Raikos N.(l>. Christopoulou K.<2>, Theodoridis G.(2), Tsoukali H.( , ), Psaroulis D.<»
(1) Laboratory of Forensic Medicine & Toxicology, Faculty of Medicine, Aristotle University, 540 06 Thessaloniki, Greece
(2) Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University, 540 06 Thessaloniki, Greece
Solid phase microextraction (SPME) is under investigation for its usefulness in the determination of a widening variety of volatiles and semivolatiles analytes in biological fluids and materials. Semivolatiles are increasingly under study as analytical targets, and difficulties with small partition coefficients and long equilibration times have been identified. Amphetamines were selected as semivolatiles exhibiting these limitations and methods to optimize their determination were investigated. A polydimethylsiloxane (PDMS) -coated SPME fiber 100pm was used for the extraction of the amphetamines from human urine.
Amphetamines determination was made by using gas chromatography (GC) with flame-ionization detec-tion. Temperature, time and salt saturation were optimized to obtain consistent extracdetec-tion.
A simple procedure for the analysis of amphetamine and methamphetamine in urine was developed and another for MDA, MDMA and MDEA using HS-SPME and GC/FID. Higher recoveries were obtained for amphetamine (19.5-47 %) and methamphetamine (20-38.1 %) than MDA (5.1-6.6 %), MDMA (7-9.6 %) and MDEA (5.4-9.6 %).
The developed methods were applied to the analysis of human urine to detect amphetamines.
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Evaluation of OnTrak TesTcard 9 panel drug-testing device for rapid immunoassay scree-ning of nine drugs of abuse in urine
Tsai J.S.C.. Deng D.Z., Terrett L., Warnecke N,, Henckel D., Demirtzoglou D., Adams L, Huang J., Kobetic R., Gatin M., Landis D.
Roche Diagnostics Corporation, 9115 Hague Road, Indianapolis, IN 46250, USA
The OnTrak TesTcard 9 device is an onsite drug-testing tool for the qualitative detection of nine abused drugs or drug metabolites in urine. In this study we evaluated three lots of OnTrak TesTcard 9 devices and assessed the precision and accuracy performance of the following nine drug assays: amphetamines, barbi-turates, benzodiazepines, cocaine, methamphetamine, morphine, phencyclidine, TCA (tricyclic antide-pressants), and THC. The cutoff concentrations for the NIDA-5 assays are the same as those mandated by the current SAMHSA guidelines. The respective target analytes and cutoff concentrations for the other 4 assays (barbiturates, benzodiazepines, methamphetamine, and TCA) are as follows: secobarbital (200 ng/rnL), oxazepam (100 ng/mL), d-methamphetamine (500 ng/mL), and imipramine (1000 ng/mL).
The precision performance of OnTrak TesTcard 9 was determined by testing three different lots of devices using multi-analyte urine standards containing drugs or drug metabolites at various concentrations. Each lot was tested over 5 days at six drug levels using 105 replicates per level each day. Test results were inter-preted by three analysts who were "blind" as to the origin of the samples. All three lots showed precision performance in the range of 98 %-10Q % when tested at drug levels of 0 %, 25 %, and 150 % of their res-pective cutoff concentrations for all nine assays. For near-cutoff performance, precision data at 50 % and 75 % of the cutoff concentrations showed lot and assay-dependent variations whereas precision results at 125% of the cutoff concentrations were in the range of 96 % to 100 % for all assays.
The accuracy performance of OnTrak TesTcard 9 was evaluated using 730 clinical specimens that have been pre-screened for the nine drug assays in clinical drug-testing laboratories. All clinical urine speci-mens were screened by automated immunoassays; 100 % of the screened-positive samples and 15 % of the screened-negative specimens were also analyzed by GC/MS. All three lots of OnTrak TesTcard 9 sho-wed good accuracy for differentiating negative specimens and positive specimens that contained drugs at 25 % or higher above their assay cutoff levels as determined by GC/MS analysis.
The OnTrak TesTcard 9 panel drug-testing device has a built-in reservoir that allows easy sample appli-cation such as instantaneous sample dipping or simple pipetting. The device provides rapid, qualitative, immunoassay screenings that differentiate negative from positive samples and, subsequently, the positive screening results should be confirmed by quantitative methods such as LC/MS or GC/MS.
Assay range [ng/mL] 0 - 2000 0 - 1000 d-Methamphetamine [ng/mL] 300 500 1000 300 500 1000
Accuracy (%) 106 105 109 104 114 107
Repeatability (%) 23.9 2.9 1.6 3.5 5.0 3.0
Intermediate precision (%) 31.2 4.4 4.4 5.4 10.1 2.1 Sensitivity and specificity compared with GC/MS, for different cutoff levels were :
Standard protocol Low cutoff protocol
Cutoff [ng/mL] 300 500 1000 300 500 1000
Sensitivity (%) 90 90 71 90 76 71
Specificity (%) 55 85 96 77 95 96
The same performance (sensitivity and specificity) can be achieved with a lower cutoff (500 ng/mL) by this way of method optimisation.
EMIT II Plus amphetamine immunoassay method optimization and validation with res-pect to a low cutoff
Van Hoof F.W.J.M.. Langen M.C.J., Olíjslager E.J.H., Rommers M.K., Egberts A.C.G
Dutch Laboratory for Drugs and Doping, Hospital Pharmacy Midden-Brabant, TweeSteden Hospital, 5000 LA Tilburg, The Netherlands
In our laboratory we perform DOA screening for prisons, detoxification centers, psychiatric hospitals and clinical toxicology. The immunoassay screening is performed using EMIT II Plus. The amphetamine assay has a cutoff of 1000 ng/mL. EU guidelines state that in workplace testing a cutoff of 300 ng/mL should be used. The immunoassay has a limited capability to detect ecstasy-analogues like MDMA, MDEA and MDA.
To optimize the Emit II plus amphetamine immunoassay and increase the performance by lowering the cutoff.
EMIT II Plus analysis is based on competition for amphetamine antibody binding sites. Amphetamines in the sample compete with amphetamine in the Antibody Reagent (AR) that is labelled with rG6PHDH.
Active (unbound) rGóPDH enzyme converts the oxidized NAD in the Enzyme Reagent (ER) to NADH, resulting in a kinetic absorbance change that can be measured spectrophotometrically. The standard pro-tocol of Dade Behring was compared to a propro-tocol which uses a double sample volume :
Standard protocol : Sample volume 7 uL; AR 125 uL; ER 55 uL; calibrator d-methamphetamine:
Low cutoff protocol : Sample volume 14 uL; AR 125 pL; ER 55 uL; calibrator d-methamphetamine.
We analysed 95 urine samples from our routine DOA tests which were above the limit of detection of the immunoassay. All samples were analysed with GC/MS. In case GC/MS analysis showed an amphetamine above the limit of detection we considered the sample as positive.
Both protocols were validated according to regulatory guidelines :
Standard protocol Low cutoff protocol
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