Immunological screening of drugs of abuse and gas chromatographic-mass spectrometric confirmation of opiates and cocaine in hair

Abstract

The work presents an analytical strategy to detect drugs of abuse in hair. It involves two sequential steps: a screening by a simple enzyme-linked immunosorbent assay (ELISA) methodology to detect opiates, cocaine and its metabolites, and benzodiacepines, followed by confirmation of opiates and cocaine metabolites in positive samples by gas chromatography coupled to mass spectrometry (GC-MS). In the same GC-MS run other drugs for substitution therapy (e.g. methadone and its main metabolite) can also be detected. After a double washing of hair samples with dichloromethane, hair specimens were cut into small pieces and 10 mg samples were incubated in 2 ml of methanol-trifluoroacetic acid (9:1) mixture, overnight at 37 degrees C. Aliquots of the extract were then evaporated, reconstituted in buffer and analysed according to the ELISA procedure. Confirmation involved solid-phase extraction of another fraction of the extract kept at -20 degrees C, derivatization with heptafluorobutyric anhydride and hexafluoroisopropanol and detection of cocaine, benzoylecgonine, ecgonine methylester, cocaethylene, morphine, codeine, 6-monoacetylmorphine, methadone and 2-ethylidene-1.5-dimethyl-3,3-diphenylpirrolidine (methadone metabolite) by selective ion monitoring after gas chromatographic separation. During the development of the method it was verified that no more than 10% of cocaine, opiates and benzodiacepines were lost when dichloromethane was used to wash real samples. The results also confirmed the increase of extractability power of TFA when it was added to methanol: the recovery for the analytes (cocaine and its metabolites and opiates) added to methanol-TFA alone was of the order of 90% except for benzoylecgonine (75%), and the recovery for the analytes added to methanol-TFA extract of drug-free hair was about 90% for all analytes except for benzoylecgonine and 6-MAM (around 70%). Regarding the stability of labile compounds, only small amounts of ecgonine methylester (2.3%) and morphine (7.2%) were produced, from cocaine and 6-MAM respectively, after the whole extraction procedure and two weeks of storage of methanol-TFA extracts at -20 degrees C. Satisfactory results were obtained when the procedures were applied to the analysis of external proficiency testing hair samples and actual specimens from drug addicts.