Molecular analysis of Francisella tularensis subspecies tularensis and holarctica.

Rapid methods are needed for public health and military applications to specifically identify Francisella tularensis, the causative agent of tularemia in humans. A comparative analysis of the capabilities of multiple technologies was performed using a well-defined set of organisms to determine which approach would provide the most information in the shortest time. High-resolution molecular techniques, including pulsed-field gel electrophoresis, amplified fragment length polymorphism, and ribotyping, provided subspecies level identification within approximately 24 hours after obtaining an isolate, whereas multilocus variable number tandem repeat analysis with 8 or 25 targets provided strain level discrimination within about 12 hours. In contrast, Raman spectroscopy provided species level identification in 10 minutes but could not differentiate between subspecies tularensis and holarctica.

Blood, brain, and hair GHB concentrations following fatal ingestion.

Despite the increasing incidence of illicit use of gamma-hydroxybutyrate (GHB), little information is available documenting levels of the drug in GHB fatalities. We measured GHB levels in postmortem blood, brain and hair specimens from a suspected overdose case by gas chromatography/mass spectrometry (GC/MS) following solid phase extraction (SPE) and derivatization with bis(trimethyl-silyl) trifluoroacetamide (BSTFA). Examination found 330 microg/mL GHB in femoral blood and 221 ng/mg GHB in frontal cortex brain tissue, values higher than those typically reported in the literature. The hair shaft was negative for GHB whereas the plucked root bulbs with outer root sheath attached (2,221 ng/mg) and root bulbs after washing and removal of the outer root sheath (47 ng/mg) contained the drug. Our results are consistent with an acute single dose of GHB and, as the toxicology screen was negative for other drugs of abuse, emphasize the significant danger of this drug.

Striatal dopaminergic and serotonergic markers in human heroin users.

To establish whether chronic opiate exposure might impair brain dopaminergic or serotonergic function in humans, we assessed biochemical indices of monoaminergic neurotransmitter activity and integrity in post mortem striatum of nine chronic heroin users and 14 control subjects. Striatal levels of the vesicular monoamine transporter were normal, suggesting that the density of dopamine nerve terminals is not reduced in heroin users. In nucleus accumbens, levels of tyrosine hydroxylase protein (-25%) and those of the dopamine metabolite homovanillic acid (-33%) were reduced significantly together with a trend for decreased dopamine (-32%) concentration. These changes could reflect either a compensatory downregulation of dopamine biosynthesis in response to prolonged dopaminergic stimulation caused by heroin, or reduced axoplasmic transport of tyrosine hydroxylase. Striatal levels of serotonin were either normal or elevated whereas concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were decreased by 27-38%. Our data suggest that chronic heroin exposure might produce a modest reduction in dopaminergic and serotonergic activity that could affect motivational state and impulse control, respectively.

Regional distribution of methamphetamine in autopsied brain of chronic human methamphetamine users.

We measured levels of methamphetamine and those of its metabolite amphetamine in 15 autopsied brain regions of 14 human methamphetamine users. Only slight regional differences were observed in drug concentrations among the brain areas. Although, some redistribution of the drugs probably occurred postmortem, these data suggest that methamphetamine might not be preferentially retained in dopamine-rich brain areas but is heterogenously distributed in brain of chronic human users of the drug. The possible pharmacological actions of methamphetamine in both dopamine-rich and poor brain areas of chronic drug users need to be considered.

Dopamine D1 receptor protein is elevated in nucleus accumbens of human, chronic methamphetamine users.

Animal data have long suggested that an adaptive upregulation of nucleus accumbens dopamine D1 receptor function might underlie part of the dependency on drugs of abuse. We measured by quantitative immunoblotting protein levels of dopamine D1 and, for comparison, D2 receptors in brain of chronic users of methamphetamine, cocaine, and heroin. As compared with the controls, brain dopamine D1 receptor concentrations were selectively increased (by 44%) in the nucleus accumbens of the methamphetamine users, whereas a trend was observed in this brain area for reduced protein levels of the dopamine D2 receptor in all three drug groups (-25 to -37%; P < 0.05 for heroin group only). Our data support the hypothesis that aspects of the drug-dependent state in human methamphetamine users might be related to increased dopamine D1 receptor function in limbic brain.

Assessment of the ion-trap mass spectrometer for routine qualitative and quantitative analysis of drugs of abuse extracted from urine.

The ion-trap mass spectrometer (MS) has been available as a detector for gas chromatography (GC) for nearly two decades. However, it still occupies a minor role in forensic toxicology drug-testing laboratories. Quadrupole MS instruments make up the majority of GC detectors used in drug confirmation. This work addresses the use of these two MS detectors, comparing the ion ratio precision and quantitative accuracy for the analysis of different classes of abused drugs extracted from urine. Urine specimens were prepared at five concentrations each for amphetamine (AMP), methamphetamine (METH), benzoylecgonine (BZE), delta9-carboxy-tetrahydrocannabinol (delta9-THCCOOH), phencyclidine (PCP), morphine (MOR), codeine (COD), and 6-acetylmorphine (6-AM). Concentration ranges for AMP, METH, BZE, delta9-THCCOOH, PCP, MOR, COD, and 6-AM were 50-2500, 50-5000, 15-800, 1.5-65, 1-250, 500-32000, 250-21000, and 1.5-118 ng/mL, respectively. Sample extracts were injected into a GC-quadrupole MS operating in selected ion monitoring (SIM) mode and a GC-ion-trap MS operating in either selected ion storage (SIS) or full scan (FS) mode. Precision was assessed by the evaluation of five ion ratios for n = 15 injections at each concentration using a single-point calibration. Precision measurements for SIM ion ratios provided coefficients of variation (CV) between 2.6 and 9.8% for all drugs. By comparison, the SIS and FS data yielded CV ranges of 4.0-12.8% and 4.0-11.2%, respectively. The total ion ratio failure rates were 0.2% (SIM), 0.7% (SIS), and 1.2% (FS) for the eight drugs analyzed. Overall, the SIS mode produced stable, comparable mean ratios over the concentration ranges examined, but had greater variance within batch runs. Examination of postmortem and quality-control samples produced forensically accurate quantitation by SIS when compared to SIM. Furthermore, sensitivity of FS was equivalent to SIM for all compounds examined except for 6-AM.

Regional distribution of cocaine in postmortem brain of chronic human cocaine users.

We measured concentrations of cocaine and its major metabolites (benzoylecgonine, ecgonine methylester, norcocaine, and cocaethylene) in 15 autopsied brain regions of 14 human chronic cocaine users. Only slight differences were observed in concentrations of cocaine and its metabolites amongst the examined brain areas. Although it is likely that some postmortem redistribution of the drug must have occurred, our data are consistent with the possibility that behaviorally relevant doses of cocaine are widely distributed throughout the brain of humans who use the drug on a chronic basis. Consideration should therefore be given to the possible pharmacological and toxicological actions of cocaine in both striatal and extra-striatal brain areas in human users of the drug.

Striatal serotonin is depleted in brain of a human MDMA (Ecstasy) user.

The authors found that striatal levels of serotonin and those of its metabolite 5-hydroxyindoleacetic acid were severely depleted by 50 to 80% in brain of a chronic user of methylenedioxymethamphetamine (MDMA) whereas concentrations of dopamine were within the normal control range. Our data suggest that MDMA exposure in the human can cause decreased tissue stores of serotonin and therefore some of the behavioral effects of this drug of abuse could be caused by massive release and depletion of brain serotonin.

The human nucleus accumbens is highly susceptible to G protein down-regulation by methamphetamine and heroin.

Although the nucleus accumbens is assumed to be a critical brain "pleasure center," its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down-regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of G(alpha)1 and/or G(alpha)2 were reduced by 32-49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug-dependent state (e.g., tolerance) might be related to inhibition of G(alpha)1-linked receptor activity.

Detection of lysergic acid diethylamide (LSD) in urine by gas chromatography-ion trap tandem mass spectrometry.

A confirmatory method for the detection and quantitation of lysergic acid diethylamide (LSD) is presented. The method employs gas chromatography-tandem mass spectrometry (GC-MS-MS) using an internal ionization ion trap detector for sensitive MS-MS-in-time measurements of LSD extracted from urine. Following a single-step solid-phase extraction of 5 mL of urine, underivatized LSD can be measured with limits of quantitation and detection of 80 and 20 pg/mL, respectively. Temperature-programmed on-column injections of urine extracts were linear over the concentration range 20-2000 pg/mL (r2 = 0.999). Intraday and interday coefficients of variation were < 6% and < 13%, respectively. This procedure has been applied to quality-control specimens and LSD-positive samples in this laboratory. Comparisons with alternate GC-MS methods and extraction procedures are discussed.

Brain choline acetyltransferase activity in chronic, human users of cocaine, methamphetamine, and heroin.

Cognitive impairment has been reported in some chronic users of psychostimulants, raising the possibility that long-term drug exposure might damage brain neuronal systems, including the cholinergic system, which are responsible for normal cognition. We measured the activity of choline acetyltransferase (ChAT), the marker enzyme for cholinergic neurones, in autopsied brain of chronic users of cocaine, methamphetamine, and, for comparison, heroin. As compared with the controls, mean ChAT levels were normal in all cortical and subcortical brain areas examined. However, the two of 12 methamphetamine users, who had the highest brain/blood drug levels at autopsy, had a severe (up to 94%) depletion of ChAT activity in cerebral cortex, striatum, and thalamus. Based on the subjects examined in the present study, our neurochemical data suggest that brain cholinergic neurone damage is unlikely to be a typical feature of chronic use of cocaine, methamphetamine, or heroin, but that exposure to very high doses of methamphetamine could impair, at least acutely, cognitive function requiring a normal nucleus basalis cholinergic neuronal system. Reduced brain ChAT might be explained in part by a hyperthermia-related mechanism as low ChAT levels have also been observed in brain of some patients with neuroleptic drug-associated hyperthermia. Studies of cognitive and brain cholinergic status in high dose users of MA are warranted.

Drug distribution in human hair by infrared microscopy.

Localization of drug metabolites within human hair is important in determining the pharmacokinetics of drug incorporation in hair. This information is critical to validate drug testing data from hair. Microspectroscopic probing of small areas within longitudinally microtomed hair sections provides a profile of the deposition of drug along a growth line and thus indicates localization as a function of time. Probing across individual hairs may reveal the hydrophobic/hydrophilic characteristics of the substance. Hydrophobic drugs tend to bind to the central core or medulla of the hair while hydrophilic drugs tend to be spread throughout the cortex of the hair and appear, generally, in lower concentrations per dose. Profiles of distribution with high spatial resolutions of the regions of the hair are necessary for these determinations. This information is available to a certain extent in normal infrared microscopy and enhanced in synchrotron powered infrared microscopy.

Striatal dopamine nerve terminal markers in human, chronic methamphetamine users.

Methamphetamine is a drug that is significantly abused worldwide, Although long-lasting depletion of dopamine and other dopamine nerve terminal markers has been reported in striatum of nonhuman primates receiving very high doses of the psychostimulant, no information is available for humans. We found reduced levels of three dopamine nerve terminal markers (dopamine, tyrosine hydroxylase and the dopamine transporter) in post-mortem striatum (nucleus accumbens, caudate, putamen) of chronic methamphetamine users. However, levels of DOPA decarboxylase and the vesicular monoamine transporter, known to be reduced in Parkinson's disease, were normal. This suggests that chronic exposure to methamphetamine does not cause permanent degeneration of striatal dopamine nerve terminals at the doses used by the young subjects in our study. However, the dopamine reduction might explain some of the dysphoric effects of the drug, whereas the decreased dopamine transporter could provide the basis for dose escalation occurring in some methamphetamine users.

Forensic application of an automated drug-profiling system.

The rapid emergency drug identification (REMEDi) system is an automated drug-profiling system that employs high-performance liquid chromatography with a multicolumn design. It has previously proven successful in emergency toxicology situations and in the clinical analysis of urine and serum. Its capabilities include the broad spectrum identification of more than 500 basic, neutral, and slightly acidic drugs and metabolites. Forensic applications, including analysis of whole blood and tissue, were investigated, and comparisons with more traditional laboratory methods are reported. The whole blood and tissue samples require offline sample extraction prior to system analysis. Approximately 50 drugs were used as standards to test the preparation method, analytical system, and limit of detection. More than 50 cases from the medical examiner's office were analyzed with the combination extraction and automated drug-profiling system; these cases were compared with previously reported findings. Results showed that the REMEDi system is a useful complimentary tool for screening forensic cases; the current range of detectable drugs was expanded by using the system.

Study of drug distribution in hair by infrared microscopy visualization.

Infrared microscopy has been shown to be a unique method of analysis for drugs-of-abuse determinations in hair, with the ability to analyze only the central core, or medulla region, of sectioned hair. By spectrally mapping infrared functional groups related to the various drugs, a three-dimensional image of the drug location can be obtained from cross-sectional and laterally microtomed hairs. This technique eliminates the question of externally contaminated hair by analyzing only that portion of the hair that is formed from within the root where ingested material would be transported. Results from these tests have shown a direct correlation between drug and medulla presence in the hair. There are occasional occurrences of drug in nonmedullated hair, but the drug distribution across the hair is entirely different in these cases. Spiked or doped hair has been shown to be the exact opposite of drug-ingested hair; the drugs spike into the hair better in nonmedullated portions. From these mapped images, two routes of drug incorporation into the hair can be postulated. The more prominent would be through the root, where the hair shaft is formed, with the drug binding to the medulla material. A second less prevalent route would be from the exterior through the cuticle via perspiration, as in spiking. The spectral data have also shown that different drugs bind differently to the various hair materials.

Hair analysis by infrared microscopy for drugs of abuse.

Hair analysis overcomes many of the drug testing problems associated with urinalysis. However, hair analysis has its own unique problem in that passive contamination of the exterior surface of the hair can taint the analysis with false positive. Infrared microscopy can examine the interior of the hair and differentiate passive contamination from drugs absorbed into the hair from ingestion. By microtoming the hair either cross-sectionally or laterally, infrared spectra can be obtained of the cortex and medulla of a single hair with a nominal spatial resolution of 10 microns. Three dimensional infrared imaging is possible with Fourier transform infrared (FT-IR) microscopy and yields a plot that can be understood by the layman.

Detection of amphetamine and methamphetamine in urine by gas chromatography/Fourier transform infrared (GC/FTIR) spectroscopy.

New methods for identification of amphetamines have been employed using gas chromatography/Fourier transform infrared spectroscopy (GC/FTIR). These methods have provided identification of the drug and its metabolites, with detection at the low picogram levels or less than 10 ng/mL. Developments in cryogenic sample deposition for GC/FTIR spectroscopy have increased the sensitivity of GC/FTIR to levels that match or surpass that of gas chromatography/mass spectrometry (GC/MS). This advancement in technology has allowed the highly selective ability of infrared spectroscopy to be used for identification and quantitation in studies where the analytes are in low concentrations. The limits of detection (LOD), quantitation (LOQ), and linearity (LOL), and precision have been determined in this study, and these instrumental parameters have been compared with those of established techniques.

Feasibility of using GC/FT-IR for drug analysis in the forensic toxicology laboratory.

New developments in cryogenic sample deposition for gas chromatography/Fourier transform infrared (GC/FT-IR) spectroscopy have increased the sensitivity of the technique 100-1000-fold, to match or surpass that of gas chromatography/mass spectrometry (GC/MS). The current methods of GC/MS have led to some false positive identifications in drug testing labs. New methods employing GC/FT-IR will provide absolute identification through infrared fingerprinting with routine detection in the ppb range. GC/FT-IR methods are being developed which indicate low picogram amounts of material are detectable. Some preliminary data have shown that reference-quality spectra can be obtained from samples containing 200 ng/mL of amphetamines, and spectra from samples below the 25 ng/mL level for amphetamines can be obtained for identification purposes. These and other applications will be addressed along with limit of detection (LOD), limit of quantitation (LOQ), and linearity in comparison to commonly used techniques.

Measurement of azacyclonol in urine and serum of humans following terfenadine (Seldane) administration using gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method is presented for the analysis of azacyclonol (AZA), a metabolite of terfenadine in serum and urine specimens. Following an alkaline extraction, AZA and an internal standard were derivatized using heptafluorobutyric anhydride. Fourier transform infrared spectrometry suggested that two sites on the AZA molecule were derivatized. GC-MS of the extracts had a limit of quantitation (LOQ) of 1 ng/ml and linear range of 1-1000 ng/ml in urine. Four volunteers were administered a therapeutic regimen of terfenadine followed by urine and serum specimen collection(s) during the next seven days. The results indicated that following a 60-mg dose of terfenadine each 12 h for five days, (1) AZA appears in urine within 2 h, (2) urine AZA concentrations were above the LOQ 72 h following the last dose, (3) peak urine concentrations were as high as 19,000 ng/ml, and (4) mean serum concentration following the ninth dose was 59 ng/ml.