SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

C31
DETECTION OF NEW MINOR METABOLITES BY LC/MS AND CHARACTERIZATION BY
LCIMS/MS AFTER COMPELLED INGESTION OF COCAINE
L. Humbert l , R. EljaoudLI, F. Orisel 2 , T. Kargar-OriseI 2 , D. Mathieu 3 , M. Lhermitte i
I Laboratoire de Biochimie & Biologie Moleculaire, H6pital Calmette, 59037 Lille Cedex France.
2 Waters Corporation, European Headquarters & Waters S.A.S., Rue Jacques Monod, 78280 Ouyancourt
France. 3 Urgence respiratoire et reanimation medicale, H6pital Calmette, 59037 Lille Cedex France.
A well-known cocaine addict presented spontaneously to the emergency service of the Lille Hospital
(France) and reported that he was forced to ingest a large quantity of a white powder. Surprisingly, the
patient did not present any intoxication symptoms but was placed under medical observation. Immunoassay
screening of a blood sample did not reveal the presence of any drugs and immunoassays performed on
urine led to positive results for cocaine only. LCIMS was used to confirm the immunoassay results and the
anticipated metabolites of cocaine were found. In addition, many other minor metabolites were detected
and characterized by LCIMSIMS.
Materials and methods: The urine sample was extracted at alkaline pH using a chloroform/isopropyl
alcohol mixture (9: 1) and evaporated to dryness. 100 ,.d of mobile phase was added to the extract and 20 J.l.1
injected onto the LCIMS system. Chromatographic separation was performed using a Waters Alliance
HPLC equipped with a Waters Xterra MS C18 column (ISO x 2.1, 3.5 J.l.m). Mass detection was achieved
using a Waters ZQ mass spectrometer operated in the electrospray mode in both positive and negative ion
polarities. Multi-functional full scan acquisitions were performed at different cone voltages. Well-known
urinary metabolites of cocaine were identified through their fragmentation patterns and an additional
LCIMSIMS analysis was performed to elucidate the structure of other analytes previously detected in MS
mode that could correspond to new non-documented metabolites. Exhaustive LC/MSIMS study (neutral
loss, parent and daughters, MRM) was performed using a Quattro Premier (Waters) operated in
electrospray combined to modified chromatographic conditions leading to greater retention for the polar
compounds and subsequent better separation efficiency.
Results: The LC/MS analysis of the gastric content revealed the presence of cocaine. Subsequent LCIMS
analysis of the urine samples led to the detection of many minors metabolites The oral ingestion was
confirmed by the positive identification of characteristic metabolites such as m- and p­
hydroxybenzoylecgonine and norbenzoylecgonine l which has also been found metabolised as N­
hydroxynorbenzoylecgonine. It was possible to change the selectivity ofthe chromatographic separation by
using a Xterra column at basic pH (10 mM ammonium bicarbonate) allowing the separation of several new
polar metabolites which usually coelute when analysed in acidic conditions.
Conclusion: Thanks to the spontaneous presentation of the patient, the delay between urine sample
collection and cocaine oral absorption was very short thus allowing the detection of many minor
metabolites of cocaine not usually found because of their very low concentrations. Combining the
sensitivity and the selectivity of tandem mass spectrometry with enhanced chromatographic separation, the
expected major metabolites were unambiguously identified and many other new metabolites were detected
and assigned to compounds such as N-hydroxynorbenzoylecgonine, methoxyecgonine and norecgonine
methyl ester.
References: 1. Klette KL, Poch OK, Czarny R, Lau CO. Simultaneous OC-MS analysis of m- et p­
hydroxybenzoylecgonine and norbenzoylecgonine: a secondary method to corroborate cocaine ingestion
using nonhydrolytic metabolites. J. Anal toxicol. 2000 Oct ;24(7) : 482-8.
Keywords: Forced ingestion, cocaine, metabolite, mass spectrometry
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