Amphetamine Screening Research Articles

Diagnostic efficiency of different amphetamine screening tests – the search for an optimal cutoff

Increased use of designer drugs (amphetamines and amphetamine-like substances) raises the need for fast screening tests in urine in clinical settings, workplace and drug rehabilitation. Immunological assays currently used are subject to unwanted crossreactivities, partly depending on the cutoff concentrations used. The values recommended in Europe and the USA are 500 and 1000 ng/ml, respectively. In Switzerland, the recommended concentration of 300 ng/ml results in a high rate of false-positive urine samples and expensive, time-consuming confirmation testing. Using the Abbott Axsym analyzer, we found numerous false positives from patients in rehabilitation centers due to concomitant medication. Therefore, the diagnostic sensitivity and specificity of the Abbott test at different cutoff concentrations and the sensitivity of the Roche Cobas Integra, Beckman Synchron and Biosite Triage point-of-care test were examined. HPLC BioRad Remedi was chosen as the method of higher hierarchical order. The specificity of the Axsym analyzer (300 ng/ml) was 86%. At 500 ng/ml or 1000 ng/ml the specificity was increased to 99 or 100%, respectively, while the sensitivity only decreased from 97 to 91 or 81%, respectively. In summary, the cutoff concentration for amphetamine screening tests should not be below 500 ng/ml to avoid a high rate of false-positive results.

Urine Amphetamine Screening, Informed Consent Is Useful Or Useless?

Urine Amphetamine Screening, Informed Consent Is Useful Or Useless?

Liquid-phase microextraction combined with flow-injection tandem mass spectrometry Rapid screening of amphetamines from biological matrices

Liquid-Phase microextraction based on polypropylene hollow fibres was combined with flow-injection tandem mass spectrometry for rapid screening of drugs in biological matrices. Amphetamine and analogues were utilised as model compounds. These drugs were extracted from 0.5 mL samples of whole blood or urine. The samples were made alkaline with 0.5 mL of 1 M NaOH and the urine samples were diluted with 3 mL water to reduce the salt concentration. The uncharged analytes were then extracted through a hollow fibre impregnated with dihexyl ether into 25 μL of 0.01 M HCl inside the hollow fibre. Parallel extraction of 20–30 samples was performed for 15 min. After extraction 20 μL of the extract was injected directly into the flow-injection tandem mass spectrometry system. Atmospheric pressure ionisation operated in positive mode was used as ion spray. All analytes were detected simultaneously after 0.1 min, utilising a combination of selected ion monitoring mass spectrometry and selected reaction monitoring tandem mass spectrometry. Limits of detection (S/N = 5) varied between the compounds and were estimated to be 2–100 ng/mL in urine and 0.4–14 ng/mL in whole blood. Comparison of injection of pure acceptor solution with urine and whole blood extracts demonstrated the efficient sample clean-up by LPME. Ion suppression due to matrix effects was not seen, rendering LPME-FIA-APCI-MS-MS a promising alternative for rapid screening of drugs in biological matrices.

Development of a Redox Mediated Amperometric Detection System for Immunoassay. Application to Urinary Amphetamine Screening

Development of a Redox Mediated Amperometric Detection System for Immunoassay. Application to Urinary Amphetamine Screening

Development of a Redox Mediated Amperometric Detection System for Immunoassay. Application to Urinary Amphetamine Screening

Development of a Redox Mediated Amperometric Detection System for Immunoassay. Application to Urinary Amphetamine Screening

On-site testing of saliva and sweat with Drugwipe and determination of concentrations of drugs of abuse in saliva, plasma and urine of suspected users.

Potential drug users participated voluntarily in a Belgian study on the usefulness of the non-instrumental immunoassay Drugwipe (Securetec, Germany) for the screening of cocaine, opiates, amphetamine and cannabinoids in saliva and sweat. If one of the screening assays (urine, oral fluid, sweat) showed a positive result, blood and saliva were collected. The on-site Drugwipe results were correlated with the Drugwipe results for saliva in the laboratory and with the GC/MS results of the corresponding saliva, plasma and urine samples and pharmacological effects at the time of sampling. The Drugwipe assay proved to be sufficiently sensitive for the detection of recent cocaine (n = 6) and amphetamine (n = 15) abuse, whether the device was wiped on the tongue or on the surface of the body, or when a saliva sample was applied to the wiping part. In five of the six potential cocaine users, the saliva concentrations of cocaine exceeded 1,000 ng/ml. In the amphetamine group, the saliva concentrations of amphetamine, MDMA or both were high (> 1,000 ng/ml) in 13 subjects. For cocaine and amphetamine, the positive scores for Drugwipe matched the GC/MS results for the three body fluids. Recent heroin abuse (n = 5) could be demonstrated to some extent with Drugwipe on samples from the tongue but only the two subjects with the highest saliva concentrations of MAM (> 500 ng/ml) and morphine (> 500 ng/ml) were positive. If the legal cut-off value for driving under the influence of opiates in Belgium (20 ng/ml of free morphine in plasma) was taken into account, only three subjects would have been legally positive. For cannabinoids (n = 15), false negatives and even some false positives were observed. Saliva can be considered as a useful analytical matrix for the detection of drugs of abuse after recent abuse when analysed with GC/MS.

Development and evaluation of an improved method for screening of amphetamines.

We developed a homogeneous immunoassay method to eliminate false-positive amphetamine results caused by cross-reactive substances, including over-the-counter allergy and cold medications. This method uses a neutralizing antibody that binds to amphetamines but does not bind to the labeled amphetamine conjugate used in the assay. The amount of neutralizing antibody is sufficient to reduce the assay signal resulting from authentic amphetamine and methamphetamine, but not the signal resulting from cross-reactants. This concept was implemented using the CEDIA DAU Amphetamines assay on Hitachi 747 and 717 clinical chemistry analyzers. Urine samples were tested using the standard, unmodified reagents in one channel and reagents containing the neutralizing antibody in a second channel. The difference in rate between the two tests was calculated by the analyzer; true-positive samples showed a significantly greater decrease in assay signal in response to neutralizing antibody as compared with false-positive samples. The neutralization method was evaluated in two studies using 448 samples that tested positive in the initial CEDIA DAU Amphetamines screening test. The samples were separated into categories of 154 true-positive samples and 294 false-positive samples based upon a secondary screen with the Abbott FPIA Amphetamines assay followed by gas chromatography-mass spectrometry (GC-MS) testing using the HHS (SAMHSA) cutoff criteria. The CEDIA neutralization test successfully identified all 154 of the GC-MS confirmed positive samples. The test successfully identified as false positive 251 out of the 294 (85.4%) samples that failed to confirm by GC-MS.

Stereoselective screening for and confirmation of urinary enantiomers of amphetamine, methamphetamine, designer drugs, methadone and selected metabolites by capillary electrophoresis

Data presented in this paper demonstrate that a competitive binding, electrokinetic capillary-based immunoassay previously used for screening of urinary amphetamine and analogs cannot be employed to distinguish between the enantiomers of amphetamine and methamphetamine. However, capillary zone electrophoresis with a pH 2.5 buffer containing (2-hydroxypropyl)-beta-cyclodextrin as chiral selector is shown to permit the enantioselective analysis of urinary extracts containing methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (Ecstasy) and other designer drugs, and methadone together with its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. In that approach, enantiomer identification is based upon comparison of extracted polychrome UV absorption data and electropherograms obtained by rerunning of spiked extracts with spectra and electropherograms monitored after extraction of fortified blank urine. The suitability of the described chiral electrokinetic capillary method for drug screening and confirmation is demonstrated via analysis of unhydrolyzed quality control urines containing a variety of drugs of abuse. Furthermore, in a urine of a patient under selegiline pharmacotherapy, the presence of the R-(-)-enantiomers of methamphetamine and amphetamine could be unambiguously identified. Direct intake of an R-enantiomer or ingestion of drugs that metabolize to the R-enantiomers can be distinguished from the intake of S-(+)-enantiomers (drug abuse) or prescribed drugs that metabolize to the S-enantiomers of methamphetamine and amphetamine. The described approach is simple, reproducible, inexpensive and reliable (free of interferences of other major basic drugs that are frequently found in toxicological urines) and could thus be used for screening for and confirmation of urinary enantiomers in a routine laboratory.

Direct semiquantitative screening of drugs of abuse in serum and whole blood by means of CEDIA DAU urine immunoassays.

The purpose of this study was to test the direct applicability of CEDIA DAU urine immunoassays to serum or whole blood. The performance of the urine assays for sensitive screening of amphetamines (AMP), benzoylecgonine (BZE), benzodiazepines (BENZ), methadone (MET), opiates (OPI), and tetrahydrocannabinol carboxylic acid (THCCOOH) was evaluated on the BM/Hitachi 911 analyzer with unpretreated serum and whole blood. The limit of detection was 0 ng/mL for all tests. Cutoff values were set from 10 to 40 ng/mL for the different assays. The assays were found to be linear between the following concentrations: AMP 0-2500 ng/mL, BZE 0-1200 ng/mL, BENZ 0-1600 ng/mL, MET 0-600 ng/mL, OPI 0-720 ng/mL, and THCCOOH 24-60 ng/mL. Precision results (within run) for different concentrations were as follows: AMP 3.1-5.7%, BZE 2.4-6.6%, BENZ 4.3-8.0%, MET 2.0-5.5%, OPI 2.8-7.6%, and THCCOOH 1.4-2.4%. Between-run results were as follows: AMP 8.7-15.5%, BZE 6.4-7.5%, BENZ 8.2-15.8%, MET 2.7-5.1%, OPI 4.3-11.2%, and THCCOOH 2.6-7.4%. Sensitivity, specificity, and comparison of CEDIA semiquantitation with GC-MS quantitative results were performed on 500 original serum and whole blood samples. The data provided sufficient documentation to use the CEDIA urine-screening technique without any adaptation as a sensitive serum/whole blood screening for BZE, BENZ, MET, OPI, and THCCOOH. Serum screening for amphetamines is not sensitive enough in the unchanged urine mode. It will require some adaptation to a serum mode (probably a higher sample volume [BM/Hitachi 911] combined with protein precipitation of the sample).

Automated trace enrichment for screening and/or determination of primary, secondary and tertiary amphetamines in biological samples by liquid chromatography

A rapid and simple liquid chromatographic method for the automated determination of amphetamines in biological fluids was developed. The proposed procedure is based on the injection of 250 microL of sample into a 20 x 2.1 mm id precolumn (packed with a 30 microns Hypersil C18 stationary phase) for enrichment and purification of the analytes. Next, the analytes are transferred to a 5 microns LiChrospher 100 RP18, 125 x 4 mm id analytical column for their separation under reversed-phase conditions. Water was used to eliminate the matrix components from the precolumn and a 0.2 M phosphate buffer (pH 3) containing 2% triethylamine was the mobile phase for the resolution of the amphetamines. The UV detector was set at 210 nm. The method was applied to the determination of different primary, secondary and tertiary amphetamines in plasma and urine: beta-phenylethylamine, norephedrine, ephedrine, N-methylpseudoephedrine, pseudoephedrine, N-methylephedrine, amphetamine, 3-phenylpropylamine and methamphetamine. The method provides satisfactory linearity and reproducibility within the tested concentration range (1.0-10.0 micrograms mL-1) and limits of detection of 50-500 ng/mL-1.

Screening for urinary amphetamine and analogs by capillary electrophoretic immunoassays and confirmation by capillary electrophoresis with on-column multiwavelength absorbance detection.

This paper characterizes competitive binding, electrokinetic capillary-based immunoassays for screening of urinary amphetamine (A) and analogs using reagents which were commercialized for a fluorescence polarization immunoassay (FPIA). After incubation of 25 microL urine with the reactants, a small aliquot of the mixture is applied onto a fused-silica capillary and unbound fluorescein-labeled tracer compounds are monitored by capillary electrophoresis with on-column laser-induced fluorescence detection. Configurations in presence and absence of micelles were investigated and found to be capable of recognizing urinary D-(+)-amphetamine at concentrations > about 80 ng/mL. Similar responses were obtained for racemic methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA). The electrokinetic immunoassay data suggest that the FPIA reagent kit includes two immunoassay systems (two antibodies and two tracer molecules), one that recognizes MA and MDMA, and one that is geared towards monitoring of A. For confirmation analysis of urinary amphetamines and ephedrines, capillary electrophoresis in a pH 9.2 buffer and multiwavelength UV detection was employed. The suitability of the electrokinetic methods for screening and confirmation is demonstrated via analysis of patient and external quality control urines.

Determination of morphine analogues, caffeine and amphetamine in biological fluids by capillary electrophoresis with the marker technique

A reliable method was sought for the fast screening and simultaneous determination of amphetamine, morphine, heroin (acetomorphine), codeine (methylmorphine) and caffeine in biological fluids and drugs seizures. Capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC), with detection at 200 and 220 nm, were investigated for analytes in human serum and urine. When adequae separation was not achieved in preliminary studies with CZE, further development was focused on the MEKC method. Glycine buffer containing sodium lauryl sulfate (pH 10.5) was usedd for the MEKC separations. The analytes and carboxylic acids used as marker compounds could be screened by a short-capillary method in lessthan 2 min. In the simultaneous determination of the drugs in urine and serum and longer separation of 18 min was preferred so that all the compounds, the markers and the endogenous compounds absorbing at the detection wavelenght could be adequately separated in a single run The migration times of the compounds increased in the order caffeine, morphine, heroin, codeine and amphetamine. The repeatability of the separation was tested by using two carboxylic acids as marker compounds in the determination of the migration indices of the analytes. The relative standard deviations for the migration indices were less than 1%, which is accurate enough for the determination of the drugs in biological fluids.

Screening and determination of methamphetamine and amphetamine in the blood, urine and stomach contents in emergency medical care and autopsy cases

Methamphetamine (MA) and amphetamine (AMP) were screened and their levels were determined using the Toxi-Lab ® thin-layer chromatography system and gas chromatography-mass spectrometry, respectively, in the blood, urine and stomach contents from 211 emergency medical care and 417 autopsy cases. MA and AMP were detected in 5 emergency medical cases, and the blood MA and AMP concentrations ranged from 0.697-0.041 μmol/100 g and from 0.0944-0.0003 μmol/100 g, respectively. MA and AMP were detected in 19 autopsy cases, in which blood MA and AMP concentration ranged from 14.3-0.123 μmol/100 g and from 0.256-0.0017 μmol/100 g, respectively. The autopsy cases included 5 cases of sudden death with blood MA concentration of less than 3 μmol/100 g. MA and AMP screening and determination in emergency medical care and autopsy cases provide useful information and are indispenable in clarifying the dimensions of MA abuse in Japan.

Polarization fluoroimmunoassays for the specific determination of amphetamine and methamphetamine in urine with the abbott TD x-analyzer

Two polarization fluoroimmunoassays for the screening of amphetamine and methamphetamine in urine were adapted for use with the Abbott TD x-Analyzer running in the automatic mode. The detection limits of amphetamine and methamphetamine in 10 μl of urine were 0.03 and 0.05 mg l −1, respectively. These assays were specific for the determination of amphetamine (cross-reactivity with methamphetamine 1%) or for the determination of methamphetamine (cross-reactivity with amphetamine 2.5%). No significant cross-reaction was observed with other drugs of abuse (ephedrine, morphine and butabarbital).

Screening of amphetamines by gradient microbore liquid chromatography and pre-column technology

Amphetamine-type drugs with a wide polarity range have been screened in both human and horse urine using on-line pre-concentration on pre-columns packed with hydrophobic and cation-exchange sorbents in series and gradient microbore high-performance liquid chromatography. The underivatized amphetamines were identified by UV detection at 210 nm. The method has potential for the automated liquid chromatographic screening of amphetamines in urine, e.g., for doping control.

Rapid screening and confirmation of amphetamine, methamphetamine, methadone, and methadone metabolite in urine by gas/thin layer chromatography.

A method suitable for large scale screening and confirmation of urine speciments for amphetamine, methamphetamine, methadone, and its primary metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenlypyrrolidine) is described. The drugs are extracted from alkaline urine into an organic solvent. The amphetamine drugs are then back-extracted into a small volume of acid and identified by gas chromatography both as free bases on a 10% Apiezon L-10% KOH column and as their trifluoracetamide derivatives on a 3% OV-17 column. The organic layer, which still contains methadone and its primary metabolite, is analyzed by split-sample thin-layer chromatography using two solvent systems: ethyl acetate: methylene cloride: concentrated ammonium hydroxide (90:10:0.7) and methanol: chloroform: concentrated ammonium hydroxide (74:25:0.8). These solvent systems separate methadone from its primary metabolite without interference from other drugs or urinary substances.

Rapid mass screening and confirmation of urinary amphetamine and methamphetamine by gas chromatography.

A rapid and simple procedure is described for the mass screening and confirmation of amphetamine and methamphetamine in urine using gas chromatography with flame ionization detector. The amphetamine drugs are screened as free drugs on a 10% Apiezon L-10% KOH column and confirmed as their trifluoroacetamide derivatives on a 3% OV-17 column. This dual approach eliminates false positives and interfering substances that may be present in the urine and is, therefore, specific for amphetamine and methamphetamine.