PMID- 12871044
OWN - NLM
STAT- MEDLINE
DCOM- 20040316
LR  - 20191026
IS  - 1389-2002 (Print)
IS  - 1389-2002 (Linking)
VI  - 4
IP  - 4
DP  - 2003 Aug
TI  - Metabolite characterization in drug discovery utilizing robotic liquid-handling, 
      quadruple time-of-flight mass spectrometry and in-silico prediction.
PG  - 259-71
AB  - An assay method for identification of metabolites from in vitro microsomal 
      incubations was developed for use in the early stage of drug discovery. We have 
      developed a practical approach which involves integrated sample generation, 
      sample preparation, bioanalysis, and data handling to maximize sample throughput 
      and speed up the process for identification of metabolites. The assay system 
      consisted of a robotic liquid handler (Genesis workstation) to generate and 
      process samples, PALLAS MetabolExpert software to predict possible metabolites, 
      exact mass measurement via a tandem quadrupole time-of-flight mass spectrometer 
      (QTOF-MS) coupled with liquid chromatography to analyze samples, MetaboLynx 
      software to find potential metabolites and Advanced Chemistry Development/MS 
      (ACD/MS) software to provide guidance to the most likely hypothetical metabolite 
      chemical structures. For purposes of evaluating this new method, 
      dextromethorphan, alprenolol, and propranolol were incubated separately for up to 
      60 minutes with rat and human hepatic microsomes. The incubation and sample 
      preparation were carried out in 96-well plates using the Genesis workstation. The 
      bioanalysis was performed by LC-MS/MS using QTOF with MetaboLynx software to find 
      metabolites. Metabolic products formed in vitro by rat and human microsomes were 
      separated using an analytical column C18 with gradient elution at flow rate of 
      250 micro l/min. The internal mass calibration was performed by continuous 
      postcolumn infusion of Haloperidol. The mass spectra from incubations containing 
      NADPH were compared to those without NADPH (control) using the MetaboLynx 
      software to find potential metabolites. Finally, the MS/MS spectra were processed 
      by the ACD/MS software to predict the chemical structure. MetaboLynx software 
      successfully identified metabolites for each of the drugs studied by 
      automatically discerning expected metabolites. Exact differences in masses 
      between each metabolite and parent drug were measured from five replicate sample 
      injections. All measured values are accurate to less than 0.001Da or 3.8 ppm with 
      the standard deviation within 0.0015 Da, which allowed good 
      prediction/confirmation of empirical formulae. Hypothetical chemical structures 
      were achieved by the ACD/MS software and provided a useful tool to assist in 
      prediction of the metabolic pathways of the drugs. The metabolites identified 
      were in good agreement with previously published results for all three compounds. 
      This new method will greatly enhance throughput, which in turn will facilitate 
      our ability to rapidly provide this guidance to the synthetic chemist.
FAU - Nassar, A-E F
AU  - Nassar AE
AD  - Department of Pharmacokinetics and Drug Metabolism, Bayer Corporation, 400 Morgan 
      Lane, West Haven, CT 06516, USA. NassarAL@aol.com
FAU - Adams, P E
AU  - Adams PE
LA  - eng
PT  - Journal Article
PL  - Netherlands
TA  - Curr Drug Metab
JT  - Current drug metabolism
JID - 100960533
RN  - 0 (Pharmaceutical Preparations)
RN  - 7355X3ROTS (Dextromethorphan)
RN  - 877K5MQ27W (Alprenolol)
RN  - 9Y8NXQ24VQ (Propranolol)
SB  - IM
MH  - Alprenolol/chemistry/metabolism/pharmacokinetics
MH  - Animals
MH  - Chromatography, Liquid/methods
MH  - Dextromethorphan/chemistry/metabolism/pharmacokinetics
MH  - Drug Design
MH  - Humans
MH  - In Vitro Techniques
MH  - Male
MH  - Mass Spectrometry/*methods
MH  - Microsomes, Liver/metabolism
MH  - Pharmaceutical Preparations/*metabolism
MH  - *Pharmacokinetics
MH  - Propranolol/chemistry/metabolism/pharmacokinetics
MH  - Rats
MH  - Reproducibility of Results
MH  - Robotics/instrumentation/*methods
MH  - Sensitivity and Specificity
MH  - Software
EDAT- 2003/07/23 05:00
MHDA- 2004/03/17 05:00
CRDT- 2003/07/23 05:00
PHST- 2003/07/23 05:00 [pubmed]
PHST- 2004/03/17 05:00 [medline]
PHST- 2003/07/23 05:00 [entrez]
AID - 10.2174/1389200033489406 [doi]
PST - ppublish
SO  - Curr Drug Metab. 2003 Aug;4(4):259-71. doi: 10.2174/1389200033489406.