Christie Hunter


How does automatic calibration work on the QTOF, TripleTOF and ZenoTOF systems?

When you think about calibration of a mass spectrometer, there are actually two aspects to consider. There is the accuracy of the calibration (that it is correct when calibration is performed) and the stability or precision of the mass calibration (that it remains correct over time).

For calibration precision or stability in quadrupole Time of Flight (QTOF) instruments, it is critical that your instrument is thermally stable and in a room with good environmental controls.  Mass spectrometers use many different high voltage power supplies which take time to “warm up” and TOF instruments require stable temperature for stable mass calibration. Avoid placing your system directly under the HVAC vent or near large windows with exposure to direct sunlight. For highest overall mass accuracy performance, you always want to make sure you keep the covers on your instrument and that the instrument has had sufficient time to warm up after the instrument has been turned off and pumped down before performing mass calibration.

For calibration accuracy, we have two automatic ways to calibrate the instrument.

  • For higher flow applications, typically we use the Calibration Delivery System (CDS).
    • On TripleTOF systems, this is an external device that works with the DuoSpray source and the APCI probe to deliver the calibrant solution. On the X500 series instruments and the ZenoTOF 7600 system, the TwinSprayer electrodes are used with the Turbo V source to the solution and the CDS system is integrated in the MS system. Additionally on the ZenoTOF 7600 system operating in microflow LC, the calibration solution can be delivered using an additional low flow probe.
    • From the introduced calibrant solution, we can perform the automatic calibration using infusion periodically during the batch and the calibration of the system will be automatically updated. There are optimized standard solutions to use for calibration in both positive and negative mode:
      • For TripleTOF systems, APCI Positive Calibration Solution P/N 4460131 and APCI Negative Calibration Solution P/N 4460134).
      • For the X500 QTOF systems and the ZenoTOF 7600 system, use
    • Note that for the CDS Calibration to work automatically within a batch the instrument mass accuracy has to be within ±100ppm and 100% of the peaks selected in the reference table must be found. If these criteria are not met, you’ll see the calibration failure symbol in the acquisition queue.
  • Alternatively, an LC AutoCalibration can be performed. Here a calibration sample is injected by LC-MS regularly during the batch. Peaks are detected and once the final peak elutes, the new mass calibration is computed and applied. This is typically used for low flow chromatography applications like nanoflow proteomics. Note that for the LC AutoCalibration to be completed successfully the instrument mass accuracy has to be within ±100ppm and 80% of specified peaks must be found. There are also retention time and minimum intensity criteria that must be met. If these criteria are not met, you’ll see the calibration failure symbol in the acquisition queue. There is a new peptide standard (PepCalMix) available for running LC calibration and information on peptides masses can be found in the documentation provided with the kit.

In either strategy, both MS and MS/MS modes are monitored and recalibrated. For more detailed information on calibration please refer to the Mass Calibration Tutorial for Analyst software that can be found C:\Program Files\Analyst\Help\Software Guides\Mass Calibration Tutorial.pdf.

In addition to all of the engineering work that goes into making SCIEX QTOF systems extremely mass stable, there are also two functions that run continuously behind the scenes and correct for any micro-fluctuations: Dynamic Background Calibration (DBC); and Inter-Sample Calibration.

With DBC, the background ions present in the MS data are constantly monitored and the calibration equation for the instrument is adjusted every 0.8 secs  to ensure these background ions stay at a constant m/z.  These ions are not fixed; they are determined dynamically for every run and for every sample and are constantly updated. This means that a separate calibrant does not need to be added during the LC-MS run to maintain a stable calibration, which simplifies instrument acquisition. Note that for DBC to be functional, a TOF MS experiment needs to be included in the method.

With inter-sample calibration, the software assesses the first 30 cycles of MS data of the previous file and determines the background ions. Then this is compared to the first 30 cycles of the next file and the calibration is adjusted to account for any drift between runs.

Note that in both cases the adjustments that are made to the calibration based on the MS data are automatically translated to the MS/MS calibration to ensure all acquisition modes are maintained in calibration. For MS/MS, only the acquisition mode (high sensitivity vs high resolution) that was monitored during the calibration runs or the previous files will be re-calibrated.



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