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  • 1.Some relevant data regarding the detection of 1-benzyl-piperazine Carmo Manzoni ()1 , Douwe de Boer1, Marta Calçada1, Irina J. Póvoa1 Carla Martins1, João M. Franco2, Mário J. Dias2, Lesseps J.A.L. dos Reys1 1-Laboratório de Análises de Dopagem e Bioquímica 2-Instituto de Medicina Legal de Lisboa Laboratório de Análises de Dopagem e Bioquímica - Instituto Nacional do Desporto Centro de Medicina Desportiva Av. Prof. Egas Moniz (Estádio Universitário), 1600-190 Lisbon - Portugal Tel: +351 217954000 Fax: +351 217977529 Laboratório de Análises de Dopagem e Bioquímica Introduction Several examples of compounds that contain a piperazine moiety in their molecule bind to serotonin receptors. Apparently the structures of piperazine-like compounds have good interaction possibilities with these receptors, which can be understood if orientations of for example 1-aryl-piperazines are compared with that of serotonin (Figure 1). Although as a group, the piperazine-like compounds can not be considered as selective compounds for serotonin receptors, they may be made more site selective with the appropriate substituents. An illustration of the phenomenon of the piperazine-like compounds is 1-benzyl-piperazine, which roughly mimics the psychoactive effects of the phenylalkylamine (S)-amphetamine (Figure 1), although at a higher dosage. 1-Benzyl-piperazine is also of special interest, because it is being promoted as a drug-of-abuse on the Internet. It is since January 2000 commercially available on a European Internet website as a so-called synthetic stimulant. It is sold in capsules under the name of A2 in a dosage of 125 mg of its dihydrochloride salt. As this development may have consequences for drug-of abuse and forensic cases, we studied some relevant analytical aspects of 1-benzyl-piperazine. Studied were the cross reactivity in several amphetamine immunoassays and the potential use of the GC/NPD screening and GC/MS confirmation analysis. Figure1. Structure similarities The immunoassays evaluated were the Amphetamines Abuscreen Online® (Roche Diagnostics), the Enzyme-multiple Immunoassay Technique (EMIT®) d.a.u. Amphetamine assay (Dade Behring Diagnostics), the AxSYM® Fluorescence Polarization Immunoassay (FPIA) Amphetamine/Methamphetamine II Immunoassay (Abbott Diagnostics Systems) and the Amphetamine MICRO-PLATE Enzyme Immunoassay (EIA) (OraSure Technologies). GC/NPD analysis was performed after basic extraction with tert.-butyl methyl ether at pH 12. GC/MS analysis was performed after N,O-trifluoroacetylation => 50 L of trifluoroacetic anhydride + 50 L of ethyl acetate => incubation for 30 min at 65 C Experimental Figure 2. Cross reactivity between 1-benzyl-piperazine and amphetamine in the Abuscreen Online® assay Results Figure 3. GC/NPD analytical conditions and chromatogram Immunoassay 1-Benzyl-piperazine showed a low cross reactivity (< 1%) using the Amphetamines Abuscreen Online® assay (Figure 2) and the EMIT® d.a.u. Amphetamine assay. The FPIA Amphetamine/Methamphetamine II Immunoassay and the Amphetamine MICRO-PLATE Enzyme Immunoassay did not show any cross reactivity. GC/NPD analysis The analysis of 1-benzyl-piperazine resulted in a good response and peak shape. The extraction recovery was > 80% (Figure 3). GC/MS confirmation The analysis of the N-TFA derivative of 1-benzyl-piperazine resulted in a characteristic mass spectrum (Figure 4). Conclusion Not every immunoassays showed cross reactivity for 1- benzyl-piperazine The implementation of 1-benzyl-piperazine in relevant analytical procedures can easily be achieved using a GC/NPD analysis. As a confirmation procedure the use of the N,O-trifluoroacetylation derivatisation step results in a characteristic mass spectrum Figure 4. Mass spectrum and fragments of 1- benzyl-piperazine