Validation of an ELISA-based screening assay for the detection of amphetamine, MDMA and MDA in blood and oral fluid

Laloup, Marleen; Tilman, Gaëlle; Maes, Viviane; Boeck, Gert De; Wallemacq, Pierre; Ramaekers, Jan; Samyn, Nele

doi:10.1016/j.forsciint.2005.04.019

« Previous Next »Forensic Science International
Volume 153, Issue 1 , Pages 29-37, 4 October 2005

Validation of an ELISA-based screening assay for the detection of amphetamine, MDMA and MDA in blood and oral fluid

Marleen Laloup
- Federal Public Service Justice, National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Vilvoordsesteenweg 98, 1120 Brussels, Belgium
- Corresponding author. Tel.: +32 2 240 05 00; fax: +32 2 242 47 61.
Gaëlle Tilman
- Federal Public Service Justice, National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Vilvoordsesteenweg 98, 1120 Brussels, Belgium
- Laboratory of Clinical Chemistry and Toxicology, St. Luc University, Hospital–UCL, Avenue Hippocrate 10, 1200 Brussels, Belgium
Viviane Maes
- Department of Clinical Chemistry–Toxicology, Academic Hospital, Free University of Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium
Gert De Boeck
- Federal Public Service Justice, National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Vilvoordsesteenweg 98, 1120 Brussels, Belgium
Pierre Wallemacq
- Laboratory of Clinical Chemistry and Toxicology, St. Luc University, Hospital–UCL, Avenue Hippocrate 10, 1200 Brussels, Belgium
Jan Ramaekers
- Experimental Psychopharmacology Unit, Brain and Behaviour Institute, Maastricht University, Universiteitssingel 50, 6229 ER, Maastricht, The Netherlands
Nele Samyn
- Federal Public Service Justice, National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Vilvoordsesteenweg 98, 1120 Brussels, Belgium

published online 26 May 2005.

Abstract

The use of amphetamine and ‘ecstasy’ (MDMA) has increased exponentially in many European countries since the late nineties, leading to a rapid growth in the number of clinical and forensic analyses. Therefore, a rapid screening procedure for these substances in biological specimens has become an important part of routine toxicological analysis in forensic laboratories. The objective of this study was to evaluate the Cozart^® amphetamine enzyme-linked immunosorbent assay (ELISA) for the screening of plasma samples and oral fluid samples (collected with the Intercept^® device).

Authentic plasma samples from drivers (n=360) were screened, using an 1:5-fold dilution. True positive, true negative, false positive and false negative results were determined relative to the in-house routine GC–MS analysis. Samples consisted of 144 amphetamine-only positives, 141MDMA/MDA-only positives, and 74 negatives when using the limit of quantitation as the cut-off level for confirmation (10ng/mL). Using these results, receiver operating characteristic (ROC) curves were generated and optimal cut-off values for the screening assay were calculated. Analysis showed that the ELISA is able to predict the presence of either amphetamine or *MDMA/MDA (*MDMA as its metabolite MDA) in plasma samples with 98.3% sensitivity and 100% specificity at a cut-off value of 66.5ng/mL d-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 (100mg) or low (75mg) dose of MDMA. Oral fluid samples were collected at 1.5 and 5.5h after administration. Combined results of the analysis of the high and low dose oral fluid samples indicated a screening cut-off of 51ng/mL d-amphetamine equivalents with both a sensitivity and specificity of 98.6% (using a LC–MS/MS confirmation cut-off of 10ng/mL). In conclusion, these data indicate that the Cozart^® AMP EIA plates constitute a fast and accurate screening technique for the identification of amphetamine and MDMA/MDA positive plasma samples and oral fluid specimens (collected with Intercept^®). It should be emphasized that method validation should be performed for each type of biological matrix.

Keywords: ELISA, Amphetamine, MDMA, Blood, Oral fluid