Easy and convenient millimole‐scale synthesis of new, potential biomarkers for gamma‐hydroxybutyric acid (GHB) intake: Feasible for analytical laboratories

New biomarkers indicating the abuse of drugs and alcohol are still of major interest for clinical and forensic sciences. The endogenous neurotransmitter and approved drug, gamma-hydroxybutyric acid (GHB), is often illegally used for drug-facilitated crimes by spiking GHB into alcoholic beverages. Analytical detection windows of only 6 h in blood and 12 h in urine are often too short to provide reliable proof of GHB ingestion. Therefore, new biomarkers are needed to prove exogenous GHB administration. Previously, amino acid GHB conjugates were discovered in an untargeted metabolomics screening and fatty acid esters with GHB were recently discussed as promising biomarkers to enlarge the analytical detection time windows. However, the development of analytical methods is still slowed down since reference compounds for targeted screenings are still missing. In this paper, we describe simple procedures for the rapid synthesis and purification of amino acid GHB conjugates as well as fatty acid esters, which can be adopted in analytical and clinical/forensic laboratories. Structural characterization data, together with IR, $^1$H-nuclear magnetic resonance (NMR), $^{13}$C-NMR, high-resolution mass spectra (MS), and MS/MS spectra in positive and negative ionization mode are reported for all obtained GHB conjugates and GHB conjugate precursors

Metabolomic Strategies in Biomarker Research–New Approach for Indirect Identification of Drug Consumption and Sample Manipulation in Clinical and Forensic Toxicology?

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

Do dried blood spots have the potential to support result management processes in routine sports drug testing?—Part 3: LC–MS/MS‐based peptide analysis for dried blood spot sampling time point estimation

Along with the recent acknowledgement of the World Anti-Doping Agency to use dried blood spot (DBS) samples for routine doping control purposes, there have been propositions to use DBS as a matrix that allows regular proactive remotely supervised self-sampling, providing potential longitudinal monitoring of an athlete's exposure to doping agents. However, several organizational aspects have to be considered before implementation, such as the verification of the sample collections time point. Based on a previous untargeted proteomics workflow utilizing liquid chromatography–high-resolution mass spectrometry (LC–HRMS) to identify protein/peptide markers to define the time since deposition of a bloodstain, the aim of the current study was to develop a targeted LC–HRMS/MS analytical method for promising peptidic target analytes. A long-term DBS storage experiment was carried out over a 3-month period (sample collection time points: 0, 2, 4, 7, 14, 21, 28, 42, 56, 70, 84 and 91 days) with DBS samples of 10 volunteers for longitudinal investigation of signal abundance changes of targeted peptide sequences at different storage temperatures (room temperature [RT], 4°C and −20°C). Prior to experimental analysis, LC–HRMS/MS method characteristics were successfully assessed, including intraday precision, carryover and sample extract stability. For estimation of DBS sample collection time points, ratios of two peptides that originate from the same protein prior to tryptic digestion were created. Two targeted peptide area ratios were found to significantly increase after being stored at RT for 28 days, representing potential markers for future use in routine doping controls that contribute to advancing complementary avenues in anti-doping

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

A fatal case of butyrfentanyl poisoning was investigated at the Zurich Institute of Forensic Medicine. At admission at the institute approx. 9 h 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 66 ng/mL in femoral blood, 39 ng/mL in heart blood, 110 ng/g in muscle, 57 ng/g in liver, 160 ng/g in kidney, 3100 ng/g in lung, 590 ng/g in spleen and 550 ng/g in adipose tissue. At t2, butyrfentanyl concentration in urine was 1100 ng/mL, in gastric content 2000 ng/mL, in hair 11,000 pg/mg and brain concentrations ranged between 200⿿340 ng/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 19 h. 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

Preliminary Investigation of Side Effects of Polymyxin B Administration in Hospitalized Horses

Neuro- and nephrotoxicity of polymyxins are known but clinical studies in horses are lacking. The aim of this study was to describe neurogenic and nephrogenic side effects of hospitalized horses receiving Polymyxin B (PolyB) as part of their treatment plan. Twenty horses diagnosed with surgical colic (n = 11), peritonitis (n = 5), typhlocolitis (n = 2), pneumonia, and pyometra (each n = 1) were included. Antimicrobial treatment was randomized to GENTA (gentamicin 10 mg/kg bwt q24 h IV, penicillin 30.000 IU/kg q6 h IV) or NO GENTA (marbofloxacin 2 mg/kg bwt q24 h IV, penicillin 30.000 IU/kg q6 h IV). The duration of PolyB treatment ranged from 1 to 4 days. Clinical and neurological examinations were performed, and serum PolyB concentrations were measured daily during and three days following PolyB treatment. Urinary analysis, plasma creatinine, urea and SDMA were assessed every other day. Video recordings of neurological examinations were graded by three blinded observers. All horses showed ataxia during PolyB treatment in both groups (median maximum ataxia score of 3/5, range 1–3/5). Weakness was detected in 15/20 (75%) horses. In 8/14 horses, the urinary γ-glutamyltransferase (GGT)/creatinine ratio was elevated. Plasma creatinine was mildly elevated in 1/16 horses, and SDMA in 2/10 horses. Mixed-model analysis showed a significant effect of time since last PolyB dose (p = 0.0001, proportional odds: 0.94) on the ataxia score. Ataxia and weakness should be considered as reversible adverse effects in hospitalized horses receiving PolyB. Signs of tubular damage occurred in a considerable number of horses; therefore, the nephrotoxic effect of polymyxins should be considered and urinary function monitored

Human Metabolome Changes after a Single Dose of 3,4-Methylenedioxymethamphetamine (MDMA) with Special Focus on Steroid Metabolism and Inflammation Processes

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

Modern Clinical Research on LSD

All modern clinical studies using the classic hallucinogen lysergic acid diethylamide (LSD) in healthy subjects or patients in the last 25 years are reviewed herein. There were five recent studies in healthy participants and one in patients. In a controlled setting, LSD acutely induced bliss, audiovisual synesthesia, altered meaning of perceptions, derealization, depersonalization, and mystical experiences. These subjective effects of LSD were mediated by the 5-HT2A receptor. LSD increased feelings of closeness to others, openness, trust, and suggestibility. LSD impaired the recognition of sad and fearful faces, reduced left amygdala reactivity to fearful faces, and enhanced emotional empathy. LSD increased the emotional response to music and the meaning of music. LSD acutely produced deficits in sensorimotor gating, similar to observations in schizophrenia. LSD had weak autonomic stimulant effects and elevated plasma cortisol, prolactin, and oxytocin levels. Resting-state functional magnetic resonance studies showed that LSD acutely reduced the integrity of functional brain networks and increased connectivity between networks that normally are more dissociated. LSD increased functional thalamocortical connectivity and functional connectivity of the primary visual cortex with other brain areas. The latter effect was correlated with subjective hallucinations. LSD acutely induced global increases in brain entropy that were associated with greater trait openness 14 days later. In patients with anxiety associated with life-threatening disease, anxiety was reduced for 2 months after two doses of LSD. In medical settings, no complications of LSD administration were observed. These data should contribute to further investigations of the therapeutic potential of LSD in psychiatry