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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. This article is a U.S. Government work and is in the public domain in the USA. [ABSTRACT FROM AUTHOR]

N-(Adamantan-1-yl)-1-(5-fluoropentyl)-1 H-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. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Drug induced liver injury (DILI) accounts for more than 50% of the cases of acute liver failure in this country, and is the major cause of drug withdrawal from the market. DILI has been associated with the induction of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α). Pro-inflammatory cytokines activate the mitogen activated protein kinase, c-Jun-N-terminal kinase (JNK) in the liver. Recent studies have shown that JNK can regulate the hepatotoxicity of the analgesic, acetaminophen (APAP). Several reports have shown that inflammation induced by the endotoxin, lipopolysaccharide (LPS) augments the toxic response to hepatotoxicants in vivo. However, the mechanism by which inflammation alters drug-induced hepatotoxicity is not known. This study investigated the role of inflammatory mediators in regulating the toxicity of the hepatotoxic drugs, APAP or chlorpromazine (CPZ) in primary mouse hepatocytes. We found that, pre-treatment with TNF-α resulted in ∼50 to 60% increase in alanine aminotransferase (ALT) levels by APAP or CPZ, while interleukin-1β (IL-1β) or IL6 treatments showed only 15-20% increase in ALT release. The bacterial components, LPS or lipoteichoic acid (LTA) increased ALT release by ∼35 to 38% upon drug treatment of the hepatocytes. The JNK inhibitor, SP600125 significantly diminished APAP and CPZ toxicity with or without TNF-α. Pre-treatment with TNF-a resulted in prolonged activation of JNK (upto 2 hr) in the presence of APAP or CPZ. These results show that TNF-α is the major cytokine involved in sensitizing hepatocytes to APAP- or CPZ-induced hepatotoxicity, likely by a mechanism involving sustained activation of JNK. [ABSTRACT FROM AUTHOR]

Although a marked decrease in mortality associated with bacterial infections is attributed to the discovery of antibiotics, antibiotic resistance has become a global health concern due to their misuse. A dynamic in vitro hollow-fiber system was used to study antibiotic resistance in Escherichia coli using ampicillin. An LC-MS/MS assay was validated for quantitative analysis of ampicillin in Luria-Bertani broth. The assay was linear from 0.10-50.00 μg/ml. The assay met acceptance criteria for inter- and intra-assay precisions and accuracies across three quality controls. Stability of ampicillin was confirmed at three different storage conditions. In vitro data were similar to simulated plasma PK data further confirming the appropriateness of the experimental design to quantify antibiotics and study occurrence of antimicrobial resistance in real-time., Competing Interests: Financial & competing interests disclosure The authors would like to thank the Center for Drugs Evaluation and Research for funding this study. All the work presented in this manuscript was supported by intramural grants from the FDA. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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., (Copyright © 2014 John Wiley & Sons, Ltd.)