SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

A50
V ALIDA nON OF AN EIA-BASED SCREENING ASSAY FOR THE DETECTION OF
AMPHETAMINE AND MDMAIMDA IN BLOOD AND ORAL FLUID
Marleen Laloup·' I, Gaelle Tilman l ,2, Gert De Boeck!, Pierre Wallemacq2, Viviane Maes 3 and Nele Samyn l
INational Institute of Crimina lis tics and Criminology (NICC), Section Toxicology, Brussels, Belgium;
2Labo Clinical Chemistry and Toxicology, St. Luc University, Hospital-UCL, Brussels, Belgium
3Department of Clinical Chemistry-Toxicology, Academic Hospital, Free University ofBrussels, Belgium.
The number of seizures and the use of amphetamine and 'ecstasy' (MDMA) have increased exponentially
in Belgium since the late nineties. Therefore, screening for these substances in biological specimens has
become an important part of routine analysis in forensic toxicology laboratories. The use of a reliable
sensitive immunological assay for the screening of blood and oral fluid samples is less widespread than for
the preliminary analysis of urine. Such an assay would save cost and labor time in comparison to a more
specific analysis as gas chromatography-mass spectrometry (GC-MS).
The objective of this study was to evaluate the suitability of the Cozart® AMP enzyme-linked
immunoassays (EIA) for the screening of blood and plasma samples, collected with sodium fluoride and
potassium oxalate as anticoagulant, and oral fluid samples, collected with the Intercept® device.
Authentic blood samples (n "" 260) were assayed on the EIA plate, using an optimal 1 :5-fold dilution. True
positive, true negative, false positive and false negative results were determined relative to our routine GC­
MS analysis. The EIA readily detects MDA but shows minimal crossreactivity with MDMA « 0.1%), so
the interpretation of the GC-MS result of the MDMA-only samples was based on the combined
MDAIMDMA concentrations. Samples consisted of 100 amphetamine-only positives, 100 MDMAIMDAonly
positives, and 60 negatives, using the limit of quantitation as the cut-off level for confirmation (10
nglml). Using these results, receiver operating curves (ROC) were generated and optimal cut-off values for
the screening assay were calculated.
For the amphetamine positive samples, the analysis showed an optimal cut-off value at 66.5 nglml
amphetamine equivalents with a sensitivity of 99.0 % and a specificity of 96.9 %. For MDMA/MDA
positive samples, 97.0 % sensitivity and 96.9 % specificity were reached at the same cut-off value of 66.5
nglml amphetamine equivalents. The area under the ROC curve exceeded 0.97. When combining the
results, the EIA assay is able to predict the presence of either amphetamine or MDMAIMDA in plasma
samples with 98.0 % sensitivity and 96.9 % specificity at a cut-off value of 66.5 nglml amphetamine
equivalents.
A similar analysis was conducted on 216 oral fluid specimens collected from a controlled double blind
study. Subjects received placebo or a high (l00 mg) or low (75 mg) dose ofMDMA. Plasma and oral fluid
samples were collected at 1.5 and 5.5 hours after administration. Preliminary analysis of the oral fluid
samples indicated a screening cut-off of 51 ng/ml amphetamine equivalents with a sensitivity of 97.9 % and
a specificity of 98.6 %, using the plasma data for confirmation. LC-MSIMS confirmation of the oral fluid
samples is in progress.
In conclusion, these data indicate that the Cozart® AMP EIA plates constitute a fast and accurate screening
technique for the identification of amphetamine and MDA/MDMA positive blood samples and oral fluid
specimens collected with Intercept®.
Keywords: EIA, Amphetamine, Blood
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