both groups albeit significantly increased through GHB intake. Untargeted metabolomics proved as a suitable tool for the non-hypothesis driven identification of new GHB markers. However, more studies on actual concentrations, detection windows, and stability will be necessary to assess the suitability of these markers for routine application., (© 2018 John Wiley & Sons, Ltd.)

Drug of abuse (DOA) consumption is a growing problem worldwide, particularly with increasing numbers of new psychoactive substances (NPS) entering the drug market. Generally, little information on their adverse effects and toxicity are available. The direct detection and identification of NPS is an analytical challenge due to their ephemerality on the drug scene. An approach that does not directly focus on the structural detection of an analyte or its metabolites, would be beneficial for this complex analytical scenario and the development of alternative screening methods could help to provide fast response on suspected NPS consumption. A metabolomics approach might represent such an alternative strategy for the identification of biomarkers for different questions in DOA testing. Metabolomics is the monitoring of changes in small (endogenous) molecules (<1,000 Da) in response to a certain stimulus, e.g., DOA consumption. For this review, a literature search targeting "metabolomics" and different DOAs or NPS was conducted. Thereby, different applications of metabolomic strategies in biomarker research for DOA identification were identified: (a) as an additional tool for metabolism studies bearing the major advantage that particularly a priori unknown or unexpected metabolites can be identified; and (b) for identification of endogenous biomarker or metabolite patterns, e.g., for synthetic cannabinoids or also to indirectly detect urine manipulation attempts by chemical adulteration or replacement with artificial urine samples. The majority of the currently available literature in that field, however, deals with metabolomic studies for DOAs to better assess their acute or chronic effects or to find biomarkers for drug addiction and tolerance. Certain changes in endogenous compounds are detected for all studied DOAs, but often similar compounds/pathways are influenced. When evaluating these studies with regard to possible biomarkers for drug consumption, the observed changes appear, albeit statistically significant, too small to reliably work as biomarker for drug consumption. Further, different drugs were shown to affect the same pathways. In conclusion, metabolomic approaches possess potential for detection of biomarkers indicating drug consumption. More studies, including more sensitive targeted analyses, multi-variant statistical models or deep-learning approaches are needed to fully explore the potential of omics science in DOA testing.

Determination of the ethanol concentration in corpses with MRS would allow a reproducible forensic assessment by which evidence is collected in a noninvasive manner. However, although MRS has been successfully used to detect ethanol in vivo, it has not been applied to postmortem ethanol quantification in situ. The present study examined the feasibility of the noninvasive measurement of the ethanol concentration in human corpses with MRS. A total of 15 corpses with suspected alcohol consumption before demise underwent examination in a 3 T whole body scanner. To address the partial overlap of the ethanol and lactate signal in the postmortem spectrum, non-water-suppressed single voxel spectra were recorded in the cerebrospinal fluid (CSF) of the left lateral ventricle via the metabolite cycling technique. The ethanol signals were quantified using the internal water as reference standard, as well as based on a reference signal acquired in a phantom. The measured values were compared with biochemically determined concentrations in the blood (BAC) and CSF (CSFAC). In 8 of the 15 corpses a BAC above zero was determined (range 0.03-1.68 g/kg). In all of these 8 corpses, ethanol was measured in CSF with the proposed MRS protocol. The two applied MRS calibration strategies resulted in similar concentrations. However, the MRS measurements generally overestimated the ethanol concentration by 0.09 g/kg (4%) to 0.72 g/kg (45%) as compared with the CSFAC value. The presented MRS protocol allows the measurement of ethanol in the CSF in human corpses and provides an estimation of the ethanol concentration prior to autopsy. Observed deviations from biochemically determined concentrations are mainly explained by the approximate correction of the relaxation attenuation of the ethanol signal., (© 2019 John Wiley & Sons, Ltd.)

Over the past few years, the interest in metabolomics has increased in various fields including forensic toxicology. Forensic analysis typically requires a high degree of accuracy, which is often a problem in metabolomics applications. We aimed for a systematic evaluation of different analytical considerations of a metabolomics workflow allowing a targeted approach within an untargeted setup. Samples with 69 metabolites from different chemical classes were qualitatively and quantitatively analyzed on a high resolution quadrupole time of flight mass spectrometer coupled to liquid chromatography (UHPLC-QTOF). Three issues were addressed: (a) Two different approaches on "blind matrix" a simulated body fluid (SBF) and plasma-filtrate, were tested for calibration samples; (b) comparison of two different HPLC columns, reverse-phase (RP) and hydrophilic interaction chromatography (HILIC); and (c) comparison of three different acquisition modes (TOF-MS, information dependent data acquisition (IDA), and sequential window acquisition of all theoretical fragment-ion spectra (SWATH). Samples were measured repeatedly for method comparison based on sensitivity, accuracy, precision, and detection robustness. The blind matrices showed similar accuracy for most analytes, while SBF provided an easier preparation with satisfying results. To cover a wide part of the human metabolome, a combination of RP and HILIC showed the best results. The different scan modes performed equally regarding metabolite quantification while TOF-MS was more sensitive but lacked MS/MS spectra generation. IDA and SWATH files were aligned to various databases where IDA showed good MS/MS spectra matches. SWATH seemed to be beneficial in detection rate but was incompatible with many important software tools in metabolomics., (© 2018 John Wiley & Sons, Ltd.)

Reliable detection of urine adulteration attempts to circumvent positive drug testing represents a critical step for laboratories in abstinence control settings. An ideal workflow for high-throughput testing would involve simultaneous detection of adulteration attempts in the same run with drug detection. Monitoring of degraded or oxidized endogenous urinary compounds as indirect markers has been previously evaluated for that purpose exemplified for the adulterant potassium nitrite (KNO 2 ). Fifteen, previously identified endogenous markers should now be evaluated for their general applicability to detect adulteration attempts for the adulterants hypochlorite-based bleach (NaOCl), peroxidase and peroxide (H 2 O 2 ), pyridinium chlorochromate (PCC), and iodine (I 2 ). Initial experiments revealed similar results for the tested adulterants regarding degradation of indolylacryloylglycine (IAG), uric acid (UA), or UA derivatives. 5-Hydroxyisourate (HIU), the oxidation product of UA, was however only formed by KNO 2 , PCC, and H 2 O 2 . Amino acids showed larger adulterant-dependent differences. All reactions were shown to be influenced by the adulterant concentration and the urinary pH with large variances depending on compound and adulterant. Except for HIU/PCC, all markers were stable within +/- 30% variation for all adulterants at -20°C. Receiver operating characteristics indicated best sensitivity and specificity over all adulterants for IAG (specificity 0.9, sensitivity 1.0) and UA (specificity 1.0, sensitivity 0.9). HIU gave best results for KNO 2 , PCC, and H 2 O 2 and N-acetylneuraminic acid for PCC and H 2 O 2 , respectively. When integrating a limited number of targets into existing screening methods, monitoring of UA, IAG, N-acetylneuraminic acid, and HIU is recommended., (© 2018 John Wiley & Sons, Ltd.)

On healthy cells the non-classical HLA class Ib molecule HLA-E displays the cognate ligand for the NK cell receptor NKG2A/CD94 when bound to HLA class I signal peptide sequences. In a pathogenic situation when HLA class I is absent, HLA-E is bound to a diverse set of peptides and enables the stimulatory NKG2C/CD94 receptor to bind. The activation of CD8⁺ T cells by certain p:HLA-E complexes illustrates the dual role of this low polymorphic HLA molecule in innate and adaptive immunity. Recent studies revealed a shift in the HLA-E peptide repertoire in cells with defects in the peptide loading complex machinery. We recently showed that HLA-E presents a highly diverse set of peptides in the absence of HLA class Ia and revealed a non-protective feature against NK cell cytotoxicity mediated by these peptides. In the present study we have evaluated the molecular basis for the impaired NK cell inhibition by these peptides and determined the cell surface stability of individual p:HLA-E complexes and their binding efficiency to soluble NKG2A/CD94 or NKG2C/CD94 receptors. Additionally, we analyzed the recognition of these p:HLA-E epitopes by CD8⁺ T cells. We show that non-canonical peptides provide stable cell surface expression of HLA-E, and these p:HLA-E complexes still bind to NKG2/CD94 receptors in a peptide-restricted fashion. Furthermore, individual p:HLA-E complexes elicit activation of CD8⁺ T cells with an effector memory phenotype. These novel HLA-E epitopes provide new implications for therapies targeting cells with abnormal HLA class I expression.

Contamination is a highly controversial issue in hair analysis. Therefore, hair testing protocols typically include wash steps to remove contamination. However, recent studies claim that washing could also lead to permanent incorporation of contaminants into hair, thus questioning the validity of hair testing at all. In the present study, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with longitudinal sectioning of single hairs and different decontamination protocols was used to reveal differences between the incorporation of a substance into hair from external sources and an incorporation via bloodstream. Single hairs were longitudinally sectioned using a custom-made sample holder. Data were acquired with MALDI-MS by rastering each hair individually. Single hair samples from drug users, blank hairs, and zolpidem- and zolpidem-D6-soaked hairs were investigated. Different published washing protocols were tested, and an in-house washing protocol was developed. For images with higher spatial resolution, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used. Longitudinal sectioning of hairs dramatically increased sensitivity; even single-dose administrations of zolpidem in single hairs could thus be detected using MALDI-MS. Zolpidem from external sources could be detected in large quantities in superficial hair structures. Zolpidem from consumer hairs, proposed to be strongly bound to inner hair structures, could not be completely removed even by the strongest tested decontamination protocol, whereas zolpidem-soaked hairs could be cleared almost completely with the developed in-house wash protocol. The applied methods allowed a first insight into the connection of decontamination protocols and wash-in phenomena in hair analysis. Further studies with other drugs are necessary to assess the general validity of these findings.

Recent studies indicate that not only the anthelminthic levamisole but also the racemate tetramisole (R-/S-phenyltetraimidazothiazole, PTHIT) was found as an adulterant for cocaine. We herein report on the investigation of the prevalence of PTHIT among cocaine-positive hair samples and the discrimination of the presence of its stereoisomers levamisole and dexamisole. Cocaine-positive hair samples were collected in a forensic context in 2015 and mainly 2017 (n = 724). Cocaine and PTHIT concentrations have been determined by achiral liquid chromatography-tandem mass spectrometry (LC-MS/MS). For distinction of levamisole/dexamisole chiral LC-MS/MS was performed. Cocaine hair concentrations ranged from 500 (cut-off) to approximately 800 000 pg/mg. The study demonstrates a strong prevalence of PTHIT in cocaine users' hair (87%, n = 627). PTHIT hair concentrations ranged from below LLOQ 3.5 to approximately 61 000 pg/mg (median: 260 pg/mg). Surprisingly, enantiomeric ratios of levamisole/dexamisole ranged from 0.17 to 1.34 (median: 0.63). Therefore, PTHIT-adulterated street cocaine samples (n = 24) seized between 2013 and 2016 were tested. Samples mainly contained racemic tetramisole (87.5%), only one sample contained levamisole only and two samples contained non-racemic PTHIT. Our experiments suggest that the presence of tetramisole in biological samples may have hitherto been underestimated. Most probably higher dexamisole than levamisole concentrations in hair specimens arise from stereoselective metabolism and/or elimination. This is particularly important in light of the different pharmacological activities of the two enantiomers and potentially different adverse effects. Toxicological interpretations in intoxication cases with adulterated cocaine should not only consider levamisole but also tetramisole and terminology in scientific contributions should be used accordingly., (© 2018 John Wiley & Sons, Ltd.)

Urine adulteration to circumvent positive drug testing is a fundamental challenge for toxicological laboratories all over the world. Untargeted mass spectrometry (MS) methods used in metabolomics had previously revealed uric acid (UA), histidine, methylhistidine, and their oxidation products, for example 5-hydroxyisourate (HIU) as potential biomarkers for urine adulteration using potassium nitrite (KNO 2 ). These markers should be further evaluated for their reliability, stability, and routine applicability. Influence of KNO 2 concentration, urinary pH, reaction time, and stability at room temperature, 4°C, and - 20°C was determined in urine under varying conditions. Analysis was performed after protein precipitation with acetonitrile by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Receiver operating characteristics (ROC) analysis was applied for cut-off evaluation after biomarker quantification (n = 100 per group). Blinded measurements (n = 50) were performed to check the general applicability to identify adulterated samples under routine conditions. The higher the adulterant concentration, the lower the concentrations of histidine, methylhistidine, and UA. In return, amounts of their oxidation products increased. Highest changes were observed under weak acid conditions (pH 4-5). Storage at -20°C ensured sufficient stability for all oxidative markers over one month. ROC evaluated biomarker performance and application to unknown samples revealed satisfying results, with HIU as the most suitable biomarker (positive predictive value (PPV) 100%), followed by UA (PPV 93%). HIU and UA proved suitable markers to identify urine adulteration using KNO 2 and are ready for implementation into routine MS procedures., (© 2018 John Wiley & Sons, Ltd.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The retrospective analysis of endogenous steroid hormones in nails can be used to elucidate endocrine diseases and thus help with their diagnosis and treatment. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method was developed for the simultaneous identification and quantification of 12 steroid hormones (aldosterone, cortisone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, dehydroepiandrosterone (DHEA), 17α-hydroxyprogesterone (17-OHP), dihydrotestosterone (DHT) and progesterone) in human fingernails. Steroid hormones were extracted from 0.5 mg to 10 mg pulverized nail clippings by methanolic extraction, followed by a liquid-liquid extraction. The analysis was conducted with LC-MS/MS in electrospray ionization positive mode. The method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect, recovery and robustness. It was successfully applied for steroid profiling in nails of mothers and their infants where cortisol, cortisone, testosterone, progesterone, androstenedione and 11-deoxycorticosterone could be detected. Furthermore, it could be shown that there is no significant difference in concentrations between left and right hand for cortisol, cortisone and progesterone. A positive linear correlation between cortisol and cortisone in nails was found. In conclusion, it could be shown that nails are a suitable matrix for the retrospective monitoring of cumulative steroid hormone levels., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Despite irrefutable evidence of its negative impact on animal behaviour and physiology, lethal and sublethal lead poisoning of wildlife is still persistent and widespread. For scavenging birds, ingestion of ammunition, or fragments thereof, is the major exposure route. In this study, we examined the occurrence of lead in four avian scavengers of Switzerland and how it differs between species, regions, and age of the bird. We measured lead concentration in liver and bone of the two main alpine avian scavengers (golden eagle Aquila chrysaetos and bearded vulture Gypaetus barbatus) over the entire area of the Swiss Alps and two of the main avian scavengers occurring in the lowlands of Switzerland (red kite Milvus milvus and common raven Corvus corax). Of those four species, only the bearded vulture is an obligate scavenger. We found that lead burdens in the two alpine avian scavengers were higher than those found for the same species elsewhere in Europe or North America and reached levels compatible with acute poisoning, whereas lead burdens of the two lowland avian scavengers seemed to be lower. Several golden eagles, but only one red kite with abnormally high bone lead concentrations were found. In all four species, a substantial proportion of birds had elevated levels which presumably represent recent (liver lead levels) or past (bone lead levels) uptake of sublethal doses of lead.

In Ancient Egypt it was common practice to embalm corpses and specific internal organs to ensure eternal life. The exact nature of the employed embalming fluids, particularly for organ preservation within the canopic jars, is debated. Therefore, the aim of the current study, was to chemically characterize and differentiate canopic jars (n = 28) and mummies (n = 6) using gas chromatography - high resolution mass spectrometry (GC-HR MS) with a new untargeted metabolomics-like screening approach; as part of a larger minimal-invasive transdisciplinary study on Ancient Egyptian human tissues. Post-analytical data processing included deconvolution, screening against the NIST 14 spectral database as well as a high resolution metabolomics library, and positive peak evaluation. In the majority of samples the presence of a coniferous resin was indicated by the detection of longiborneol in combination with abietadiene acid derivatives and guajacol. Beeswax, proposedly used for symbolic reasons and/or as a binding agent, was detected in 10 samples. Previously not mentioned in the literature, but identified in the current sample set, were medical-related substances like aniseed constituents, salicylic acid, chamazulene and jacobine. By applying an untargeted metabolomics-like approach to archaeological samples for the first time, extensive statistical analysis was made possible (using both identified and non-identified features; adding up to 4381 features), which showed significant differences in the overall chemical composition of canopic jar and mummy samples using principle component analysis (PCA) and partial least square-discriminant analysis (PLS-DA). This emphasizes the necessity for more extensive canopic jar studies in the future in order to interpret findings correctly.

Purpose To evaluate whether gadolinium penetrates human cerebrospinal fluid (CSF) after MR imaging (MRI) with a gadolinium-based contrast agent (GBCA). Materials and Methods For this retrospective study, the authors analyzed 60 CSF samples from 57 patients (median age, 50 years; range, 3-92 years) who underwent one contrast material-enhanced MRI examination with gadoterate meglumine within 60 days of CSF extraction between January and December 2016. CSF samples from patients who underwent MRI without contrast material administration (n = 22) or those who underwent contrast-enhanced MRI at least 1 year before extraction (n = 2) were analyzed and used as control samples. CSF measurements were performed with inductively coupled plasma mass spectrometry by monitoring the gadolinium 158 isotope. Statistical analyses were performed by using a preliminary Kruskal-Wallis test. Results Higher CSF gadolinium concentrations were detected within the first 8 hours after GBCA administration (mean concentration, 1152 ng/mL ± 734.6). Concentrations were lower between 8 and 48 hours (872 ng/mL ± 586). After 48 hours, gadolinium was almost completely cleared from CSF (121 ng/mL ± 296.3). All but two samples from the 24 control patients (median age, 60.5 years; range, 19-79 years) were negative for the presence of gadolinium. Those samples were from patients who had undergone GBCA-enhanced MRI examination more than a year before CSF extraction (0.1 and 0.2 ng/mL after 1 and 3 years, respectively). The concentrations in patients with chronic renal insufficiency (n = 3), cerebral toxoplasmosis (n = 1), and liver cirrhosis (n = 1) were higher than the mean concentrations. Conclusion Gadoterate meglumine can be detected in human CSF after intravenous administration., (© RSNA, 2018.)

The intake of 3,4-methylenedioxymethamphetamine (MDMA) is known to increase several endogenous substances involved in steroid and inflammation pathways. Untargeted metabolomics screening approaches can determine biochemical changes after drug exposure and can reveal new pathways, which might be involved in the pharmacology and toxicology of a drug of abuse. We analyzed plasma samples from a placebo-controlled crossover study of a single intake of MDMA. Plasma samples from a time point before and three time points after the intake of a single dose of 125 mg MDMA were screened for changes of endogenous metabolites. An untargeted metabolomics approach on a high-resolution quadrupole time-of-flight mass spectrometer coupled to liquid chromatography with two different chromatographic systems (reversed-phase and hydrophobic interaction liquid chromatography) was applied. Over 10 000 features of the human metabolome were detected. Hence, 28 metabolites were identified, which showed significant changes after administration of MDMA compared with placebo. The analysis revealed an upregulation of cortisol and pregnenolone sulfate 4 h after MDMA intake, suggesting increased stress and serotonergic activity. Furthermore, calcitriol levels were decreased after the intake of MDMA. Calcitriol is involved in the upregulation of trophic factors, which have protective effects on brain dopamine neurons. The inflammation mediators hydroxyeicosatetraenoic acid, dihydroxyeicosatetraenoic acid, and octadecadienoic acid were found to be upregulated after the intake of MDMA compared with placebo, which suggested a stimulation of inflammation pathways.

Hair samples are increasingly used for measuring the long-term stress mediator cortisol. However, hair is not always available and nails (finger or toe), as a keratinized matrix, may be an alternative to hair. In order to measure cortisol and cortisone in the nail matrix, an LC-MS/MS method has been developed and validated using 13 C 3 -labeled surrogate analytes. Both analytes were measured in ESI negative mode as formic acid adducts. Different sample preparation techniques were assessed, and single-step extraction in methanol was established for determination of cortisone and cortisol in the nail matrix. The method was successfully validated with limits of detection (LOD) and limits of quantification (LOQ) of 0.5 and 1.0 pg/mg for cortisol and cortisone, respectively. The calibration curve was linear up to a concentration of 500 pg/mg. Recovery was good for both analytes and showed values over 50%. Matrix effects with ion suppression occurred for both substances but could be corrected by the use of internal standard. Accuracy and precision were in the accepted range of ± 20% for both substances. The method was successfully applied to determine cortisol and cortisone concentrations in authentic nail samples. Cortisol and cortisone concentrations varied significantly among different fingernails, being highest in the little fingernails and lowest in the thumbnails. It could be shown that even in only 1 mg nail sample cortisol and cortisone can be reliably quantified. No correlation between hair and nail cortisol and cortisone concentrations could be found. Furthermore, cortisol and cortisone concentrations were significantly higher in hair. Graphical abstract.

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.

The number of new psychoactive substances (NPS) increases rapidly, harming society and fuelling the need for alternative testing strategies. These should allow the ever-increasing number of drugs to be tested more effectively for their toxicity and psychoactive effects. One proposed strategy is to complement rodent models with zebrafish ( Danio rerio ) larvae. Yet, our understanding of the toxicokinetics in this model, owing to the waterborne drug exposure and the distinct physiology of the fish, is incomplete. We here explore the toxicokinetics and behavioral effects of an NPS, meta-chlorophenylpiperazine (mCPP), in zebrafish larvae. Uptake kinetics of mCPP, supported by toxicokinetic modeling, strongly suggested the existence of active transport processes. Internal distribution showed a dominant accumulation in the eye, implying that in zebrafish, like in mammals, melanin could serve as a binding site for basic drugs. We confirmed this by demonstrating significantly lower drug accumulation in two types of hypo-pigmented fish. Comparison of the elimination kinetics between mCPP and previously characterized cocaine demonstrated that drug affinities to melanin in zebrafish vary depending on the structure of the test compound. As expected from mCPP-elicited responses in rodents and humans, zebrafish larvae displayed hypoactive behavior. However, significant differences were seen between zebrafish and rodents with regard to the concentration-dependency of the behavioral response and the comparability of tissue levels, corroborating the need to consider the organism-internal distribution of the chemical to allow appropriate dose modeling while evaluating effects and concordance between zebrafish and mammals. Our results highlight commonalities and differences of mammalian versus the fish model in need of further exploration.

Background: The empathogen 3,4-methylenedioxymethamphetamine (MDMA) is the prototypical prosocial club drug inducing emotional openness to others. It has recently been shown that acutely applied 3,4-MDMA in fact enhances emotional empathy and prosocial behavior, while it simultaneously decreases cognitive empathy. However, the long-term effects of 3,4-MDMA use on socio-cognitive functions and social interactions have not been investigated yet. Therefore, we examined emotional and cognitive empathy, social decision-making, and oxytocin plasma levels in chronic 3,4-MDMA users., Methods: We tested 38 regular but recently abstinent 3,4-MDMA users and 56 3,4-MDMA-naïve controls with the Movie for the Assessment of Social Cognition, the Multifaceted Empathy Test, and the Distribution Game and the Dictator Game. Drug use was objectively quantified by 6-month hair analyses. Furthermore, oxytocin plasma levels were determined in smaller subgroups (24 3,4-MDMA users, 9 controls)., Results: 3,4-MDMA users showed superior cognitive empathy compared with controls in the Multifaceted Empathy Test (Cohen's d=.39) and in the Movie for the Assessment of Social Cognition (d=.50), but they did not differ from controls in emotional empathy. Moreover, 3,4-MDMA users acted less self-serving in the Distribution Game. However, within 3,4-MDMA users, multiple regression analyses showed that higher 3,4-MDMA concentrations in hair were associated with lower cognitive empathy (βMDMA=-.34, t=-2.12, P<.05). Oxytocin plasma concentrations did not significantly differ between both groups., Conclusions: We conclude that people with high cognitive empathy abilities and pronounced social motivations might be more prone to 3,4-MDMA consumption. In contrast, long-term 3,4-MDMA use might nevertheless have a detrimental effect on cognitive empathy capacity.

High resolution mass spectrometry and modern data independent acquisition (DIA) methods enable the creation of general unknown screening (GUS) procedures. However, even when DIA is used, its potential is far from being exploited, because often, the untargeted acquisition is followed by a targeted search. Applying an actual GUS (including untargeted screening) produces an immense amount of data that must be dealt with. An optimization of the parameters regulating the feature detection and hit generation algorithms of the data processing software could significantly reduce the amount of unnecessary data and thereby the workload. Design of experiment (DoE) approaches allow a simultaneous optimization of multiple parameters. In a first step, parameters are evaluated (crucial or noncrucial). Second, crucial parameters are optimized. The aim in this study was to reduce the number of hits, without missing analytes. The obtained parameter settings from the optimization were compared to the standard settings by analyzing a test set of blood samples spiked with 22 relevant analytes as well as 62 authentic forensic cases. The optimization lead to a marked reduction of workload (12.3 to 1.1% and 3.8 to 1.1% hits for the test set and the authentic cases, respectively) while simultaneously increasing the identification rate (68.2 to 86.4% and 68.8 to 88.1%, respectively). This proof of concept study emphasizes the great potential of DoE approaches to master the data overload resulting from modern data independent acquisition methods used for general unknown screening procedures by optimizing software parameters.

The measurement of hair cortisol is increasingly used to measure long-term cumulative cortisol levels and investigate its role as an important stress mediator. In this study a comparative statistical analysis of five independent studies (all analyzed in our laboratory) was performed to investigate baseline ranges of cortisol values in hair and evaluate potential influences of sex, age and hair color. Cortisol concentrations in hair of 554 subjects were measured and a comparative statistical analysis was performed. The analysis showed that cortisol levels significantly differ depending on age. The toddler group (7 months (0.6 years) to 3 years) showed significantly higher values (median 10pg/mg, p-value<0.0001, d=0.78) than the adolescent group. The adolescent groups showed significantly lower (p-value<0.0001, d=0.58 and p<0.0001, d=0.13) values (median 2.4pg/mg and 2.8pg/mg) than the adult group (median 5.8pg/mg). Furthermore, in the adult group men showed significantly higher cortisol values than women (p-value<0.05, d=0.17). This effect could not be seen in the adolescent group. Black hair showed higher cortisol concentrations than blond hair (p-value<0.0001, d=1.3). In addition, two rounds of interlaboratory comparisons for hair cortisol samples between four laboratories revealed very consistent results. Our results demonstrate that baseline cortisol levels are generally low in hair thus making a standardized and well-elaborated analytical method indispensable for accurate determination. Age-dependent normative baseline cortisol levels (toddlers, adolescents and adults) are highly recommended based on the comparative analysis comprising five independent studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Amphetamine (AMP) is used as an illicit drug and also for the treatment of attention deficit hyperactivity disorder (ADHD). Respective drugs most often contain the enantiomer (S)-AMP as active compound or (S)-AMP is formed from the prodrug lisdexamfetamine (Elvanse®) whereas the illicit drug is usually traded as racemate ((R/S)-AMP). A differentiation between the use of the medically prescribed drug and the abuse of illicit street amphetamine is of great importance, for example in retrospective consumption monitoring by hair analysis. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the chiral separation and quantitation of (S)- and (R)-AMP in hair was developed. For this purpose, 20 mg hair was extracted and derivatized with N-(2,4-dinitro-5-fluorophenyl)-L(S)-valinamide L(S)-(DNPV) to yield amphetamine diastereomers. Baseline separation of the resulting diastereomers was achieved on a high-pressure liquid-chromatography system (HPLC) coupled to a Sciex QTRAP® 5500 linear ion trap quadrupole mass spectrometer. The method was successfully validated. Analysis of hair samples from nine Elvanse® patients revealed only (S)-AMP in eight cases; one subject showed both enantiomers indicating a (side-) consumption of street amphetamine. The analysis of the 16 amphetamine users' samples showed only racemic amphetamine. Furthermore, it could be shown in a controlled study that (S)-AMP can be detected after administration of even very low doses of lisdexamfetamine and dexamphetamine, which can be of interest in forensic toxicology and especially in drug-facilitated crime (DFC). The method now enables the retrospective compliance-monitoring of ADHD patients and the differentiation between medically prescribed intake of (S)-amphetamine and abuse of illicit street amphetamine., (Copyright © 2017 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 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., (Copyright © 2017 John Wiley & Sons, Ltd.)

Hair analysis has been established as a prevalent tool for retrospective drug monitoring. In this study, different extraction solvents for the determination of drugs of abuse and pharmaceuticals in hair were evaluated for their efficiency. A pool of authentic hair from drug users was used for extraction experiments. Hair was pulverized and extracted in triplicate with seven different solvents in a one- or two-step extraction. Three one- (methanol, acetonitrile, and acetonitrile/water) and four two-step extractions (methanol two-fold, methanol and methanol/acetonitrile/formate buffer, methanol and methanol/formate buffer, and methanol and methanol/hydrochloric acid) were tested under accurately equal experimental conditions. The extracts were directly analyzed by liquid chromatography-tandem mass spectrometry for opiates/opioids, stimulants, ketamine, selected benzodiazepines, antidepressants, antipsychotics, and antihistamines using deuterated internal standards. For most analytes, a two-step extraction with methanol did not significantly improve the yield compared to a one-step extraction with methanol. Extraction with acetonitrile alone was least efficient for most analytes. Extraction yields of acetonitrile/water, methanol and methanol/acetonitrile/formate buffer, and methanol and methanol/formate buffer were significantly higher compared to methanol. Highest efficiencies were obtained by a two-step extraction with methanol and methanol/hydrochloric acid, particularly for morphine, 6-monoacetylmorphine, codeine, 6-acetylcodeine, MDMA, zopiclone, zolpidem, amitriptyline, nortriptyline, citalopram, and doxylamine. For some analytes (e.g., tramadol, fluoxetine, sertraline), all extraction solvents, except for acetonitrile, were comparably efficient. There was no significant correlation between extraction efficiency with an acidic solvent and the pka or log P of the analyte. However, there was a significant trend for the extraction efficiency with acetonitrile to the log P of the analyte. The study demonstrates that the choice of extraction solvent has a strong impact on hair analysis outcomes. Therefore, validation protocols should include the evaluation of extraction efficiency of drugs by using authentic rather than spiked hair. Different extraction procedures may contribute to the scatter of quantitative results in inter-laboratory comparisons. Harmonization of extraction protocols is recommended, when interpretation is based on same cut-off levels., (Copyright © 2017 Elsevier B.V. All rights reserved.)