Detectability of oxandrolone, metandienone, clostebol and dehydrochloromethyltestosterone in urine after transdermal application.

Situations of both, intentional and inadvertent or accidental doping, necessitate consideration in today's doping controls, especially in the light of the substantial consequences that athletes are facing in case of so-called adverse analytical findings. The aim of this study was to investigate, whether a transdermal uptake of doping substances would be possible. In addition to the period of detectability of the particular substances or respective characteristic metabolites, the possibility of deducing the route of administration by metabolite patterns was also assessed. Twelve male subjects were included in the study. Four common anabolic androgenic steroids (AAS) were dissolved in dimethylsulfoxide to facilitate transdermal administration on different skin regions. One half of the test persons received only oxandrolone (17α-methyl-2-oxa-4,5α-dihydrotestosterone), and the other half were applied a mixture of oxandrolone, metandienone (17ß-hydroxy-17α-methylandrosta-1,4-dien-3-one), clostebol (4-chlorotestosterone-17ß-acetate) and dehydrochloromethyltestosterone (DHCMT). Urine samples were collected 1 h, 6 h and one sample per day for the next 14 consecutive days. Measurements were conducted on a tandem-gas chromatography-mass spectrometry (GC-MS/MS) or tandem-liquid chromatography-MS/MS (LC-MS/MS) system. Substance findings were obtained at least 1 day after application on nearly all skin locations. The results indicated inter-individual variability in detection windows, also varying between the different analytes and possible impact of skin location and skin thickness, respectively. Nevertheless, a rapid and rather long detectability of all substances (or respective metabolites) was given, in some cases within hours after administration and for up to 10-14 days. Hence, the transdermal application or exposure to the investigated AAS is a plausible scenario that warrants consideration in anti-doping.

Quantification of direct-acting oral anticoagulants: Application of a clinically validated liquid chromatography-tandem mass spectrometry method to forensic cases.

In certain forensic cases, a quantification of direct-acting oral anticoagulants (DOACs) can be necessary. We evaluate the applicability of a previously described liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for the determination of DOACs in plasma to postmortem specimen. Postmortem internal quality control (PIQC) samples were prepared in pooled blank postmortem heart blood, femoral blood, cerebrospinal fluid (CSF), and urine as well in plasma. To examine the application of the clinical method to forensic cases, the main validation parameters were reinvestigated using PIQC samples. Postmortem samples of 12 forensic cases with evidence of previous rivaroxaban intake and unknown bleeding disorders were analyzed. Interday variability remained within the acceptance criterion of ±15%. Matrix effects were comparable in blank plasma and postmortem matrix extracts. After 4 weeks of storage in the refrigerator, no relevant decrease of DOACs was evident. After 96 h of storage at room temperature, a slight decrease in edoxaban concentration was observed in CSF and urine, while plasma edoxaban decreased by about 50%. Median (range) rivaroxaban concentrations determined in specimen of forensic cases were as follows: heart blood (n = 6), 17.2 ng/ml (

Drinking Ethanol Has Few Acute Effects on CYP2C9, CYP2C19, NAT2, and P-Glycoprotein Activities but Somewhat Inhibits CYP1A2, CYP2D6, and Intestinal CYP3A: So What?

We quantified the effect of acute ethanol exposure (initial blood concentrations 0.7 g/L) on major drug metabolizing enzymes and p-glycoprotein. Sixteen healthy Caucasians participated in a randomized crossover study with repeated administration of either vodka or water. Enzyme/transporter activity was assessed by a cocktail of probe substrates, including caffeine (CYP1A2/NAT2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and digoxin (P-glycoprotein). The ratio of AUC0-t of dextromethorphan for ethanol/water coadministration was 1.95 (90% confidence interval (CI) 1.48-2.58). The effect was strongest in individuals with a CYP2D6 genotype predicting high activity (n = 7, ratio 2.66, 90% CI 1.65-4.27). Ethanol increased caffeine AUC0-t 1.38-fold (90% CI 1.25-1.52) and reduced intestinal midazolam extraction 0.77-fold (90% CI 0.69-0.86). The other probe drugs were not affected by ethanol. The results suggest that acute ethanol intake typically has no clinically important effect on the enzymes/transporters tested.

Not only smoking is deadly: fatal ingestion of e-juice-a case report.

A fatal case of nicotine intoxication by oral intake of a nicotine solution, sold via the Internet, is reported. The concentrated nicotine solution (72 mg/mL) is usually diluted with polypropylene, polyethylene glycol or glycerine, respectively, in order to allow the user to generate their own solution for vaporisation in electronic cigarettes (e-juice). A 34-year-old man was found lifeless by his parents, who reported that their son had been in good health and had shown no hints of suicidal behaviour. The medicolegal autopsy revealed unspecific findings. Toxicological analysis revealed nicotine concentrations of 5.5 mg/L in femoral venous blood, 136 mg/L in heart blood, 12.0 mg/kg in brain tissue, 42.6 mg/kg in kidney tissue, 89.5 mg/kg in lung tissue and a total amount of 3,950 mg in the gastric contents. Cotinine concentrations were 0.9 mg/L in femoral venous blood, 7.6 mg/L in heart blood, 0.4 mg/kg in brain tissue, 0.9 mg/kg in kidney tissue and 0.8 mg/kg in lung tissue. No cotinine was detected in the gastric contents. The nicotine level measured in the femoral blood was in good accordance with the levels reported in other fatal cases caused by oral or patch application of nicotine. Moreover, the high level of nicotine in lung and kidney tissue, compared to that within femoral blood, strikingly emphasises the strong effect of post-mortem redistribution, underlined by the comparably low concentration of nicotine in the brain. The extremely high level of nicotine in the heart blood is more likely due to the high concentration in the gastric contents, due to oral intake, and by accumulation of the basic substance in the acidic gastric contents. This further highlights the effect of post-mortem redistribution. The mother of the deceased later admitted that her son had been suffering from psychosis and that she found a package containing five nicotine solution vials of the brand "Titanium Ice" (of 50 mL each). Three of the vials were empty. The nicotine concentration in the e-juice Titanium Ice was confirmed by HPLC analysis.

Assessing Cognitive and Psychomotor Performance in Patients with Fibromyalgia Syndrome.

INTRODUCTION: Patients with fibromyalgia syndrome (FMS) generally present with chronic widespread pain, accompanied by a range of additional and non-specific symptoms, such as fatigue, disturbed sleep, and cognitive dysfunction, which tend to increase with overall severity. Previous studies have shown moderate cognitive impairment in patients with FMS, but there are few valid data explicitly assessing the relevance of these findings to everyday functions, such as driving ability. Therefore, we studied patients with FMS to assess the impact of FMS on tests that predict driving ability. METHODS: Female patients with FMS were prospectively compared to a historical control group of healthy volunteers. The test battery comprised assessments of visual orientation, concentration, attention, vigilance, motor coordination, performance under stress, and reaction time. RESULTS: A total of 43 patients were matched to 129 controls. The results indicated that the patients' psychomotor and cognitive performances were significantly non-inferior when compared to healthy controls (with 0.05% alcohol), with the exception of motor coordination. Patients and healthy controls showed an age-related decline in test performance. Correlations were smaller in patients and reversed for vigilance which was linked to a greater FMS symptom load in younger patients. CONCLUSION: The results of the present study demonstrate that, in general, the driving ability of patients with FMS was not inferior to that of healthy volunteers based on a standardized computer-based test battery. However, variables, such as younger age, depression, anxiety, fatigue, pain, and poor motor coordination, likely contribute to the subjective perception of cognitive dysfunction in FMS.

Insights into mechanisms and proteomic characterisation of Pseudomonas aeruginosa adaptation to a novel antimicrobial substance.

Antibiotic resistance has been reported since the introduction of synthetic antibiotics. Bacteria, such as one of the most common nosocomial pathogens P. aeruginosa, adapt quickly to changing environmental conditions, due to their short generation time. Thus microevolutional changes can be monitored in situ. In this study, the microevolutional process of Pseudomonas aeruginosa PAO1 resistance against a recently developed novel antibacterial zinc Schiff-base (ZSB) was investigated at the proteome level. After extended exposure to ZSB the passaged strain differed in tolerance against ZSB, with the adapted P. aeruginosa PAO1 exhibiting 1.6 times higher minimal inhibitory concentration. Using Two-dimensional Difference Gel Electrophoresis, the changes in the proteome of ZSB adapted P. aeruginosa PAO1 were examined by comparison with the non-adapted P. aeruginosa PAO1. The proteome of the adapted P. aeruginosa PAO1 strain differed significantly from the non-adapted in the abundance of two proteins when both strains were grown under stressing conditions. One protein could be identified as the outer membrane protein D that plays a role in uptake of basic amino acids as well as in carbapeneme resistance. The second protein has been identified as alkyl peroxide reductase subunit F. Our data indicated a slight increase in abundance of alkyl peroxide reductase F (AhpF) in the case of ZSB passaged P. aeruginosa PAO1. Higher abundance of Ahp has been discussed in the literature as a promoter of accelerated detoxification of benzene derivatives. The observed up-regulated AhpF thus appears to be connected to an increased tolerance against ZSB. Changes in the abundance of proteins connected to oxidative stress were also found after short-time exposure of P. aeruginosa PAO1 to the ZSB. Furthermore, adapted P. aeruginosa PAO1 showed increased tolerance against hydrogen peroxide and, in addition, showed accelerated degradation of ZSB, as determined by HPLC measurements.

Inhibition of 1,4-butanediol metabolism in human liver in vitro.

The conversion of 1,4-butanediol (1,4-BD) to gamma-hydroxybutyric acid (GHB), a drug of abuse, is most probably catalyzed by alcohol dehydrogenase, and potentially by aldehyde dehydrogenase. The purpose of this study was to investigate the degradation of 1,4-BD in cytosolic supernatant of human liver in vitro, and to verify involvement of the suggested enzymes by means of gas chromatography-mass spectrometry. The coingestion of 1,4-BD and ethanol (EtOH) might cause complex pharmacokinetic interactions in humans. Therefore, the effect of EtOH on 1,4-BD metabolism by human liver was examined in vitro. Additionally, the influence of acetaldehyde (AL), which might inhibit the second step of 1,4-BD degradation, was investigated. In case of a 1,4-BD intoxication, the alcohol dehydrogenase inhibitor fomepizole (4-methylpyrazole, FOM) has been discussed as an antidote preventing the formation of the central nervous system depressing GHB. Besides FOM, we tested pyrazole, disulfiram, and cimetidine as possible inhibitors of the formation of GHB from 1,4-BD catalyzed by human liver enzymes in vitro. The conversion of 1,4-BD to GHB was inhibited competitively by EtOH with an apparent K(i) of 0.56 mM. Therefore, the coingestion of 1,4-BD and EtOH might increase the concentrations and the effects of 1,4-BD itself. By contrast AL accelerated the formation of GHB. All antidotes showed the ability to inhibit the formation of GHB. In comparison FOM showed the highest inhibitory effectiveness. Furthermore, the results confirm strong involvement of ADH in 1,4-BD metabolism by human liver.

Screening for the synthetic cannabinoid JWH-018 and its major metabolites in human doping controls.

Referred to as 'spice', several new drugs, advertised as herbal blends, have appeared on the market in the last few years, in which the synthetic cannabinoids JWH-018 and a C(8) homologue of CP 47,497 were identified as major active ingredients. Due to their reported cannabis-like effects, many European countries have banned these substances. The World Anti-Doping Agency has also explicitly prohibited synthetic cannabinoids in elite sport in-competition. Since urine specimens have been the preferred doping control samples, the elucidation of the metabolic pathways of these substances is of particular importance to implement them in sports drug testing programmes. In a recent report, an in vitro phase-I metabolism study of JWH-018 was presented yielding mainly hydroxylated and N-dealkylated metabolites. Due to these findings, a urine sample of a healthy man declaring to have smoked a 'spice' product was screened for potential phase-I and -II metabolites by high-resolution/high-accuracy mass spectrometry in the present report. The majority of the phase-I metabolites observed in earlier in vitro studies of JWH-018 were detected in this urine specimen and furthermore most of their respective monoglucuronides. As no intact JWH-018 was detectable, the monohydroxylated metabolite being the most abundant one was chosen as a target analyte for sports drug testing purposes; a detection method was subsequently developed and validated in accordance to conventional screening protocols based on enzymatic hydrolysis, liquid-liquid extraction, and liquid chromatography/electrospray tandem mass spectrometry analysis. The method was applied to approximately 7500 urine doping control samples yielding two JWH-018 findings and demonstrated its capability for a sensitive and selective identification of JWH-018 and its metabolites in human urine.

In vitro phase I metabolism of the synthetic cannabimimetic JWH-018.

A potent synthetic cannabinoid receptor agonist, JHW-018, was recently detected as one of the most prominent active agents in abusively used incenses such as Spice and other herbal blends. The high pharmacological and addictive potency of JWH-018 highlights the importance of elucidating the metabolism of JWH-018, without which a meaningful insight into its pharmacokinetics and its toxicity would not be possible. In the present study, the cytochrome P450 phase I metabolites of JWH-018 were investigated, after in vitro incubation of the drug with human liver microsomes, followed by liquid chromatography-tandem mass spectrometry analysis. This revealed monohydroxylation of the naphthalene ring system, the indole moiety, and the alkyl side chain. In addition, observations were made of dihydroxylation of the naphthalene ring system, and the indole moiety, or as result of a combination of monohydroxylations of both the naphthalene ring system and the indole moiety or the alkyl side chain, or a combination of monohydroxylations of both the indole ring system and the alkyl side chain. There is also evidence of trihydroxylation at different locations of the hydroxyl groups in the molecule. Furthermore, dehydration of the alkyl side chain, in combination with both monohydroxylation and dihydroxylation as well as arene oxidation of the naphthalene ring system, combined with both monohydroxylation and dihydroxylation at different sites of oxidation were found. N-dealkylation also in combination with both monohydroxylation and dihydrodiol formation of the N-dealkylated metabolite was detected. Finally, a metabolite was found carboxylated at the alkyl side chain.

The novel beta-defensin DEFB123 prevents lipopolysaccharide-mediated effects in vitro and in vivo.

Defensins are a family of secreted antimicrobial peptides proposed to directly interfere with bacterial membranes. Here we show a functional analysis of the novel beta-defensin DEFB123. A peptide comprising the beta-defensin core region was synthesized and used for our analysis. Like other beta-defensins, DEFB123 exerted antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, which was assessed by microbroth dilution assay and radial diffusion zone assay. In addition, the peptide showed lipopolysaccharide (LPS)-binding activity in a Limulus amoebocyte lysate (LAL) assay. Moreover, DEFB123 prevented LPS-induced tumor necrosis factor (TNF)-alpha secretion in a murine monocyte cell line (RAW264.7). Accordingly, DEFB123 abolished LPS-mediated MAPK induction in these cells. Protection against LPS-mediated effects was then investigated in a murine model of acute sepsis. Our experiments show that synthetic beta-defensin DEFB123 prevents LPS-induced mortality in C57BL/6 mice in a therapeutic approach. We propose that the physiological role of beta-defensins may include interference with LPS-action on macrophages, a function formerly thought to be restricted to the family of cathelicidins, a structurally unrelated group of antimicrobial peptides.