Strategies for Developing Optimal Synchronous SIM-Scan Acquisition Methods AutoSIM/Scan Setup and Rapid SIM. Technical Overview.

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1 Strategies for Developing Optimal Synchronous SIM-Scan Acquisition Methods AutoSIM/Scan Setup and Rapid SIM Technical Overview Introduction The 5975A and B series mass selective detectors (MSDs) provide the user with the ability to create MS methods that continually alternate between scanning and selected ion monitoring (SIM) acquisitions in what is known as SIM-Scan. The ability to obtain scan information and SIM information almost simultaneously throughout the chromatographic run can be a powerful tool. For example, in pesticide screening the analyst may have a list of target compounds that must be assayed at very low detection limits and is therefore conducted by SIM. To avoid missing compounds of concern that may not be included in the SIM target list, a second analysis (duplicate injection) by scan acquisition is no longer necessary since this scan acquisition can be added to the SIM method. Similarly, in some environmental analyses, certain legislated methods (for example, some U.S. EPA methods) require scan acquisition of target compounds that provides data for comparison to spectral libraries as a form of compound confirmation and simultaneously requires quantitative (scan) acquisitions of these target compounds. Because the scan quantitation is typically based on a subset of the ions in each compound s spectrum (usually three or four), the SIMscan method can provide SIM acquisition of these ions yet also generate full scan data to satisfy the library match requirements. The advantage of this approach is target compound ion ratios provided by the SIM portion of the SIM-scan acquisition will have better statistics (better reproducibility and accuracy) than a scan-only method. This results in improved detection limits, linearity, etc., while the scan portion of the SIM-Scan acquisition can be arranged to provide compound spectra of high quality. This technical overview will provide guidance on how to make these arrangements and cite the tools available in the MSD Productivity Software package G1701DA (D.03 revision) of the 5975B MSD software to assist SIM-Scan method development. SIM-Scan Function In synchronous SIM-Scan, the instrument rapidly and sequentially switches between a scan acquisition and a SIM acquisition. This is best illustrated in the diagram shown in Figure 1. Over the course of the elution of the chromatographic peak, spectra are recorded according to the arranged SIM or scan parameters. The bar beneath the peak indicates an important idea; the time spent in scan or SIM acquisition modes is not the same and is determined by the acquisition parameters in those modes. Success in SIM-Scan is determined by optimizing the time spent in either mode relative to the chromatographic peak width.

2 Figure 1. SIM-Scan Data Chromatographic peak time Scan time SIM time The SIM-Scan situation. Scan data points SIM data points Figure 2 shows the SIM-Scan data file. The upper window (2) contains the scan data and the next lower window the SIM data. Either signal can be loaded independently by the menu item File\Select Signals. In the scan data window, the cursor allows spectra to be selected and displayed in the lowest window. In the SIM data window, the cursor performs integrations. SIM-Scan Considerations There are two existing application notes that are critical to an understanding of the considerations needed for SIM-scan methods: 1. New Approaches to the Development of GC/MS Selected Ion Monitoring Acquisition and Quantitation Methods, Agilent Technologies, publication EN Inert Performance Electronics: Considerations for GC/MS Methods in Scan and Selected Ion Monitoring Modes, Agilent Technologies, publication EN It is assumed that these publications have been read and understood in the discussion that follows. If users wish to add SIM to their existing scan method or add scan to their SIM method, the final SIM-Scan method should be arranged to provide Scan data SIM data Figure 2. SIM-Scan data. 2

3 quantitative quality data from the SIM data and qualitative data from the scan signal in the vast majority of cases. The scenario of using both SIM and scan acquisitions for quantitation should immediately be understood as a less optimal approach. This is because the guiding rule of mass spectroscopy is sample all chromatographic data as slowly as possible or sample only as rapidly as necessary. The best quality data is always obtained at the lowest possible speeds because there is more opportunity to obtain good sampling of the peaks. So good qualitative data (higher quality library matches) is obtained at low speeds and the rule of thumb is four or five scan data points over the peak. The rule in SIM is generally eight to 12 scans over the peak. A refinement is worth mentioning. If you are using SIM ion areas to quantitate the peak, then 6 to <12 SIM data points will suffice. However, if quantitation by peak height is used, then eight or more are required. The situation is always checked by examining the lowest concentration standard. The objective of the SIM-scan method then is four scan data points over the peak and eight SIM data points over the peak. The MSD Productivity Software package G1701DA (D.03 revision) has some enhanced tools to assist method development. AutoSIM Method. This will present the panel shown in Figure 3 and described in detail in the previously cited publication ( EN). The key parameters from a SIM-Scan point of view are: a) The minimum time between peak and SIM ion group start. Shorter time intervals are required for target lists with many compounds. A guiding rule is to look at the chromatographic peak width at the peak base for the highest concentration standard and multiply that 1.5 to get a starting parameter. Shorten this time if there are too many compounds as this will divide the chromatogram into more SIM groups. b) The minimum number of scan or SIM cycles on a peak. Since this is a higher concentration standard then the typical analysis and the method will be a SIM-Scan method, choose a higher number like 10 or 12. This parameter will determine the ion dwell times. SIM-Scan Method Development The more detailed description of setting up SIM methods ( EN) applies here as well. In brief: 1. Apply Retention Time Locking (RTL) to your GC-MS method. RTL provides two important characteristics to your compound retention times (RTs) and your method: compound RTs become permanent and universal. Permanent meaning that they can be reproduced by your method after column maintenance or replacement; and universal meaning that compound RTs can be replicated by any similar instrument anywhere within your lab or elsewhere on the planet or beyond for that matter. 2. Under this method acquire a high concentration standard using scan. Use the resulting data file to select SIM ions for each desired target compound and add these to your quantitation database. 3. With the acquired data file loaded in Data Analysis, select under Method\Generate Note: The minimum dwell time used in the automated SIM calculations is 10 ms, while in the Instrument Control the minimum dwell time is 1 ms. Figure 3. AutoSIM-Scan setup. 4. After a panel appears to print reports, another panel will provide three options (Figure 4) for the new method: make no changes, make a SIM method, or make a SIM-Scan method. Select the SIM-Scan method option. 3

4 Figure 4. Method selection parameters. 5. The following panel asks which signal will be used for quantitation in the database. One should always select the SIM signal as the target option. This means the database will use the SIM data file to build calibration tables and generate quantitative data (Figure 5). 6. Lastly, acquire another standard and check the following: In the scan data, see if there are four cycles across the chromatographic peaks. If too many scans are across the peak, reduce the samples; if too few, increase the samples. In the SIM data, similarly check the data sampling over the chromatographic peaks. Use the SIM table Editor (Method\Edit SIM Parameters) to modify or refine the dwell times for the Select SIM ion Figure 5. SIM-Scan compound quantitation database. 4

5 groups (Figure 6). SIM ion dwell times as short as 1 msec are supported by the 5975B MSD (see below in Employing Rapid SIM ) but are accessible in Instrument Control. Frequently Asked Questions Will I lose signal in SIM-Scan? If you examine the extracted ion signals in scan and SIM for a particular ion, you should find the areas and heights comparable if you are not oversampling in either mode. This is because the same ion is being measured in both modes. What can SIM-Scan tell me? When properly configured, a SIM-Scan method will produce high-quality scan data providing good matches to library spectra. The scan peaks may look less smooth or a little rough but this improves the quality and intensity of the peaks over those that have been scanned more rapidly and show more Gaussian shapes. The SIM data will result in good ion ratio reproducibilities that can be confirmed by replicate injections. These are the criteria for a suitable method. Additionally, typical SIM-only acquisitions of prepared samples may suffer from shifting retention times, poor peak shapes, issues with rapid deterioration of signal, etc., due to the properties of the injected samples themselves; however, because SIM-Scan provides a scan acquisition to examine, problems in sample preparation that produce these effects can be rapidly and readily apprehended. Even a cursory examination of the scan data can be helpful in these cases, so the addition of the scan component to a SIM method can provide highly valuable quality control to SIM work that would otherwise be invisible to a SIM-only method. When do methods require Fast Scan and very fast SIM? Over-sampling chromatographic peaks will result in degraded spectral data in both scan and SIM modes but for very fast analysis such as with microbore capillary columns or hydrogen carrier gas, the fastest speed may be required as discussed below. Employing Rapid SIM The 5973 and 5975 series MSDs have always allowed SIM ion dwell times to be adjusted in 1-msec increments; however, the new 5975B MSD allows ion dwell times to be set as short as 1 msec. For fast chromatography, the combination of rapid scanning and very rapid SIM can be necessary. Figure 7 shows relatively narrow peaks of 600 msec (base width) acquired in SIM-Scan. Because scan cycling usually takes longer than SIM cycling, the enhanced flexibility of very rapid dwells, here 2 msec for the 10 ions in the group, allows very tight peaks to be quantitatively studied. The reader should immediately recognize that the scan peak is oversampled and know that two Edit SIM group ion dwell times to increase or reduce scans over peak Edit SIM group start times to prevent peak clipping Note: The minimum dwell time used in the Edit SIM Parameters is 5 msec. In the Instrument Control, the minimum dwell time is 1 msec. Figure 6. SIM Table editor. 5

6 opportunities exist: 1) expand the mass range being scanned with no change in the speed or 2) decrease the speed by a factor of two. To determine whether the SIM method should be similarly slowed, the lowest concentration standard should be injected. If six or more SIM cycles of each peak are obtained, quantitation by area will be good. To use peak heights, the rule of eight or more SIM cycles must be applied. In this way, all MS methods are refined. Enhanced SIM Data Analysis An annoyance of SIM work in the past is shown in Figure 8. In this case, an ion of interest is superimposed upon a rising baseline, here due to column bleed. Figure 9 shows the normalization of the SIM ions at the time of the beginning of the SIM ion group. To produce this, a separate data file is created in the data file directory: dataorig.ms. This file contains the non-normalized or original data while a new data.ms is written and automatically loaded from the data file. This adjustment aids visual evaluations of the SIM data but will not affect the results. This is a feature of the new MSD Productivity Software package G1701DA (D.03 revision). 10 scans of the peak for a mass range >350 m/z 600-msec wide peaks 10 SIM scans for 10 ions in the SIM group Figure 7. Applying rapid SIM and scan. 6

7 Figure 8. SIM ion offset due to baseline effects. Figure 9. SIM ion offset adjustment. 7

8 For More Information For more information on our products and services, visit our Web site at 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 September 20, EN