Marie Mardal

Studies on the microbial biotransformation of the novel psychoactive substance methylenedioxypyrovalerone (MDPV) in wastewater by means of liquid chromatography-high resolution mass spectrometry/mass spectrometry

Sewage profiling as a mean to estimate consumption of drugs of abuse is gaining increasing attention. However, only scarce data are available so far on the impact of microbial biotransformation on the presence and hence detectability of drugs of abuse and their metabolites in wastewater (WW) samples. The aim of this work was therefore to study the biotransformation pathways of the novel psychoactive substance 3,4-methylenedioxypyrovalerone (MPDV) in WW by incubating it, based on the OECD guideline 314 A. MDPV was incubated (100 μg/L) for 10d at 22 °C in WW from a local WW treatment plant. Furthermore, urine and feces collected from rats administered 20mg MDPV/kg BW were incubated correspondingly. Samples were worked-up either by centrifugation/filtration and solid-phase (HCX) extraction or QuEChERS. High resolution (HR) mass spectra (MS) were recorded using an Orbitrap mass spectrometer. All products were identified via their HR-MS(2) spectra and chromatographic properties. The observed biotransformations in WW were: demethylenation and subsequent O-methylation, hydroxylation at the phenyl part, hydroxylation at the pyrrolidine part with subsequent methylation or oxidation, N-demethylation, and hydroxylation at the alkyl part as well as combination of them. In total, 12 biotransformation products were identified after 10 days of incubation. Three of these biotransformation products were previously reported to be also rat and human metabolites. No additional MDPV biotransformation products could be found after incubating the rat urine and feces samples. Instead, the urinary phase II glucuronides were nearly completely cleaved after one day of WW incubation. The presented study indicates that demethylenyl-methyl MDPV, the most abundant metabolite in human urine, should be the best indicator in WW to estimate its use.

Targeted and non-targeted drug screening in whole blood by UHPLC-TOF-MS with data-independent acquisition.

High-resolution mass spectrometry (HRMS) is widely used for the drug screening of biological samples in clinical and forensic laboratories. With the continuous addition of new psychoactive substances (NPS), keeping such methods updated is challenging. HRMS allows for combined targeted and non-targeted screening. First, peaks are identified by software algorithms, and identifications are based on reference standard data. Attempts are made to identify the remaining unknown peaks with in silico and literature data. However, several thousand peaks remain where most are unidentifiable or uninteresting in drug screening. The aims of the study were to apply a combined targeted and non-targeted screening approach to authentic driving-under-the-influence-of-drugs (DUID) samples (n = 44) and further validate the approach using whole-blood samples spiked with 11 low-dose synthetic benzodiazepine analogues (SBAs). Analytical data were acquired using ultra-high-performance liquid chromatography coupled with a time-of-flight mass spectrometer (UHPLC-TOF-MS) with data-independent acquisition (DIA). We present a combined targeted and non-targeted screening, where peak deconvolution and filtering reduced the number of peaks to inspect by three orders of magnitude, down to four peaks per DUID sample. The screening allowed for tentative identification of metabolites and drugs not included in the initial screening; 3 drugs and 14 metabolites were tentatively identified in the authentic DUID samples. Running targeted-screening true-positive identifications through the filters retained 73% of identifications. In the non-targeted screening, nine of the spiked SBAs were identified in the concentration range of 0.005-0.1 mg/kg, of which three were tentatively identified at concentrations below those reported in the literature. Copyright

Toxicokinetics of new psychoactive substances: plasma protein binding, metabolic stability, and human phase I metabolism of the synthetic cannabinoid WIN 55,212-2 studied using in vitro tools and LC-HR-MS/MS

The new psychoactive substance WIN 55,212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone) is a potent synthetic cannabinoid receptor agonist. The metabolism of WIN 55,212-2 in man has never been reported. Therefore, the aim of this study was to identify the human in vitro metabolites of WIN 55,212-2 using pooled human liver microsomes and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS) to provide targets for toxicological, doping, and environmental screening procedures. Moreover, a metabolic stability study in pooled human liver microsomes (pHLM) was carried out. In total, 19 metabolites were identified and the following partly overlapping metabolic steps were deduced: degradation of the morpholine ring via hydroxylation, N- and O-dealkylation, and oxidative deamination, hydroxylations on either the naphthalene or morpholine ring or the alkyl spacer with subsequent oxidation, epoxide formation with subsequent hydrolysis, or combinations. In conclusion, WIN 55,212-2 was extensively metabolized in human liver microsomes incubations and the calculated hepatic clearance was comparably high, indicating a fast and nearly complete metabolism in vivo. This is in line with previous findings on other synthetic cannabinoids. Copyright

Screening for illicit drugs in pooled human urine and urinated soil samples and studies on the stability of urinary excretion products of cocaine, MDMA, and MDEA in wastewater by hyphenated mass spectrometry techniques

Monitoring population drug use through wastewater-based epidemiology (WBE) is a useful method to quantitatively follow trends and estimate total drug consumption in communities. Concentrations of drug biomarkers might be low in wastewater due to dilution; and therefore analysis of pooled urine (PU) is useful to detect consumed drugs and identify targets of illicit drugs use. The aims of the study were (1) to screen PU and urinated soil (US) samples collected at festivals for illicit drug excretion products using hyphenated techniques; (2) to develop and validate a hydrophilic interaction liquid chromatography - mass spectrometry / mass spectrometry (HILIC-MS/MS) method of quantifying urinary targets of identified drugs in wastewater; and (3) to conduct a 24 h stability study, using PU and US to better reflect the chemical environment for targets in wastewater. Cocaine (COC) and ecstasy-like compounds were the most frequently detected illicit drugs; an analytical method was developed to quantify their excretion products. Hydroxymethoxymethamphetamine (HMMA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), HMMA sulfate (HMMA-S), benzoylecgonine (BE), and cocaethylene (CE) had 85-102% of initial concentration after 8 h of incubation, whereas COC and ecgonine methyl ester (EME) had 74 and 67% after 8 h, respectively. HMMA showed a net increase during 24 h of incubation (107% ± 27, n = 8), possibly due to the cleavage of HMMA conjugates, and biotransformation of MDMA. The results suggest HMMA as analytical target for MDMA consumption in WBE, due to its stability in wastewater and its excretion as the main phase I metabolite of MDMA. Copyright

3-Fluorophenmetrazine, a fluorinated analogue of phenmetrazine:Studies on in vivo metabolism in rat and human, in vitro metabolism in human CYP isoenzymes and microbial biotransformation in Pseudomonas Putida and wastewater using GC and LC coupled to(HR)-MS techniques

Wastewater-based epidemiology (WBE) as means to estimate illicit drug and new psychoactive substance (NPS) consumption with spatial and temporal resolution is gaining increasing attention. In order to evaluate a given NPS using WBE, in vivo metabolism and microbial biotransformation of excretion products and unchanged compounds need evaluation. The aims of this study were to identify in vivo phase I and II metabolites of the NPS 3-fluorophenmetrazine (3-FPM) in human and rat urine and study the in vitro contribution of Cytochrome P450 (CYP) isoenzymes in phase I metabolism. Additionally, to study microbial biotransformation products (MBPs) of 3-FPM from incubations in wastewater and in a wastewater isolated Pseudomonas Putida strain. To these aims gas chromatography and liquid chromatography coupled to mass spectrometry were applied. Metabolites and MBPs were isolated from urine and microbial incubations after solid phase extraction and precipitation with or without enzymatic conjungate cleaving. The main transformation pathways were N-oxidation, aryl hydroxylation and subsequent O-methylation, alkyl hydroxylation, oxidation, and degradation of the ethyl-bridge yielding the O/N-bis-dealkylated metabolite, combinations thereof and further glucuronidation or sulfations. The main excretion products in the human urine sample were the unchanged compound and the N-oxide, and the main MBPs were the N-oxide and hydroxylation with subsequent oxidations on the alpha-methyl position. Based on these findings, the proposed strategy for WBE analysis of 3-FPM is quantitative determination of unchanged 3-FPM together with qualitative verification of a number of selected metabolites to verify consumption and rule out discharge.

A new approach towards biomarker selection in estimation of human exposure to chiral chemicals: a case study of mephedrone

Wastewater-based epidemiology is an innovative approach to estimate public health status using biomarker analysis in wastewater. A new compound detected in wastewater can be a potential biomarker of an emerging trend in public health. However, it is currently difficult to select new biomarkers mainly due to limited human metabolism data. This manuscript presents a new framework, which enables the identification and selection of new biomarkers of human exposure to drugs with scarce or unknown human metabolism data. Mephedrone was targeted to elucidate the assessment of biomarkers for emerging drugs of abuse using a four-step analytical procedure. This framework consists of: (i) identification of possible metabolic biomarkers present in wastewater using an in-vivo study; (ii) verification of chiral signature of the target compound; (iii) confirmation of human metabolic residues in in-vivo/vitro studies and (iv) verification of stability of biomarkers in wastewater. Mephedrone was selected as a suitable biomarker due to its high stability profile in wastewater. Its enantiomeric profiling was studied for the first time in biological and environmental matrices, showing stereoselective metabolism of mephedrone in humans. Further biomarker candidates were also proposed for future investigation: 4′-carboxy-mephedrone, 4′-carboxy-normephedrone, 1-dihydro-mephedrone, 1-dihydro-normephedrone and 4′-hydroxy-normephedrone.

Microbial Biotransformation of Five Pyrrolidinophenone‐type Psychoactive Substances in Wastewater and a Wastewater Isolated Pseudomonas putida Strain

Wastewater-based epidemiology (WBE) employs the analysis of wastewater to detect and quantify drug use and discharge within a community. In this work, transformation products (TP) by microbes in the environment were identified after incubations in wastewater and an isolated microbial strain. The microbial strain was isolated from an enrichment culture of wastewater supplemented with 3,4-methylenedioxy-pyrovalerone, and identified by matrix assisted laser desorption - time of flight mass spectrometry as Pseudomonas putida (P. putida). Five pyrrolidinophenone-type psychoactive substances (PPPS) were then incubated in wastewater and in P. putida tryptic soy broth (TSB) growth cultures. TPs were identified using liquid chromatography coupled to mass spectrometry techniques. All TPs observed in P. putida TSB growth cultures were also identified in wastewater incubations. The main TP for all PPPSs in P. putida TSB growth cultures, and two PPPSs incubated in wastewater, were the N-desalkyl-carboxy-TPs. The study showed P. putida TSB growth cultures used for identification of TPs in wastewater, represent parts of the microbial community. With data provided in this type of experiments more information will be available to select targets for monitoring drug use by WBE. Copyright

Metabolism of the synthetic cannabinoid 5F-PY-PICA by human and rat hepatocytes and identification of biliary analytical targets by directional efflux in sandwich-cultured rat hepatocytes using UHPLC-HR-MS/MS

Highlights24 metabolites of 5F‐PY‐PICA identified by LC‐HR‐MS/MS after hepatocytes incubation.Analytical targets identified with directional efflux from 3 d cultured SCRH.Two metabolites proposed as biliary analytical targets.Results help further understand overall disposition of synthetic cannabinoids. ABSTRACT Analytical strategies for detecting drugs in biological samples rely on information on metabolism and elimination. 5F‐PY‐PICA belongs to the group of synthetic cannabinoids that are known to undergo excretion into the bile. The aims of this study were the in vitro identification of metabolites of 5F‐PY‐PICA and to determine which analytical targets are excreted into the bile and urine. Metabolites identified after incubation of 5F‐PY‐PICA with pooled human liver microsomes (pHLM), pooled human hepatocytes (pHH), or suspended and sandwich‐cultured rat hepatocytes (SCRH). Rat hepatocytes were harvested following a two‐step perfusion protocol and the SCRH were prepared between layers of rat‐tail collagen. The biliary efflux of 5F‐PY‐PICA and its metabolites was determined in three‐day–cultured SCRH by differential efflux into either standard buffer from intact bile canaliculi or standard buffer without divalent cations, which disrupts the bile canaliculi. The metabolites were identified using liquid chromatography–high resolution mass spectrometry/mass spectrometry (LC‐HR‐MS/MS). The main metabolites were the COOH‐ω‐metabolite (M4) in pHH, the defluoro‐HO‐ω‐metabolite (M3) in pHLM, and the COOH‐pyrrolidine‐metabolite (M6) in rat hepatocytes. Efflux into standard buffer without divalent cations was significantly higher (p < 0.050) for 5F‐PY‐PICA, M4, and the HO‐indole‐glucuronide‐metabolite (M22). M6 did not undergo significant biliary efflux, indicating that basolateral efflux dominates for this metabolite. 5F‐PY‐PICA, M4, and M22 are proposed as analytical targets for bile analysis in forensic screening protocols, whereas M6 should be one of the main urinary targets for 5F‐PY‐PICA analysis.

Advantages of analyzing postmortem brain samples in routine forensic drug screening—Case series of three non-natural deaths tested positive for lysergic acid diethylamide (LSD)

Three case reports are presented, including autopsy findings and toxicological screening results, which were tested positive for the potent hallucinogenic drug lysergic acid diethylamide (LSD). LSD and its main metabolites were quantified in brain tissue and femoral blood, and furthermore hematoma and urine when available. LSD, its main metabolite 2-oxo-3-hydroxy-LSD (oxo-HO-LSD), and iso-LSD were quantified in biological samples according to a previously published procedure involving liquid-liquid extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). LSD was measured in the brain tissue of all presented cases at a concentration level from 0.34-10.8μg/kg. The concentration level in the target organ was higher than in peripheral blood. Additional psychoactive compounds were quantified in blood and brain tissue, though all below toxic concentration levels. The cause of death in case 1 was collision-induced brain injury, while it was drowning in case 2 and 3 and thus not drug intoxication. However, the toxicological findings could help explain the decedents inability to cope with brain injury or drowning incidents. The presented findings could help establish reference concentrations in brain samples and assist in interpretation of results from forensic drug screening in brain tissue. This is to the authors knowledge the first report of LSD, iso-LSD, and oxo-HO-LSD measured in brain tissue samples.

Prediction of Collision Cross Section and Retention Time for Broad Scope Screening in Gradient Reversed-Phase Liquid Chromatography-Ion Mobility-High Resolution Accurate Mass Spectrometry

Exact mass, retention time (RT), and collision cross section (CCS) are used as identification parameters in liquid chromatography coupled to ion mobility high resolution accurate mass spectrometry (LC-IM-HRMS). Targeted screening analyses are now more flexible and can be expanded for suspect and non-targeted screening. These allow for tentative identification of new compounds, and in-silico predicted reference values are used for improving confidence and filtering false-positive identifications. In this work, predictions of both RT and CCS values are performed with machine learning using artificial neural networks (ANNs). Prediction was based on molecular descriptors, 827 RTs, and 357 CCS values from pharmaceuticals, drugs of abuse, and their metabolites. ANN models for the prediction of RT or CCS separately were examined, and the potential to predict both from a single model was investigated for the first time. The optimized combined RT-CCS model was a four-layered multi-layer perceptron ANN, and the 95th prediction error percentiles were within 2 min RT error and 5% relative CCS error for the external validation set (n = 36) and the full RT-CCS dataset (n = 357). 88.6% (n = 733) of predicted RTs were within 2 min error for the full dataset. Overall, when using 2 min RT error and 5% relative CCS error, 91.9% (n = 328) of compounds were retained, while 99.4% (n = 355) were retained when using at least one of these thresholds. This combined prediction approach can therefore be useful for rapid suspect/non-targeted screening involving HRMS, and will support current workflows.