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

Original Paper

Abstract

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.

Keywords

LC-MS Human liver microsomes Cathinones Online extraction Human metabolism 

Abbreviations

APCI

Atmospheric pressure chemical ionization

AU

Arbitrary units

bk

Beta-keto

CYP

Cytochrome P450

EDTA

Ethylenediaminetetraacetic acid

ESI

Electrospray ionization

GST

Glutathione-S-transferase

G6P

Glucose-6-phosphate

G6PD

Glucose-6-phosphate dehydrogenase

HPLC

High-pressure liquid chromatography

HLMs

Human liver microsomes

IU

International units

LC-MS

Liquid chromatography–mass spectrometry

MDPV

Methylenedioxypyrovalerone

NADP+

Nicotinic acid adenine dinucleotide phosphate (oxidized form)

NADPH

Nicotinic acid adenine dinucleotide phosphate (reduced form)

NAT

N-Acetyltransferase

References

  1. 1.
    Asha S, Vidyavathi M (2010) Role of human liver microsomes in in vitro metabolism of drugs—a review. Appl Biochem Biotechnol 160:1699–1722Google Scholar
  2. 2.
    Wintermeyer A, Moller I, Thevis M, Jubner M, Beike J, Rothschild MA, Bender K (2010) In vitro phase I metabolism of the synthetic cannabimimetic JWH-018. Anal Bioanal Chem 398:2141–2153CrossRefGoogle Scholar
  3. 3.
    Meyer MR, Du P, Schuster F, Maurer HH (2010) Studies on the metabolism of the alpha-pyrrolidinophenone designer drug methylenedioxy-pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC-MS and LC-high-resolution MS and its detectability in urine by GC-MS. J Mass Spectrom 45:1426–1442CrossRefGoogle Scholar
  4. 4.
    Zaitsu K, Katagi M, Kamata HT, Kamata T, Shima N, Miki A, Tsuchihashi H, Mori Y (2009) Determination of the metabolites of the new designer drugs bk-MBDB and bk-MDEA in human urine. Forensic Sci Int 188:131–139CrossRefGoogle Scholar
  5. 5.
    Meyer MR, Maurer HH (2011) Current status of hyphenated mass spectrometry in studies of the metabolism of drugs of abuse, including doping agents. Anal Bioanal ChemGoogle Scholar
  6. 6.
    Drexler DM, Belcastro JV, Dickinson KE, Edinger KJ, Hnatyshyn SY, Josephs JL, Langish RA, McNaney CA, Santone KS, Shipkova PA, Tymiak AA, Zvyaga TA, Sanders M (2007) An automated high throughput liquid chromatography–mass spectrometry process to assess the metabolic stability of drug candidates. Assay Drug Dev Technol 5:247–264Google Scholar
  7. 7.
    Xu R, Manuel M, Cramlett J, Kassel DB (2010) A high throughput metabolic stability screening workflow with automated assessment of data quality in pharmaceutical industry. J Chromatogr A 1217:1616–1625CrossRefGoogle Scholar
  8. 8.
    Jenkins KM, Angeles R, Quintos MT, Xu R, Kassel DB, Rourick RA (2004) Automated high throughput ADME assays for metabolic stability and cytochrome P450 inhibition profiling of combinatorial libraries. J Pharm Biomed Anal 34:989–1004CrossRefGoogle Scholar
  9. 9.
    Lai F, Khojasteh-Bakht SC (2005) Automated online liquid chromatographic/mass spectrometric metabolic study for prodrug stability. J Chromatogr B 814:225–232Google Scholar
  10. 10.
    Kool J, van Liempd SM, van Rossum H, van Elswijk DA, Irth H, Commandeur JN, Vermeulen NP (2007) Development of three parallel cytochrome P450 enzyme affinity detection systems coupled on-line to gradient high-performance liquid chromatography. Drug Metab Dispos 35:640–648CrossRefGoogle Scholar
  11. 11.
    Nicoli R, Curcio R, Rudaz S, Veuthey JL (2009) Development of an in-capillary approach to nanoscale automated in vitro cytochromes p450 assays. J Med Chem 52:2192–2195CrossRefGoogle Scholar
  12. 12.
    Curcio R, Nicoli R, Rudaz S, Veuthey JL (2010) Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies. Anal Bioanal Chem 398:2163–2171CrossRefGoogle Scholar
  13. 13.
    Mueller DM, Duretz B, Espourteille FA, Rentsch KM (2011) Development of a fully automated toxicological LC-MS(n) screening system in urine using online extraction with turbulent flow chromatography. Anal Bioanal Chem 400:89–100CrossRefGoogle Scholar
  14. 14.
    Cozzi NV, Sievert MK, Shulgin AT, Jacob P 3rd, Ruoho AE (1999) Inhibition of plasma membrane monoamine transporters by beta-ketoamphetamines. Eur J Pharmacol 381:63–69CrossRefGoogle Scholar
  15. 15.
    Mueller DM, Rentsch KM (2011) Online extraction toxicological MS(n) screening system for serum and heparinized plasma and comparison of screening results between plasma and urine in the context of clinical data. J Chromatogr BGoogle Scholar
  16. 16.
    Sohl CD, Cheng Q, Guengerich FP (2009) Chromatographic assays of drug oxidation by human cytochrome P450 3A4. Nat Protoc 4:1252–1257CrossRefGoogle Scholar
  17. 17.
    Kamata HT, Shima N, Zaitsu K, Kamata T, Miki A, Nishikawa M, Katagi M, Tsuchihashi H (2006) Metabolism of the recently encountered designer drug, methylone, in humans and rats. Xenobiotica 36:709–723CrossRefGoogle Scholar
  18. 18.
    Meyer MR, Wilhelm J, Peters FT, Maurer HH (2010) Beta-keto amphetamines: studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography–mass spectrometry. Anal Bioanal Chem 397:1225–1233Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.Institute for Clinical ChemistryUniversity Hospital ZurichZurichSwitzerland

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