Simultaneous enantioselective determination of amphetamine and congeners in hair specimens by negative chemical ionization gas chromatography-mass spectrometry

Enantioselective quantification of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers in hair using gas chromatography-mass spectrometry (GC-MS) is described. Hair specimens were digested with 1M sodium hydroxide at 100 degrees C for 30 min and extracted by a solid phase procedure using Cleanscreen ZSDAU020. Extracted analytes were derivatised with (S)-heptafluorobutyrylprolyl chloride and the resulting diastereoisomers were quantified by GC-MS operating in the negative chemical ionization mode. Extraction yields were between 73.0 and 97.9%. Limits of detection varied in the range of 2.1-45.9 pg/mg hair, whereas the lowest limits of quantification varied between 4.3 and 91.8 pg/mg hair. Intra- and inter-assay precision and respective accuracy were acceptable. The enantiomeric ratios (R versus S) of AM, MA, MDA, MDMA and MDEA were determined in hair from suspected amphetamine abusers. Only MA and AM enantiomers were detectable in this collective and the quantification data showed in most cases higher concentrations of (R)-MA and (R)-AM than those of the corresponding (S)-enantiomers.     

Sensitive and simple gas chromatographic-mass spectrometric determination for amphetamine in microdialysate and ultrafiltrate samples

A gas chromatographic-mass spectrometric (GC-MS) method is described for the measurement of amphetamine (AMP) using negative chemical ionization (NCI) mode. Without prior extraction AMP was derivatized with 2,3,4,5,6-pentafluorobenzoyl chloride (PFBC) and simultaneously extracted into toluene. The toluene extract was injected directly into GC-MS equipped with a HP-1 capillary column. The method is simple and more sensitive than most of the previously published methods. The limit of quantification of amphetamine is 25pg (1.4pg on column) with a very limited sample volume (25microl). The within-day precision was from 1.7 to 5.1% and between-day precision was from 2.2 to 7.3%. The method has been used for the measurement of several thousand microdialysate and ultrafiltrate samples and proven reliable.     

Free radicals mediate amphetamine-induced acute pulmonary edema in isolated rat lung

Intravenous amphetamine abuse may cause serious cardiopulmonary complications via unknown mechanisms. We investigated the role of free radicals in the amphetamine-induced lung injury using isolated rat lungs. Adding amphetamine into the perfusate caused dose-dependent increases in perfusion pressure and lung weight. Amphetamine increased the filtration coefficient (K(f)) by 90 +/- 20% and 210 +/- 10% at doses of 10 microM and 50 microM, respectively, as compared to the baseline level. Pretreatment with dimethylthiourea (DMTU), an oxygen radical scavenger, abolished the pulmonary hypertension, lung weight gain, and permeability changes. We also examined the effect of amphetamine on free radical generation in polymorphonuclear leukocytes (PMN). Adding phorbol myristate acetate (PMA, 1 nM) enhanced the chemiluminescence indicating the functional viability of the isolated PMN. Amphetamine (50 microM) significantly enhanced the chemiluminescence generation of PMN by 152 +/- 26% as compared with the baseline value. Combination of amphetamine and PMA increased free radical formation by 360 +/- 85%. In summary, our results showed that amphetamine may cause acute lung injury by overproduction of free radicals. Although amphetamine can activate PMN, the source of free radicals remains to be determined.     

Biphasic Effects of Selegiline on Striatal Dopamine: Lack of Effect on Methamphetamine-Induced Dopamine Depletion

We tested the hypothesis that selegiline can attenuate dopamine depletion if administered following high doses of methamphetamine that cause neurotoxicity in the striatum. Methamphetamine produced decreases of 50% or greater in both striatal concentrations of dopamine and combined concentrations of homovanillic acid and DOPAC in mice. For animals not exposed to methamphetamine, chronic treatment with selegiline over 18 days caused biphasic effects on striatal dopamine content, with decreases, no effect, or increases observed for mice receiving treatment with 0.02, 0.2, and 2.0 mg/kg, respectively. Selegiline failed to modify methamphetamine-induced reductions in striatal dopamine content or combined concentrations of homovanillic acid and DOPAC. Significant increases in mortality following the onset of selegiline treatment (24 hours after the initial dose of methamphetamine) occurred in methamphetamine-treated mice that received saline or 2.0 mg/kg of selegiline, but not for mice treated with 0.02 or 0.2 mg/kg of selegiline. These results indicate that selegiline fails to attenuate dopamine depletion when administered chronically following exposure to methamphetamine, but may attenuate methamphetamine-induced mortality. In control animals that did not receive methamphetamine, low doses of selegiline produced decreases the concentration of striatal dopamine, while high dose treatment caused increases in striatal dopamine content.     

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 &#119 l each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.     

Amphetamine affects the start of responding in the peak interval timing task

In this paper we investigate how amphetamine affects performance in a PI task by comparing two analyses of responding during peak trials. After training on 24 s fixed interval (FI-24) with 96 s peak trials, rats were given amphetamine for 4 consecutive days at doses of .5 and 1.0 mg/kg. Responses during peak trials were fitted with a Gaussian distribution to estimate the expected time of reinforcement from the peak time. A single trials analysis was also performed to determine the start time and stop time of the transition into and out of a high rate of responding on each peak trial. Amphetamine significantly decreased peak times as measured with the Gaussian curve fitting. However, in the single trials analysis, animals initiated responding significantly earlier, but did not stop responding earlier. Thus, fitting a Gaussian to the average performance across trials sometimes provides a different characterization of the timing process than does analyzing the start and stop of responding on individual trials. In the current experiment, the latter approach provided a more precise characterization of the effects of amphetamine on response timing.     

Good riddance to dopamine: Roles for the dopamine transporter in synaptic function and dopamine-associated brain disorders

The neurotransmitter dopamine (DA) plays a critical role in CNS circuits that provide for attention, executive function, reward responses, motivation and movement. DA is inactivated by the cocaine- and amphetamine-sensitive DA transporter (DAT), a protein that also provides a pathway for non-vesicular DA release. After a brief review of DAT function and psychostimulant actions, we consider the importance DAT in relation to the distinct firing patterns of DA neurons that permit awareness of novelty and reward. Finally, we review recent efforts to gather direct support for DAT-linked disorders, with a specific focus on DAT mutations recently identified in subjects with ADHD.     

Behavioral pharmacology of designer cathinones: A review of the preclinical literature

“Bath salts” is one street name for a family of synthetic cathinones that display pharmacological effects resembling cocaine and commonly abused amphetamines. Despite extensive legislation aimed at the criminalization of bath salts, several designer cathinones are gaining a foothold in the illicit drug scene; for example, in the United Kingdom, mephedrone (4-methylmethcathinone, MEPH) is highly popular among drug abusers whereas, in the United States, MDPV (methylenedioxypyrovalerone) and methylone are highly prevalent. To date, knowledge about the hazards of designer cathinones is based mostly on hospital reports and anecdotal evidence derived from online surveys. Despite the paucity of preclinical studies directed toward designer cathinones, a number of invaluable findings arising from those studies are enabling scientists to develop their neuropharmacological profiles. Despite their commonalities in chemical structures, synthetic cathinones possess distinct neuropharmacological profiles and produce different behavioral effects, including unique effects on locomotor activity, learning, anxiety, thermoregulation, and abuse liability. The present review will discuss the behavioral effects of MEPH, MDPV, and methylone and compare those effects to established psychostimulant drugs. The rise in the use of designer cathinones in the United States and abroad justifies further investigations into these compounds, both for a greater understanding of the danger that “bath salts” pose to the public, and to provide insight into replacement cathinones as they emerge onto the market.     

Intracellular Methamphetamine Prevents the Dopamine-induced Enhancement of Neuronal Firing

The dysregulation of the dopaminergic system is implicated in multiple neurological and neuropsychiatric disorders such as Parkinson disease and drug addiction. The primary target of psychostimulants such as amphetamine and methamphetamine is the dopamine transporter (DAT), the major regulator of extracellular dopamine levels in the brain. However, the behavioral and neurophysiological correlates of methamphetamine and amphetamine administration are unique from one another, thereby suggesting these two compounds impact dopaminergic neurotransmission differentially. We further examined the unique mechanisms by which amphetamine and methamphetamine regulate DAT function and dopamine neurotransmission; in the present study we examined the impact of extracellular and intracellular amphetamine and methamphetamine on the spontaneous firing of cultured midbrain dopaminergic neurons and isolated DAT-mediated current. In dopaminergic neurons the spontaneous firing rate was enhanced by extracellular application of amphetamine > dopamine > methamphetamine and was DAT-dependent. Amphetamine > methamphetamine similarly enhanced DAT-mediated inward current, which was sensitive to isosmotic substitution of Na⁺ or Cl⁻ ion. Although isosmotic substitution of extracellular Na⁺ ions blocked amphetamine and methamphetamine-induced DAT-mediated inward current similarly, the removal of extracellular Cl⁻ ions preferentially blocked amphetamine-induced inward current. The intracellular application of methamphetamine, but not amphetamine, prevented the dopamine-induced increase in the spontaneous firing of dopaminergic neurons and the corresponding DAT-mediated inward current. The results reveal a new mechanism for methamphetamine-induced dysregulation of dopaminergic neurons.     

The Dopamine D1-D2 Receptor Heteromer Localizes in Dynorphin/Enkephalin Neurons: INCREASED HIGH AFFINITY STATE FOLLOWING AMPHETAMINE AND IN SCHIZOPHRENIA*

The distribution and function of neurons coexpressing the dopamine D1 and D2 receptors in the basal ganglia and mesolimbic system are unknown. We found a subset of medium spiny neurons coexpressing D1 and D2 receptors in varying densities throughout the basal ganglia, with the highest incidence in nucleus accumbens and globus pallidus and the lowest incidence in caudate putamen. These receptors formed D1-D2 receptor heteromers that were localized to cell bodies and presynaptic terminals. In rats, selective activation of D1-D2 heteromers increased grooming behavior and attenuated AMPA receptor GluR1 phosphorylation by calcium/calmodulin kinase IIα in nucleus accumbens, implying a role in reward pathways. D1-D2 heteromer sensitivity and functional activity was up-regulated in rat striatum by chronic amphetamine treatment and in globus pallidus from schizophrenia patients, indicating that the dopamine D1-D2 heteromer may contribute to psychopathologies of drug abuse, schizophrenia, or other disorders involving elevated dopamine transmission.