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Comprehensive Guide to Reviewing GC-MS/MS Autotune Reports in EI Mode

Detailed overview of GC-MS/MS autotune report analysis using PFTBA calibration, focusing on m/z scale calibration, signal verification, contamination checks, and instrument tuning parameters.

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Reviewing the GC-MS/MS Autotune Report in EI Mode PFTBA (perfluorotributylamine) is a colorless liquid chemical compound used as a calibration standard for mass spectrometers, an electronic coolant, and a potent greenhouse gas. Chandra Prakash Singh
 Unlike other spectrometric techniques used in analytical chemistry, the abscissa of the instrument’s data output is not hard calibrated at the time of manufacture.  The m/z scale must be calibrated on a regular basis; it must be checked for accuracy at least every 8 hours of operation. In the case of instruments used for accurate mass assignments, it may be necessary to calibrate at the time the data are acquired.  The vast majority of GC/MS data are acquired at unit resolution although many of the data systems report the data to the 0.05 m/z unit. As was pointed out above, the best resolution that can be obtained with a QMF or QIT mass spectrometer is 0.3 m/z units. The scale is calibrated at integer m/z values.  Perfluorinated compounds are used for this because their ions have very little mass defect, the difference between the nominal mass and the monoisotopic mass. For example, the ion formed by fragmentation of the molecular ion of perfluorotributylamine (PFTBA) (the compound used to calibrate the m/z scale of QMF and QIT GC/MS systems) that has a nominal mass of 502 Da has a monoisotopic mass of 501.971134 Da or a mass defect of 0.03 Da.  A corresponding aliphatic ion C36H73 with a nominal mass of 505 Da would have a monoisotopic mass of 505.571225 Da or a mass defect of 0.6 Da. Most GC/MS instruments have a maximum m/z range to m/z 1,000.  The maximum m/z value with an intensity observed in the mass spectrum of PFTBA is at m/z 614. Because of the linearity of the m/z for quadrupole-based instruments, it is possible to extrapolate from m/z 614 to m/z 1,000.  PFTBA does not form a lot of fragments (m/z 50, 69, 100, 131, 164, 169, 219, 264, 414, 464, 502, and 604).  Again, the linearity in the m/z scale interpolation between these points allows for a reliable calibration. In many cases, the peak at m/z 614 is not used because the abundance of this ion is very low. m/z Scale Calibration
What It Tells You Shows you the signal for the three main ions from PFTBA that the instrument tunes on. Things to Look For 1. Check the instrument has picked the three corrections. It’s normally 69, 219 and 502 2. Check the signal intensity for each mass is in keeping with what you’d expect. These values will differ from the signals presented later in the report as the information is acquired at different times. 3. Confirm the shape of the peak is Gaussian and symmetrical. 4. Check for excessive levels of baseline noise. 5. Check for any additional extraneous masses in the region. Reviewing the GC-MS/MS Autotune Report in EI Mode - Section 1
What It Tells You Tells you which settings the instrument is using to obtain the data presented. Things to Look For 1.Some values will stay very similar in every report, some will vary considerably with every tune. Review several reports and get a feel for what ‘normal’ values your instrument typically delivers 2.Carefully check the multiplier voltage. Over time this will drift upwards as the multiplier wears. Your instrument manual or service engineer will advise when the multiplier requires replacement. Do not manually adjust the multiplier voltage. Reviewing the GC-MS/MS Autotune Report in EI Mode - Section 2
What It Tells You Shows you the full scan spectrum for your tune Things to Look For 1.Visually check that the complete tune file looks correct 2.Check that each target mass (69, 219 and 502) has been selected correctly by the instrument for tuning 3.Check the entire spectrum for unusual signals i.e. contamination. This is one indication that the source requires cleaning. 4.Carefully check the region around each target mass for any contaminating ions. The instrument could misselect a contamination ion and tune on this substance rather than the PFTBA ion. 5.Verify there is not excessive baseline noise 6.Check the signal between 17 – 45 — this is the region where ions generated by leaks are found. Reviewing the GC-MS/MS Autotune Report in EI Mode - Section 3
What It Tells You Gives the data acquired from the full scan of the PFTBA tune Things to Look For 1.Verify the three masses (69, 219 and 502) are correct and within normal tolerances typical of previous reports 2.Verify the absolute abundance (signal) values for each mass are typical. These give an indication of the level of sensitivity. The instrument will generally aim to obtain these values but may generate a noisy chromatogram. 3.Verify the relative abundances are typical. These differ significantly between instrument types but are critical in allowing the instrument to obtain representative spectra suitable for library matching 4.Ensure these C13 isotope masses are +1 on the target masses. 5.Isotope ratios. Check they are typical – they would normally be around 1, 4 and 9. These give an indication of the number of carbons present in the target mass. If the instrument has tuned on a contaminant mass, there is a good chance the isotope ratio will differ due to the contamination containing a different number of carbons to the required mass. Reviewing the GC-MS/MS Autotune Report in EI Mode - Section 4
What It Tells You The signals attributable to H2O, N2, O2 and CO2 – the substances introduced by an air leak. These are normally expressed as a ratio relative to another constant ion (e.g. 69), but this differs between instruments. Things to Look For 1.Verify that signals for these ions are below the acceptance level. Any higher and you may have a leak – usually as a result of the high vacuum pulling in air. It is common for this signal to be high if the system has been recently vented 2.A common source for a leak is the ferrule connecting the column to the transfer line. This should contain vespel, but occasionally needs tightened. 3.A high signal at 18, without the associated gas ions at 28, 32 and 44 tends to indicate contamination – either directly from water present within the GC-MS system (e.g. after cleaning), an exhausted water filter on the carrier gas line or a water fragment formed from a higher mass compound. Reviewing the GC-MS/MS Autotune Report in EI Mode - Section 5 Thanks