When very large numbers of MRM transitions are monitored in a single acquisition method, the time spent acquiring data on a single analyte drops significantly. Thus, it is typical to schedule the acquisition of MRM transitions only around their time of elution, such that fewer MRM transitions are monitored concurrently across the gradient. This is done in both Analyst software and SCIEX OS software using the Scheduled MRM algorithm. Using knowledge of the elution time of each analyte, each MRM transition is monitored only during a short retention time window. This allows many more MRM transitions to be monitored in a single LC run, while still maintaining maximized dwell times and optimized cycle times.1
Here is the MRM table for the Scheduled MRM algorithm in SCIEX OS software. To activate, first click the Apply scan schedule to expose the additional columns. The Retention Time (min) is determined from previous LC-MS runs. The Retention time tolerance can be computed for each analyte based on the peak width at base and the retention time variance observed in replicate injections. The algorithm will then compute and maximize the dwell time to be used for each analyte while maintaining the target cycle time specified by the user. Note that the user can modify the dwell time for individual analytes by clicking the Edit dwell time box in the analyte row and then manually entering a dwell time.
The table of information can be either pasted or imported into the SCIEX OS software Mass Table. By selecting Import compounds from a file… from the Import menu, you can select a *.csv file that you have previously created. The format of the *.csv file must match the columns exposed in the user interface, in the correct order, as shown. Typically it will be 11 columns, as shown above. Enter the value of FALSE for the Edit dwell time column and enter a value of 1 in the Dwell time column (even though the software will recompute this). Finally, make sure the header row is removed and save the file as a *.csv file. Close the file.
Once the method is built, the user can evaluate the dwell times that will be used across the method by clicking on the sMRM Summary to open the sMRM Plots. This tool displays a set of plots that helps the user optimize the sMRM method. Method Overview provides a view of all the MRM transitions in the method plotted as Q1 m/z vs. Time. The Concurrency of MRM transitions across the gradient (# of MRMs per cycle) is also plotted vs. retention time. Next, the optimized Cycle Time and computed Dwell Times for each MRM are plotted vs. retention time as well. The user can then adjust the target cycle time or adjust the retention time tolerances for the analytes to further optimize the method.
You can zoom in on a region of each plot by dragging in a diagonal direction on the plot. This opens the zoom sliders for both axes on the plot. Click the arrows at either end to move the zoom, then close the zoom by clicking the circle at the end of each slider.
Also, you can hover over the dots in each plot to see which analyte it represents, along with the Compound ID and the values from the x-axis and y-axis (in the above example from the dwell time plot, you can see the retention time and the dwell time). By clicking the blue icon in the top right corner, the plot graphic can be exported. Finally, you can also select a row on the table to show a vertical line where the selected analyte point is on each plot.
- The Scheduled MRM algorithm Pro – automated intelligent design of high throughput, high quality quantitation assays. SCIEX technical note RUO-MKT-02-8539-A.