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A High Throughput Approach for Metabolite Note: 369
Profiling and Characterization Using theLXQ Linear Ion Trap Mass SpectrometerMin He, Alicia Du, Gargi Choudhary, Karen Salomon and Diane Cho; Thermo Electron Corporation, San Jose, CA, USA Key Words
Within the drug discovery environment, high sample • LXQ
throughput that provides comprehensive drug metabolite Glyburide was incubated using human hepatic microsomes identification and profiling is highly desirable. Traditionally, at a concentration of 10 µM for 40 minutes and quenched • Surveyor Plus
triple quadrupole instruments running selected reaction with ACN. The sample was then centrifuged, and the monitoring (SRM or MRM) have been employed; however, supernatant was collected and re-constituted for further SRM and MRM studies do not necessarily detect all of the analysis. A 10 µL sample was injected into LC/MS drug metabolites being investigated. Additional detection system. A glyburide sample prepared at t=0 minute was • Mass Frontier
methods including precursor ion and neutral loss scanning used as a control. are often required, resulting in multiple LC-MS/MS injec- tions. This experimental multiplicity is both time and sample consuming. A better approach is to use a mass spectrom- LC System: Surveyor Plus HPLC System eter that delivers fast cycle time, high sensitivity and high Column: Hypersil GOLD™ quality MSn spectra such as the LXQ so that all the (20 × 2.1 mm, 1.9 µm particle size) structural information about potential metabolites can Mobile phase: (A) water with 0.1% formic acid (B) acetonitrile with 0.1% formic acid be collected in one run. Flow rate: 300 µL/min Glyburide is a potent sulfonylurea drug and has been Injection volume: 10 µL used widely in the treatment of non-insulin-dependentdiabetes mellitus for more than 25 years.1-2 The metabolite analysis of glyburide has been carried out using a number of LC-MS/MS approaches, and hydroxylation has been found to be the major metabolic pathway in most cases.3-6 In addition, other metabolites have also been identified.
A previous report using a quadrupole/trap hybrid mass spectrometer claimed to be able to identify 14 metabolites of glyburide incubated with human liver microsomes.
Multiple separate LC-MS/MS runs of 45 minutes eachwere required, which is not an ideal approach for high Mass Spectrometer
throughput metabolic profiling, particularly if sample The LXQ linear ion trap mass spectrometer was operated and time are limited. In this report, a rapid LC-MS/MS in positive electrospray mode. The electrospray voltage method was developed for analyzing glyburide and its was 5 kV. The capillary temperature was 275 °C, and metabolites in a single run of approximately three minutes the sheath gas flow was 25 units. An isolation width of 2 Da was used with a 30 ms activation time for MS/MS The rapid LC-MS/MS method made use of Data- experiments. All scan events were acquired with one micro Dependent™ acquisitions to study known metabolites and scan. Full scan MS spectra and Data Dependent MS/MS to uncover unexpected metabolites. In addition, data spectra were acquired with a 50 ms and 200 ms maximum analysis was facilitated by MetWorks and Mass Frontier ionization time respectively. software that enhanced the screening and characterizationof metabolites in complex matrices. Results and Discussions
Glyburide LC-MS/MS data was processed with The MS method workflow is demonstrated in Figure 1.
MetWorks software, which has an intuitive, user-friendly The m/z values of the parent drug and the predicted workflow as shown in Figure 2a. The embedded algorithm metabolites were put into the parent mass list so that the automatically searches all the possible metabolites based MS/MS analysis was preferentially performed on these on the modifications specified by the user and generates ions. This list ensured that specific metabolites were the final report showing what metabolites have been examined in a similar fashion to SRM or MRM experi- detected. For glyburide, component detection results ments. In addition, if none of the ions in the mass list in a range of metabolites that are shown in Figure 2b.
were observed, the LXQ automatically performed MS/MS The components found in both sample and control are analysis on the most abundant ion(s) in the MS survey listed in the columns on the left with their chromatograms scan, thus ensuring the analysis of unpredicted species that shown on the right. The top panel showed all the compo- could also be metabolites. nents found in the sample, while the middle panel showed those found in the control. The differentialcomponent detection results are shown in Full Scan MS
the bottom panel where all the identifiedcomponents are marked with a greentriangle. By clicking on the triangles, thecorresponding MS or MSn spectra would appear in the two spectrum placeholders Found ions in the list
at the bottom of the page. The componentdetection software automatically searched MS/MS on the
most abundant ions
all biotransformations and screened thereal metabolites from false positives.
MS/MS scan
Figure 1: Schematic of MS method setup using precursor ion inclusion list Load the acquired RAW files Specify the analyte (s) Define the expected modifications Define the user traces Configure the processing anddisplay parameters Process the data; review the results and confirmthe candidate selection in multiple views Generate the summary reports Figure 2: Using MetWorks for metabolic profiling data analysis
(a) Seven-step procedure of MetWorks;
(b) Component detection results of glyburide data using MetWorks
Mass Frontier software was used for structural identi- identities of four unpredicted metabolites were confirmed fication of the glyburide metabolites. Its database searching by using Mass Frontier to generate possible fragments and capabilities can be coupled to a fragment prediction module match against the MS/MS spectra (Figure 4). The bio- for accurate structure characterization from MSn data.
transformation pathways that produced these previously This predictive fragmentation is crucial for identifying unidentified metabolites include O-dealkylation (480, drugs and their metabolites. Another degree of confirma- M-14), loss of cyclohexyl moiety by N-dealkylation (412, tion in Mass Frontier software is the ability to do easy M-82), amide hydroxylation (369, M-125) and amide searches of a target component. For the glyburide parent hydroxylation plus ethyl hydroxylation (385, M-109).
drug, a search was performed against an in-house library Note that the ions 480, 369 and 385 are not reported in using Mass Frontier, and the top hit was the correct commonly cited literature on the metabolism of glyburide.
compound (Figure 3a). More interestingly, the spectra With the aid of Mass Frontier, the major fragments for comparison feature embedded in Mass Frontier provides all four metabolites were assigned with structures.
a handy tool to determine the possible sites of biotrans- The differentiation of isobaric metabolites was formations. For example, the MS/MS spectrum of one accomplished with the MS/MS spectra. For example, the metabolite [M+16+H]+ was compared to that of the extracted ion chromatogram of m/z 492 indicated that parent drug [M+H]+ (Figure 3b), and the comparison of there were four dehydrogenation metabolites, and the fragmentation patterns readily pointed out that hydroxy- challenge was to determine the location of the biotrans- lation occurred on the ethyl chain. formations (Figure 5a). The MS/MS spectra of the four Enhanced confidence in the structure identification of dehydrogenation metabolites are depicted in Figure 5(b) unknowns was achieved by combining a library search to (e). The metabolites eluting at 1.33 min and 2.10 min with the chromatographic elution time of the unknown have similar MS/MS spectra as shown in Figure 5(b) and and the mass of the precursor ion. As an example, the (c). The major fragments, m/z 393 and 367 indicated that Figure 3: Application of Mass Frontier for metabolism study of glyburide
(a) Library search results for glyburide; (b) Spectra comparison for identification of hydroxylated metabolites
dehydrogenation occurred at the left side of cyclohexyl Figure 5(d) and (e) depicted the MS/MS spectra for the moiety, and m/z 169 indicated that the dehydrogenation metabolites eluting at 1.58 min and 2.32 min. The major occurred at the right side of benzyl ring. Therefore, the fragments ions at m/z 395 and 369 indicated that the biotransformation very likely occurred on the ethyl group.
transformation occurred on the cyclohexyl ring. (a) O-Dealkylation Metabolite
(b) Ring Loss Metabolite
Relative Abundance Relative Abundance (c) Amide Hydroxylation Metabolite
(d) Amide Hydroxylation + Ethyl Hydroxylation Metabolite
Relative Abundance Relative Abundance Figure 4: MS/MS Spectra of four unpredicted metabolites. (a) MS/MS spectrum of O-dealkylation glyburide (m/z 480); (b) MS/MS spectrum of ring loss
metabolite (m/z 412); (c) MS/MS spectrum of metabolite by amide hydroxylation (m/z 369); (d) MS/MS spectrum of metabolite by amide hydroxylation +
ethyl hydroxylation (m/z 385).
Figure 5: Identification and characterization of dehydrogenated metabolites (m/z 492)
(a) Extracted ion chromatogram (EIC) of 492 in MS; (b)–(e) MS/MS spectra of 492
Glyburide, and 19 metabolites, were identified and tation patterns with a 2 amu shift. Considering the fact characterized in approximately three minutes. The extracted that there is one chlorine in glyburide, it is safe to conclude ion chromatograms of the parent drug and metabolites are that m/z 414 is an isotopic peak of m/z 412 (Figure 7a).
depicted in Figure 6a, and a summary table is shown in However, this ion was mistakenly identified as a unique Figure 6b. Six mono-oxidation metabolites (m/z 510, metabolite using a quadrupole/trap hybrid mass spectrom- M+16), five di-oxidation metabolites (m/z 526, M+32), eter where two different scan methods were required to and four dehydrogenation metabolites (m/z 492, M-2) detect these two ions.6 Furthermore, the application of were identified. In addition, four unpredicted metabolites MetWorks readily eliminates degradation products from were also found. These metabolites were all identified from the metabolite candidates. As an example, two species, their retention time, precursor ion m/z and MS/MS spectra.
formed from: (a) dehalogenation and oxidation (476, M-18) In addition to a more comprehensive identification of and (b) cyclohexyl loss followed by oxidation (427, M-67) the metabolites, the study with the LXQ enabled elimina- were previously reported6 as metabolites. However, both tion of false positives. For example, two ions, m/z 412 and species were observed in the control with similar abundance m/z 414 were detected at the same retention time with an as in the incubation sample. In addition, their MS/MS approximate 3:1 intensity ratio. The LXQ acquired MS/MS spectra are identical. Therefore MetWorks automatically spectra of both of them, and they share the same fragmen- eliminates them from the possible metabolite list (Figure 7b).
Drug and Metabolites Figure 6: Summary of identification and Parent Drug (494) characterization of glyburide with its 19metabolites Hydroxylation (510) (a) Extracted ion chromatograms of parent
Di-Hydroxylation (526) drug and metabolites;
(b) Summary of metabolic profiling results
Dehydrogenation (492) O-Dealkylation (480) Amide hydroxylation (369) Amide hydroxylation + Ethyl hydroxylation (385) Total Number of Metabolites
In addition to these offices, Thermo Electron Corporation maintains EIC of 412
a network of represen- NL: 6.6e4
tative organizations throughout the world.
Relative Abundance EIC of 414
+61 2 8844 9500
Relative Abundance 20 Belgium
+32 2 482 30 30
+86 10 5850 3588
m/z 476: dehalogenation and oxidation
m/z 427: cyclohexyl loss followed by oxidation
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Figure 7. Elimination of false positive (a) Identification of isotopic peak (m/z 414); (b) Identification of degradation products m/z 476 and m/z 427 using MetWorks
+44 1442 233555
A high throughput LC/MS/MS approach has been developed The authors would like to thank D. Murphy, Dr. T. McClure, and T. McLaughlin for helpful discussions and suggestions. for metabolic profiling using the LXQ linear ion trap massspectrometer. The identification and characterization of 2006 Thermo Electron glyburide along with 19 metabolites was accomplished in a Corporation. All rights 1 Dollery, C. Therapeutic Drugs, Churchill Livingstone, New York, NY, reserved. Mass Frontier is a single LC run of approximately three minutes using the fast 1991, pp. G21-26.
trademark of HighChem, Ltd.
All other trademarks are the cycle time, high sensitivity, and excellent spectral quality of 2 Kaiser, D.G. and Forist, A.A. in Micronase: Pharmacological and Clinical property of Thermo Electron this instrument. Compared to previous literature reports, Evaluation, Excerpta Medica Foundation International Congress, 1975, Corporation and its subsidiaries.
this approach provided a more comprehensive identification 382, Princeton, NJ, pp. 31-41 W. Rifkin, H. et. al edit.
Specifications, terms andpricing are subject to change.
of metabolites and eliminated false positives. The use of 3 Schaefer, W.H., Murphy, D.M.; Sozio, R.; Ayrton, A.; Chenery, R.; Not all products are available Tiller, P.R.; Land, A.P. 45th ASMS Conf., Palm Springs, June 1-5, 1997.
in all countries. Please consult MetWorks and Mass Frontier software enabled rapid and your local sales representative 4 Tiller, P.R.; Land, A.P.; Jardine, I.; Murphy, D.M.; Sozio, R.; Ayrton, A.; confident analysis of complicated metabolic profiling data. for details.
Schaefer, W.H. J. Chromatogr. A. 1998, 794 (1-2), 15-25.
5 Zhang, H.; Henion, J.; Yang, Y.; Spooner, N. Anal. Chem. 2000, 72,
Thermo Finnigan LLC, 6 Jones, E.; Du, A.; Basa, L.; Impey, G. 51st ASMS Conf., Montréal, QC, San Jose, CA USA is ISO Certified.
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Microsoft word - ampa antipsychotics biol psychiatry v01.doc

(submitted November 10, 2005) Antipsychotic drugs reverse the AMPA receptor-stimulated release of 5-HT in the medial prefrontal cortex Mercè Amargós-Bosch. Albert Adell and Francesc Artigas Department of Neurochemistry and Neuropharmacology, Institut d' Investigacions Biomèdiques de Barcelona (CSIC), IDIBAPS, 08036 Barcelona, Spain