Chromatography Forum

How to develop a LC-MS/MS method for a given compound?

Some books about it?

If you know how you need to ionize it, either APCI, ESI, APPI, ect, then you would begin with infusing each target separately diluted in mobile phase. Use about 1ppm concentration to begin, if too strong dilute, if too weak use higher concentration. During infusion you do a scan from about 50amu up to about 50amu above the molecular weight of the target you are infusing. Find the molecular ion in the spectra, it should be -1amu if negative ionization or +1 if positive ionization, but could also be higher if you are getting an adduct from your mobile phase if it is ammonium or sodium or potassium, ect based. Sodium would be molecular weight + 23amu for an example of positive ionization or +23-1amu for negative ionization.

Once you find the molecular ion, you narrow the scan range to a few amu around it and optimize your ionization and MS parameters to achieve the best sensitivity.(parameters will vary depending on which instrument you have, for ABSciex it will be Declustering Potential and such). This is done on the first quadrupole. Once you have that optimized you will then lock the first quadrupole on the mass and begin to scan third quadrupole to find your daughter ions. You can vary the collision energy in the second quadrupole(collision cell, or other names) while scanning to find the best daughter ions. Once you have three daughter ions(if it forms three or more, some compounds will not) optimize your lens settings, collision energy, collision gas flow, ect to maximize the responses of the daughter ions. When finished you have the setting for that target.

Repeat above for all targets. When you have them all, you then build them into your method, this will vary with each instruments manufacturer's software. You now have the MS/MS portion developed. Now you optimize the chromatography for separation and combine it with the MS/MS settings to give you the final method. There may be a little tweaking needed on source settings such as temperature and probe alignment to maximize sensitivity at full mobile phase flows, this too will vary with instrument manufacturer. As a side note, many instrument manufacturers have some of these steps automated in their software to make it go faster, but it is always a good idea to know how to do it yourself because sometimes the automated macros do not find the best settings.

That is the very basic explanation of how to setup a method, if you can attend a class for your instrument given by the manufacturer they will go much more in depth.

Are you looking to develop the instrument conditions or the entire method from sample to result? Instrument conditions depend to some extent on what you are able to present to the instrument as a sample or sample extract. And the more extraneous stuff you can remove before getting to the LC/MS, the eaiser it it to get good LC/MS conditions, and you may reduce instrument maintanance issues.

Thanks a lot for the information,

In fact, what I'm really really more interested on is the method development regarding the optimization of the parameters of the MS/MS detector.
I know that it is almost automatic, but how to change them to improve the selectivity, for example?
What the effect of changing them? When to do it?

....

Thanks a lot for the information,

In fact, what I'm really really more interested on is the method development regarding the optimization of the parameters of the MS/MS detector.
I know that it is almost automatic, but how to change them to improve the selectivity, for example?
What the effect of changing them? When to do it?

....

After a little training from the person who installed our LCMSMS, I just began infusing a target analyte and altering the settings one at a time to see what happened. I learned to tune GC/MS the same way, just move the settings around and watch what happens. Other than ramping the electron multiplier too high you really can't hurt the instrument by playing around with the settings, the really important ones are usually locked out for service people only. If you adjust declustering potential higher and sensitivity goes up, or goes down, then you know how that adjustment affects that analyte. The affect will be different for each analyte, but somewhat similar across the board. Adjust and observe, best way to learn it.

I agree with James, put it in monitoring mode, and see whether the signal increases or decreases as you change the various settings. One thing to note though if using ESI in particular. Higher is not always better as you also want a stable spray, and by setting the various gases too high, you might get some really large spikes, combined with fairly low valleys. Using a Thermo TSQ I can monitor spray stability and watch the trend over time. It helps me to make sure I'm as consistent as possible.

You may be able to significantly improve selectivity by using an alternative daughter ion, the largest ion is not always the best choice.

There is an excellent book with good references:

Boyd, R.K.; Basic, C.; Bethem, R.A.
Trace Quantitative Analysis by Mass Spectrometry
ISBN 978-0-470-05771-1