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Scientific References for Drug Metabolism |
Quantitation of small molecules using high-resolution accurate mass spectrometers - a different approach for analysis of biological samples
Nayan Rena Zhang1*, Sean Yu1, Philip Tiller2, Suzie Yeh1, Elizabeth Mahan1 and William Bart Emary1
Department of Drug Metabolism and Pharmacokinetics, Merc Research Laboratories, West Point, PA 19486, USA
RMI Laboratories, North Wales, PA 19454, USA
Rapid Commun. Mass Spectrom. 2009; 23: 1085–1094
The quantitative capabilities of a linear ion trap high-resolution mass spectrometer (LTQ-Orbitrap™) were investigated using full scan mode bracketing the m/z range of the ions of interest and utilizing a mass resolution (mass/FWHM) of 1500. Extracted ion chromatograms using a mass window of +/-5-10 mmu centering on the theoretical m/z of each analyte were generated and used for quantitation. The quantitative performance of the LTQ-Orbitrap™ was compared with that of a triple quadrupole (API 4000) operating using selected reaction monitoring (SRM detection. Comparable assay precision, accuracy, linearity and sensitivity were observed for both approaches. The concentrations of actual study samples from 15 Merc drug candidates reported by the two methods were statistically equivalent. Unline SRM being a tandem mass spectrometric (MS/MS)-based detection method, a high resolution mass spectrometer operated in full scan does not need MS/MS optimization. This approach not only provides quantitative results for compounds of interest, but also will afford data on other analytes present in the sample. An example of the identification of a major circulating metabolite for a preclinical development study is demonstrated.
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Rapid metabolite identification with sub parts-per-million mass accuracy from biological matrices by direct infusion nanoelectrospray ionization after clean-up on a
ZipTip and LTQ/Orbitrap mass spectrometry
John C. L. Erve*, William DeMaio and Rasmy E. Talaat, Drug Safety and Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
Rapid Commun. Mass Spectrom. 2008; 22: 3015–3026
Metabolite identification studies remain an integral part of pre-clinical and clinical drug development programs. Analysis of biological matrices, such as plasma, urine, feces and bile, pose challenges due to the large amounts of endogenous components that can mask a drug and its metabolites. Although direct infusion nanoelectrospray using capillaries has been used routinely for proteomic studies, metabolite identification has traditionally employed liquid chromatographic (LC) separation prior to analysis. A method is described here for rapid metabolite profiling in biological fluids that involves initial sample clean-up using pipette tips packed with reversed-phase material (i.e. ZipTips) to remove matrix components followed by direct infusion nanoelectrospray on an LTQ/Orbitrap mass spectrometer using a protonated polydimethylcyclosiloxane cluster ion for internal calibration. We re-examined samples collected from a prazosin metabolism study in the rat. Results are presented that demonstrate that sub parts-per-million accuracies can be achieved on molecular ions, facilitating identification of metabolites, and on product ions, facilitating structural assignments. The data also show that the high-resolution measurements (R¼100 000 at m/z 400) enable metabolites of interest to be resolved from endogenous components. The extended analysis times available with nanospray enables signal averaging for 1 min or more that is valuable when metabolites are present in low concentrations as encountered here in plasma and brain. Using this approach, the metabolic fate of a drug can be quickly obtained. A limitation of this approach is that metabolites that are structural isomers cannot be distinguished, although such information can be collected by LC/MS during follow-on experiments.
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Rapid Screening of Glutathione-Trapped Reactive Metabolites by Linear Ion Trap Mass Spectrometry with Isotope Pattern-Dependent Scanning and Postacquisition Data Mining
Li Ma et al. from Bristol-Myers Squibb, Princeton, NJ
Chemical Research in Toxicology, 21 (7), 1477-1483, 2008
The present study describes a novel integrated approach for rapid analysis of reactive metabolites with a linear ion trap mass spectrometer (LTQ). In this approach, an isotope pattern-dependent scanning method was applied to the data acquisition of glutathione (GSH)-trapped reactive metabolites. Recorded fullscan MS and MS/MS data sets were further processed with neutral loss filtering, product ion filtering, and extracted ion chromatographic analysis to search for protonated molecules and MS/MS spectra of GSH adducts. To evaluate the effectiveness and reliability of the approach, GSH adducts of carbamazepine, diclofenac, 4-ethylphenol, acetaminophen, p-cresol, and omeprazole were analyzed, which were formed in human liver microsome incubations fortified with a mixture of nonlabeled GSH and stable isotopelabeled GSH at a 1:0.8 ratio. Results demonstrate that the combination of the isotope pattern-dependent scanning with the postacquisition data mining was very effective in detecting low levels of GSH adducts, regardless of their fragmentation patterns. As compared to a neutral loss scanning method performed with a triple quadrupole mass spectrometer, the LTQ-based approach had several major advantages, including the superior selectivity and sensitivity in detecting different classes of GSH adducts and the higher throughput capability of the detection and MS/MS spectral acquisition of GSH adducts in a single LC/MS run. Overall, this analytical approach provides a simple and efficient means for screening for reactive metabolites using a linear ion trap LC/MS platform.
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LTQ (Isotope pattern triggered data dependant acquisition)
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Unbiased High-Throughput Screening of Reactive Metabolites on the Linear Ion Trap Mass Spectrometer Using Polarity Switch and Mass Tag Triggered Data-Dependent Acquisition
Zhengyi Yan et al. from Johnson & Johnson, Spring House, PA
Analytical Chemistry, 80 (16), 6410-6422, 2008
Constant neutral loss (CNL) and precursor ion (PI) scan have been widely used for the in vitro screening of glutathione conjugates derived from reactive metabolites, but these two methods are only applicable to triple quadrupole or hybrid triple quadrupole mass spectrometers. Additionally, the success of CNL and PI scanning largely depends on structure and CID fragmentation pathways of GSH conjugates. In the present study, a highly efficient methodology has been developed as an alternative approach for high-throughput screening and structural characterization of reactive metabolites using the linear ion trap mass spectrometer.
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LTQ (Polarity switching; Isotope pattern triggered data dependant acquisition)
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A Generic Method to Detect Electrophilic Intermediates Using Isotopic Pattern Triggered Data-dependant High-resolution Accurate Mass Spectrometry
Heng-Keang Lim et al. from Johnson & Johnson, Raritan, NJ
Rapid Communications in Mass Spectrometry 22 (8), 1295-1311, 2008
A need still exists for a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method that can detect broad classes of glutathione (GSH) conjugates and provide characterization of their structures. We now describe the development of a method that multiplexes high-resolution accurate mass analysis with isotope pattern triggered data-dependent product ion scans, for simultaneous detection and structural elucidation of GSH conjugates within a single analysis using a LTQ/ Orbitrap. This method was initially developed to detect GSH conjugates generated from incubating 10mM test compound with pooled human liver microsomes fortified with NADPH-regenerating system and a 2:1 ratio of 5mM glutathione and [13C15 2 N-Gly]glutathione. The GSH conjugates were detected by isotope search of mass defect filtered and control subtracted full scan accurate MS data using MetWorks software. This was followed by elucidation of reactive intermediate structures using chemical formulae for both protonated molecules and their product ions from accurate masses in a single analysis. The mass accuracies measured for the precursor and product ions by the Orbitrap were <2ppm in external mass calibration mode. Successful detection and characterization of GSH conjugates of acetaminophen, tienilic acid, clozapine, ticlopidine and mifepristone validated this method. In each case, the detected GSH conjugates were within the top five hits by isotope search.
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LTQ Orbitrap (Isotope pattern triggered data dependant acquisition)
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Metworks (MMDF, Isotope pattern search, Background subtraction)
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An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection
Qian Ruan et al. from Bristol-Myers Squibb, Princeton, NJ
JOURNAL OF MASS SPECTROMETRY, 43: 251–261, 2008
A new strategy using a hybrid linear ion trap/Orbitrapmass spectrometer and multiple post-acquisitiondata mining techniques was evaluated and applied to the detection and characterization of in vitro metabolites of indinavir. Accurate-mass, full-scan MS and MS/MS data sets were acquired with a generic data-dependent method and processed with extracted-ion chromatography (EIC), mass-defect filter (MDF), product-ion filter (PIF), and neutral-loss filter (NLF) techniques. The high-resolution EIC processwas shown to be highly effective in the detection of common metabolites with predicted molecular weights. The MDF process, which searched for metabolites based on the similarity of mass defects of metabolites to those of indinavir and its core substructures, was able to find uncommon metabolites not detected by the EIC processing. In summary, the results demonstrated that this analytical strategy enables the high-throughput acquisition of accurate-mass LC/MS data sets, comprehensive search of a variety of metabolites through the post-acquisition processes, and effective structural characterization based on elemental compositions of metabolite molecules and their product ions. Copyright 2007 John Wiley & Sons, Ltd.
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Detection and Structural Characterization of Glutathione-Trapped Reactive Metabolites Using Liquid Chromatography-High-Resolution Mass Spectrometry and Mass Defect Filtering
Mingshe Zhu et al. from Bristol-Myers Squibb, Princeton, NJ
Analytical Chemistry, 79 (21), 8333 -8341, 2007
The present study was designed to apply the mass defect filter (MDF) approach to the screening and identification of reactive metabolites using high-resolution mass spectrometry. Glutathione (GSH)-trapped reactive metabolites of acetaminophen, diclofenac, carbamazepine, clozapine, p-cresol, 4-ethylphenol, and 3-methylindole in human liver microsomes (HLM) were analyzed by HPLC coupled with Orbitrap or Fourier transform ion cyclotron resonance mass spectrometry. Through the selective removal of all ions that fall outside of the GSH adduct MDF template windows, the processed full scan MS chromatograms displayed GSH adducts as major components with no or a few interference peaks. The accurate mass LCMS data sets were also utilized for the elimination of false positive peaks, detection of stable oxidative metabolites with other MDF templates, and determination of metabolite molecular formulas. Compared to the neutral loss scan by a triple quadrupole instrument, the MDF approach was more sensitive and selective in screening for GSH-trapped reactive metabolites in HLM and rat bile and far more effective in detecting GSH adducts that do not afford the neutral loss of 129 Da as a significant fragmentation pathway. The GSH adduct screening capability of the MDF approach, together with the utility of accurate mass MS/MS information in structural elucidation, makes high-resolution LC-MS a useful tool for analyzing reactive metabolites.
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Metabolite identification by data-dependent accurate mass spectrometric analysis at resolving power of 60 000 in external calibration mode using an LTQ/Orbitrap
Heng-Keang Lim et al. from Johnson & Johnson, Raritan, NJ
Rapid Communications in Mass Spectrometry, 21, 1821–1832, 2007
Performance evaluation of accurate mass measurement by the LTQ/Orbitrap, at a resolving power of 60 000 and in external calibration mode, indicated that the Orbitrap is capable of providing high mass accuracy of <2ppm for over 24 h post-calibration. This, together with limited trade-off between sensitivity and resolving power plus a wide dynamic range for mass accuracy, suggested that the LTQ/Orbitrap is an ideal analytical tool for structural elucidation of metabolites. The application of the LTQ/Orbitrap to identification of human liver microsomal metabolites of carvedilol was evaluated, using parent mass list triggered data-dependent multiple-stage accurate mass analysis, at a resolving power of 60 000 in external calibration mode. A metabolite identification workflow was developed to utilize chemical formulas from high-resolution accurate mass measurements to confirm structures of product ions of a drug proposed by Mass Frontier, illustrated by identification of structures used to establish lineage of product ions of carvedilol, which later served as a template for identification of its metabolites. A total of 58 in vitro metabolites of carvedilol were detected using 5-ppm mass tolerance filters for theoretical m/z of protonated molecules of predicted metabolites in addition to product ions and neutral mass losses diagnostic of carvedilol. The chemical formulas with unsaturation numbers calculated from the accurate m/z of precursor and product ions can be used to assign, with a high degree of confidence, the structures of metabolites and the sites of metabolism. The mass accuracies obtained for all full scan MS and MSn spectra were <2 ppm. The majority of the metabolites identified agreed with those previously reported except for those that have not been reported before. For example, several glutathione conjugates of carvedilol were reported for the first time, which may explain the reported hepatotoxicity during clinical trials and recent clinical use. Copyright 2007 John Wiley & Sons, Ltd.
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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-FT MS 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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LTQ XL™ linear ion trap mass spectrometer
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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.
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LTQ XL™ linear ion trap mass spectrometer
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