Analytical and Bioanalytical Chemistry

, Volume 402, Issue 6, pp 2141–2151 | Cite as

Generation of metabolites by an automated online metabolism method using human liver microsomes with subsequent identification by LC-MS(n), and metabolism of 11 cathinones

  • Daniel M. Mueller
  • Katharina M. Rentsch
Original Paper


Human liver microsomes (HLMs) are used to simulate human xenobiotic metabolism in vitro. In forensic and clinical toxicology, HLMs are popularly used to study the metabolism of new designer drugs for example. In this work, we present an automated online extraction system we developed for HLM experiments, which was compared to a classical offline approach. Furthermore, we present studies on the metabolism of 11 cathinones; for eight of these, the metabolism has not previously been reported. Metabolites were identified based on MS2 and MS3 scans. Fifty-three substances encompassing various classes of drugs were employed to compare the established offline and the new online methods. The metabolism of each of the following 11 cathinones was studied using the new method: 3,4-methylenedioxy-N-benzylcathinone, benzedrone, butylone, dimethylcathinone, ethylone, flephedrone, methedrone, methylone, methylethylcathinone, naphyrone, and pentylone. The agreement between the offline and the online methods was good; a total of 158 metabolites were identified. Using only the offline method, 156 (98.7%) metabolites were identified, while 151 (95.6%) were identified using only the online method. The metabolic pathways identified for the 11 cathinones included the reduction of the keto group, desalkylation, hydroxylation, and desmethylenation in cathinones containing a methylenedioxy moiety. Our method provides a straightforward approach to identifying metabolites which can then be added to the library utilized by our clinical toxicological screening method. The performance of our method compares well with that of an established offline HLM procedure, but is as automated as possible.


LC-MS Human liver microsomes Cathinones Online extraction Human metabolism 



Atmospheric pressure chemical ionization


Arbitrary units




Cytochrome P450


Ethylenediaminetetraacetic acid


Electrospray ionization






Glucose-6-phosphate dehydrogenase


High-pressure liquid chromatography


Human liver microsomes


International units


Liquid chromatography–mass spectrometry




Nicotinic acid adenine dinucleotide phosphate (oxidized form)


Nicotinic acid adenine dinucleotide phosphate (reduced form)





The authors would like to thank Thermo Fisher Scientific for providing the LC-MS system and the online extraction instrument.


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.Institute for Clinical ChemistryUniversity Hospital ZurichZurichSwitzerland

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