Rapid screening method for determination of Ecstasy and amphetamines in urine samples using gas chromatography-chemical ionisation mass spectrometry

The need for analytical screening tests more reliable and valid to detect amphetamine and related "designer drugs" in biological samples is becoming critical, due to the increasing diffusion of these drugs on the European illegal market. The most common screening procedures based on immunoassays suffer a number of limitations, including low sensitivity, lack of specificity and limited number of detectable substances. This paper describes a screening method based on gas-chromatography-mass-spectrometry (GC/MS) using positive chemical ionisation (PCI) detection. Methanol was used as reactant gas in the ionisation chamber. Molecular ions of different compounds were monitored, allowing a sensitivity of 5-10 ng/ml with high selectivity. The sensitivity of the method gives positive results in samples taken 48-72 h after intake of one dose of 50-100 mg. The method is simple and rapid. Sample preparation was limited to one liquid-liquid extraction, without any hydrolysis and derivatisation. Hydrolysis is critical to identify metabolites excreted as conjugates. Blank urine samples spiked with known amounts of amphetamine (AM), methylamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and methylenedioxyphenyl-N-methyl-2-butanamine (MBDB) were analysed. The method was successfully tested on real samples of urine from people, whose use of amphetamine was suspected, and results were compared with results obtained with immunoassays.     

Determination of 4-alkyl 2,5 dimethoxy-amphetamine derivatives by capillary electrophoresis with mass spectrometry detection from urine samples

The methylenedioxy-derivatives of amphetamine represent the largest group of designer drugs. The 4-methyl (DOM), -ethyl (DOET) and -propyl (DOPR) derivatives of 2,5-dimethoxy-amphetamine (2,5-DMA) were found to possess quite similar serotonin receptor affinities [R.A. Glennon, D.L. Doot, R. Young, Pharmacol. Biochem. Behav. 14 (1981) 287.]. This paper describes a method to screen for and quantify DOM, DOET and DOPR in urine samples, using capillary electrophoresis coupled to electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple solid-phase extraction (SPE) was used for sample cleanup. The method was validated according to international guidelines. Data for accuracy and precision were within required limits. Calibration curves were generated ranging from 10 to 1000 ng/mL and correlation coefficients always exceeded 0.996.     

Determination of drugs of abuse and their metabolites in human plasma by liquid chromatography-mass spectrometry. An application to 156 road fatalities

A method, using 0.2 ml of plasma, was designed for the simultaneous determination of morphine, 6-monoacetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, benzoylecgonine and cocaine. The drugs were analysed by LC-MS, after solid phase extraction in the presence of the deuterated analogues. Reversed phase separation on an Atlantis dC18 column was achieved in 10 min, under gradient conditions. The method was full validated, including linearity (2-250 ng/ml, r2>0.99), recovery (>50%), within-day and between-day precision and accuracy (CV and bias <15%), limit of detection (0.5 and 1 ng/ml) and quantitation (2 ng/ml), relative ion intensities and no matrix effect was observed. The procedure showed to be sensitive and specific, and was applied to 156 real cases from road fatalities (7.1% cases positive to cocaine and 0.6% to designer drugs).     

Determination of amphetamine,methamphetamine and amphetamine-derived designer drugs or medicaments in blood and urine

This paper reviews procedures for the determination of amphetamine, methamphetamine and amphetamine-derived designer drugs or medicaments in blood and urine. Papers published from 1991 to early 1997 were taken into consideration. Gas chromatographic and liquid chromatographic procedures with different detectors (e.g., mass spectrometer or diode array) were considered as well as the seldom used thin-layer chromatography and capillary electrophoresis. Enantioselective procedures are also discussed. A chapter deals with amphetamine-derived medicaments, e.g. anoretics, antiparkinsonians or vasodilators, which are metabolized to amphetamine or methamphetamine. Differentiation of an intake of such medicaments from amphetamine or methamphetamine intake is discussed. Basic information about the biosample assayed, internal standard, work-up, GC column or LC column and mobile phase, detection mode, reference data and validation data of each procedure is summarized in Tables. Examples of typical applications are presented.     

Effects of chronic treatment of rats with "designer" amphetamines on brain regional monoamines.

(+)-Amphetamine and two structurally related analogues, 4-methoxyamphetamine and a recent "designer drug," 4-ethoxyamphetamine, were given to rats via subcutaneous osmotic minipumps for 1-14 days. Regional brain levels of the drugs as well as monoamine neurotransmitters and some of their major acidic metabolites were determined. Amphetamine produced depletions of dopamine in the striatum after at least 3 days of treatment but not in the nucleus accumbens of olfactory tubercle, even after 14 days of treatment. In contrast, the two ring-substituted amphetamine analogues increased levels of the monoamines and decreased levels of their acid metabolites. These data indicate that the two ring-substituted amphetamine analogues, at least one of which is a potent hallucinogen, have potent monoamine oxidase inhibition properties that are sustained during chronic treatment. Furthermore, these two compounds do not share amphetamine's regionally selective neurotoxic effects on dopamine-releasing terminals, even though brain and striatal drug levels are the same or higher than those of amphetamine.     

Enantiomeric Separation of 13 New Amphetamine‐Like Designer Drugs by Capillary Electrophoresis,Using Modified‐‐Cyclodextrins

An easy‐to‐prepare chiral CE method for the enantiomeric separation of 13 new amphetamine‐like designer drugs, using CDs as chiral selectors, was developed. Sulfated‐β‐CD was found to be the best chiral selector among the three used (sulfated‐β‐CD, caroboxymethyl‐β‐CD, dimethyl‐β‐CD). The separation of the analytes was achieved in a fused‐silica gel capillary at 20 °C using an applied voltage of +25 kV. The optimized background electrolyte consisted of 63.5 mM H₃PO₄ and 46.9 mM NaOH in water. Several electrophoretic parameters such as CD type, CD concentration (1 ? 40 mg/mL), buffer pH (2.6, 3.6, 5.0, 6.0), length of the capillary (70 ? 40 cm total length), amount of the organic solvent (methanol and acetonitrile) were investigated and optimized. Chirality 25:617–621, 2013. © 2013 Wiley Periodicals, Inc.     

Capillary electrophoresis with laser-induced fluorescence and pre-column derivatization for the analysis of illicit drugs

In the current paper, we report the development of a new capillary electrophoresis method using pre-column derivatization and laser-induced fluorescence detection for the determination of ephedrine and amphetamine drugs. Our new method allows for the identification and quantification of six commonly used illicit drugs namely pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethylamphetamine, respectively, as well as propafenone (internal standard). Following derivatization with fluorescein isothiocyanate, a total of six amphetamine drugs and the internal standard could readily be separated using a fused-silica 75 micromID x 60 cm length (effective length: 50.2 cm) capillary column. The mobile phase consisted of buffer containing 20mM borate (pH 12, adjusted with sodium hydroxide). Samples were injected in pressure mode with the capillary being operated at 25kV/25 degrees C, and the detection of the derivatized compounds was sought using a laser-induced fluorescence (LIF) detector (lambda(ex)=488 nm and lambda(em)=520 nm), with a run-time of 20 min. The current method was validated with regard to precision (relative standard deviation, RSD), accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ). In human blood and urine samples, detection limits were 0.2 ngmL(-1), and the linear range of the calibration curves was 0.5-100 ngmL(-1). The intra-day and inter-day precisions were both less than 13.22%.     

Differential effects of the pharmacological manipulation of serotonin systems on cocaine and amphetamine self-administration in rats

The effects of the administration of serotonergic drugs on infusion rates of rats self-administering cocaine and amphetamine on an FR-10 schedule of reinforcement in daily 4 hour sessions were compared. Pretreatment with fluoxetine (2.5, 5, and 10 mg/kg), an inhibitor of serotonin reuptake, significantly decreased rates of responding maintained by amphetamine, but had no effect on responding maintained by cocaine at any of the doses tested. Pretreatment with cinanserin (3, 10, and 17.5 mg/kg), a serotonergic receptor antagonist, decreased rates of amphetamine self-administration at the highest dose tested, and also had no effect on cocaine self-administration. These data suggest a differential sensitivity of cocaine and amphetamine self-administration to pharmacological manipulation of central serotonin systems. They are consistent with biochemical data which demonstrates a negative correlation between the reinforcing potency of amphetamine-like drugs, but not cocaine-like drugs and their potency at serotonin binding sites.     

Increased antidepressant-like effect of desipramine combined with central stimulants (caffeine and amphetamine) in mice

Desipramine is a widely used antidepressive agent that inhibits the reuptake of noradrenaline and serotonin, and central stimulants such as caffeine and amphetamine help to release noradrenaline and serotonin. This work aimed to evaluate whether the combination of these agents could produce a stronger antidepressant-like effect than either of the drugs alone. To this end, male mice were treated with different doses of desipramine, caffeine, amphetamine, desipramine-caffeine and desipramine-amphetamine. The results showed that all drugs produced decreased immobility time in the forced swimming model. The combined treatment of desipramine (0.31, 1.0 or 3.1 mg/kg i.p.) with caffeine or amphetamine (0.31 or 1 mg/kg i.p.) reduced immobility time greater than either of those drugs alone. The combined treatment of desipramine (0.31, 1 and 3.1 mg/kg i.p.) with amphetamine or caffeine (0.1 and 1 mg/kg i.p.) did not increase the motor activity significantly compared to the control. These results also suggested that drugs which promote the release of noradrenaline and serotonin could increase antidepressant-like effect of desipramine.     

Toxicological detection of the designer drug 3,4-methylenedioxyethylamphetamine (MDE, “Eve”) and its metabolites in urine by gas chromatography — mass spectrometry and fluorescence polarization immunoassay

Studies are presented on the toxicological detection of the designer drug methylenedioxyethylapphetamine [MDE, rac-N-ethyl-(3,4-methylenedioxyphenyl)-propane-2-amine] in urine after a single oral dose of 140 mg of MDE by GC-MS and fluorescence polarization immunoassay (FPIA). After acid hydrolysis, extraction and acetylation MDE and its metabolites could be detected by mass chromatography with the selected ions m/z 72, 86, 114, 150, 162 and 164, followed by identification of the peaks underlying full mass spectra by computer library search. The following metabolites could be detected: unchanged MDE and 3,4-dihydroxyethylamphetamine (DHE) for 33,62 h, 3,4-methylenedioxyamphetamine (MDA) for 32–2036 h, and 4-hydroxy-3-methoxyethylamphetamine (HME) for 7 4-hydroxy-3-methoxyamphetamine (HMA), piperonul aceton, 3,4-Dihydroxyphenyl acetone and 4-hydroxy-3-methoxy-phenyl acetone could only be detected in trace amounts within the first few hours. The Abbott TD_x FPIA assay amphetamine/metamphetamine II gave positive results in urine for 33--62 h. Therefore, positive immunoassay results could be confirmed by the GC-MS procedure which also allowed the differentiation of MDE and its homologues 3,4-methylenedioxymethamphetamine (MDMA) and MDA as well as other amphetamine derivatives interfering with the TD_x assay. Furthermore, this GC-MS procedure allowed the simultaneous detection of most of the toxicologically relevant drugs.     

Comparative metabolism of the amphetamine drugs of dependence in man and monkeys.

The use of animal models to study drug dependence and tolerance requires that the species used metabolizes the drugs like man, a condition frequently not fulfilled by non-primate species. The metabolic fate of several amphetamine drugs, namely amphetamine, norephedrine, chlorphentermine and phenmetrazine, in the rhesus monkey and the tamarin and two non-primate species has been investigated and compared to that found for man. The findings show that the two primate species metabolize these drugs in a manner similar to that in man.     

Anorectic drugs which stimulate thermogenesis

Two anorectic drugs commonly prescribed as adjuncts in weight control and a third experimental drug were studied in rats for their anorectic and possible thermogenic activities. Diethylpropion, a congener of amphetamine, mazindol, which is chemically unrelated to amphetamine, and ciclazindol, an experimental drug structurally similar to mazindol, were given in graded doses to determine their effect on food and oxygen consumption (VO₂). Anorectic effects exhibited by diethylpropion and mazindol were similar and more potent than ciclazindol. Both resting and anesthetized VO₂ measurements were done to assess the thermogenic activity of the drugs. Anesthetized VO₂ was performed in an attempt to seperate peripheral from centrally-mediated actions of the drugs. Amphetamine was also tested at 1.0 mg/kg in order to correlate relative potencies. Mazindol, but not diethylpropion or ciclazindol, produced a dose response increases in resting VO₂. At the 1.0 mg/kg dose, amphetamine produced a greater increase in resting VO₂ than mazindol. At this dose, both drugs elicited centrally-induced alertness, although amphetamine elicited greater activity than mazindol. Mazindol and diethylpropion, but not ciclazindol, caused a dose-related increase in anesthetized VO₂. The anesthetized VO₂ response to amphetamine at 1 mg/kg was greater than the responses of mazindol and diethylpropion at 3.0 mg/kg. These findings confirm the previously recognized anorectic effects of mazindol and diethylpropion and also demonstrate that mazindol and diethylpropion but not ciclazindol (at the doses used) produced dose-related increases in VO₂ (energy expenditure) by stimulating directly peripheral mechanisms and in the case of mazindol central mechanisms as well.     

Amphetamine-induced c-fos mRNA expression in the caudate-putamen and subthalamic nucleus: interactions between dose,environment, and neuronal phenotype

When administered in a novel environment relatively low doses of amphetamine induce c-fos mRNA in the subthalamic nucleus (STN) and in preproenkephalin mRNA-containing (ENK+) neurons in the caudate-putamen (CPu). When administered at home, however, low doses of amphetamine do not produce these effects. Environmental novelty also facilitates the behavioral effects of acute and repeated amphetamine, but this is dose-dependent. The purpose of the present experiment therefore was to determine if the effect of context on amphetamine-induced c-fos expression is also dose-dependent. It was found that: (i) No dose of amphetamine tested (1-10 mg/kg) induced c-fos in many ENK+ cells when given at home. (ii) When given in a novel environment low to moderate doses of amphetamine (1-5 mg/kg) induced c-fos in substantial numbers of ENK+ cells, but the highest dose examined (10 mg/kg) did not. (iii) Environmental novelty enhanced the ability of low to moderate doses of amphetamine to induce c-fos in the STN, but the highest dose of amphetamine induced robust c-fos mRNA expression in the STN regardless of context. The results do not support the idea that engaging ENK+ cells, at least as indicated by c-fos mRNA expression, is critical to produce robust behavioral sensitization, but do suggest a possible role for the STN. Furthermore, the results highlight the importance of drug-environment interactions on the neurobiological effects of drugs, and have implications for thinking about the circuits by which context modulates the acute and long-lasting consequences of amphetamine treatment.     

Genetics of novelty seeking,amphetamine self‐administration and reinstatement using inbred rats

Previous research using outbred rats indicates that individual differences in activity in a novel environment predict sensitivity to the reinforcing effect of psychostimulant drugs. The current study examined if the link between responses related to novelty and amphetamine self‐administration is heritable. Twelve inbred rat strains were assessed for locomotor activity in a novel environment, preference for a novel environment, and intravenous amphetamine self‐administration (acquisition, extinction and amphetamine‐induced reinstatement). Strain differences were observed in activity in a novel environment, novelty preference and amphetamine self‐administration, indicating a genetic influence for each of these behaviors. While there was no relation between activity in an inescapable novel environment and amphetamine self‐administration, strain‐dependent differences in novelty preference were positively correlated with the amount of amphetamine self‐administered. There was also a positive correlation between the dose‐dependent rate of amphetamine self‐administration and magnitude of reinstatement. These results show that the activity in an inescapable novel environment and the preference for a novel environment are different genetically, and thus likely to reflect different behavioral constructs. Moreover, these results implicate a genetic influence on the relation between novelty seeking and stimulant self‐administration, as well as on the relation between stimulant reward and reinstatement.     

Food reward and cocaine increase extracellular dopamine in the nucleus accumbens as measured by microdialysis

Dopamine was measured by microdialysis in the nucleus accumbens of freely moving rats while they experienced rewarding food, brain stimulation and drugs. Extracellular dopamine increased 37% when the animals pressed a lever for food reward. Electrical stimulation of a lateral hypothalamic feeding-reward (self-stimulation) site caused a similar increase in dopamine, with or without food. At the site in the nucleus accumbens where rats will administer amphetamine to themselves, injections of amphetamine or cocaine increased extracellular dopamine five-fold. Thus amphetamine and cocaine increase dopamine in a behavior reinforcement system which is normally activated by eating. Conversely, the release of dopamine by eating could be a factor in addiction to food.     

Simultaneous determination of 13 amphetamine related drugs in human whole blood using an enhanced polymer column and gas chromatography-mass spectrometry

Metamphetamine (MA) is one of the most frequently encountered abused drugs in Japan and the Triage immunoassay kit is often used to screen for this drug. However, immunoassay screening also gives positive results with other structurally related compounds, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-methoxyamphetamine (PMA), an ephedrine metabolite and beta-phenethylamine (PEA). Therefore, it is important to develop a simple and reliable method which can determine these drugs simultaneously. This paper describes a simple method for simultaneous identification and quantification of 13 amphetamine related drugs in human whole blood. The method consists of a solid phase extraction using a new polar-enhanced Focus column followed by acetylation and gas chromatography-mass spectrometry in the scan mode. Tetradeuterated MA and trideuterated methylephedrine (ME) were used as internal standards. As the Focus column required only simple extraction steps and provided a clean extract, identification of each drug was feasible even at low concentrations. The calibration curves were linear over the concentration range from 50 to 5000 ng/ml for all drugs with correlation coefficients that exceeded 0.99. The lower limits of detection of the drugs were 5-50 ng/ml. The absolute recoveries for the drugs were 65-95% and 64-89% at concentrations of 100 and 1000 ng/ml, respectively. Accuracy and precision data were satisfactory when using 2 internal standards. The applicability of the assay was proven by the analysis of blood samples in forensic cases. This method should be most useful for confirmation of positive immunoassay results for amphetamines and related drugs.     

Distinct temperature-dependent dopamine-releasing effect of drugs of abuse in the olfactory bulb

It was recently shown in the olfactory bulb (OB) that the response to olfactory stimulation might be related to local reinforcement mechanisms involved in discrimination of different odors. Therefore, it seemed interesting to study the effects of several drugs of abuse on the release of dopamine (DA) in the OB. Nicotine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), and cocaine at 37 degrees C increased the release of [3H] DA from olfactory bulb slice preparations of the rats. While nicotine, amphetamine, and MDMA directly evoked DA release, cocaine, by inhibiting the reuptake processes, enhanced the electrical stimulation-evoked release. At low temperature (17 degrees C), a condition in which the transmitter uptake carriers of the plasma membrane in both the normal and reverse mode of operation are inhibited, the nicotine-evoked [3H] DA release was potentiated, whereas those evoked by amphetamine and MDMA were inhibited. At low temperature the field stimulation-evoked [3H] DA release was potentiated, but under this condition cocaine failed to increase the release. Our results show that low temperature (a) increases the concentration of extracellular DA released by Ca(2+)-dependent vesicular exocytosis elicited by nicotine, (b) inhibits the extracellular Ca(2+)-independent amphetamine- and MDMA-induced release of DA that occurs by the reverse operation of membrane carriers transporting DA from the cytoplasm of presynaptic terminals to the extraneuronal space, and (c) does not alter the inhibitory effect of cocaine on DA uptake that increases the concentration of extracellular DA released by field stimulation. The findings that the drugs of abuse tested all enhanced the release of DA in the olfactory bulb suggest that local reinforcing mechanisms may also exist in this brain area. In addition, we also show that lowering the temperature in in vitro experiments is an easy and straightforward method for separating vesicular and cytoplasmic release of transmitters, and is suitable for studying the mechanism of catecholamine release evoked by drugs of abuse. This technique may be applicable in other neurochemical studies that need inhibition of the uptake carriers without the blockade of the ligand-gated ion channels caused by reuptake inhibitor drugs.     

The effect of adren- and serotoninergic substances on the behavior of amygdalectomized rats and on the aggressiveness evoked by intra-amygdaloid administration of acetylcholine]

The effect of serotoninergic (serotonin, 1-trytophane, imipramime, methysergide), catecholaminergic (noradrenaline, amphetamine, dopamine, 1-DOPA, iproniazid) and cholinergic drugs (physostigmine, atropine, benactyzine) on emotional reactions and orienting-motor activity, as well as the effect of these drugs on shock-elicited aggressiveness enhanced by intraamygdaloid microinjection of acetylcholine was investigated in experiments on amygdalectomized male albino rats. In amygdalectomized animals, as compared to control false-operated rats, the stimulating effect of amphetamine, imipramine, tryptophane and m-cholinoblockators was enhanced and their inhibitory effect was weakened. Bilateral microinjection of cholinergic drugs (acetylcholine, physostigmine and carbacholine) and noradrenaline into the amygdaloid body intensified emotional reactivity and aggressiveness. Microinjection of serotonin and dopamine inhibited aggressiveness and caused facilitaion of orienting-motor activity. It is suggested that the adrenergic system intensifies and serotoninergic system depresses the m-cholinergic trigger mechanism of aggressive behavior in limbico-diencephalic structures.     

Effect of psychotropic drugs on the development of experimental gastric ulcer produced by different stress-inducing factors in rats

The anti-ulcer activity of three different doses (1/10, 1/30 and 1/90 of LD50) of imipramine, amitryptyline, chlorpromazine, amphetamine, ephedrine, chlordiazepoxide and meprobamate was studied in two types of stress-produced gastric ulcers in rats. It was found that these drugs given in doses of 1/10 or 1/30 LD50 inhibited in the same degree the development of gastric ulcer-produced by the method of Senay, in spite of differences in their psychotropic activity. On the other hand, when the method of Rossi was used for ulcer production the ulcer-preventing activity of these drugs has been varied. Thymoleptics were most effective and ataractics least effective against ulcers produced by the method of Rossi.     

Chiral separation and quantification of R/S-amphetamine, R/S-methamphetamine, R/S-MDA, R/S-MDMA, and R/S-MDEA in whole blood by GC-EI-MS

The enantioselective composition of the amphetamines is of interest, as the enantiomers show differences in their pharmacological effects and several methods for chiral separation of amphetamines have been described. Only a few methods have used whole blood as matrix and none of these separates both classic amphetamines (amphetamine and methamphetamine) and designer amphetamines (MDA, MDMA and MDEA). The aim of this study was, therefore, to develop a method for enantioselective analysis of AM, MA, MDA, MDMA, and MDEA in whole blood. The amphetamines were extracted from 0.5 g of whole blood by liquid-liquid extraction. After derivatization with R-MTPCl, the resulting diastereomers were separated by GC on a HP-5MS column and detected by SIM-MS. R-MTPCl was used as derivatization reagent because of the stability of this reagent and good separation of these analytes. Through the method, development time and temperature of the derivatization were optimized, and by admixture of 0.02% triethylamine it became possible to detect the amphetamines in adequately low concentrations as more analytes were derivatized. The method was validated and it was linear from 0.004 to 3 microg/g per enantiomer. The accuracy was within 91-115%, while the repeatability and reproducibility were < or =15% R.S.D. A method suitable for enantioselective separation and analysis of the amphetamines has been achieved, and the method was applied to analysis of whole blood samples originating from traffic and criminal cases and post mortem cases.