What is a Tune Report and Why is it important in GC-MS work?

Would Eric Clapton ever play a concert or for a recording on an out-of-tune guitar? Of course not.

The tune on a GC-MS instrument is as important if not more than the tune on Eric Clapton’s guitar. When the result matters, Clapton makes sure that his guitar is in tune prior to preforming so that he has confidence that the result of his using his instrument. When the results matter in a criminal case (which is in every case), and a GC-MS instrument is used, the GC-MS instrument must likewise be proven to be in tune or the results may be compromised.

Background

We have written on this blog a lot about the Gas Chromatography with Mass Spectrometer instrument because it truly is the workhorse in forensic science. You can see our main post on how it works here:

How does a GC-MS machine know that there’s a drug in the blood or urine?

And we have an entire category devoted to this great piece of technology here:

GC-MS posts

In all sincerity I state emphatically that is one great instrument. When I write about GC-MS, I am referring to the classic single quadrupole device. I think it is just scientifically the cat’s pajamas. In fact, in a laboratory, the GC-MS device and the LC-MS-MS are by far my favorite devices to operate, maintain, and run. When the 5 Q’s [i.e., Design Qualification (DQ), Installation Qualification (IQ), Operational Qualification (OQ), scheduled repeat OQ, Performance Qualification (PQ), and Re-Qualification after Repair (RQ). See more about the 5 Q’s in this post “A Forensic Measurement Device is Not an X Box”] of putting a GC-MS into service are followed, it can be a very powerful device of discovery. There is a reason it has earned its reputation as “the gold standard.” Sadly, not many Quality Assurance Managers or Quality Risk Management (QRM) officers in forensic laboratories have ever heard about the 5 Q’s. True equipment qualification, which is required in the pharmaceutical field, is lacking in all but a few laboratories in forensic science.

So, in a GC-MS context with or without adherence to the 5 Q’s, how can we all be sure that we have good quality results?

The sad truth is without adherence to the 5 Q’s, you may not have a valid result… ever.

One of the 5 Q’s is PQ or Performance Qualification.PQ refers to the test or validation protocol carried out by the user, offering documentary evidence that the instrument is maintaining the agreed values.

In the GC-MS world, a big part of any sort of validated instructions (sometimes called Standard Operating Procedures or SOPs) is the tune of the Mass Spectrometer.

Is the tune and the tune report evidence that the system is working well and will develop valid results every time?

No. The tune of the MS is a check of the MS system only. It is vital to note that if an instrument passes the tune and is functional, it does not mean the the Gas Chromatograph is working appropriately. The tune compound is directly injected into the MS and does not go through the GC system at all. While a tune is important, it is one part out of many that concerns quality control (QC). Proper QC is only a small part of what comprises a valid result.

What is a tune and why is it important to tune the MS? Why do we need to perform a tune on the MS?

The power of the GC-MS system is its multiple points of information that it gathers. From the GC, we get the retention time. From the MS we get ions and a spectrum. The key to the qualitative identification is in the ability to take the retention time, and combine it with the fragmentation pattern that results in the spectrum and compare it against a library of results from Certified Reference Materials that have been tested on that instrument in that environment to come up with some metric of similarity, whether it be a match factor, quality score, probability score or something similar. As this is a function of comparison between a standard and an unknown, the key then is in the reproducibility of the results. If the results are not reproducible, then the computerized library qualitative identification may be wrong or the metric used to determine similarity will be skewed. So this is why we need a means to assure that the fragmentation pattern is performing as it should. We use a compound, usually PFTBA (perfluorotributyl-amine), that is directly injected into the MS device. PFTBA is almost the universal choice in tune compounds because it is a very stable compound with a very well known and uniform fragmentation pattern. It also has masses that clearly fragment at low, medium and high ranges.

A comprehensive tune of the MS evaluates the RF/DC frequency at several masses, relative abundances, isotopic ratios, the Electron Multiplier voltage, various temperatures and pressures as well as for leaks in the system. If needed it refocuses the lens or makes adjustments in these variables to tune it correctly so that the results are reproducible. At the end of the tune, a tune report and maybe a tune evaluation is printed out. These tune reports and evaluations must be kept to prove adherence to the QC protocol and tracked over time to evaluate changes in the system.

How frequently should a tune be performed?

When results matter and critical decisions are made, the QC system has to be comprehensive and frequent. A frequent QC regimen that is robust reduces the risk of being wrong.

A good guide into best practices for MS work can be found in “Best Practice Guide for Generating Mass Spctra” by Vicki Barwick, John Langley, Tony Mallet, Bridget Stein, and Ken Webb. This was a consensus document produced by mass spectrometers that are part of the UK’s Department of Trade and Industry’s VAM Programme which forms part of the UK National Measurement System. According to the authors:

The Guide arose from discussions held at the VAM Mass Spectrometry Working Group and was prepared by LGC in collaboration with the members. In addition to major contributions by the authors, other member s of the Working Group provided suggestions and comments. The idea for this work came about during preparation of an earlier guidance document concerning accurate mass (“AccMass”) applications of mass spectrometry. It became clear that users of mass spectrometry instrumentation or services, including both specialists and research chemists, frequently have little understanding of the instrumentation or the meaning of the spectra they produce. Often, they will obtain or request an accurate mass determination for confirmation of identity on the basis of spectra which are meaningless or which could not possibly have originated from the target molecule. Discussion of this problem highlighted the changes which have taken place in teaching chemistry and analytical science and the rapid expansion in the application of mass spectrometry. The latter has been fueled by a number of factors, including advances in the automation and performance of instrumentation and recent rapid growth in the use of mass spectrometry for the biosciences. The outcome has been widespread use of complex instrumentation, often as a “walk up” service, by staff with little education or training relevant to the task. The main aim of the Guide is to enable those unfamiliar with mass spectrometry to generate mass spectra that are fit for purpose, primarily for qualitative analysis of small molecules. We have done this by providing a clear and concise summary of the essential steps in obtaining reliable spectra.

Later in the Guide, we find the following outline of the essential steps in obtaining reliable spectra:

5.1 General sequence

• The general sequence of actions when acquiring a mass spectrum is as follows: tune instrument, mass calibrate, acquire a background spectrum, analyse a test compound, analyse the sample. This sequence requires a number of essential checks on aspects of instrument performance before acquiring a mass spectrum, to ensure that spectra obtained for samples will be of acceptable quality. Initial instrument performance should be checked by acquiring the mass spectrum of a test compound using a defined protocol.

So in short, a tune must be performed (along with other aspects of QC) every single time before a batch is run. Best practices dictate a bracketed approach whereby a tune is run before a batch and then after. We evaluate the two tunes and look for meaningful differences. If there are none, then we can infer that the instrument was in tune during the batch testing. If there are meaningful differences, then we can infer that there may have been an error during the analysis and the validity of all results are compromised.

The laboratory says that it did a tune, is that good enough?

Not all tunes are alike. There are three major types: (1) Quicktune, (2) Autotune, and (3) Standard tune. A Quicktune only examines the Electron Multiplier and the peak width. No lens corrections are made. It can be thought of as an act of verification, not a true robust tune. A Standard tune does all of the acts that the Quicktune does, but it adds in standard response values over the mass range. The Autotune performs a total system verification and adjustment if needed. It is the most comprehensive of the three. It is the autotune that should be performed and evaluated every day as well as evaluated over time. And even beyond the simple fact that a tune was performed, is the major question of is this particular tune any good? Unlike courthouse and courtroom folklore, the MS will operate if it is out of tune. A good number of GC-MS operators that I have encountered have no idea how to interpret an autotune report or know what acceptable values are.
In conclusion, don’t let your crime laboratory be like this guy:

and have them try to tell your jury that it is like Eric Clapton and/or Kirk Hammett and/or some really talented guitar player who is in tune.

Tuning matters.