Using the ACD/MS Manager Software with Agilent 1100 Series LC/MS Systems Application Note Bryan Miller, Christine Miller, Paul Goodley, Masahiko Takino 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 substructures, and extract enhanced spectral data from noisy data sets. Importing Agilent MS Data into ACD/MS Manager Software It is fast and easy to import data from Agilent mass spectrometers into the MS Manager software. Data from Agilent 1100 Series LC/MSD quadrupole mass spectrometers and from Agilent gas chromatograph/mass selective detector (GC/MSD) instruments can be imported directly into the MS Manager software. Data from Agilent 1100 Series LC/MSD Trap ion trap mass spectrometers can be imported through a simple and rapid process of conversion to Agilent MS ChemStation format, followed by import to MS Manager. During the conversion step, LC/MSD Trap MS n data can be prefiltered in order to simplify subsequent processing within the MS Manager software. For example, rather than converting an entire MS 3 data set, the data can be prefiltered to include only MS/MS or MS 3 data. It is also possible to transfer individual mass spectra directly from the LC/MSD Trap data analysis software to the MS Manager software as ASCII files.
ACD/MS Manager permits import and display of chromatographic and spectral data from Agilent LC UV detectors. Since Agilent data files support multiple data types (e.g., MS and DAD), MS and UV data can be imported simultaneously. This makes it straightforward to compare chromatograms and perform integrated data processing. Figure 1 shows an example of integrated display of both MS and DAD data from an analysis of sulfa drugs. ACD/MS Manager Tools for Mass Spectral Data Analysis Tools for structure drawing and structure-spectrum correlation The ACD/MS Manager software allows the analyst to draw a chemical structure, attach it to a spectrum, and assign structures to fragments in the spectrum. Given a chemical structure, the software also has tools for spectral prediction. These tools are compatible with positive and negative ions, singly and multiply charged ions, and protonated and deprotonated ions. This flexibility ensures that the MS Manager software is compatible with data from both LC/MS and GC/MS instrumentation. The ChemSketch structure drawing program is a component of the ACD/MS Manager software. It can be used to draw structures, or to find needed structures in the ACD dictionary of over 85,000 structures. Structures can also be imported. Windows metafiles (*.wmf) and Molecular Design Limited (*.mol) files are among the supported formats. The following example shows how easy it is to draw a chemical structure, attach it to a spectrum, and assign structures to fragments in the spectrum. The example uses an LC/MSD analysis of four sulfa drugs. The data were collected in Figure 1. Simultaneous display of MS and UV chromatograms and spectra from a common Agilent data file 2
positive ion electrospray mode, with in-source collision-induced dissociation (CID). First, the analyst starts the MS Manager software and imports 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. After finding the structure for sulfadimethoxine in the dictionary (Figure 2), and placing it in the Chem- Sketch Window (Figure 3), the user clicks to go Figure 2. The structure of sulfadimethoxine is located easily in the dictionary within the ACD ChemSketch program Figure 3. Structure of sulfadimethoxine placed in the ChemSketch window. The Spectrum button on the lower left switches back to the Spectrum Window. 3
back to the Spectrum Window and attaches the structure to the spectrum (Figure 4). Next, the analyst clicks the Assignment button to assign structures to the fragments in the spectrum. The resulting Table of Fragments is shown in Figure 5 Scrolling down the list of fragments automatically highlights the corresponding peaks in the spectrum and the correlated portions of the molecule. This makes it easy to see the structures associated with the major peaks in the spectrum. Figure 6 shows the Table of Fragments assigned for the MS/MS analysis of the pseudo-molecular ion (372.2) of the cancer drug tamoxifen. This full scan MS/MS spectrum was obtained using the SmartFrag feature of the Agilent 1100 Series LC/MSD Trap. SmartFrag automatically ramps the applied ion excitation energy between low and high amplitude limits during fragmentation, to ensure optimal CID spectrum information content and signal-to-noise. Figure 4. Spectrum of sulfadimethoxine with attached structure Figure 5. Part of the table of assigned fragments for sulfadimethoxine. Scrolling through the table highlights the corresponding substructures and mass spectral peaks. 4
Figure 6. Assigned fragments for ion trap MS/MS analysis of tamoxifen Software options provide precise control over the assignment of structures to mass spectral peaks. Figure 7 shows the Reactions Options dialog box, where one specifies the ionization type, depth of fragmentation, and restrictions on the fragmentation reactions. These flexible settings allow the software to handle a variety of experimental conditions. There is also a Spectrum Options dialog box, where one specifies limits on the mass and abundance range, or requests that the fragment table be simplified to include only fragments related to a specific parent or product ion. In addition, there is a Specific Fragmentation Options dialog box, where one specifies particular cleavages and rearrangements. MS Manager software is useful for spectral prediction as well as spectral analysis. Given a molecular formula, ionization mode, and number of charges, the software can predict the molecular Figure 7. Ionization type, depth of fragmentation, and restrictions on reactions can be set when automatically assigning structures to mass spectral peaks. 5
ion region of the mass spectrum (Figure 8). This is useful for comparing experimental molecular ion isotope cluster data with theoretical isotope cluster patterns. Tools for mass spectral interpretation The ACD/MS Manager software has a number of tools to assist with mass spectral interpretation. For example, mass spectra can be reformatted to show the losses associated with each ion (neutral loss spectra). Spectra can be searched for peak pairs having a fixed mass difference. The Formulae Generator can be used to obtain and refine a list of possible molecular formulae for a spectrum. After tentative spectral identification, the presence of a substructure in the spectrum can be confirmed. The following example shows how some of these tools can be applied to analysis of the MS/MS spectrum of prednisone (Figure 9). This full scan CID MS/MS spectrum was obtained using the SmartFrag feature of the LC/MSD Trap. It is Figure 8. Calculated molecular ion region for sulfachlorpyridazine, based on molecular formula. Note that the M+2 isotope peak at 287 nicely shows the contributions from chlorine and sulfur. 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. 6
easy to use the MS Manager software to calculate the major losses from the 359.1 precursor ion for this spectrum. One simply clicks a couple of buttons and enters the precursor mass. The result is shown in Figure 10. The neutral loss spectrum helps 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). The MS Manager software allows the analyst to see quickly which peak pairs result from a given mass loss. For example, Figure 11 shows a table of peak pairs differing in mass by 18. Scrolling down the list of peak pairs highlights the corresponding peaks in the spectrum. The MS Manager software has a Formulae Generator to help a scientist obtain a list of molecular ion formulae. The Formulae Generator allows refinement of the list of possibilities using knowledge of the chemistry pertaining to the unknown. 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. 7
Once a tentative spectral identification has been made, the MS Manager software can be used to search for a substructure in a spectrum. For example, an analyst tentatively identifies a spectrum for sulfachlorpyridazine. After drawing the structure and attaching it to the spectrum, the user could lasso a portion of the molecule to see if it was present in the spectrum. One might predict that the resonance-stabilized aminophenylsulfonyl group would form a relatively stable positive ion and lasso it (Figure 12). In Figure 13, where both the selected substructure and the corresponding ion are highlighted, one can see immediately that the predicted ion is present as the base peak. Create and search mass spectral databases with attached structures and interpreted substructures The Database Module of the ACD/SpecManager software allows the scientist to create and search spectral databases. While many library search packages allow effective database spectral searching, the SpecManager software has advanced capabilities that permit creating and searching databases containing spectra, structures, mass spectral peaks with assigned substructures, spectrum parameters, and user data. Standard libraries such as NIST and Wiley can also be searched. The following example shows how easy it is to create a database entry in a new database of environmental contaminants. First, the analyst creates a new, empty database. The Database Module is started from the MS Manager window. The user creates a new database and gives it a name, and sets up optional passwords for database security. The analyst switches back to the MS Manager window and imports a data file from the LC/MSD analysis of phenylurea pesticides. The data were collected in negative electrospray mode, with Figure 12. Using the lasso tool to select a portion of the sulfachlorpyridazine molecule so the MS Manager program can check for its presence in the mass spectrum 8
in-source CID using alternating high and low CID energies within the same analytical run. To create a database entry comprised of spectra, structures, and assigned fragments, the scientist first obtains a mass spectrum of one of the analytes and attaches a structure, as was described earlier. For negative ion spectra, the user can manually assign structural fragments corresponding to spectral peaks, as shown in Figure 14. This information is transferred to the database with the click of a button. Figure 15 shows the database entry containing the spectrum, structure, and assigned peaks. The scientist can add user information to this record by double-clicking on an empty space in the User Data subwindow on the lower right, or right-clicking in the subwindow and choosing the New Item command from the shortcut menu. Up to 16,000 fields of user data can be added. Additional spectra can be appended to the record. For example, the analyst could add positive electrospray spectra using high and low CID energies, to complement the negative electrospray spectrum. With the appropriate ACD software modules, IR, UV-Vis, and NMR spectra can be appended as well. Once created, databases can be searched by spectrum, subspectrum, chemical structure, substructure, formula, molecular weight, or user data. Spectra can be searched in multiple databases (both user-created databases and standard libraries such as NIST and Wiley) with a single setup. The user databases can be built from data from multiple instrument vendors, and can be organized by department, project, etc. They can be explored record-by-record, if desired, and can be updated easily. Databases can be shared throughout a company, facilitating rapid information exchange and time savings. Figure 13. Verification that a substructure appears in the mass spectrum. Both the substructure and the corresponding ion are highlighted. 9
Figure 14. Chlorfluazuron spectrum with assigned substructures Figure 15. Chlorfluazuron database record with table of assigned fragments displayed in upper right 10
The following example illustrates spectral searching in a user database. The spectrum to be searched is a positive electrospray MS/MS spectrum obtained using an LC/MSD Trap. The database can be searched for the entire spectrum, a single ion, or a group of ions. A dialog box alerts the user when a match is found, and clicking OK displays the database window with the match, in this case the antihypertensive labetalol (Figure 16). Each database entry includes the spectrum, structure, and user information. The analyst can search user databases by a variety of parameters other than spectrum. For example, a scientist who has just started working with the antibiotic chlortetracycline can check whether prior data already exists in the company database by searching on the chemical structure. The user can draw or find the structure in ChemSketch and then click the Database button to display a dialog box to search for the structure in an open database (Figure 17). After finding a hit, the scientist can examine the database record for useful information, such as spectra, analysis conditions, etc. Figure 16. Database hit shows that the ion trap MS/MS spectrum matches the database spectrum of the antihypertensive Labetalol 11
Figure 17. Preparing to search a user database for a chemical structure Tools for extraction of spectral data from noisy data sets The ACD/MS Manager software has a powerful algorithm to reduce random noise and high background. The algorithm, called COmponent Detection Algorithm (CODA), 1 was developed specifically to extract useful data from noisy LC/MS data files. It is described in detail in an ACD application note. 2 An example illustrates the use of CODA to find minor impurities in the total ion chromatogram from the LC/MSD analysis of salbutamol, a bronchodilator (Figure 18). First, the user subtracts a spectrum from the leading edge of the salbutamol peak from the entire data file, resulting in the total ion chromatogram in Figure 19. Next, the user runs the CODA processing, and obtains ion chromatograms of the minor impurities in the salbutamol (Figure 20). This example shows how quick and easy it is to find minor components which initially were in the background noise of the chromatogram. The MS Manager software s COMPARE LC/MS 3 algorithm permits identification of differences between two or more LC/MS data sets. This is useful for pinpointing degradation products, metabolites, or other minor sample differences. In the following example, the COMPARE LC/MS algorithm is used to find differences between the LC/MSD analyses of extracts from two types of blueberries. The extracts contain anthocyanins, a family of flavonoid pigments. These extracts were analyzed using an LC/MSD in the positive electrospray mode. Figure 21 shows total ion chromatograms from the two analytical runs, while Figure 21 shows the results of the COMPARE LC/MS algorithm. The algorithm has automatically 12
Figure 18. LC/MSD analysis of salbutamol, prior to CODA processing Figure 19. Salbutamol total ion chromatogram after subtracting salbutamol spectrum 13
Figure 20. CODA processing has revealed mass chromatograms of minor impurities in salbutamol LC/MSD analysis. The mass chromatograms are shown in color. Figure 21. LC/MSD total ion chromatograms of extracts of anthocyanins from European blueberry (top) and Lowbush blueberry (bottom) 14
determined and generated a set of mass chromatograms which highlight subtle spectral data differences between the samples. It is possible to view specific mass chromatograms by unchecking masses in the Table of Mass Chromatograms (shown in Figure 22). Figure 23, for example, shows only the chromatograms for mass 579. By referring back to the total ion chromatograms in Figure 21, one can see that the COMPARE algorithm has located sample differences that were not at all obvious at the outset. The COMPARE LC/MS algorithm is discussed in greater detail in an ACD application note. 4 Conclusions The ACD/MS Manger software provides a powerful set of mass spectral data reduction tools more than can be discussed in this technical note. The integrated ChemSketch program makes it easy to draw a structure and attach it to a mass spectrum. Substructures can be mapped to ions in the spectrum. The spectrum, chemical structure and interpreted mass spectrum can be added to a user database. The database can be enriched with additional mass spectra, IR or other spectra, and a wealth of user data. The database can be searched by spectrum, subspectrum, chemical structure, substructure, formula, molecular weight, or user data. Figure 22. COMPARE LC/MS provides a table of mass chromatograms showing differences between the extracts from the two different types of blueberries (European blueberry at top and Lowbush blueberry at bottom). Mass chromatograms are plotted in color for both samples. Figure 23. The COMPARE LC/MS algorithm identified the mass 579 chromatogram as one showing differences between extracts from two types of blueberries (European blueberry at top and Lowbush blueberry at bottom) 15
Other MS Manager tools include the ability to reformat mass spectra to show neutral losses, the ability to find peak pairs having fixed mass differences, the capability of generating tables of possible molecular formulae for mass spectra, and the ability to search a spectrum for a particular substructure. Finally, the software offers an algorithm for extraction of useful data from noisy data sets, as well as one for comparison of two or more similar LC/MS data files to find minor differences. The tools provided in the MS Manager software make it easier for less experienced scientists to interpret their own mass spectra, and allow experienced mass spectrometrists to save time in their interpretations. The tools for database creation and searching extend beyond the usual spectrum search capabilities and allow users to archive and retrieve a wealth of useful information. Databases can be shared company-wide, saving time when scientists are dealing with new compounds and new analyses. The ACD software works with data from a number of instrument vendors and offers modules for a number of additional spectral techniques (NMR, IR, UV-Vis). This reduces training time and provides a common platform to analyze data and build databases for of a variety of laboratory data. The Agilent LC/MSD quadrupole and ion trap systems provide sensitivity, ruggedness, and easeof-use. Their associated data systems provide facile instrument control, automation capabilities, and many data analysis features. Data from both types of instruments can easily be imported into the ACD software for processing which extends beyond the capabilities of the instrument-control data systems. This powerful combination provides the user with both high-quality LC/MS data and an excellent means for advanced mass spectral data analysis. 16
References 1. Willem Windig, J. Martin Phalp, and Allan W. Payne. A Noise and Background Reduction Method for Component Detection in Liquid Chromatography/Mass Spectrometry, Anal. Chem., 1996, 68, 3602 3606. 2. Antony Williams. An Introduction to CODA: Integration into ACD/MS Manager, ACD/MS Manager Version 5.0 for Windows Application Note, http://www.acdlabs.com/appnotes/ms/, Advanced Chemistry Development, 2001. 3. Willem Windig, William F. Smith, William F. Nichols. Fast Interpretation of Complex LC/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 Windows Application Note, http://www.acdlabs.com/ appnotes/ms/, Advanced Chemistry Development, 2001. Authors Bryan Miller, Christine Miller, Paul Goodley, are scientists at in Santa Clara, California U.S.A. Masahiko Takino is a scientist at in Tokyo, Japan. For more information about ACD/MS Manager software, see http://www.acdlabs.com. For additional ACD application notes, see http://www. acdlabs.com/appnotes/ms/ www.agilent.com/chem Copyright 2003 Information, descriptions and specifications in this publication are subject to change without notice. shall not be liable for errors contained herein or for incidental or consequential damages in connection 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 the copyright laws. Printed in the U.S.A. March 7, 2003 5988-5204EN