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.
lowest window. In the SIM data window, the cursor
performs integrations.
Scan data points
SIM data points
SIM-Scan Considerations
There are two existing application notes that are
critical to an understanding of the considerations
needed for SIM-scan methods:
Chromatographic peak time
Scan time
Figure 1.
SIM time
The SIM-Scan situation.
SIM-Scan Data
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
1. “New Approaches to the Development of GC/MS
Selected Ion Monitoring Acquisition and
Quantitation Methods,” Agilent Technologies,
publication 5988-4188EN
2. “5973 Inert Performance Electronics: Considerations for GC/MS Methods in Scan and Selected
Ion Monitoring Modes,” Agilent Technologies,
publication 5989-1574EN
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.
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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 (5988-4188EN).
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 (5988-4188EN) 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.
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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.
Figure 4.
Method selection parameters.
Select SIM ion
Figure 5.
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SIM-Scan compound quantitation database.
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
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.
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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
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).
An annoyance of SIM work in the past is shown
in Figure 8. In this case, an ion of interest is
10 scans of the peak for
a mass range >350 m/z
10 SIM scans for 10 ions in
the SIM group
600-msec wide peaks
Figure 7.
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Applying rapid SIM and scan.
Figure 8.
SIM ion offset due to baseline effects.
Figure 9.
SIM ion offset adjustment.
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Information, descriptions, and specifications in this publication are subject to change
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© Agilent Technologies, Inc. 2006
Printed in the USA
September 20, 2006
5989-5669EN