Giacomo Musile

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.

Chiral separation and determination of ketamine and norketamine in hair by capillary electrophoresis.

Ketamine, traditionally available as racemic mixture, has recently become available in the form of the single S-enantiomer, due to its higher anaesthetic potency associated with faster recovery times. The different pharmaceutical forms and the different pharmacodynamics of the two enantiomers imply the need for a chiral method, since most available analytical methods for biological matrices are not enantioselective. The method herein showed consists of simple capillary zone electrophoresis (CZE) for the chiral separation of ketamine and its major metabolite, norketamine, in hair specimens. After liquid-liquid extraction, the samples were electrokinetically injected and analysed in CE (running buffer: 15mM Tris phosphate pH 2.5, containing HS-γ-CDs, 0.1%, w/v). A complete separation of both racemic ketamine and norketamine in the respective enantiomers was obtained in less than 10minutes. Limit of detection (LOD) and limit of quantification (LOQ) were 0.08ng/mg and 0.25ng/mg, respectively. Percent recovery varied from 49% to 91% for all four enantiomers. Matrix effect on spiked hair samples demonstrated values ranging from 63% to 119%. Linearity was estimated using a calibration curve consisting of five concentration levels for each enantiomer (0.5-8.0ng/mg); the regression coefficients (R(2)) of weighted (1/x(2)) linear regression were all >0.988. The method is suitable for the analysis of real-world hair samples in order to investigate ketamine chronic abuse and to discriminate between the type of abused drug, either single enantiomer or racemic drug.

Dextromethorphan/levomethorphan issues in a case of opiate overdose.

Dextromethorphan is the d-isomer of 3-methoxy-N-methylmorphinan (methorphan), a synthetic analogue of codeine. It interacts with the σ-opioid receptors with resulting antitussive activity, while it does not show any significant affinity for the μ and δ opioid receptors, which are responsible for analgesic and central nervous system depressant effects. On this basis, d-methorphan is widely used all over the world as an over-the-counter cough suppressant drug. Indeed, there are hundreds of cough and cold preparations containing d-methorphan alone or in combination with analgesic (acetaminophen, acetyl salicylic acid), decongestant (phenylephrine, pseudoephedrine), anthistaminic (chlorpheniramine, brompheniramine, pheniramine), and/or expectorant mucolytic agents. In large doses, d-methorphan and its metabolite dextrorphan antagonize the actions of excitatory amino acids on N-methyl-d-aspartate (NMDA) receptors as do phencyclidine and ketamine. This action is responsible for the hallucinogenic and dissociative effects experienced by the subjects who abuse d-methorphan. Moreover, both d-methorphan and its metabolite dextrorphan are reported to inhibit reuptake of serotonin, and to have competitive 5HT1 agonist activity. On this basis, d-methorphan abuse could lead also to serotonin syndrome. Actually, many papers report fatal and non-fatal cases of abuse of d-methorphan especially among teenagers. Levomethorphan is the l-isomer of 3-methoxy-N-methylmorphinan and, unlike d-methorphan, is a potent narcotic analgesic. For this reason it is not commercially available and in many countries, including Italy, it is listed as a controlled substance. Moreover, the hepatic O-demethylation of l-methorphan produces levorphanol, which is a pure opioid agonist used to treat severe pain. Levorphanol has the same properties as morphine with respect to the potential for habituation, tolerance, physical dependence and withdrawal syndrome, but it is 4–5 times more potent than morphine and has a longer half-life. Starting from 2010, the Italian National Early Warning System (NEWS, Department of Antidrug Policy, Presidency of the Council of Ministers) collected numerous warnings from national collaborating centres (forensic laboratories, law enforcement, health services, research centres) related to heroin containing methorphan. Moreover, the use of methorphan as adulterant is reported in recent scientific literature. Both notifications and scientific papers often report methorphan as an adulterant, since the analytical methods used for heroin analysis cannot distinguish between the two stereoisomers. On the other hand, some papers report the identification of dextromethorphan because the gas chromatography–mass

An aptamer-based paper microfluidic device for the colorimetric determination of cocaine

A method utilizing paper microfluidics coupled with gold nanoparticles and two anticocaine aptamers has been developed to detect seized cocaine samples. The ready‐to‐use format involves the use of a paper strip that produces a color change resulting from the salt‐induced aggregation of gold nanoparticles producing a visible color change indicating the presence of the drug. This format is specific for the detection of cocaine. The visual LOD for the method was 2.5 μg and the camera based LOD was 2.36 μg. The operation of the device is easy and rapid, and does not require extensive training or instrumentation. All of the materials utilized in the device are safe and environmental friendly. This device should prove a useful tool for the screening of forensic samples.

The alcohol used for cleansing the venipuncture site does not jeopardize blood and plasma alcohol measurement with head-space gas chromatography and an enzymatic assay

Introduction This study aimed to establish whether an alcoholic antiseptic, wiped or not before venipuncture, may jeopardize alcohol testing with a commercial enzymatic assay and a reference head-space gas chromatography (GC) technique. Materials and methods Venous blood was collected from 23 healthy volunteers, with two sequential procedures. In the first blood collection, 2 mL of alcoholic antiseptic (0.5% chlorhexidine, 70% ethanol) were place on a gauge pad, the venipuncture site of right arm was cleaned but the antiseptic was not let to dry before phlebotomy. In the second blood collection, 2 mL of the same alcoholic antiseptic were placed on another gauge pad, the venipuncture site of left harm was cleaned and the antiseptic was accurately cleansed before phlebotomy. Ethanol was measured with a reference GC technique in whole blood and EDTA plasma, and a commercial enzymatic assay in EDTA plasma. Results No subject complained about feeling a particular itchy sensation when the alcohol was not wiped before puncturing the vein. The concentration of alcohol in all EDTA plasma samples was always lower than the limit of detection of the enzymatic assay (i.e., 2.2 mmol/L; 0.1 g/L). Similarly, alcohol concentration was also undetectable using a reference GC technique (i.e., < 0.22 mmol/L; 0.01 g/L) in EDTA plasma and whole blood. Conclusion It seems reasonable to conclude that using ethanol-containing antiseptics before venipuncture may not be causes of spurious or false positive results of alcohol measurement at least when ideal venipunctures can be performed.

Development of an in-house mixed-mode solid-phase extraction for the determination of 16 basic drugs in urine by High Performance Liquid Chromatography-Ion Trap Mass Spectrometry

The aim of the present work was to develop a novel in-house mixed-mode SPE sorbent to be used for the HPLC-Ion TrapMS determination of 16 basic drugs in urine. By using a computational modelling, a virtual monomer library was screened identifying three suitable functional monomers, methacrylic acid (MAA), itaconic acid (IA) and 2-acrylamide-2-methylpropane sulfonic acid (AMPSA), respectively. Three different sorbents were then synthetized based on these monomers, and using as cross-linker trimethylolpropane trimethacrylate (TMPTMA). The sorbent characterization analyses brought to the selection of the AMPSA based phase. Using this novel in-house sorbent, a SPE-HPLC-Ion TrapMS method for drug analysis in urine was validated proving to be selective and accurate and showing a sensitivity adequate for toxicological urine analysis. The comparison of the in-house mixed-mode SPE sorbent with two analogous commercial mixed-mode SPE phases showed that the first one was better not only in terms of process efficiency, but also in terms of quality-price rate. To the best of our knowledge, this is the first time in which an in-house SPE procedure has been applied to the toxicological analysis of a complex matrix, such as urine.