Scientific References for Drug Metabolism

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Utility of the hybrid LTQ-FTMS for drug metabolism applications.

Sanders M, Shipkova PA, Zhang H, Warrack BM. Curr Drug Metab. 2006 Jun;7(5):547-55.

Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb Pharmaceutical Research Institute, Lawrenceville, NJ, USA.

Thermo Scientific LTQ-FT™ instruments are compatible with HPLC flow rates, have high throughput and automation compatibility, and also provide data dependant MSn. The ability to maintain the fidelity of an externally calibrated accurate mass measurement across an HPLC peak, where the analyte concentrations are rapidly changing, is a significant advance for this technology, as is the ability to perform data dependent MS/MS experiments on the chromatographic time scale. The MSn and accurate mass capabilities are routinely utilized to rapidly confirm the identification of expected metabolites or to elucidate the structures of unusual or unexpected metabolites. The combination of traditional high-flow chromatography and robust, externally calibrated accurate mass determination for both parent and product ions makes the LTQ-FTMS a very powerful analytical tool for the characterization of metabolites, identification of metabolic soft-spots and for metabonomics studies.

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Determination of therapeutics with growth-hormone secretagogue activity in human urine for doping control purposes

Thevis M., Wilkens F, Geyer H. Schanzer W. Rapid Commun Mass Spectrom. 2006;20(22):3393-402

Center for Preventive Doping Research-Institute of Biochemistry, German Sport University Cologne, Carl-Diem Weg 6, 50933 Cologne, Germany.

The administration of growth-promoting agents is prohibited in sports. Preventive antidoping strategies include method development for emerging drugs and potentially misused compounds. The mass spectrometric dissociation behavior of four such compounds was studied using high-resolution/high-accuracy Thermo Scientific LTQ Orbitrap™ mass spectrometry. These data provided substantial information for screening procedures, complementing common methods of sports drug testing. The four target analytes were determined at urinary concentrations of 15-20 ng/mL, recoveries ranged from 55-97%, and assay precisions were calculated at 5.2-15.8% (intraday) and 10.2-21.6% (interday) for all compounds. In all tested cases, the administered drug and the respective desmethylated metabolites were detected.

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Overexpression of Superoxide Dismutase or Glutathione Peroxidase Protects against the Paraquat + Maneb-induced Parkinson Disease Phenotype

Mona Thiruchelvam, Olga Prokopenko, Deborah A. Cory-Slechta, Eric K. Richfield, Brian Buckley, and Oleg Mirochnitchenko. J. Biol. Chem., Vol. 280, Issue 23, 22530-22539, June 10, 2005

Department of Biochemistry and Environmental and Occupational Health Sciences Institute, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

This study analyzed the role of two drugs, paraquat and maneb, in the dopaminergic neurotoxicity of a Parkinson disease model system. Mice were chronically exposed to saline (control) and a combination of the two drugs for 9 weeks. The mice treated with these two drugs exhibited a significant reduction in locomotor activity, levels of dopamine and its metabolites and dopaminergic neuronal death. Brain tissue extracts from mice exposed to toxic levels of the two drugs was analyzed by LC-ESI MS using the Thermo Scientific LCQ™ mass spectrometer. The mass spectrum was acquired in full scan (150-250 m/z and 150-1000 m/z) mode and easily identified the parent drug and related adducts.

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Cytochrome P450 3A-mediated metabolism of Buspirone in human liver microsomes

Mingshe Zhu, Weiping Zhao, Humberto Jimenez, Donglu Zhang, Suresh Yeola, Renke Dai, Nimish Vachharajani, and James Mitroka. Drug Metab Dispos. Vol. 33, No. 4 2005

Pharmaceutical Candidate Optimization (M.Z., W.Z., H.J., D.Z., S.Y., R.D., J.M.), and Clinical Discovery (N.V.), Bristol- Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey

This study was carried out to determine the metabolic pathways of buspirone and cytochrome P450 (P450) isoform(s) responsible for buspirone metabolism in human liver microsomes (HLMs). Buspirone mainly underwent N-dealkylation, N-oxidation on the piperazine ring, and hydroxylation to 3-OH-Bu, 5-OH-Bu, and 6-OHBu. CYP3A inhibitor ketoconazole completely inhibited the formation of all major metabolites in HLMs (0–16% of control), whereas the chemical inhibitor selective to other P450 isoforms had little or no inhibitory effect. Metabolites were identified using the Thermo Scientific TSQ Quantum™ triple quadrupole mass spectrometer. In a panel of HLMs from 16 donors, buspirone metabolism correlated well CYP3A activity, but not the activities of other P450 isoforms.  These data suggest that CYP3A, mostly likely CYP3A4, is primarily responsible for the metabolism of buspirone in HLMs.

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Identification of human liver cytochrome P450 enzymes responsible for the metabolism of Ionafamib (Sarasar).

Anima Ghosal, Swapan K. Chowdhury, Wei Tong, Neil Hapangama, Yuan Yuan, Ai-Duen Su, and Shmuel Zbaida Drug Metab Dispos. 2006 Apr;34(4):628-35. 2006

Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, 2015 Galloping Hill Rd., K-15-1945, Kenilworth, NJ 07033

Lonafarnib (Sarasar), a farnesyl transferase inhibitor, is under development for the treatment of solid tumors. Incubation of lonafarnib with human liver microsomes resulted in the formation of four oxidative metabolites (M1, M2, M3, and M4). Minor to trace levels of these metabolites were detected in humans after multiple-dose administration. LC ESI-MS analyses using the Thermo Scientific LCQ™ mass spectrometer exhibited a mass to charge ratio (m/z) for the (M+H)(+) ion of M1, M2, M3, and M4 at 653, 635, 669, and 653 Th, respectively. These metabolites, respectively, resulted from changes of +O, -2H, +2O, and +O relative to lonafarnib. Recombinant human CYP3A4 and CYP3A5 exhibited catalytic activity with respect to the formation of M1, M2, and M3, whereas CYP2C8 exhibited catalytic activity with respect to the formation of M4. In conclusion, the formation of metabolites M1, M2, and M3 from lonafarnib was mediated via CYP3A4 and CYP3A5.

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Application of the LTQ Orbitrap MS in metabolite characterization studies: examination of the human liver microsomal metabolism of the anti-depressant nefazodone using data-dependent accurate mass measurements.

Peterman SM, Duczak N Jr, Kalgutkar AS, Lame ME, Soglia JR. J Am Soc Mass Spectrom. 2006 Mar;17(3):363-75.

Thermo Fisher Scientific, New Jersey, and Pfizer Global Research

We report an easy metabolite identification workflow for the anti-depressant nefazodone. The LTQ Orbitrap™ mass spectrometer enabled accurate mass full scan MS and MS/MS in a data-dependent single run to eliminate the reliance on a parent mass list. A 10 ppm mass tolerance mass defect filter was used for known nefazodone metabolites to reduce background noise in detecting metabolites as well as isobaric forms. Metabolites were characterized using spectral correlation of parent and product ion m/z values to filter all MS/MS spectra for identification of precursor ions yielding similar product ions. Identified metabolite and parent accurate masses were subjected to chemical formula calculator and bond saturation. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of Mass Frontier™ predictive software in determining product ion structure.

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Characterization of Penicillin-G Instability in Equine Plasma by egative Ion Electrospray MSn Ion Tree Experiments Using a Linear Ion Trap

Jeffrey Rudy1, Rongfang Xu2, Cornelius Uboh2 and Joseph Dibussolo3. ASMS 2007 Abstract

1PA Equine Toxicology, West Chester, PA; 2University of Pennsylvania New Bolton Vet Center, Kennet Square, PA; 3Thermo Fisher Scientific, Franklin, MA

Instability of penicillin-G has been extensively reported, which lead to evaluation of possible sources of instability to eliminate or minimize penicillin-G degradation in equine plasma. Ion tree MSn identification of degradation products and descriptions of the fragmentation pathways were conducted using the Thermo Scientific LXQ™ linear ion trap and Mass Frontier version 5.0 software. This resulted in development of a simple, sensitive (LOD ~ 1 ng/mL), linear, reproducible method that avoided analyte degradation.

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In Vitro Metabolism of L-768242, an Aminoalkylindole CB2 Receptor Agonist

Qiang Zhang, Peng Ma and Guangdi Wang. ASMS 2007 Abstract

Xavier University of Louisiana, New Orleans, LA

L768242, an aminoalkylindole, selectively binds the human cannabinoid receptor, CB2. No study has been reported on its metabolism, and it is unknown whether its metabolites retain any receptor binding properties. The in vitro metabolism of L768242 in rat liver microsomes was examined. A total of 11 novel metabolites of L768242 were detected and structurally characterized using ESI LC/MS/MS and MS3 analyses performed on a Thermo Scientific LTQ XL™ linear ion trap mass spectrometer. Possible metabolic pathways responsible for the identified metabolites are described.

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Clozapine Distribution in Rat Brain and Lung: A Comparison of Imaging by DESI-MS vs LC MS/MS Analysis of Brain Homogenates

Justin M. Wiseman1, Candice Kissinger2, Demian R. Ifa3, Candace Rohde2, James Burleigh2, Simon Katner2, Bruce Solomon2, Yongxin Zhu2 and R. Graham Cooks3. ASMS 2007 Abstract

1Prosolia, Inc., Indianapolis, IN; 2Bioanalytical Systems Inc., West Lafayette, IN; 3Purdue University, West Lafayette, IN

The distribution of the antipsychotic drug, clozapine, was compared using two complementary analytical methods: DESI imaging mass spectrometry of lung and brain tissue recorded using a Thermo Scientific LTQ XL™ instrument equipped with an automated DESI ion source and HPLC MS/MS analysis of tissue homogenates and plasma using a Thermo Scientific Quantum Ultra mass spectrometer. Tissue homogenization reveals that the drug is present and provides a concentration, but does not show where it is located whereas DESI provides two-dimensional imaging of clozapine throughout the lung and substructures of the brain. The identity of the drug was confirmed using tandem MS. These studies extend the application of DESI imaging throughout the course of a typical pharmacokinetic experiment and include distribution in a new tissue, rat lung.