Peschier L.J.C., Nunes Roque J., Lusthof K.J., Zweipfenning P.G.M.

Forensic Science Laboratory, Volmerlaan 17, NL-2288 GD, Rijswijk, The Netherlands

Amphetamine, methamphetamine and the structurally and pharmacologically related designer drugs as methylendioxy-methamphetamine (MDMA), methylendioxy-ethylamphetamine (MDEA) and methylendioxy-amphetamine (MDA) have become widely used-abused as recreational drugs. Death and life threatening hyperthermia, as well as a decreased driving performance is associated with these substances. Therefore, forensic toxicologists need selective and sensitive methods for their determination in body fluids, especially whole blood. Most methods published, meeting forensic standards, are based on gaschromatography/mass spectrometry after extraction and derivatisation. Mostly derivatization is done with short chain perfluoro-carboxylic acid anhydrides (TFAA, PFPA, HFBA). The excess of highly reactive reagent is harmful for the GC column and causes chromatographic problems with other applications on the system.
We developed a Solid-Phase Extraction procedure for whole blood, using Bond-Elut Certify columns, after addition of deuterated internal standards, followed by derivatization with HFBA (HeptaFluoroButyric-acid Anhydride).
After application of the sample, the extraction column is washed with HCl-solution and methanol. The compounds are eluted with triethylamine (TEA) in toluene. To the dried extract HFBA is added. This is allowed to react for at least 15 seconds at room temperature. The extract is then injected in GC/MS (SIM mode) for quantification and reinjected (Full-Scan mode) to identify other related substances and/or metabolites.
Analysis is performed on a HP 6890 gas chromatograph with a HP 5973 Mass Selective Detector. The column is HP Ultra-1. For the SIM analysis, three ions have been selected per compound (except for the deuterated internal standard of amphetamine, where two ions have been selected). The Limit of Detection in blood is about 1 ng/mL and the Limit of quantitation is about 10 ng/mL for all compounds. The method is linear from 0-4000 ng/mL. The coefficient of variation is less than 15% at concentrations of 50 ng/mL and higher, for all compounds.
MBDB has not been considered as compound of choice, because we hardly see cases with this compound. However, the method can also be used for screening in the full scan mode and MBDB will then be detected.
Validation was performed in order to request accreditation in The Netherlands.