Ariane Wohlfarth

Bioanalysis of new designer drugs

Since the late 1990s the illicit drug market has undergone considerable change: along with the traditional drugs of abuse that still dominate, more than 100 psychotropic substances designed to bypass controlled substances legislation have appeared and led to intoxications and fatalities. Starting from the huge class of phenylalkylamines, containing many subgroups, the spectrum of structures has grown from tryptamines, piperazines, phenylcyclohexyl derivates and pyrrolidinophenones to synthetic cannabinoids and the first synthetic cocaine. Due to the small prevalence and high number of unknown substances, the detection of new designer drugs is a challenge for clinical and forensic toxicologists. Standard screening procedures might fail because a recently discovered or yet unknown substance has not been incorporated in the library used. Nevertheless, many metabolism studies, case reports, screening methods and substance-profiling papers concentrating on single compounds have been published. This review provides an overview of the developed bioanalytical and analytical methods, the matrices used, sample-preparation procedures, concentration of analytes in case of intoxication and also gives a résumé of immunoassay experiences. Additionally, six screening methods for biological matrices with a larger spectrum of analytes are described in more detail.

Synthetic cannabinoids pharmacokinetics and detection methods in biological matrices

Abstract Synthetic cannabinoids (SC), originally developed as research tools, are now highly abused novel psychoactive substances. We present a comprehensive systematic review covering in vivo and in vitro animal and human pharmacokinetics and analytical methods for identifying SC and their metabolites in biological matrices. Of two main phases of SC research, the first investigated therapeutic applications, and the second abuse-related issues. Administration studies showed high lipophilicity and distribution into brain and fat tissue. Metabolite profiling studies, mostly with human liver microsomes and human hepatocytes, structurally elucidated metabolites and identified suitable SC markers. In general, SC underwent hydroxylation at various molecular sites, defluorination of fluorinated analogs and phase II metabolites were almost exclusively glucuronides. Analytical methods are critical for documenting intake, with different strategies applied to adequately address the continuous emergence of new compounds. Immunoassays have different cross-reactivities for different SC classes, but cannot keep pace with changing analyte targets. Gas chromatography and liquid chromatography mass spectrometry assays – first for a few, then numerous analytes – are available but constrained by reference standard availability, and must be continuously updated and revalidated. In blood and oral fluid, parent compounds are frequently present, albeit in low concentrations; for urinary detection, metabolites must be identified and interpretation is complex due to shared metabolic pathways. A new approach is non-targeted HRMS screening that is more flexible and permits retrospective data analysis. We suggest that streamlined assessment of new SC’s pharmacokinetics and advanced HRMS screening provide a promising strategy to maintain relevant assays.

Metabolic profiling of new synthetic cannabinoids AMB and 5F‐AMB by human hepatocyte and liver microsome incubations and high‐resolution mass spectrometry

RATIONALE AMB (methyl (1-pentyl-1H-indazole-3-carbonyl)-L-valinate)) and its fluoro analog 5F-AMB (methyl (1-(5-fluoropentyl)-1H-indazole-3-carbonyl)-L-valinate) are two new synthetic cannabinoids that are structural analogs of AB-PINACA and 5F-AB-PINACA, respectively. 5F-AMB is scheduled as an illicit drug in China, Germany, Singapore and Japan, and no metabolism data are currently available for either drug. The aim of the present work was to investigate the metabolism of AMB and 5F-AMB and propose appropriate markers to identify their intake in clinical or forensic cases. METHODS AMB and 5F-AMB were incubated in human hepatocytes (10 μmol/L) to generate phase I and II metabolites, which were identified with a TripleTOF 5600(+) high-resolution mass spectrometer. AMB and 5F-AMB metabolic stability studies also were performed with human liver microsomes (HLM) to evaluate metabolic clearances, and to adequately design the human hepatocyte experiment. RESULTS AMB and 5F-AMB were quickly metabolized in HLM with a 1.1 ± 0.1 and 1.0 ± 0.2 min T1/2, respectively. The predominant metabolic pathway for AMB and 5F-AMB in hepatocytes was ester hydrolysis, and further oxidation and/or glucuronidation. In total, 19 metabolites were identified for AMB and 17 for 5F-AMB. We describe metabolites to differentiate AMB from 5F-AMB, and metabolites that are common to both analytes due to oxidative defluorination of 5F-AMB. CONCLUSIONS For the first time, AMB and 5F-AMB metabolism profiles were characterized, providing valuable data for identifying these two novel psychoactive substances. The difficulties of differentiating AMB and 5F-AMB from AB-PINACA/5F-AB-PINACA metabolites also were examined. These data improve the interpretation of urinary markers after AMB and 5F-AMB intake. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

Urine tested positive for ethyl glucuronide and ethyl sulfate after the consumption of yeast and sugar

BACKGROUND To an increasing degree, EtG and EtS are routinely used for the proof of abstinence for purposes of traffic, occupational, addiction and social medicine. This routine use demands further investigations on the sensitivity and specificity of these analytes and the examination of possible genesis of positive EtG and EtS concentrations even without the consumption of ethanol. In vivo fermentation with consecutive formation of EtG and EtS was addressed by experiments with yeast products. METHODS Two experiments with bakers yeast and brewers yeast tablets were performed. The ethanol concentrations in urine of the 2 and 4 volunteers, respectively, were detected by HS-GC-FID, EtG and EtS analysis was performed by LC-ESI-MS/MS, and the creatinine concentration was determined using a method based on the Jaffé reaction. RESULTS AND CONCLUSIONS After the consumption of bakers yeast the maximum concentrations of EtG and EtS normalised to creatinine were found to be 0.67 and 1.41mg/L, respectively, and therefore clearly above the commonly applied cut-off value for the proof of abstinence of 0.1mg/L. In contrast, in this study the, uptake of yeast tablets did not result in a detection of EtG and EtS in urine.

High-resolution mass spectrometric metabolite profiling of a novel synthetic designer drug, N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135), using cryopreserved human hepatocytes and assessment of metabolic stability with human liver microsomes

N-(Adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a new synthetic cannabinoid in herbal incense products discussed on Internet drug user forums and identified in police seizures. To date, there are no STS-135 clinical or in vitro studies identifying STS-135 metabolites. However, characterizing STS-135 metabolism is critical because synthetic cannabinoid metabolites can possess pharmacological activity and parent compounds are rarely detectable in urine. To characterize the metabolite profile, human hepatocytes were incubated with 10 µmol/L STS-135 for up to 3 h. High-resolution mass spectrometry with software-assisted data mining identified 29 STS-135 metabolites. Less than 25% of STS-135 parent compound remained after 3 h incubation. Primary metabolites were generated by mono-, di- or trihydroxylation with and without ketone formation, dealkylation, and oxidative defluorination of N-fluoropentyl side chain or possible oxidation to carboxylic acid, some of them further glucuronidated. Hydroxylations occurred mainly on the aliphatic adamantane ring and less commonly on the N-pentyl side chain. At 1 h, phase I metabolites predominated, while at 3 h, phase II metabolites were present in higher amounts. The major metabolites were monohydroxy STS-135 (M25) and dihydroxy STS-135 (M21), both hydroxylated on the adamantane system. Moreover, metabolic stability of STS-135 (1 µmol/L) was assessed in human liver microsomes experiments. The in vitro half-life of STS-135 was 3.1 ± 0.2 min and intrinsic clearance (CLint ) was 208.8 mL · min(-1)  · kg(-1) . This is the first report characterizing STS-135 hepatic metabolic pathways. These data provide potential urinary targets to document STS-135 intake in clinical and forensic settings and potential candidates for pharmacological testing.

In vitro, in vivo and in silico metabolic profiling of α-pyrrolidinopentiothiophenone, a novel thiophene stimulant

BACKGROUND Little or no pharmacological or toxicological data are available for novel psychoactive substances when they first emerge, making their identification and interpretation in biological matrices challenging. MATERIALS & METHODS A new synthetic cathinone, α-pyrrolidinopentiothiophenone (α-PVT), was incubated with hepatocytes and samples were analyzed using liquid chromatography coupled to a Q Exactive™ Orbitrap mass spectrometer. Authentic urine specimens from suspected α-PVT cases were also analyzed. Scans were data mined with Compound Discoverer™ for identification and structural elucidation of metabolites. RESULTS/CONCLUSION Seven α-PVT metabolites were identified in hepatocyte incubations, and in the authentic urine samples, also with an additional monohydroxylated product and a glucuronide of low intensity. α-PVT dihydroxypyrrolidinyl, α-PVT 2-ketopyrrolidinyl, α-PVT hydroxythiophenyl and α-PVT thiophenol had the most intense in vivo signals.

Ethyl sulphate and ethyl glucuronide in vitreous humor as postmortem evidence marker for ethanol consumption prior to death.

To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices.

Quantitative urine confirmatory testing for synthetic cannabinoids in randomly collected urine specimens.

Synthetic cannabinoid intake is an ongoing health issue worldwide, with new compounds continually emerging, making drug testing complex. Parent synthetic cannabinoids are rarely detected in urine, the most common matrix employed in workplace drug testing. Optimal identification of synthetic cannabinoid markers in authentic urine specimens and correlation of metabolite concentrations and toxicities would improve synthetic cannabinoid result interpretation. We screened 20 017 randomly collected US military urine specimens between July 2011 and June 2012 with a synthetic cannabinoid immunoassay yielding 1432 presumptive positive specimens. We analyzed all presumptive positive and 1069 negative specimens with our qualitative synthetic cannabinoid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which confirmed 290 positive specimens. All 290 positive and 487 randomly selected negative specimens were quantified with the most comprehensive urine quantitative LC-MS/MS method published to date; 290 specimens confirmed positive for 22 metabolites from 11 parent synthetic cannabinoids. The five most predominant metabolites were JWH-018 pentanoic acid (93%), JWH-N-hydroxypentyl (84%), AM2201 N-hydroxypentyl (69%), JWH-073 butanoic acid (69%), and JWH-122 N-hydroxypentyl (45%) with 11.1 (0.1-2,434), 5.1 (0.1-1,239), 2.0 (0.1-321), 1.1 (0.1-48.6), and 1.1 (0.1-250) µg/L median (range) concentrations, respectively. Alkyl hydroxy and carboxy metabolites provided suitable biomarkers for 11 parent synthetic cannabinoids; although hydroxyindoles were also observed. This is by far the largest data set of synthetic cannabinoid metabolites urine concentrations from randomly collected workplace drug testing specimens rather than acute intoxications or driving under the influence of drugs. These data improve the interpretation of synthetic cannabinoid urine test results and suggest suitable urine markers of synthetic cannabinoid intake.

Metabolism of RCS-8, a synthetic cannabinoid with cyclohexyl structure, in human hepatocytes by high-resolution MS.

BACKGROUND Since 2008, synthetic cannabinoids are major new designer drugs of abuse. They are extensively metabolized and excreted in urine, but limited human metabolism data are available. As there are no reports on the metabolism of RCS-8, a scheduled phenylacetylindole synthetic cannabinoid with an N-cyclohexylethyl moiety, we investigated metabolism of this new designer drug by human hepatocytes and high resolution MS. METHODS After human hepatocyte incubation with RCS-8, samples were analyzed on a TripleTOF 5600+ mass spectrometer with time-of-flight survey scan and information-dependent acquisition triggered product ion scans. Data mining of the accurate mass full scan and product ion spectra employed different data processing algorithms. RESULTS & CONCLUSION More than 20 RCS-8 metabolites were identified, products of oxidation, demethylation, and glucuronidation. Major metabolites and targets for analytical methods were hydroxyphenyl RCS-8 glucuronide, a variety of hydroxycyclohexyl-hydroxyphenyl RCS-8 glucuronides, hydroxyphenyl RCS-8, as well as the demethyl-hydroxycyclohexyl RCS-8 glucuronide.

Urinary prevalence, metabolite detection rates, temporal patterns and evaluation of suitable LC-MS/MS targets to document synthetic cannabinoid intake in US military urine specimens.

Abstract Background: Identifying synthetic cannabinoid designer drug abuse challenges toxicologists and drug testing programs. The best analytical approach for reliably documenting intake of emerging synthetic cannabinoids is unknown. Primarily metabolites are found in urine, but optimal metabolite targets remain unknown, and definitive identification is complicated by converging metabolic pathways. Methods: We screened 20,017 US military urine specimens collected from service members worldwide for synthetic cannabinoids between July 2011 and June 2012. We confirmed 1432 presumptive positive and 1069 presumptive negative specimens by qualitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis including 29 biomarkers for JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, RCS-4, AM2201 and MAM2201. Specimen preparation included enzyme hydrolysis and acetonitrile precipitation prior to LC-MS/MS analysis. We evaluated individual synthetic cannabinoid metabolite detection rates, prevalence, temporal patterns and suitable targets for analytical procedures. Results: Prevalence was 1.4% with 290 confirmed positive specimens, 92% JWH-018, 54% AM2201 and 39% JWH-122 metabolites. JWH-073, JWH-210 and JWH-250 also were identified in 37%, 4% and 8% of specimens, respectively. The United States Army Criminal Investigation Command seizure pattern for synthetic cannabinoid compounds matched our urine specimen results over the time frame of the study. Apart from one exception (AM2201), no parent compounds were observed. Conclusions: Hydroxyalkyl metabolites accounted for most confirmed positive tests, and in many cases, two metabolites were identified, increasing confidence in the results, and improving detection rates. These data also emphasize the need for new designer drug metabolism studies to provide relevant targets for synthetic cannabinoid identification.