False negative results in testosterone doping in forensic cases: Sensitivity of the urinary detection criteria T/E and T/LH.

At the Swedish national forensic toxicology laboratory, a measured testosterone/epitestosterone (T/E) ratio ≥ 12 together with testosterone/luteinizing hormone (T/LH) in urine > 400 nmol/IU is considered as a proof of exogenous testosterone administration. However, according to the rules of the World Anti-Doping Agency (WADA), samples with T/E ratio > 4 are considered suspicious and shall be further analysed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to confirm the origin of testosterone and its metabolites. The aim of this study was to investigate the possibility of false negative results and to estimate the frequency of negative results using the current criteria for detection of abuse of testosterone in forensic investigations. Urine and serum samples were collected by the police at suspected infringement of the doping law in Sweden. Fifty-eight male subjects were included in the study. Urinary testosterone was determined by gas chromatography-mass spectrometry (GC-MS), serum testosterone and LH-by immunoassay. The origin of testosterone and its metabolites was confirmed by means of GC-C-IRMS. Twenty-six of the 57 analysed subjects tested positive for exogenous testosterone using the criteria T/E ≥ 12 combined with T/LH > 400 nmol/IU. The IRMS analyses confirmed 47 positives; thus, 21 were considered false negatives. Negative predictive value was 32% (95% confidence interval [CI]: 16%-50%) and sensitivity 55%. No false positive subjects were found. The number of false negative cases using the current criteria for the detection of testosterone abuse and hence the low sensitivity indicates a need to discuss introduction of new strategies in forensic doping investigations.

Study on the phase I metabolism of novel synthetic cannabinoids, APICA and its fluorinated analogue.

The data are reported for an in vitro metabolism study of two novel synthetic cannabinoids, N-(1-adamantyl)-1-pentyl-1H-indole-3-carboxamide (APICA) and its fluorinated analog N-(1-adamantyl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-APICA, STS-135), which are active ingredients of smoking mixtures sold in Russia since 2012. The cannabinoids were isolated from herbal mixtures using preparative liquid chromatography and then incubated with human liver microsomes (HLMs). The formed metabolites were characterized by liquid chromatography - triple quadrupole mass spectrometry and high-resolution mass spectrometry with electrospray ionization in positive ion mode. It was found that HLMs produce mono-, di-, and trihydroxylated metabolites, as well as N-desalkyl metabolites, which can be further hydroxylated; the amide bond resisted the metabolic cleavage. For 5F-APICA, a series of oxidative defluorination products formed as well. For in vivo confirmation of the formed in vitro metabolites, spot urine samples from drug users were analyzed with the created method. It was shown that for the detection of APICA abuse, the preferred metabolites are the di- and tri-hydroxylated species, while in case of 5F-APICA, a monohydroxy metabolite is a better target. The N-despentyl (desfluoropentyl) hydroxyadamantyl metabolite also provides good retrospectivity to confirm the administration of any of these cannabinoids.

Detection of urinary metabolites of AM-2201 and UR-144, two novel synthetic cannabinoids.

Synthetic cannabinoids are the psychotropic compounds frequently identified as active components of smoking mixtures easily available via the Internet in several countries. These herbal blends have become extremely popular as a legal alternative to cannabis-based products and are difficult to detect by regular drug tests. Here we report on an in vitro and in vivo metabolism of AM-2201, 1-[(5-fluoropentyl)-1H-indol-3-yl]-(naphthalen-1-yl)methanone, and UR-144 (KM-X1), (1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone isolated using preparative liquid chromatography from the smoking mixtures sold in Russia. After incubation with human liver microsomes (HLM) as well as with cytochrome isoenzymes 3A4 and 2B6, the metabolic pathways were identified by means of liquid chromatography - triple quadrupole and high resolution mass spectrometry with electrospray ionization in positive mode. It was found that the in vitro reactions include mono- and dihydroxylation, loss of N-alkyl side chain and formation of dihydrodiol metabolites in case of AM-2201. The HLM were found to be superior over the other two isoenzymes for generation of cannabinoid metabolites. Finally, forensic urine samples were analyzed to validate the in vitro data and it has been shown that for both cannabimimetics the recommended screening targets are the monohydroxylated metabolites.

Detection of JWH-018 metabolites in smoking mixture post-administration urine.

Smoking mixtures containing the cannabimimetic indoles may still be available over-the-counter in several countries. Due to the high affinity of these compounds to the cannabinoid receptors, their effective dose is lower than that of the marijuana products resulting in a low concentration of the excreted metabolites accompanied by a higher psychoactive potency. Up to now the in vivo metabolism of the cannabimimetic indoles seems to be insufficiently investigated and no data have been published on an assay of JWH-018 in urine. In this publication the urinary metabolites of JWH-018 are reported. Using gas and liquid chromatography combined with tandem mass spectrometry two main monohydroxylated metabolites were identified in the forensic urine samples. Based on the differences in their electron ionization MS/MS spectra it is supposed that one is formed by hydroxylation of the indole ring whilst the other by hydroxylation of the N-alkyl chain. The main metabolites are almost completely glucuroconjugated, whereas minor ones (N-despentyl hydroxy-, carboxy-, dihydroxy-, and reduced di- and trihydroxy metabolites) were also present in the free fraction. The parent compound was not detected in urine.