Chiral analysis of methorphan in opiate-overdose related deaths by using capillary electrophoresis.

An enantioselective CE-based determination of methorphan and its main metabolites in blood is described. Enantiomeric separations were carried out in 50cm×50µm (ID) uncoated fused silica capillaries, using a background electrolyte composed of 150mM sodium phosphate pH 4.4 added with 5mM 2-(hydroxypropyl)-ß-cyclodextrin and methanol 20% (v/v), at a constant voltage of 25kV. Sample injections were performed under field amplified sample stacking conditions. Detection was by recording UV absorbance at the wavelength of 200nm. Linearity of response was assessed within a concentration range from 25 to 500ng/mL for dextrometorhan, levomethorphan and their main metabolites (namely dextrorphan and levorphanol, respectively). Folcodine was used as internal standard. Under these conditions, the limit of quantification resulted 25ng/mL for each one of the analytes. The intra-day and inter-day precision, in terms of coefficient of variation (CV) were below 3.7% and 14.9 % for migration times and peak areas, respectively. The present method was successfully applied to the analysis of post-mortem blood samples from ten subjects died for heroin overdoses. Among the samples "positive" for methorphan (n=4), the d-enantiomer was found in concentrations ranging from 214 to 1282ng/mL. The concentration of its main metabolite dextrorphan in the same samples ranged from 49 to 389ng/mL.

Screening for synthetic cannabinoids in hair by using LC-QTOF MS: a new and powerful approach to study the penetration of these new psychoactive substances in the population.

The current analytical technology for the determination of New Psychoactive Substances in biological samples is still largely inadequate, because the immunoassays are unsuitable for the detection of most of these compounds and the use of traditional gas chromatography-mass spectrometry techniques is hampered by the lack of chromatographic standards and mass fragmentation patterns. Taking advantage of the molecular recognition capability of high-resolution mass spectrometry, the present work aimed to apply liquid chromatography-quadrupole-time of flight mass spectrometry for the rapid identification of New Psychoactive Substances in the hair, a peculiar tissue which "keeps memory" of the recent history of drug intake of the subject. All the samples were screened for the presence of 50 different New Psychoactive Substances (synthetic cannabinoids, cathinones and phenethylamines), substances that had been reported officially by the National Early Warning System in the period 2009-2011. Among the 435 samples analyzed, 8 were found "positive" for the following compounds: JWH-018, JWH-073, JWH-081, JWH-250, JWH-122, in a broad range of concentrations (0.010-1.28 ng/mg). Results strongly support the use of hair analysis to monitor the diffusion of new psychoactive drugs in the community.

Micellar electrokinetic chromatography: a new simple tool for the analysis of synthetic cannabinoids in herbal blends and for the rapid estimation of their logP values.

For the first time a capillary separation based on micellar electrokinetic chromatography (MEKC) with diode array detection (DAD) was developed and validated for the rapid determination of synthetic cannabinoids in herbal blends. Separations were carried out on a 30 µm(ID) × 40 cm uncoated fused silica capillaries. The optimized buffer electrolyte was composed of 25 mM sodium tetraborate pH 8.0, 30 mM SDS and n-propanol 20% (v/v). Separations were performed at 30 kV. Sample injection conditions were 0.5 psi, 10s. Diazepam and JWH-015 were used as internal standards. The determination of the analytes was based on the UV signal recorded at 220 nm, corresponding to the maximum wavelength of absorbance of the molecules, whereas peak identification and purity check were also performed on the basis of the acquisition of UV spectra between 200 and 400 nm wavelengths. Under the described conditions, the separation of the compounds was achieved in 25 min without any significant interference from the matrix. Linearity was assessed within a concentration range from 5 to 100 µg/mL. The intra-day and inter-day imprecision values were below 2.45% for relative migration times and below 10.75% for relative peak areas. The present method was successfully applied to the direct determination of synthetic cannabinoids in 15 different herbal blend samples requiring only sample dilution. In addition, the developed MEKC separation was also applied to estimate the octanol/water partition coefficients (logP) of these new and poorly known molecules.

Rapid analysis of caffeine in "smart drugs" and "energy drinks" by microemulsion electrokinetic chromatography (MEEKC).

A novel method based on microemulsion electrokinetic chromatography (MEEKC) with diode array detection (DAD) for rapid determination of caffeine in commercial and clandestine stimulants, known as "energy drinks" and "smart drugs", is described. Separations were carried out in 50 cm × 50 µm (ID) uncoated fused silica capillaries. The optimized buffer electrolyte was composed of 8.85 mM sodium tetraborate pH 9.5, SDS 3.3% (w/v), n-hexane 1.5% (v/v) and 1-butanol 6.6% (v/v). Separations were performed at a voltage of 20 kV. Sample injection conditions were 0.5 psi, 3 s. Diprofilline was used as internal standard. The determination of the analytes was based on the UV signal recorded at 275 nm, corresponding to the maximum wavelength of absorbance of caffeine, whereas peak identification and purity check was performed on the basis of the acquisition of UV radiation between 200 and 400 nm wavelengths. Under the described conditions, the separation of the compounds was achieved in 6 min without any interference from the matrix. Linearity was assessed within a caffeine concentration range from 5 to 100 µg/mL. The intra-day and inter-day precision values were below 0.37% for migration times and below 9.86% for peak areas. The present MEEKC method was successfully applied to the direct determination of caffeine in smart drugs and energy drinks.

Analysis of drugs of forensic interest with capillary zone electrophoresis/time-of-flight mass spectrometry based on the use of non-volatile buffers.

The present work is aimed at investigating the influence of the background electrolyte composition and concentration on the separation efficiency and resolution and mass spectrometric detection of illicit drugs in a capillary zone electrophoresis-electrospray ionization-time of flight mass spectrometry (CZE-ESI-TOF MS) system. The effect of phosphate, borate and Tris buffers on the separation and mass spectrometry response of a mixture of 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, methadone, cocaine, morphine, codeine and 6-monoacetylmorphine was studied, in comparison with a reference ammonium formate separation buffer. Inorganic non-volatile borate and Tris buffers proved hardly suitable for capillary electrophoresis-mass spectrometry (CE-MS) analysis, but quite unexpectedly ammonium phosphate buffers showed good separation and ionization performances for all the analytes tested. Applications of this method to real samples of hair from drug addicts are also provided.

Direct screening of herbal blends for new synthetic cannabinoids by MALDI-TOF MS.

Since 2004, a number of herbal blends containing different synthetic compounds mimicking the pharmacological activity of cannabinoids and displaying a high toxicological potential have appeared in the market. Their availability is mainly based on the so-called "e-commerce", being sold as legal alternatives to cannabis and cannabis derivatives. Although highly selective, sensitive, accurate, and quantitative methods based on GC-MS and LC-MS are available, they lack simplicity, rapidity, versatility and throughput, which are required for product monitoring. In this context, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers a simple and rapid operation with high throughput. Thus, the aim of the present work was to develop a MALDI-TOF MS method for the rapid qualitative direct analysis of herbal blend preparations for synthetic cannabinoids to be used as front screening of confiscated clandestine preparations. The sample preparation was limited to herbal blend leaves finely grinding in a mortar and loading onto the MALDI plate followed by addition of 2 µl of the matrix/surfactant mixture [α-cyano-4-hydroxy-cinnamic acid/cetyltrimethylammonium bromide (CTAB)]. After drying, the sample plate was introduced into the ion source for analysis. MALDI-TOF conditions were as follows: mass spectra were analyzed in the range m/z 150-550 by averaging the data from 50 laser shots and using an accelerating voltage of 20 kV. The described method was successfully applied to the screening of 31 commercial herbal blends, previously analyzed by GC-MS. Among the samples analyzed, 21 contained synthetic cannabinoids (namely JWH-018, JWH-073, JWH-081, JWH-250, JWH-210, JWH-019, and AM-694). All the results were in agreement with GC-MS, which was used as the reference technique.

Stereospecific synthesis and structure-activity relationships of unsymmetrical 4,4-diphenylbut-3-enyl derivatives of nipecotic acid as GAT-1 inhibitors.

Two complementary stereospecific synthetic approaches for the preparation of unsymmetrical ortho-substituted N-(4,4-diphenylbut-3-enyl) derivatives of nipecotic acid are described. Determination of the activity of the prepared compounds at the GAT-1 transporter highlighted differing SAR requirements of the E- and Z-phenyl rings, and led to the discovery of a compound with comparable potency to tiagabine. Some attempts to replace nipecotic acid with alternative novel amino acids are also described.

Design, synthesis and SAR of a novel series of benzimidazoles as potent NPY Y5 antagonists.

A novel class of benzimidazole NPY Y5 receptor antagonists was prepared exploiting a privileged spirocarbamate moiety. The structure-activity relationship of this series and efforts to achieve a profile suitable for further development and an appropriate pharmacokinetic profile in rat are described. Optimisation led to the identification of the brain penetrant, orally bioavailable Y5 antagonist 9b which significantly inhibited the food intake induced by a Y5 selective agonist with a minimal effective dose of 30mg/kg po.

Discovery and structure-activity relationship of a novel spirocarbamate series of NPY Y5 antagonists.

A novel series of trans-8-aminomethyl-1-oxa-3-azaspiro[4.5]decan-2-one derivatives was identified with potent NPY Y5 antagonist activity. Optimization of the original lead furnished compounds 23p and 23u, which combine sub-nanomolar Y5 activity with metabolic stability, oral bioavailability, brain penetration and strong preclinical profile for development. Both compounds significantly inhibited the food intake induced by a Y5 selective agonist with minimal effective doses of 3mg/kg po.

Synthesis and structure-activity relationship of N-(3-azabicyclo[3.1.0]hex-6-ylmethyl)-5-(2-pyridinyl)-1,3-thiazol-2-amines derivatives as NPY Y5 antagonists.

A novel class of small molecule NPY Y5 antagonists based around an azabicyclo[3.1.0]hexane scaffold was identified through modification of a screening hit. Structure-activity relationships and efforts undertaken to achieve a favourable pharmacokinetic profile in rat are described.