Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard.

The identification of a predominate metabolite found in urine specimens which test positive for naphthoylindole-based synthetic cannabinoids is reported. The presence of this new metabolite was detected at the Air Force Drug Testing Lab Investigations Division during screening analysis for metabolites of JWH-018 and JWH-073, because it shares the same MRM transitions as the JWH-073 N-(3-hydroxybutyl) metabolite. However, the detected peak is chromatographically distinguished from other metabolites due to differences in retention time. This metabolite appears to be a common metabolite for select naphthoylindole-based synthetic cannabinoids that could potentially be used as a common biomarker for their qualitative and quantitative analyses. The new metabolite has been successfully identified as 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid (1, JWH 072 N-propanoic acid metabolite, Fig. 1) by using various mass spectrometric and liquid chromatographic techniques as well as chemical derivatization. The metabolite identity was confirmed through the comparison of authentic positive urine and a chemically synthesized metabolite standard. Both materials shared the same chromatographic retention time on two separate chromatographic systems, mass fragmentation pattern and exact mass. Full characterization of the synthetic reference material and intermediates by (1)H NMR, (13)C NMR, IR and HRMS was also conducted.

High-throughput bioanalytical method for analysis of synthetic cannabinoid metabolites in urine using salting-out sample preparation and LC-MS/MS.

Herbal smoking mixtures which are sold as incense or potpourri and often referred to as 'Spice' are actually inactive plant matter adulterated with alkylamino indole based synthetic cannabinoids such as JWH-018 and JWH-073. Due to the inclusion of five synthetic cannabinoids, including JWH-018 and JWH-073, as Schedule I drugs by the Drug Enforcement Agency (DEA) in March 2011, it has become necessary for forensic laboratories to develop analytical methods to test for the presence of metabolites of synthetic cannabinoids. When a new analyte of interest emerges, most laboratories strive to develop a sample preparation procedure and validate an analytical method as quickly as possible and therefore, rely on effective but time consuming traditional protocols such as solid phase and liquid-liquid extraction. This research focuses on the examination of all aspects of sample preparation and analytical method development to streamline the analysis of four urinary metabolites of JWH-018 and JWH-073. A detailed evaluation of the ß-glucuronide hydrolysis step lead to the reduction of time required for hydrolysis from 1h at 50°C to only 10min at room temperature. By utilizing a salting-out assisted liquid-liquid extraction (SALLE) in place of traditional liquid-liquid extraction with a volatile solvent, processing time was saved and waste was reduced. The analysis run time was also shortened to one-third of a typical published run time by utilizing UPLC with isocratic conditions in place of conventional HPLC running a gradient method.

Formal synthesis of angiogenesis inhibitor NM-3.

We report the formal synthesis of angiogenesis inhibitor NM-3 (1) in six steps from either of the 2,4-dimethoxyhalobenzenes 13a,b or 3,5-dimethoxychlorobenzene (13c). The first key reaction is the regiospecific alkylation/rearrangement between the aryne derived from 13a-c with sodium diethylmalonate in THF to produce diester 11, which after hydrolysis and cyclization affords homophthalic anhydride 3. The second is the reaction of anhydride 3 with either ethyl 2-methylmalonate (28a), in the presence of 1,1'-carbonyldiimidazole, or ethyl-2-methylmalonyl chloride (28b) under basic conditions to afford key isocoumarin 27. The conversion of 27 constitutes a formal synthesis of NM-3.