Building Agilent GC/MSD Deconvolution Reporting Libraries for Any Application Technical Overview
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1 Building Agilent GC/MSD Deconvolution Reporting Libraries for Any Application Technical Overview Authors Xiaofei Ping Agilent Technologies (Shanghai) Co Ltd 412 Yinglun Road, Shanghai, P.R. China Chin-Kai Meng and Michael Szelewski Agilent Technologies, Inc Centerville Road Wilmington, DE USA Abstract This technical note describes the steps to build deconvolution reporting software (DRS) libraries for any application. The starting point is either the data file(s) for all the chemical standards or a customer-built Agilent mass spectral library (*.L). The DRS library-building process would take 3 4 hours following the steps in this technical note. Introduction Agilent Technologies developed methods and libraries to screen for 567 pesticides and suspected endocrine disrupters in a single gas chromatography/mass spectrometry (GC/MS) analysis using the techniques of retention time locking (RTL) [1, 2] and spectral deconvolution [3]. Spectral deconvolution helps to identify compounds even when they are buried under co-eluting matrix compounds. The deconvolution process is fully automated and only takes about 1 to 2 minutes for a total ion chromatogram (TIC). The process not only allows the analysts to get reliable and reproducible results fast, false positives and false negatives are also minimized. However, not all compounds are included in a commercial mass spectral library ready for deconvolution process, especially the unique compounds for a special study. In this case, the analysts need to build their own deconvolution libraries in order to take advantages of DRS for the specific analyses in their laboratories. Prerequisites GC/MSD ChemStation (G1701DA) revision D or higher Microsoft Excel NIST05 Mass Spectral Library Automatic Mass Spectral Deconvolution and Identification System (AMDIS, version 2.62) from the National Institute of Standards and Technology (NIST). This software is included on the NIST05 CD. GC/MS data file(s) for all the chemical standards acquired using GC/MSD (gas chromatography/mass selective detector)
2 This article describes the steps required to construct the needed files and libraries for DRS. (Note: oxymix is just an example used in this article to be substituted with a name relevant to your application. The operating system may affect the graphics shown.) Here is an outline for the process: Create a *.tab file (for example, oxy_original.tab) using Microsoft Excel, see reference 4. File Locations (Files types: *.d, *.tab, *.L, *.SCD, *.MSP, *.MSL, and *.CAL) Data files (*.d) in C:\MSDChem\1\DATA *.TAB, *.SCD and *.L in C:\Database *.MSP in C:\NIST05\MSSEARCH *.MSL and *.CAL in C:\NIST05\AMDIS32\LIB Build a Mass Spectral Library (MSL) (for example, oxymix.l) Convert oxymix.l to oxymix.msp (Simple Mass Spectrum) Create oxymix.msl from oxymix.msp Create oxymix.csl (Calibrated Spectral Library) from oxymix.msl Create oxymix.cal (Retention Time Calibration File) for RTL applications Use MSD-DRS Configuration program to associate oxymix.msl and oxymix.cal with oxyquant.m Please refer to Technical Note Building and Editing RTL Screener/Quant Database and Libraries [4] for further information. 2
3 Procedure for Building Databases and Libraries* Create the *.TAB File 1. Load a data file (for this example, oxymix_2.d, see Figure below) which consists of the peaks (spectra) of standard compounds for the library entries. Make sure no peaks were overlapped in the data file causing contaminated spectra. In situations where clean spectra of target compounds cannot be obtained (for example, overlapping peaks), refer to the DRS User Information section Adding Compounds to Existing AMDIS_32 Libraries for instructions to deconvolute the spectra before adding them to the library. Load the default.m, turn off library searching and reporting, and save the method under a unique name. When you complete the steps in this section, refer to Appendix A to build a quant database for this new method. *A manual procedure for building the library is also available; please refer to application note Retention Time Locking: Creating Custom Retention Time Locked Screener Libraries [5] for further information. 3
4 2. Create a *.tab file using Microsoft Excel. Refer to Table 1 for explanation of columns in the *.tab file [4]. For column F, sort all entries in *.tab file into ascending retention time (RT) order before saving, this is especially important if the library is to be created from multiple datafiles. See the Figure below. Table 1. Explanation of Columns in *.tab File Column Header A Open (column A is used in GC for RT, not used in MSD) B Name (compound name, for example isobutanol) C CAS (CAS number without dashes, for example, 12345, not ) D Molecular formula (for example, C6H14N2O, no commas, dashes, quotes, etc., must use capital letters) (Note: for chlorine, use Cl, not CL) E Molecular weight (for example, 32.04) F RT (GC/MSD, in minutes, for example, 3.33) G Open (not used) H Company ID (an unique identifier, for example, cmpd01 - more explanation on this later) I File name (complete file path for *.d acquired such as: C:\MSDChem\1\data\oxymix_2.d) The first two lines of the spreadsheet are ignored in the library-building process; however, they cannot be left blank. All information used for building libraries will start from the third line. The columns B, C, D, E, F and I are required; column H is optional but recommended. All other columns are left blank. Use one compound per row. The formula is case sensitive, only capital letters can be used. Note : Sort all entries in.tab file into ascending RT order before saving especially important if the library is to be created from multiple datafiles Only capital letters could be used for Molecular Formula 4 Use single sheet template only
5 3. Save the file in *.tab type in C:\database. Go to File Save as and choose Text (Tab delimited) (*.txt). The file name must be enclosed in as shown. Path C:\DATABASE Type in oxy_original.tab We recommend adding the word original in the tab file name so the file won t get overwritten in the future. 4. Click Yes to keep the format. 5
6 5. Close Excel, otherwise you will get an error message (Could not open file C:\database\oxy_original.tab) from MSD ChemStation. 6. Click No. Build the Library and SCD 1. Go to Enhanced Data Analysis. Load the proper method and data file. Click the Data Analysis Options icon to toggle/display the command line. On the command line type rtl_import and then click Execute. 2. Select Library and SCD and then click OK. 6
7 3. Select oxy_original.tab in C:\database, click Open. 4. Type in the Library name (such as oxymix.l - the library name is limited to eight characters) and SAV folder name (for example C:\DATABASE\oxymix.SAV), click OK. Make sure your library name is different from your tab-file name. Type in oxymix.l Type in C:\DATABASE\oxymix.SAV Enhanced DA will call each of the files in Column I of your *.tab file. It will then go to the RT you specified in Column F and create a Library entry using that spectrum and the information from Columns B, D, and E. You should see flashing chromatograms. If you fill in column H of the *.tab file, you will get a *.SAV directory when the library is built. 7
8 The figure below shows the new library, oxymix.l, oxymix.scd, oxy_original.tab files and the *.sav directory. The *.sav is a folder created in Database folder when you create a Library from a *.tab. The *.sav folder contains one *.sav file for each entry in the Library you just created as shown in the Figure below. Each *.sav file contains the spectrum of the Library entry. The *.sav file name is taken from Column H. If Column H is left blank you will get no usable *.sav files. You can rebuild the Library using the *.sav files if you lost the original data files. In the dialogue box where you entered the library name when you first created it, there is a more button. Clicking more gives you a field to specify whether you are using original data or *.sav files. 8
9 The *.scd file, found in the database folder, could be used to create Calibration Table (quantitation database) automatically; refer to Appendix A for further information. Use these steps to confirm the MSL was built successfully: 1. Type listlib on the command line and then click Execute. 2. Select the library name you just created (that is, oxymix.l), then click OK. 9
10 3. Click No to the detailed graphics question, as shown below. If all went well, you will see a list of your library entry, compound name, MW, CAS, etc as shown in the Figure below. If you see only a few lines of text at the top, the library was not built properly. Go back to repeat steps 1 to 6 (starting on page 3) carefully, especially entering the correct data file name and the data path in column I of the *.tab file. 10
11 Create *.MSP from *.L 1. Go to Windows Start, Programs, NIST Mass Spectral Database and click Lib2NIST Converter. 2. Choose the *.L library you created from previous steps (that is, oxymix.l ), select HP Lib[*.L], Text[*.SDF, *.MSP], NIST Lib[*.*] type, then click Open. 11
12 3. Select folder C:\NIST05\MSSEARCH for NIST Library and Output, select Text File [.MSP] for Output Format, highlight the library in the left panel, then click Convert. 4. The oxymix.msp file will be created in C:\NIST05\MSSEARCH, click Exit. The.MSP file will be created even though it is not displayed in the above graphic. 12
13 Convert *.MSP to *.MSL 1. As shown below, go to Windows Start, Programs, NIST Mass Spectral Database and then click AMDIS_32. While in AMDIS, pay special attention to the file extensions, that is, *.msp, *.msl, and *.csl. 2. Click File and Open to open a data file (for example, oxymix_2.d). 3. Click Library and then click Library Transfer. 13
14 4. You will be prompted with a Transfer from Library to Library window, click Files. A subsequent Files window will display. 14
15 5. In Files window, click Load Library, change the file type to *.MSP, select OXYMIX.MSP in C:\NIST05\MSSEARCH, then click Open. Back to Files window, click Create New Library, select folder C:\NIST05\AMDIS32\LIB, save files as type Target Library(*.MSL), type in oxymix.msl for File name, click OK. Path C:\NIST05\MSSEARCH Type in proper file name Select oxymix.msp Path C:\NIST05\AMDIS32\LIB 6. Click Close. 15
16 7. Highlight all compounds on left and click Transfer->. The *.MSL file is saved in C:\NIST05\AMDIS32\LIB. Highlight all compounds Convert *.MSL to *.CSL 1. Continue from previous step, click Files. 16
17 2. In Files window, click Load Library, select oxymix.msl in C:\NIST05\AMDIS32\LIB, then click Open. Back to Files window, click Create New Library, select folder C:\NIST05\AMDIS32\LIB, save files as type RI Calib Library(*.CSL), type in oxymix.csl for File name, click OK. Path C:\NIST05\AMDIS32\LIB Type in proper file name Select oxymix.msl Path C:\NIST05\AMDIS32\LIB 3. Click Close. 17
18 4. Highlight all compounds on left and click Transfer->. Highlight all compounds 5. Click Exit. The *.CSL file is saved in C:\NIST05\AMDIS32\LIB. So far, all libraries and files required by DRS were saved in C:\NIST05\AMDIS32\LIB. 18
19 Using AMDIS Interactively 1. Open AMDIS, click Analyze and then Settings shown below. 2. Click the Libraries tab, highlight Target Compound Library, and then click Select New. 19
20 Select oxymix.msl in C:\NIST05\AMDIS32\LIB and then click Open. Path C:\NIST05\AMDIS32\LIB Confirm the proper *.MSL is selected 20
21 3. Highlight RI Calibration Library and then click Select New. Select oxymix.csl in C:\NIST05\AMDIS32\LIB and then click Open. Path C:\NIST05\AMDIS32\LIB 21
22 Confirm the proper *.CSL is selected 4. Highlight RI Calibration Data, and then click Select New. 22
23 Do not pick one of the existing file names. Type in a unique name for a *.cal file to be created. Type in oxymix.cal as filename, then click Open. Path C:\NIST05\AMDIS32\LIB Type in proper file name Confirm the proper *.CAL is selected 23
24 5. Click the Identif. tab, type in Minimum Match Factor suitable for your analysis (for example, 60) and highlight RI Calibration/Performance in Type of analysis. This setting is used to create the *.CAL file. 6. Click Save. Refer to the User Information section of DRS for selecting all the proper settings. 24
25 7. Click Yes. 8. Close AMDIS. In order to perform DRS using the libraries just built, you need to associate the libraries (that is, oxymix.msl and oxymix.cal) with the quantitative method (that is, oxyquant.m). The CSL is only needed for building the CAL file. 1. Go to Windows Start, Programs, MSD Deconvolution Reporting Software, and Configuration Utility, launch the Compound Identification Configuration program. 25
26 2. Click Method Associate Settings, and then New Method Associate Settings. 3. Type in the MSD ChemStation quantitative method name (that is, oxyquant) and then click the icon. Do not add.m after the method name! Type in proper method name 26
27 4. Select oxymix.msl as AMDIS target library, and click Open. 5. Check Use RI Calibration Data, and then click the icon. 27
28 6. Select oxymix.cal as RI Calibration Data, and click Open. 7. Check Open Report and Print Report, and then click icon. 28
29 8. Select onsite.ini as AMDIS Initialization Settings File. Select this file 9. Click Add to complete the configuration. 29
30 10.Click Exit, and then Exit and Save. Note : AMDIS only recognizes and uses the onsite.ini settings file when it runs automatically. The DRS software copies the settings file specified here to onsite.ini before it deconvolutes the chromatogram Now you can use the new DRS libraries to generate reports as shown in reference 3 for your own applications. References 1. V. Giarocco, B. Quimby, and M. Klee, Retention Time Locking: Concepts and Applications, Agilent Technologies, publication E, 2. H. Prest, P. Wylie, K. Weiner, and D. Agnew, Efficient Screening for Pesticides and Endocrine Disrupters Using the 6890/5973 GC/MSD System, Agilent Technologies, publication E, 3. P. Wylie, M. Szelewski, and C.K. Meng, Comprehensive Pesticide Screening by GC/MSD Using Deconvolution Reporting Software, Agilent Technologies, publication EN, 4. Michael J. Szelewski, Kenneth R. Weiner, and Chin-Kai Meng, Building and Editing RTL/Screener/Quant Databases and Libraries, Agilent Technologies, publication EN, 5. Kenneth R. Weiner and Harry F. Prest, Retention Time Locking: Creating Custom Retention Time Locked Screener Libraries, Agilent Technologies, publication E, 30
31 Appendix A Creating Quantitation Database Automatically Using a *.SCD File The information in *.SCD file could be used to build a quantitative method automatically following steps 1 to Find qdb.mth in relevant *.M directory (for example, oxyquant.m). 2. Rename qdb.mth to qdb.mth.ori. 31
32 3. Copy oxymix.scd from the C:\database directory into relevant *.M (oxyquant.m) directory. 4. Rename oxymix.scd to qdb.mth. For More Information For more information on our products and services, visit our Web site at The resulting calibration table in the method has the Quant Ion set to the m/z of the most abundant ion in the target peak spectrum. The three Qualifying ions were set to the next three most abundant ions from the target peak spectrum. Ion ratio criteria were also set automatically. Reload the method from MSD DataAnalysis program to review the calibration table. Make sure that the CAS number is entered on page 2 for each compound - no leading zeroes, no dashes, etc. Agilent 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. Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies, Inc Printed in the USA October 3, EN
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