High-performance liquid chromatography-tandem mass spectrometry method for the analysis of N-oleoyl glycine and N-oleoyl alanine in brain and plasma.

Interest has increased in the role of N-acyl amino acids in a variety of disease states and as potential pharmacotherapies. Recently, N-oleoyl glycine and N-oleoyl alanine have shown promise in reducing the rewarding effects of drugs of abuse and alleviating withdrawal signs in rodent models. Previously published methods for the quantitation of these analytes by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in tissue were part of extensive lipidomic panels which may result in limited sensitivity and selectivity and also reported low recovery. Presented is a method for the extraction and HPLC-MS/MS analysis of N-oleoyl glycine and N-oleoyl alanine. The bias and precision of the assay were determined to be within ± 20%. The method was shown to be reliable and robust, with over 90% recovery for the low-level analytes. Increasing concentrations of N-oleoyl glycine and N-oleoyl alanine were quantitated in mouse brain and plasma following exogenous administration. This method was developed to serve to support studies investigating the pharmacokinetics and involvement of N-oleoyl glycine and N-oleoyl alanine in drug dependence and other diseases.

Characterization of E-cigarette coil temperature and toxic metal analysis by infrared temperature sensing and scanning electron microscopy - energy-dispersive X-ray.

INTRODUCTION: Electronic cigarettes (e-cigarettes) have rapidly evolved since their introduction to the U.S. market. The rebuildable atomizer (RBA) offers user-driven modification to the heating element (coil) and wicking systems. Different coil materials can be chosen based on user needs and preferences. However, the heating element of an e-cigarette is believed to be one-source for toxic metal exposure. METHODS: E-cigarette coils from Kanthal and nichrome wires were constructed in a contact and non-contact configuration and heated at four voltages. The maximum temperatures of the coils were measured by infrared temperature sensing when dry and when saturated with 100% vegetable glycerin or 100% propylene glycol. The metal composition of each coil was analyzed with Scanning Electron Microscopy-Energy-Dispersive X-Ray (SEM-EDX) when new, and subsequently after 1, 50, and 150 heat cycles when dry. RESULTS: The coils reached temperatures above 1000 °C when dry, but were below 300 °C in both liquid-saturated mediums. Metal analysis showed a decrease of 9-19% chromium and 39-58% iron in Kanthal wire and a decrease of 12-14% iron and 39-43% nickel in nichrome wire after 150 heat cycles. Significant metal loss was observed after one heat cycle for both coil alloys and configurations. CONCLUSIONS: The loss of metals from these heat cycles further suggests that the metals from the coils are potentially entering the aerosol of the e-cigarette, which can be inhaled by the user.

Investigation of Cannabidiol in the Mouse Drug Discrimination Paradigm.

Introduction: Cannabidiol (CBD) has gained considerable public and scientific attention because of its known and potential medicinal properties, as well as its commercial success in a wide range of products. Although CBD lacks cannabimimetic intoxicating side effects in humans and fails to substitute for cannabinoid type-1 receptor (CB1R) agonists in laboratory animal models of drug discrimination paradigm, anecdotal reports describe it as producing a "pleasant" subjective effect in humans. Thus, we speculated that this phytocannabinoid may elicit distinct subjective effects. Accordingly, we investigated whether mice would learn to discriminate CBD from vehicle. Additionally, we examined whether CBD may act as a CB1R allosteric and whether it would elevate brain endocannabinoid concentrations. Materials and Methods: C57BL/6J mice underwent discrimination training of either CBD or the high-efficacy CB1R agonist CP55,940 from vehicle. Additionally, we examined whether CBD or the CB1R-positive allosteric modulator ZCZ011 would alter the CP55,940 discriminative cue. Finally, we tested whether an acute CBD injection would elevate endocannabinoid levels in brain, and also quantified blood and brain levels of CBD. Results: Mice failed to discriminate high doses of CBD from vehicle following 124 training days, though the same subjects subsequently acquired CP55,940 discrimination. In a second group of mice trained to discriminate CP55,940, CBD neither elicited substitution nor altered response rates. A single injection of 100 or 200 mg/kg CBD did not affect brain levels of endogenous cannabinoids and related lipids and resulted in high drug concentrations in blood and whole brain at 0.5 h and continued to increase at 3 h. Discussion: CBD did not engender an interoceptive stimulus, did not disrupt performance in a food-motivated operant task, and lacked apparent effectiveness in altering brain endocannabinoid levels or modulating the pharmacological effects of a CB1R agonist. These findings support the assertions that CBD lacks abuse liability and its acute administration does not appear to play a functional role in modulating key components of the endocannabinoid system in whole animals.

N-oleoyl alanine attenuates nicotine reward and spontaneous nicotine withdrawal in mice.

BACKGROUND: As nicotine dependence represents a longstanding major public health issue, new nicotine cessation pharmacotherapies are needed. Administration of N-oleoyl glycine (OlGly), an endogenous lipid signaling molecule, prevents nicotine-induced conditioned place preference (CPP) through a peroxisome proliferator-activated receptor-alpha (PPARα) dependent mechanism, and also ameliorated withdrawal signs in nicotine-dependent mice. Pharmacological evidence suggests that the methylated analog of OlGly, N-oleoyl alanine (OlAla), has an increased duration of action and may offer translational benefit. Accordingly, OlAla was assessed in nicotine CPP and dependence assays as well as its pharmacokinetics compared to OlGly. METHODS: ICR female and male mice were tested in nicotine-induced CPP with and without the PPARα antagonist GW6471. OlAla was also assessed in nicotine-dependent mice following removal of nicotine minipumps: somatic withdrawal signs, thermal hyper-nociception and altered affective behavior (i.e., light/dark box). Finally, plasma and brain were collected after administration of OlGly or OlAla and analyzed by high-performance liquid chromatography tandem mass spectrometry. RESULTS: OlAla prevented nicotine-induced CPP, but this effect was not blocked by GW6471. OlAla attenuated somatic and affective nicotine withdrawal signs, but not thermal hyper-nociception in nicotine-dependent mice undergoing withdrawal. OlAla and OlGly showed similar time-courses in plasma and brain. CONCLUSIONS: The observation that both molecules showed similar pharmacokinetics argues against the notion that OlAla offers increased metabolic stability. Moreover, while these structurally similar lipids show efficacy in mouse models of reward and dependence, they reduce nicotine reward through distinct mechanisms.

Cannabinoid-based vaping products and supplement formulations reported by consumers to precipitate adverse effects.

Cannabinoid-based products submitted by consumers experiencing adverse effects were analyzed to identify and quantitate ingredients. Product testing identified several synthetic cannabinoids and products with inaccurate or incomplete labeling.

Rate of onset of dopamine transporter inhibitors assessed with intracranial self-stimulation and in vivo dopamine photometry in rats.

Drug self-administration and intracranial self-stimulation (ICSS) are two preclinical behavioral procedures used to predict abuse potential of drugs, and abuse-related drug effects in both procedures are thought to depend on increased mesolimbic dopamine (DA) signaling. Drug self-administration and ICSS yield concordant metrics of abuse potential across a diverse range of drug mechanisms of action. The "rate of onset," defined as the velocity with which a drug produces its effect once administered, has also been implicated as a determinant of abuse-related drug effects in self-administration procedures, but this variable has not been systematically examined in ICSS. Accordingly, this study compared ICSS effects produced in rats by three DA transporter inhibitors that have different rates of onset (fastest to slowest: cocaine, WIN-35428, RTI-31) and that produced progressively weaker metrics of abuse potential in a drug self-administration procedure in rhesus monkeys. Additionally, in vivo photometry using the fluorescent DA sensor dLight1.1 targeted to the nucleus accumbens (NAc) was used to assess the time course of extracellular DA levels as a neurochemical correlate of behavioral effects. All three compounds produced ICSS facilitation and increased DA levels assessed by dLight. In both procedures, the rank order of onset rate was cocaine > WIN-35428 > RTI-31; however, in contrast to monkey drug self-administration results, maximum effects did not differ across compounds. These results provide additional evidence that drug-induced increases in DA drive ICSS facilitation in rats and illustrate the utility of both ICSS and photometry to evaluate the time course and magnitude of abuse-related drug effects in rats.

Evaluation of extraction methods for pharmacologically active compounds from anticonvulsant traditional Chinese medicines: Gou Teng, Tian Ma, Jiang Can using DART-TOF-MS.

Chinese herbal medicines (CHMs) are classified as dietary supplements. Interactions with western medications, the presence of contaminants or adulterants, or a mis-labeled or mis-used CHM may lead to toxicological emergencies that can be undetected in death investigations. Laboratories must be able to efficiently analyze cases in which CHMs are suspected. Five extractions were evaluated for their ability to extract pharmacologically active compounds from herbal matrices: water, ethanol, microwave-assisted (MAE), ethanol : chloroform, and acid-wash. Anticonvulsive and other pharmacologically active compounds in Gou Teng, Tian Ma, and Jiang Can purchased from Beijing, China and New York were compared in the powder and the extracts using Direct Analysis in Real Time-Mass Spectrometry (DART-MS). Approximately 0.25 g of macerated herb was used per extraction. The water and ethanol extractions were simple liquid extractions. For the MAE, powdered herb was soaked in 65% ethanol, microwaved, and concentrated. The ethanol : chloroform extraction involved soaking in 1 : 1 ethanol : chloroform, sonication, and concentration. In the acid-wash extraction, powdered herb was soaked in acetic acid, followed by addition of sodium hydroxide, hexane extraction, and reconstitution in ethyl acetate. The powdered herbs and extracts were analyzed using a Jeol JMS T100LC AccuTOF DART-MS in positive and negative mode. Of the evaluated methods, no single extraction worked for all active compounds from the three CHMs. The MAE extract contained the most pharmacologically active compounds, while the acid-wash contained the least for the three products. Gou Teng purchased from different sources did exhibit a difference in pharmacologically active compounds, potentially from different species.