Chromatography Forum

Hello

I have seen a few LC/MS threads recently, but I don't think any of them have addressed the following question.

I am wondering how "LC/high-resolution mass spec" compares with "LC/2-stage mass spec". The high resolution systems can apparently give the molecular ion peak to a resolution of 4 decimal places. This allows the elemental composition to be determined fairly easily. From there you can narrow things down to just a few possible structures.

It seems to good to be true. Apparently you can get a structure with minimal detective work (no need to strain your brain trying to interpret a complex spectra).

Anyone want to share any experiences with this. And I'm wondering how it compares to LC/MS/MS: which would seem to me to be a little bit more difficult to work with.

Thanks
Adam

Adam,

Sorry to be the one to tell you this, but....yes it is too good to be true !!!

Accurate mass from a high resolution MS gives you ONLY a choice of several (to several dozen or hundred, depending on MW) elemental compositions that match your measured accurate mass. Usually, only in the simplest cases can you deduce structure.

Two-stage MS (MS/MS or tandem MS), which leads to fragmentation of the selected (usually molecular) ion will give you structural information.

If you have the $$ choose an instrument that can do both, otherwise go for MS/MS.

JMB

The number of possible formulae depend on the accuracy and precision of the instrument. Usually the lower the MW, the fewer the possibilities. Also can use sample history and isotope ratios to minimize possibilities.

Another approach is to use in-source fragmentation instead of MS/MS. We have a TOF MS (single stage MS system, Micromass LCT TOF) and use in-source fragmentation to get substructural information. Thus the instrument does "pseudo MS/MS" and accurate mass. Accurate mass can be done on the parent ion and the fragments.

Normally run five experiments in one LC separation. One function is low energy in source for MW in positive ion, another at higher energy for fragments in positive ion. The third a low energy at negative for MW, the fourth at higher negative energy for substructural fragments. The last is the diode array. Thus can get a lot of information in one LC injection.

In-source depends on doing the separation of the components on the column since not multistage separation with two mass specs in tandem. Also, some molecules can give solvent adducts with the parent molecule and with the fragment ions. Acetonitrile seems to be worst than methanol in electrospray positive ion mode.

Need to be aware of the limitation of dead time correction on TDC type TOF instruments. We have some info in accurate mass section at http://users.chartertn.net/slittle. A ADC system discussion can be found at http://www.agilent-biomarkers.com/docs/pro_3.pdf and is compared to TDC type system. I have heard in the past that the TDC is more sensitive than ADC, but the former less dynamic range. Would just have to look at an individual systems performance to determine which is best approach.

Thanks for the feedback.

I have been hearing more and more about the time of flight in-source fragmentation option. It would be very interesting to get my hands on some papers that talk about the uses and limitation of this technology.

For example, I wonder if it can be used for bioanalysis instead of "true" 2-stage mass spec.

Any good reading material on this topic?

Thanks
Adam

page 11 of

http://littledomain.com/james/files/pit ... raries.pdf

gives some references in a document I wrote.

We routinely used multi function approach, examples of use in ID of surfactants noted in

http://littledomain.com/james/files/pit ... _point.ppt

I agree with everything that have been said, I'll just add my two cents from some recent developments on the field...

On top of the mass accuracy vs. MW of the analytes, it has been demonstrated that if you take in to account the isotopic abundance patterns as an additional filter you can really narrow down your possibiliites. For example a 3 ppm mass accuracy and 2% error for isotopic abundance pateterns outperforms mass spectrometers with less than 0.1 ppm (actually none of the mass spectrometers can achieve 0.1 ppm under LC-MS condidtions, only under infusion... the best that can be achieved in LC-MS is about 1 ppm for the high end FT-MS instruments). For example for MW up to 900, 5ppm mass accuracy gives you 1712 molecular choices, 0.1 ppm mass accuracy gives you 32 and 3 ppm mass accuracy and 2 % isotopic abundance accuracy gives you only 18 choices... (see reference at the end...)

Another dimension of discrimination that I foresee in the future is ion-mobility (Waters introduced an instrument with their T-Wave technology which is actually a relatively low resolution IMS) as their flying time can be predicted with high accuracy as it is directly proposional in the "molecular volume".

A third dimension of discrimination could be retention time in C18 although it is quite difficult to predict retention time just from the molecular stucture.

I suggest to have a look at BMC Bioinformatics 2006, 7, 234. The article can be find at http://www.biomedcentral.com/content/pd ... -7-234.pdf and normally is open access for everybody but if not I can send a pdf if you contact me directly...

Kostas

How is the ion mobility interface different from other interfaces. In particularm what are the benefits.

Thanks
Adam

Adam,

Ion mobility could be used as an additional to the LC separation tool (in the gas phase). The Waters ion mobility resolving power/resolution is about 10 which might not offer too much separation but still usefull if you are doing structural studies with the MS or as I mentioned before could be used as an additional filter due to the direct correlation of the ion mobility flying time of ions to their cross-section area (before I said "molecular volume"...).


To put things in to perspective our in house, home build, ion mobility provides a resolving power/resolution of 120 but it uses a 2.5m flying tube while the waters one with only 20 cm they achieve about 10. The addition of the ion mobility might impact the overall sensitivity which we came around it in a certain way. I do not know how the Waters deal with it (maybe 20 cm is too short to be a problem) but I give them credit for introducing the first commercial ion mobility-TOF instrument (hopefully I am not overlooking someone else...).