Amphetamine Concentrations Research Articles

Determination of amphetamine in rat brain by in vivo microdialysis and ion-pairing liquid chromatography with electrospray tandem mass spectrometry.

An ion-pairing liquid chromatography/electrospray tandem mass spectrometry (LC/ES-MS/MS) method with in vivo microdialysis for the determination of amphetamine in rat brain has been developed. A microdialysis probe was surgically implanted into the striatum of the rat and artificial cerebrospinal fluid (aCSF) was used as the perfusion medium. Samples were collected and then analyzed off-line by LC/ES-MS/MS. A reversed-phase C18 column was employed for LC separation and MS/MS was utilized for detection. Trifluoroacetic acid (TFA) was added to the mobile phase (acetonitrile/water) as an ion-pairing reagent. Detection was by ES-MS/MS directly, and no post-column addition of organic modifier was needed. Dual linear ranges were determined from 0.1-0.5 microg/mL and 0.005-0.1 microg/mL, respectively. The detection limit, based on a signal-to-noise ratio of 3, was 0.001 microg/mL (5 nM). Good precision and accuracy were obtained. The applicability of this newly developed method was demonstrated by continuous monitoring of amphetamine concentrations in rat brain. Amphetamine reached a maximum concentration of 0.086 +/- 0.017 microg/mL over 20-40 min after a single 3.0 mg/kg intraperitoneal administration.

Miniaturized sample preparation method for determination of amphetamines in urine

A simple and miniaturized sample preparation method for determination of amphetamines in urine was developed using on-column derivatization and gas chromatography–mass spectrometry (GC–MS). Urine was directly applied to the extraction column that was pre-packed with Extrelut and sodium carbonate. Amphetamine (AP) and methamphetamine (MA) in urine were adsorbed on the surface of Extrelut. AP and MA were then converted to a free base and derivatized to N-propoxycarbonyl derivatives using propylchloroformate on the column. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 100 and 102%, respectively. The calibration curves showed linearity in the range of 0.50–50 μg/mL for AP and MA in urine. When urine samples containing two different concentrations (0.50 and 5.0 μg/mL) of AP and MA were determined, the intra-day and inter-day coefficients of variation were 1.4–7.7%. This method was applied to 14 medico-legal cases of MA intoxication. The results were compared and a good agreement was obtained with a HPLC method.

Development of a liquid chromatography–tandem mass spectrometric method for the determination of methamphetamine and amphetamine using small volumes of rat serum

The aim of this paper was to develop LC/MS/MS methodology for the determination of methamphetamine (METH) and amphetamine (AMP) using low microliter volumes (20–150 μl) of rat serum and demonstrate the use of this method for the study of serum pharmacokinetics in the rat. The analytes were extracted from rat serum using solid-phase extraction followed by an isocratic separation on a narrow-bore Hypersil C 18 column. Lower limits of quantitation for METH and AMP were 0.3 ng/ml using positive ion electrospray tandem mass spectrometry. The accuracy of the method was within 20% of the actual values over a wide range of serum concentrations. The within-day and between-day precision was better than 20% (R.S.D.). Ion-suppression matrix effects on electrospray ionization were evaluated for extracted rat serum. The LC/MS/MS method was further validated by comparing serum concentrations of METH and AMP to serum concentrations previously determined using an LC/[ 3 H ]-METH assay with radiochemical detection. Finally, the LC/MS/MS method was used to study the pharmacokinetics of METH and AMP after a 1 mg/kg intravenous bolus dose of METH to female Sprague–Dawley rats.

Determination of free-form amphetamine in rat brain by ion-pair liquid chromatography–electrospray mass spectrometry with in vivo microdialysis

An ion-pair liquid chromatography–electrospray mass spectrometry (LC–ESI-MS) method with in vivo microdialysis for the determination of free-form amphetamine in rat brain has been developed. A microdialysis probe was surgically implanted into the striatum of the rat and artificial cerebrospinal fluid (aCSF) was used as the perfusion medium. Samples were collected and then analyzed off-line by LC–ESI-MS. A reversed phase C 18 column was employed for LC separation. Trifluoroacetic acid (TFA) was added in the mobile phase (acetonitrile–water, 10:90, v/v) as an ion-pair reagent. The ion-pair process disguises the protonated amphetamine cations from the ESI-MS electric field as neutral molecules. Post-column addition of volatile organic acid was utilized to minimize TFA signal suppression effect on ESI-MS detection. More than six-fold enhancement of ESI-MS response was achieved by the post-column addition of propionic acid. Good linearity (0.01–1.00 μg/ml, r 2=0.99) and detection limit (0.002 μg/ml) were determined. Good precision and accuracy were obtained. The applicability of this newly developed method was demonstrated by continuous monitoring of amphetamine concentrations in rat brain after a single 3.0 mg/kg i.p. administration.

Metabolic Profile of Amphetamine and Methamphetamine Following Administration of the Drug Famprofazone

There are a several drugs that lead to the production of methamphetamine and/or amphetamine in the body which are subsequently excreted in the urine. These drugs raise obvious concerns when interpreting positive amphetamine drug testing results. Famprofazone is an analgesic found in a multi-ingredient medication (Gewodin) used for pain relief. Two Gewodin tablets (50 mg of famprofazone) were administered orally to healthy volunteers with no history of amphetamine, methamphetamine, or famprofazone use. Following administration, urine samples were collected ad lib for up to six days, and pH, specific gravity, and creatinine values were determined. In order to determine the quantitative excretion profile of amphetamine and methamphetamine, samples were extracted using liquid-liquid extraction, derivatized with heptafluorobutyric anhydride, and analyzed by gas chromatography-mass spectrometry (GC-MS). The ions monitored were 91, 118, 240 for amphetamine and 254, 210, 118 for methamphetamine. Amphetamine-d(6) and methamphetamine-d(11) were used as internal standards. Peak concentrations for amphetamine ranged from 148 to 2271 ng/mL and for methamphetamine 615 to 7361 ng/mL. Concentrations of both compounds peaked between 3 and 7 h post-dose. Amphetamine and methamphetamine could be detected (limit of detection = 5 ng/mL) at 121 and 143 h post-dose, respectively. Using a cutoff of 500 ng/mL, all subjects had individual urine samples that tested positive. One subject had 14 samples above the cutoff with the last positive being detected over 48 h post-dose. The profile of methamphetamine and amphetamine enantiomers was also determined using liquid-liquid extraction, derivatization with N-trifluoroacetyl-l-prolyl chloride and analysis by GC-MS. Data showed the famprofazone metabolites amphetamine and methamphetamine to be both d- and l-enantiomers. The proportion of l-methamphetamine exceeded that of its d-enantiomer from the first sample collected. Initially, the proportion was approximately 70% l-methamphetamine and this proportion increased over time. Amphetamine results showed l- and d-amphetamine were virtually the same in the early samples with the proportion of l-amphetamine increasing as time progressed. Forensic interpretation of drug testing results is a challenging critical part of forensic drug testing area because of the potential repercussions the results found may have on an individual's life. The finding of each enantiomers by itself differentiates famprofazone use from the most commonly abused form of methamphetamine and all medicinal methamphetamine available in the U.S., which is either d-methamphetamine (prescription medication) or l-methamphetamine (Vicks inhaler). Coupling this information with the concentrations of amphetamine and methamphetamine helps to determine the potential for use of this drug.

Amphetamine Enantiomer Excretion Profile Following Administration of Adderall

Amphetamine remains a widely abused drug throughout the world. It is also used therapeutically for weight loss, narcolepsy, and attention deficit disorder with hyperactivity (ADHD). ADHD has grown dramatically recently both in terms of diagnosis and treatment. Increasingly, older individuals are diagnosed and treated for ADHD, and treatment often continues into adulthood. Of the available treatments for ADHD, Adderall is widely prescribed. Despite its widespread use, there are no published data regarding the expected amphetamine excretion profile following its use. This is problematic because, in this case, medical review officers (MRO) and forensic toxicologists are asked to assess results in terms of use pursuant to valid medical prescription without specific data on which to base a sound decision. To address this situation, a study to determine the concentration and enantiomer composition of amphetamine excretion following administration of Adderall was undertaken. Adderall (20 mg) was administered to five healthy subjects with all subsequent ad lib urine samples (total urine void) collected for seven days. Adderall is a 3:1 mixture of d- and l-enantiomers of amphetamine salts. Peak amphetamine concentrations ranged from 2645 to 5948 ng/mL. Samples containing > or = 500 ng/mL of amphetamine (the administrative cutoff for a positive result by gas chromatography-mass spectrometry) were seen up to 47:30 h post dose. The number of samples that contained amphetamine concentrations of > or = 500 ng/mL ranged among individuals from 7 to 13. As anticipated, analysis showed the d-enantiomer to be in excess of the l-enantiomer, with the proportion of l-enantiomer increasing over time. Because of the mixture of enantiomers, not all samples that contained > or = 500 ng/mL of amphetamine were positive when tested by immunoassay. The drug concentration profiles were quite variable within and between subjects because of dilution and fluctuations in pH of the samples. These results are the first to describe the excretion of amphetamine following administration of Adderall. The presence of the l-enantiomer separates this drug from other preparations of the drug that are composed of only the d-enantiomer (i.e., dexedrine and much illicit amphetamine), thus readily differentiating them from Adderall use. Some illicit and medicinal amphetamine is, however, a mixture of amphetamine enantiomers. Because the enantiomers are metabolized at different rates, their proportion offers the opportunity to describe excretion versus time. Coupling this data with drug concentration makes it possible for forensic toxicologists and MROs to come to an informed decision about the involvement of this drug in a positive drug test result. Using the combination of enantiomer composition and quantitative data will allow MROs and forensic toxicologists to better assess the use of this drug from abuse of amphetamine.

Routine analysis of amphetamine and methamphetamine in biological materials by gas chromatography–mass spectrometry and on-column derivatization

A simple determination method of amphetamine (AP) and methamphetamine (MA) in biological materials was developed using on-column derivatization and gas chromatography–mass spectrometry (GC–MS). AP and MA in biological materials were adsorbed on the surface of Extrelut and then extracted and derivatized simultaneously on the Extrelut column. AP and MA were derivatized to the N-propoxycarbonyl derivatives using propylchloroformate. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 88.2 and 92.5%, and those from blood were 89.7 and 90.3%, respectively. The calibration curves showed linearity in the range of 12.5–2000 ng/ml (ng/g) for AP and MA in urine and blood, and 0.25–20 ng/mg in hair. When urine samples containing two different concentrations (200 and 1000 ng/ml) of AP and MA, blood samples containing two different concentrations (200 and 1000 ng/g) of AP and MA, hair samples containing two different concentrations (0.5 and 5.0 ng/mg) of AP and MA, the coefficients of variation of intra-day and inter-day were 0.68–3.60% in urine, 0.42–4.58% in blood, and 1.20–13.1% in hair. Furthermore, this proposed method was applied to a medico-legal case of MA intoxication.

Amphetamine concentrations in human urine following single-dose administration of the calcium antagonist prenylamine-studies using fluorescence polarization immunoassay (FPIA) and GC-MS.

Prenylamine (R,S-N-(3,3-diphenylpropyl-methyl-2-phenethylamine), a World Health Organization class V calcium antagonist, is known to be metabolized to amphetamine. In this study, amphetamine concentrations after a single-dose administration of prenylamine were determined to check if they reached values that could be of analytical and/or pharmacological importance in clinical and forensic toxicology. Enantiomeric composition of amphetamine was also studied. Five volunteers received a single 120-mg oral dose of prenylamine. Urine samples were analyzed using the Abbott TDx immunoassay Amphetamine/Methamphetamine II and using our routine systematic toxicological analysis (STA) gas chromatography-mass spectrometry (GC-MS) procedure. For quantitation purposes, GC-MS was used in the selected-ion monitoring (SIM) mode (ions m/z 118, 122, 240, 244) after solid-phase extraction (Isolute Confirm HCX) and derivatization (heptafluorobutyric anhydride). Amphetamine-d5 was used as internal standard (IS). Chiral separation of the heptafluorobutyrated amphetamine enantiomers was achieved using an Astec Chiraldex G-PN column. The TDx results showed a great variability for the different volunteers. A urine sample of one volunteer showed results as high as 3200 ng/mL, whereas the urine samples of another volunteer never gave results greater than the TDx detection limit (100 ng/mL). Using the STA procedure, the presence of amphetamine could be confirmed in all urine samples with TDx results greater than the cutoff value (300 ng/mL). Using the GC-MS SIM method, amphetamine concentrations up to 1280 ng/mL were determined. Chiral analysis revealed that both enantiomers of amphetamine were present in the samples with a surplus of the S(+)-enantiomer in the early phase of excretion. Forensic implications are discussed.

Effects of amphetamine on steroidogenesis in MA-10 mouse Leydig tumor cells

Amphetamine influences plasma and testicular testosterone levels. However, there is no evidence that amphetamine can directly influence Leydig cell functions. In the present study, a MA-10 mouse Leydig tumor cell line was used to determine whether and how amphetamine affected Leydig cell steroidogenesis. MA-10 cells were treated with different concentrations of amphetamine without or with human chorionic gonadotropin (hCG) and/or enzyme precursors over different time durations. Steroid production, enzyme activities and StAR protein expression were determined. Amphetamine alone had no any effect on MA-10 cell steroidogenesis. However, amphetamine (10 −11M and 10 −10M) significantly enhanced hCG-treated progesterone production at 3 hr in MA-10 cells (p < 0.05). Furthermore, amphetamine significantly induced more progesterone production upon treatment with 22R-hydroxycholesterol (p < 0.05), a precursor of P450 side-chain cleavage enzyme (P450scc). However, amphetamine did not induce more progesterone production when treated with pregnenolone (p > 0.05), a precursor of 3β-hydroxysteroid dehydrogenase. In addition, the expressions of StAR protein and P450scc enzyme were not significantly different between hCG alone and hCG plus amphetamine treatment in MA-10 cells (p > 0.05). These results suggested that amphetamine enhanced hCG-induced progesterone production in MA-10 cells by increasing P450scc activity without influencng StAR protein and P450scc enzyme expression or 3β-HSD enzyme activity.

Dopamine efflux via wild-type and mutant dopamine transporters: alanine substitution for proline-572 enhances efflux and reduces dependence on extracellular dopamine, sodium and chloride concentrations

The dopamine transporter (DAT) can mediate not only inward uptake of dopamine, but also its outward efflux by mechanisms that have been only partially elucidated. DAT-dependent dopamine efflux can be studied kinetically and apparent substrate affinity and V max values determined. We now report that wild-type DAT displays apparent affinities for efflux more than 300-fold lower than those for uptake. Efflux rates are enhanced by increased extracellular concentrations of dopamine or amphetamine and by lowered extracellular concentrations of Na + or Cl −. Alanine substitutions for six proline residues located in or near DAT transmembrane domains increase apparent affinity and decrease V max values for dopamine efflux mediated by these mutant transporters. Mutant 12P572A displays increased DAT efflux with reduced dependence on ion or dopamine concentrations. These data add to evidence for the specificity of transporter-mediated efflux processes and begin to elucidate DAT candidate domains that may be preferentially involved with efflux activities.

Duration of Detectable Methamphetamine and Amphetamine Excretion in Urine after Controlled Oral Administration of Methamphetamine to Humans

Confirmation of a workplace drug test requires urinary methamphetamine (MAMP) and amphetamine (AMP) concentrations > or = 500 and 200 micro g/L, respectively, but cutoffs at half those values (250/100 micro g/L) have been proposed. We determined the urinary excretion of MAMP after oral ingestion and examined the effect of using lower cutoffs on detection of exposure. Volunteers (n = 8) ingested four 10-mg doses of MAMP. HCl daily over 7 days, and five of them ingested four 20-mg doses 4 weeks later. After ingestion, the volunteers collected all urine specimens for 2 weeks. After solid-phase extraction, MAMP and AMP were measured by gas chromatography-positive chemical ionization mass spectrometry with dual silyl derivatization. MAMP and AMP were generally detected in the first or second void (0.7-11.3 h) collected after drug administration, with concentrations of 82-1827 and 12-180 micro g/L, respectively. Peak MAMP concentrations (1871-6004 micro g/L) after single doses occurred within 1.5-60 h. MAMP > or = 500 micro g/L was first detected in the first or second void (1-11 h) at 524-1871 micro g/L. Lowering the MAMP cutoff to 250 micro g/L changed the initial detection time little. AMP > or = 200 micro g/L was first detected in the 2nd-13th (7-20 h) post-administration voids. At a cutoff of 100 micro g/L, AMP was first confirmed in the second to eighth void (4-13 h). Reducing the cutoff to 250/100 micro g/L extended terminal MAMP detection by up to 24 h, increased total detection time by up to 34 h, and increased the total number of positive specimens by 48%. At the lower cutoff, initial detection times are earlier, detection windows are longer, and confirmation rates are increased. Elimination of the AMP requirement would increase detection rates and allow earlier detection.

Multiplex assay of amphetamine, methamphetamine, and ecstasy drug using CEDIA technology.

Using Microgenics unique technology, cloned enzyme donor immunoassay (CEDIA), the multiplex assay (CEDIA Amphetamines/Ecstasy) has been developed for the detection of amphetamine, methamphetamine, and ecstasy drugs at cutoff level either of 500 ng/mL or 1000 ng/mL applicable for either qualitative screening or semiquantitative measurement. The multiplex assay detects the total concentration of amphetamine, methamphetamine, and ecstasy drugs in urine samples. In addition, the assay detects metabolites of parent drugs and structurally related drugs including d,l-amphetamine (67.2%), d,l-methamphetamine (58.4%), MDA (113%), MDMA (199%), MDEA (207%), MBDB (123%), BDB (72%), PMA (24%), and PMMA (100%). The assay is highly specific and exhibits minimal cross-reactivity with the undesirable, structurally related over-the-counter amphetaminelike drugs. Imprecision results (n = 120) demonstrate an intra-assay < 6.3% CV and an interassay < 9.2% CV as performed by a modified NCCLS protocol. Using the SAMHSA mandatory guidelines of a 500-ng/mL cutoff for the immunoassay and a 250-ng/mL cutoff for GC-MS, the assay detects 164 out of 165 GC-MS-positive samples. For drug abuse testing, the multiplex assay presents an excellent method for the detection of multiple drugs with greater sensitivity and broader detection.

Plasma, oral fluid and sweat wipe ecstasy concentrations in controlled and real life conditions

In a double-blind placebo controlled study on psychomotor skills important for car driving (Study 1), a 75mg dose of ±3,4-methylenedioxymethamphetamine (MDMA) was administered orally to 12 healthy volunteers who were known to be recreational MDMA-users. Toxicokinetic data were gathered by analysis of blood, urine, oral fluid and sweat wipes collected during the first 5h after administration. Resultant plasma concentrations varied from 21 to 295ng/ml, with an average peak concentration of 178ng/ml observed between 2 and 4h after administration. MDA concentrations never exceeded 20ng/ml. Corresponding MDMA concentrations in oral fluid, as measured with a specific LC-MS/MS method (which required only 50μl of oral fluid), generally exceeded those in plasma and peaked at an average concentration of 1215ng/ml. A substantial intra- and inter-subject variability was observed with this matrix, and values ranged from 50 to 6982ng/ml MDMA. Somewhat surprisingly, even 4–5h after ingestion, the MDMA levels in sweat only averaged 25ng/wipe.In addition to this controlled study, data were collected from 19 MDMA-users who participated in a driving simulator study (Study 2), comparing sober non-drug conditions with MDMA-only and multiple drug use conditions. In this particular study, urine samples were used for general drug screening and oral fluid was collected as an alternative to blood sampling. Analysis of oral fluid samples by LC-MS/MS revealed an average MDMA/MDEA concentration of 1121ng/ml in the MDMA-only condition, with large inter-subject variability. This was also the case in the multiple drug condition, where generally, significantly higher concentrations of MDMA, MDEA and/or amphetamine were detected in the oral fluid samples. Urine screening revealed the presence of combinations such as MDMA, MDEA, amph, cannabis, cocaine, LSD and psilocine in the multiple-drug condition.

On-fiber derivatization for direct immersion solid-phase microextraction. Part II. Acylation of amphetamine with pentafluorobenzoyl chloride for urine analysis.

On-fiber derivatization was used for solid-phase microextraction (SPME) in order to increase the detectability and extractability of drugs in biological samples. Amphetamine, which was used as a model compound, was derivatized with pentafluorobenzoyl chloride (PFBCl) and subjected to gas chromatography with electron capture or mass spectrometric detection. Extraction was performed by direct immersion of a 100 microm polydimethylsiloxane-coated fiber into buffered human urine. On-fiber derivatization was performed either after or simultaneously with extraction. The former procedure gave cleaner chromatograms but the latter turned out to be superior with respect to linearity and repeatability. For the on-fiber derivatization of amphetamine an excess of reagent is required. Because a considerable part of the PFBCl loaded on to the fiber is used up by reaction with matrix compounds and water, a reagent loading time of 5 min was needed to obtain a linear range (r = 0.9756) from 250 pg mL(-1) to 15 ng mL(-1). Due to an interfering matrix compound, the limit of detection was also found to be dependent on the reagent loading time, i.e., the limit of detection for a PFBCl loading time of 5 min is 250 pg mL(-1) whereas that for a 1 min loading time it is 100 pg mL(-1). The relative standard deviation (n = 7) of the method was about 11% at an amphetamine concentration of 1 ng mL(-1). The applicability of the method for the determination of drugs in biological samples is shown.

Effect of Food on Early Drug Exposure from Extended-Release Stimulants: Results from the Concerta®, Adderall XR™ Food Evaluation (CAFÉ) Study

SummaryStimulant therapy is the mainstay of treatment for children, adolescents and adults with attention-deficit/hyperactivity disorder (ADHD). Once-daily, extended-release oral formulations offer long acting control of symptoms by modifying drug delivery and absorption. In particular, consistency in early drug exposure is important for symptom control during school or work hours. Because these once-daily formulations are usually taken in the morning, the timing of the doses with breakfast is important. This study compared the effect of a high-fat breakfast on early drug exposure from a morning dose of two extended-release stimulant formulations: the osmotic-controlled OROS® tablet of methylphenidate HCl (Concerta®) and the capsule containing extended-release beads of mixed amphetamine salts (Adderall XR™). The study had a single-dose, open-label, randomised, four-treatment, crossover design in which healthy subjects received either 36 mg Concerta® or 20 mg Adderall XR™ in the morning after an overnight fast or a high-fat breakfast. Serial blood samples were collected over 28 h to determine plasma concentrations of methylphenidate and amphetamine. The food effect on early drug exposure and the pharmacokinetic profiles up to 8 h after dosing of the two extended-release stimulants were directly compared using partial area (AUCp4h, AUCp6h and AUCp8h) fed/fasted ratios. Amphetamine concentrations were markedly lower when the subjects had eaten breakfast, resulting in lower early drug exposures (p < 0.0001). By contrast, methylphenidate concentrations over the same 8 h were unaffected by breakfast, providing consistent levels of early drug exposure. Therefore, as a child's or adult's eating pattern varies, methylphenidate exposure over the first 8 h would be expected to have less day-to-day variation compared with amphetamine exposure. The osmotic-controlled OROS® tablet provides a reliable and consistent delivery of methylphenidate HCl, independent of food, for patients with ADHD.

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 w 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.

Evaluating the separation of amphetamines by electrospray ionization ion mobility spectrometry/MS and charge competition within the ESI process.

The rapid increase in amphetamine abuse for recreational purposes has created a need for fast analysis and detection methodologies. For the first time, we show the separation of six amphetamines by ESI-IMS/MS. A complete analysis can be performed in 70 s, which is faster than traditional chromatographic techniques. In addition, ESI-IMS/MS was found to provide low detection limits for the six compounds (15.4 ppb for ethylamphetamine). Charge competition between amphetamines was found to occur at high amphetamine concentrations. The degree of preferential ionization was dependent on the functional group placed on the amine. Both one-analyte and two-analyte calibration curves were evaluated on the basis of the ion evaporation model. Evaporation rates were determined for the six amphetamines, and the rates were correlated with the degree of selective ionization. Evaluation of three typical ESI solvent compositions showed that the addition of a modifier (acetic acid and formic acid) enhanced the degree of preferential ionization for some amphetamines and increased the effect of charge competition. The solvent studies show the complexity of ESI and provide possible strategies for altering the amount of charge competition between analytes. Overall, ESI-IMS/MS appears to be a promising technique because of its sensitivity and rapid separation times for the amphetamines in aqueous samples; however, further research employing biological samples is required before it can be recommended as a mainstream technique.

Highly sensitive analysis of methamphetamine and amphetamine in human whole blood using headspace solid-phase microextraction and gas chromatography–mass spectrometry

A simple and highly sensitive method for analysis of derivatized methamphetamine (MA) and amphetamine (AM) in whole blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry electron impact ionization selected ion monitoring (GC–MS-EI-SIM). A whole blood sample, deuterated-MA (d 5-MA), as an internal standard (IS), tri- n-propylamine and pentafluorobenzyl bromide were placed in a vial. The vial was heated and stirred at 90°C for 30 min. Then the extraction fiber of the SPME was exposed at 90°C for 30 min in the headspace of the vial while being stirred. The derivatives adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC–MS. The calibration curves showed linearity in the range of 0.5–1000 ng/g for both MA and AM. The time for analysis was about 80 min per sample. In addition, this proposed method was applied to two autopsy cases where MA ingestion was suspected. In one case, MA and AM concentrations in the mixed left and right heart blood were 165 and 36.9 ng/g, respectively. In the other case, MA and AM concentrations were 1.79 and 0.119 μg/g in the left heart blood, and 1.27 and 0.074 μg/g in the right heart blood, respectively.

A curious autopsy case of accidental carbon monoxide poisoning in a motor vehicle

A 26-year-old man was found dead in his car. All doors and windows were locked inside. The ignition key was in the “on” position; but the engine was not running and the fuel tank was empty. His post-mortem lividity was cherry-pink, and marked congestion was observed in the lungs and brain macrocopically. Massive intracardiac blood containing a small amount of cruor was found in the heart. In histological examination of the heart, partial disarrangement or necrosis was found in the myocardium. The liver cells showed derangement and degenerative changes, with focal lymphocyte infiltration in the portal regions, although they were not severe. The chemical tests showed that the blood concentration of carboxyhemoglobin was 46.6%. Stimulants were also detected from his blood and urine; the concentrations of methamphetamine and amphetamine were 3.25 and 0.84 μg/ml, respectively, for his cardiac blood. Therefore, it seemed reasonable to judge that the cause of his death was carbon monoxide poisoning; the cardiomyopathy and the presence of stimulants in blood might facilitate his death. Upon careful investigation of his car, it was disclosed that exhaust gas, leaked from small holes of the exhaust pipe due to rust-through, invaded the interior through four holes on the floor of the car during parking with the engine being on for the purpose of air-conditioning of the interior. It is very common to commit suicide by introducing exhaust gas into an interior of a closed motor car, but the present accidental case of carbon monoxide poisoning in a car seems rare and worthwhile reporting.

Effect of amphetamine-induced dopamine release on radiotracer binding to D1 and D2 receptors in rat brain striatal slices.

The in vivo binding of positron emission tomography (PET) and single photon emission computer tomography (SPECT) radiotracers to dopamine D2 receptors in the striatum can be influenced by competition with endogenous dopamine. The present study was undertaken to determine if a similar inhibition of radiotracer binding to dopamine receptors could be observed following pharmacologically-evoked dopamine release in rat brain striatal slices. Striatal slices were incubated in a large volume of oxygenated Krebs saline and exposed to amphetamine or methamphetamine to evoke dopamine release within the slice. Amphetamine and methamphetamine, at concentrations up to 30 microM, reduced [3H]raclopride binding in the slices by 77% and 86%, respectively, with 50% inhibition at 1.6 microM amphetamine or 3.0 microM methamphetamine. Neither drug produced a significant effect on binding of [3H]SCH 23390 in the slices. This suggests that dopamine was able to interfere with radiotracer binding to D2 but not D1 receptors. The dopamine uptake blockers, cocaine and methylphenidate, had relatively little effect by themselves on [3H]raclopride binding but, by inhibiting amphetamine-induced dopamine release, significantly reduced inhibition of [3H]raclopride binding by a low (3 microM) amphetamine concentration. At a higher (30 microM) amphetamine concentration the inhibition of [3H]raclopride binding was not antagonized by uptake blockers and data obtained from homogenate binding experiments indicated a direct displacement of [3H]raclopride binding by amphetamine at this concentration. In conclusion the data obtained in the present study demonstrate that the effects of amphetamine on striatal radiotracer accumulation observed in PET and SPECT can also be observed in brain slices in vitro and, at least at low amphetamine concentrations, are mediated by competition with released dopamine.