using the acd/ms manager software with agilent 1100 series ... · tools for mass spectral...

Introduction

ACD/MS Manager software from Advanced

Chemistry Development, Inc. (ACD) offers a

powerful tool kit for sophisticated analysis of

mass spectral data. This tool kit is now available

for processing data from Agilent quadrupole and

ion trap mass spectrometer systems. Data generated

with Agilent MS instruments can be easily imported

into MS Manager. The MS Manager software

includes tools to draw structures and correlate

them with mass spectra, interpret mass spectra,

create and search mass spectral databases with

attached structures and interpreted substruc-

tures, and extract enhanced spectral data from

noisy data sets.

Using the ACD/MS ManagerSoftware with Agilent 1100 SeriesLC/MS Systems

Application Note

Bryan Miller, Christine Miller, Paul Goodley, Masahiko TakinoAgilent Technologies

Importing Agilent MS Data into ACD/MSManager Software

It is fast and easy to import data from Agilentmass spectrometers into the MS Manager soft-ware. Data from Agilent 1100 Series LC/MSDquadrupole mass spectrometers and from Agilentgas chromatograph/mass selective detector(GC/MSD) instruments can be imported directlyinto the MS Manager software.

Data from Agilent 1100 Series LC/MSD Trap iontrap mass spectrometers can be imported througha simple and rapid process of conversion toAgilent MS ChemStation format, followed byimport to MS Manager. During the conversionstep, LC/MSD Trap MSn data can be prefiltered inorder to simplify subsequent processing withinthe MS Manager software. For example, ratherthan converting an entire MS3 data set, the datacan be prefiltered to include only MS/MS or MS3 data. It is also possible to transfer individualmass spectra directly from the LC/MSD Trap dataanalysis software to the MS Manager software asASCII files.

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Using the ACD/MS Manager Software with Agilent 1100 Series LC/MS Systems

Agilent Technologies

ACD/MS Manager permits import and display ofchromatographic and spectral data from AgilentLC UV detectors. Since Agilent data files supportmultiple data types (e.g., MS and DAD), MS andUV data can be imported simultaneously. Thismakes it straightforward to compare chromato-grams and perform integrated data processing.Figure 1 shows an example of integrated displayof both MS and DAD data from an analysis of sulfa drugs.

ACD/MS Manager Tools for Mass SpectralData Analysis

Tools for structure drawing and structure-spectrumcorrelation

The ACD/MS Manager software allows the analystto draw a chemical structure, attach it to a spec-trum, and assign structures to fragments in thespectrum. Given a chemical structure, the soft-ware also has tools for spectral prediction. These

tools are compatible with positive and negativeions, singly and multiply charged ions, and proto-nated and deprotonated ions. This flexibilityensures that the MS Manager software is com-patible with data from both LC/MS and GC/MSinstrumentation.

The ChemSketch structure drawing program is acomponent of the ACD/MS Manager software. Itcan be used to draw structures, or to find neededstructures in the ACD dictionary of over 85,000structures. Structures can also be imported.Windows metafiles (*.wmf) and Molecular DesignLimited (*.mol) files are among the supportedformats.

The following example shows how easy it is todraw a chemical structure, attach it to a spec-trum, and assign structures to fragments in thespectrum. The example uses an LC/MSD analysisof four sulfa drugs. The data were collected in

Figure 1. Simultaneous display ofMS and UV chromatograms andspectra from a common Agilentdata file

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Figure 3. Structure of sulfa-dimethoxine placed in theChemSketch window. TheSpectrum button on thelower left switches back tothe Spectrum Window.

positive ion electrospray mode, with in-sourcecollision-induced dissociation (CID). First, theanalyst starts the MS Manager software andimports the data file into the Spectrum Window.After obtaining an averaged, background subtracted

spectrum for one of the peaks, sulfadimethoxine,the user starts the ChemSketch program. Afterfinding the structure for sulfadimethoxine in thedictionary (Figure 2), and placing it in the Chem-Sketch Window (Figure 3), the user clicks to go

Figure 2. The structure ofsulfadimethoxine is locatedeasily in the dictionarywithin the ACD ChemSketchprogram

back to the Spectrum Window and attaches thestructure to the spectrum (Figure 4).

Next, the analyst clicks the Assignment button toassign structures to the fragments in the spec-trum. The resulting Table of Fragments is shownin Figure 5 Scrolling down the list of fragmentsautomatically highlights the corresponding peaksin the spectrum and the correlated portions of themolecule. This makes it easy to see the structuresassociated with the major peaks in the spectrum.

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Figure 5. Part of the table of assigned fragments forsulfadimethoxine. Scrollingthrough the table highlightsthe corresponding substruc-tures and mass spectralpeaks.

Figure 4. Spectrum of sulfadimethoxine with attached structure

Figure 6 shows the Table of Fragments assignedfor the MS/MS analysis of the pseudo-molecularion (372.2) of the cancer drug tamoxifen. This fullscan MS/MS spectrum was obtained using the“SmartFrag” feature of the Agilent 1100 SeriesLC/MSD Trap. SmartFrag automatically ramps theapplied ion excitation energy between low andhigh amplitude limits during fragmentation, toensure optimal CID spectrum information contentand signal-to-noise.

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Software options provide precise control over theassignment of structures to mass spectral peaks.Figure 7 shows the Reactions Options dialog box,where one specifies the ionization type, depth offragmentation, and restrictions on the fragmenta-tion reactions. These flexible settings allow thesoftware to handle a variety of experimental con-ditions. There is also a Spectrum Options dialogbox, where one specifies limits on the mass andabundance range, or requests that the fragment



Figure 7. Ionization type,depth of fragmentation, andrestrictions on reactions canbe set when automaticallyassigning structures to massspectral peaks.

Figure 6. Assigned fragmentsfor ion trap MS/MS analysisof tamoxifen

table be simplified to include only fragmentsrelated to a specific parent or product ion. Inaddition, there is a Specific FragmentationOptions dialog box, where one specifies particularcleavages and rearrangements.

MS Manager software is useful for spectral predic-tion as well as spectral analysis. Given a molecu-lar formula, ionization mode, and number ofcharges, the software can predict the molecular

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ion region of the mass spectrum (Figure 8). This isuseful for comparing experimental molecular ionisotope cluster data with theoretical isotope clus-ter patterns.

Tools for mass spectral interpretation

The ACD/MS Manager software has a number oftools to assist with mass spectral interpretation.For example, mass spectra can be reformatted toshow the losses associated with each ion (neutralloss spectra). Spectra can be searched for peak

pairs having a fixed mass difference. The Formu-lae Generator can be used to obtain and refine alist of possible molecular formulae for a spectrum.After tentative spectral identification, the pres-ence of a substructure in the spectrum can beconfirmed.

The following example shows how some of thesetools can be applied to analysis of the MS/MSspectrum of prednisone (Figure 9). This full scanCID MS/MS spectrum was obtained using the“SmartFrag” feature of the LC/MSD Trap. It is

Figure 9. ACD/MS Manager window showing positive-ion electrospray MS/MS spectrum of prednisone.The (M + H)+ ion (359.1) was selected for this MS/MS analysis.

Figure 8. Calculated molecular ion region for sulfachlorpyridazine, based on molecular formula. Note that theM+2 isotope peak at 287 nicely shows the contributions from chlorine and sulfur.

The MS Manager software allows the analyst tosee quickly which peak pairs result from a givenmass loss. For example, Figure 11 shows a table ofpeak pairs differing in mass by 18. Scrolling downthe list of peak pairs highlights the correspondingpeaks in the spectrum.

The MS Manager software has a Formulae Genera-tor to help a scientist obtain a list of molecularion formulae. The Formulae Generator allowsrefinement of the list of possibilities using knowl-edge of the chemistry pertaining to the unknown.

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easy to use the MS Manager software to calculatethe major losses from the 359.1 precursor ion forthis spectrum. One simply clicks a couple of but-tons and enters the precursor mass. The result isshown in Figure 10. The neutral loss spectrumhelps the user to see (without using a calculator)that some of the losses include water (losses of 18,36, and 54) and water plus carbon monoxide(total loss of 46).

Figure 10. Neutral loss spectrum for prednisone

Figure 11. MS/MS spectrum of prednisone, showing table of peak pairs having a fixed mass difference of 18.Clicking on rows in the table highlights the corresponding peak pairs in the spectrum.

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Once a tentative spectral identification has beenmade, the MS Manager software can be used tosearch for a substructure in a spectrum. Forexample, an analyst tentatively identifies a spec-trum for sulfachlorpyridazine. After drawing thestructure and attaching it to the spectrum, theuser could lasso a portion of the molecule to see ifit was present in the spectrum. One might predictthat the resonance-stabilized aminophenylsulfonylgroup would form a relatively stable positive ionand lasso it (Figure 12). In Figure 13, where boththe selected substructure and the correspondingion are highlighted, one can see immediately thatthe predicted ion is present as the base peak.

Create and search mass spectral databases withattached structures and interpreted substructures

The Database Module of the ACD/SpecManagersoftware allows the scientist to create and searchspectral databases. While many library searchpackages allow effective database spectral search-

ing, the SpecManager software has advancedcapabilities that permit creating and searchingdatabases containing spectra, structures, massspectral peaks with assigned substructures,spectrum parameters, and user data. Standardlibraries such as NIST and Wiley can also besearched.

The following example shows how easy it is tocreate a database entry in a new database of envi-ronmental contaminants.

First, the analyst creates a new, empty database.The Database Module is started from the MSManager window. The user creates a new databaseand gives it a name, and sets up optional pass-words for database security.

The analyst switches back to the MS Managerwindow and imports a data file from the LC/MSDanalysis of phenylurea pesticides. The data werecollected in negative electrospray mode, with

Figure 12. Using the lassotool to select a portion of the sulfachlorpyridazinemolecule so the MS Man-ager program can check forits presence in the massspectrum

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in-source CID using alternating high and low CIDenergies within the same analytical run.

To create a database entry comprised of spectra,structures, and assigned fragments, the scientistfirst obtains a mass spectrum of one of the ana-lytes and attaches a structure, as was describedearlier. For negative ion spectra, the user canmanually assign structural fragments correspond-ing to spectral peaks, as shown in Figure 14. Thisinformation is transferred to the database withthe click of a button. Figure 15 shows the data-base entry containing the spectrum, structure,and assigned peaks.

The scientist can add user information to thisrecord by double-clicking on an empty space inthe User Data subwindow on the lower right, orright-clicking in the subwindow and choosing theNew Item command from the shortcut menu. Upto 16,000 fields of user data can be added.

Additional spectra can be appended to the record.For example, the analyst could add positive elec-trospray spectra using high and low CID energies,to complement the negative electrospray spec-trum. With the appropriate ACD software mod-ules, IR, UV-Vis, and NMR spectra can be appendedas well.

Once created, databases can be searched by spec-trum, subspectrum, chemical structure, substruc-ture, formula, molecular weight, or user data.Spectra can be searched in multiple databases(both user-created databases and standardlibraries such as NIST and Wiley) with a singlesetup. The user databases can be built from datafrom multiple instrument vendors, and can beorganized by department, project, etc. They can beexplored record-by-record, if desired, and can beupdated easily. Databases can be shared through-out a company, facilitating rapid informationexchange and time savings.



Figure 13. Verification that a substructure appears in themass spectrum. Both the sub-structure and the correspond-ing ion are highlighted.

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Figure 14. Chlorfluazuronspectrum with assignedsubstructures

Figure 15. Chlorfluazurondatabase record with table ofassigned fragments displayedin upper right

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Agilent Technologies Using the ACD/MS Manager Software with Agilent 1100 Series LC/MS Systems

The following example illustrates spectral search-ing in a user database. The spectrum to besearched is a positive electrospray MS/MS spec-trum obtained using an LC/MSD Trap. The data-base can be searched for the entire spectrum, asingle ion, or a group of ions. A dialog box alertsthe user when a match is found, and clicking OKdisplays the database window with the match, inthis case the antihypertensive labetalol (Figure16). Each database entry includes the spectrum,structure, and user information.

The analyst can search user databases by a varietyof parameters other than spectrum. For example,a scientist who has just started working with theantibiotic chlortetracycline can check whetherprior data already exists in the company databaseby searching on the chemical structure. The usercan draw or find the structure in ChemSketch andthen click the Database button to display a dialogbox to search for the structure in an open data-base (Figure 17). After finding a hit, the scientistcan examine the database record for useful infor-mation, such as spectra, analysis conditions, etc.

Figure 16. Database hitshows that the ion trapMS/MS spectrum matchesthe database spectrum of theantihypertensive Labetalol

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Tools for extraction of spectral data from noisy data sets

The ACD/MS Manager software has a powerfulalgorithm to reduce random noise and high back-ground. The algorithm, called COmponent Detec-tion Algorithm (CODA),1 was developed specificallyto extract useful data from noisy LC/MS data files. It is described in detail in an ACD applica-tion note.2

An example illustrates the use of CODA to findminor impurities in the total ion chromatogramfrom the LC/MSD analysis of salbutamol, a bron-chodilator (Figure 18). First, the user subtracts aspectrum from the leading edge of the salbutamolpeak from the entire data file, resulting in thetotal ion chromatogram in Figure 19. Next, theuser runs the CODA processing, and obtains ionchromatograms of the minor impurities in thesalbutamol (Figure 20). This example shows how

quick and easy it is to find minor componentswhich initially were in the background noise ofthe chromatogram.

The MS Manager software’s COMPARE LC/MS 3

algorithm permits identification of differencesbetween two or more LC/MS data sets. This isuseful for pinpointing degradation products,metabolites, or other minor sample differences. In the following example, the COMPARE LC/MSalgorithm is used to find differences between theLC/MSD analyses of extracts from two types ofblueberries. The extracts contain anthocyanins, afamily of flavonoid pigments. These extracts wereanalyzed using an LC/MSD in the positive electro-spray mode. Figure 21 shows total ion chro-matograms from the two analytical runs, whileFigure 21 shows the results of the COMPARELC/MS algorithm. The algorithm has automatically

Figure 17. Preparing tosearch a user databasefor a chemical structure

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Figure 18. LC/MSD analysis of salbutamol, prior to CODA processing

Figure 19. Salbutamol total ion chromatogram after subtracting salbutamol spectrum

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Figure 20. CODA processing has revealed mass chromatograms of minor impurities in salbutamol LC/MSDanalysis. The mass chromatograms are shown in color.

Figure 21. LC/MSD total ion chromatograms of extracts of anthocyanins from European blueberry (top) andLowbush blueberry (bottom)

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determined and generated a set of mass chromato-grams which highlight subtle spectral data differ-ences between the samples.

It is possible to view specific mass chromatogramsby unchecking masses in the Table of Mass Chro-matograms (shown in Figure 22). Figure 23, forexample, shows only the chromatograms for mass579. By referring back to the total ion chro-matograms in Figure 21, one can see that theCOMPARE algorithm has located sample differ-ences that were not at all obvious at the outset.The COMPARE LC/MS algorithm is discussed ingreater detail in an ACD application note.4

Conclusions

The ACD/MS Manger software provides a power-ful set of mass spectral data reduction tools—morethan can be discussed in this technical note. Theintegrated ChemSketch program makes it easy todraw a structure and attach it to a mass spectrum.Substructures can be mapped to ions in the spec-trum. The spectrum, chemical structure and inter-preted mass spectrum can be added to a userdatabase. The database can be enriched withadditional mass spectra, IR or other spectra, anda wealth of user data. The database can besearched by spectrum, subspectrum, chemicalstructure, substructure, formula, molecularweight, or user data.

Figure 23. The COMPARELC/MS algorithm identifiedthe mass 579 chromato-gram as one showing dif-ferences between extractsfrom two types of blueber-ries (European blueberryat top and Lowbush blue-berry at bottom)

Figure 22. COMPARELC/MS provides a tableof mass chromatogramsshowing differencesbetween the extractsfrom the two differenttypes of blueberries(European blueberry attop and Lowbush blue-berry at bottom). Masschromatograms areplotted in color for bothsamples.

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Other MS Manager tools include the ability toreformat mass spectra to show neutral losses, theability to find peak pairs having fixed mass differ-ences, the capability of generating tables of possi-ble molecular formulae for mass spectra, and theability to search a spectrum for a particular sub-structure. Finally, the software offers an algorithmfor extraction of useful data from noisy data sets,as well as one for comparison of two or more simi-lar LC/MS data files to find minor differences.

The tools provided in the MS Manager softwaremake it easier for less experienced scientists tointerpret their own mass spectra, and allow expe-rienced mass spectrometrists to save time in theirinterpretations. The tools for database creationand searching extend beyond the usual spectrumsearch capabilities and allow users to archive andretrieve a wealth of useful information. Databasescan be shared company-wide, saving time whenscientists are dealing with new compounds andnew analyses. The ACD software works with datafrom a number of instrument vendors and offersmodules for a number of additional spectral tech-niques (NMR, IR, UV-Vis). This reduces trainingtime and provides a common platform to analyzedata and build databases for of a variety of labora-tory data.

The Agilent LC/MSD quadrupole and ion trapsystems provide sensitivity, ruggedness, and ease-of-use. Their associated data systems providefacile instrument control, automation capabilities,and many data analysis features. Data from bothtypes of instruments can easily be imported intothe ACD software for processing which extendsbeyond the capabilities of the instrument-controldata systems. This powerful combination providesthe user with both high-quality LC/MS data andan excellent means for advanced mass spectraldata analysis.



References

1. Willem Windig, J. Martin Phalp, and Allan W.Payne. “A Noise and Background ReductionMethod for Component Detection in LiquidChromatography/Mass Spectrometry,” Anal. Chem., 1996, 68, 3602–3606.

2. Antony Williams. “An Introduction to CODA:Integration into ACD/MS Manager,” ACD/MSManager Version 5.0 for Windows ApplicationNote, http://www.acdlabs.com/appnotes/ms/,Advanced Chemistry Development, 2001.

3. Willem Windig, William F. Smith, William F.Nichols. “Fast Interpretation of ComplexLC/MS Data Using Chemometrics,” Anal.Chim. Acta., August, 2001, 446, 467–476.

4. “Using the Compare LC-MS Algorithm,”ACD/MS Manager Version 5.0 for WindowsApplication Note, http://www.acdlabs.com/appnotes/ms/, Advanced Chemistry Develop-ment, 2001.

For more information about ACD/MS Managersoftware, see http://www.acdlabs.com. For addi-tional ACD application notes, see http://www.acdlabs.com/appnotes/ms/

Authors

Bryan Miller, Christine Miller, Paul Goodley, arescientists at Agilent Technologies in Santa Clara,California U.S.A. Masahiko Takino is a scientistat Agilent Technologies in Tokyo, Japan.

www.agilent.com/chem

Copyright © 2003Agilent Technologies

Information, descriptions and specifications in this publication aresubject to change without notice. Agilent Technologies shall not be liablefor errors contained herein or for incidental or consequential damages inconnection with the furnishing, performance or use of this material.

All rights reserved. Reproduction, adaptation or translation without prior written permission is prohibited, except as allowed under thecopyright laws.

Printed in the U.S.A. March 7, 20035988-5204EN

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