Chromatography Forum

Hey, im doing my masters with GCMS and LCMS...i was talking with the so called experts here in the university and he told me LCMS is new technology and due to compounds having different intereactions with moblie phase it is near impossible to predict identify the compound unless it is in the libary!!...is this true??

Look at the information available to you from the LC/MS.

From the LC, you have a measure of the partitioning between two phases. From LC/MS you have a molecuar ion - or adduct - or in some cases fragment. Assuming you can know that you have a molecuar ion, you have a molecuar weight and the isotope cluster to give you and idea of what the molecuar formula would be. And there are likely to be several possibilities, depending on the mass accuracy and accuracy in determining ion ratios of your instrument. You know that the molecule is one that will ionize under the conditions you are using and the partition characteristics give you some information about the solution chemistry. From there to an assignment of identity typically takes more information.

With MS/MS you can get some idea of how a molecule can be broken apart. The fragmentation will give some clues, but these will only take you so far. If you slip a UV detector into the path, you can tell if there is a chormophore present.

It is all about how much information you have and howmuch you need to solve a strcuture.

(1) Just take a very simple example: a disaccharide of two glucoses. Each glucose has 5 hydroxyl groups any of which could (theoretically) be attached to any of the hydroxyl groups on the other glucose, so there are a phenomenal number of isomers. All the isomers have the same empirical formula, so they will also all have exactly the same exact mass and isotope pattern. In LCMS collision-induced dissociation fragmentation, they will break along the glycosidic bond. They'll probably have almost the same MS2 spectrum. Even with a library, they'll be almost indistinguishable! They'll also probably be hard to separate chromatographically.

(2) There are good theoretical reasons why LC-MS is more difficult than GC-MS. If you use EI to create fragments in GC-MS, you put a lot of energy in, resulting in rapid fragmentation by simple bond breakage. The precursor ion doesn't have to be in any particular conformation, and the reactions are simple and local (and well-described in text-books). CID in LC-MS is a very low energy technique, and relies on low-energy fragmentation reactions, frequently things that rely on a particular conformation of precursor ion, so that reactive groups in the molecule are appropriately placed. This is rarer, and that's why these reactions don't happen (much) in EI (and it's also why ion traps are so good at fragmentation, because they have all the time in the world to let the pseudomolecular ion fragment). The result is that LC-MS fragmentations often happen by complex and very unique mechanisms, and can involve major internal rearrangements of the molecule. This makes it harder to work out what bit was originally attached to what other bit.

(3) I sympathise with your expert. Grant applications have to look really positive, and there are many people writing applications promising to identify novel chemicals present at low abundance in samples by using high-tech LC-MS. The mass spec companies want to sell instruments, so they'll happily exaggerate the capabilities of their instrument and software. You can't publish a paper titled "MS isn't good enough to identify simple flavonoids", but you can easily publish a paper called "Application of photon-torpedo-induced dissociation spectra from LC-Megatron-MS to structural identification of novel antibiotics". The result is that some poor expert is having to live up to vastly over-blown expectations, and will occasionally rebel by pointing out the bottom line: to identify most reasonably-complex organic molecules beyond doubt, you will need NMR. To identify simple molecules, you will need comparison to an authentic standard (and a library search is basically a comparison to a standard where someone else collected the data for the standard).

(4) I also have sympathy with you, and the situation might not be as bad as your expert describes. If you have an idea of what you might be looking for, from literature/experts/wikipedia/previous experiments etc., MS evidence may be enough to tell you which peaks correspond to which likely chemical in your sample. Your expert may be trying to shock you into doing some literature searching before expecting him/her to identify everything from scratch...

lmh,
what is "glucoses"?
NMR also has its problems, to identify some compounds one may need much more than NMR and MS. Obviously, though, both are fantastic tools, take a look at some work before MS and NMR came into use.

I meant a simple disaccharide formed by condensation of two molecules of glucose, however achieved. Thanks for drawing attention to work before NMR and MS. I am full of admiration for people who worked back then. When I look at classics like Harborne's flavonoids, and the detailed chemistry that was necessary to distinguish all these (fairly complex!) natural products, I realise how shallow I am now, and how easy we have life!

I personally won't promise to identify an unknown chemical by LC-MS, because I know I will probably fail. If I succeed, it will probably involve a certain amount of guesswork and reliance on knowing what sorts of things I ought to see. It can also be depressing: I shall never forget struggling to identify a peak by every means I could; having guessed what it might be, I obtained an authentic commerical standard, ran that, compared MS2 and retention time, got a perfect match, and extatically rushed off to tell my client. They replied: "Oh, yeah, we knew it contained that. We wanted you to find out what else it contains?"

I think I should jump on this.

IMO, your expert are both right or wrong depending on what MS technique and what kinds of compounds he refers to and , so never underestimate an experienced person's capability.

Below is cited from the final report of 2009 ASMS metabolomics workshop. It doesn't necessarily represent my personal opinion.

"Another audience member echoed a similar statement. He noted that in biological samples,
rarely does one have enough material to isolate for NMR determination. However, by using a
combination of mass spec techniques, such as LC and GC, in conjunction with other treatments, such as HD exchange, one can elucidate a structure using just mass spectrometry. However, the incompatibility of different vendors’ data file formats creates a “Tower of Babel.”

For purposes of structure elucidation, an audience member stressed the importance of
distinguishing between a truly novel, unknown compound versus one that is unknown to you. In the case of the former, using just mass spectrometry alone will likely not result in success. However, in the case of the latter, then one has a chance of being successful."

Hey; whay about QToF? you can get the molecular formula with a very high confidence; beside; depending of the purity of your compound you could fragmente your analyte in order to get diferente pieces that help you to elucidate the structure. I have not worked with ToF but as far as I have read this is possible.

In QTOF you have the issue of collision energy. So make several runs at varying collision energies and scan with the quad, collect everthing with the TOF - you will have A LOT of data. Now the trick is to transform that data into information. While you have a lot of information on the fragmentation of the molecule, you have the issue of low energy fragmentation in the mix.

One thing that sometimes gets forgotten, is that solutions of structure by mass spec require that you have enough information from the fragments that retain the charge. In some molecules, a portion of the molecue makes a stable ion, with the remainder being lost as a neutral fragment. You may know how big the fragment is by looking at smaller ions and determing the mass difference. And with accurate mass, you may even be able to come up with a formula for the fragment. There may be several arrangements of those atoms that all would have the property that they would be an easily lost neutral fragment - and with the mass spec, you've hit a dead end.

I am waiting for the LC/NMR that will handle picogram quantities of analyte

I'm even sceptical about being sure of the empirical formula, armed with a typical Q-ToF and a target analyte of 1500Da. By the time you get to this sort of mass (which is fairly common for a complex natural product) there will typically be a number of empirical formulae that differ only slightly in mass, particularly if elements apart from Na/C/H/O/N might be present (and not all other elements have nice isotope peaks for recognition!). Don is also quite right in emphasising which part of the molecule retains charge. A large neutral leaving group can be a big problem to elucidate.

Hi, IMH,

I agree with you the larger the molecular mass, the more possibility of elemental composition. And it all depends on the resolution and accuracy of the mass spectrometer. We also know there is no single technique can solve all the problems, like there is no single ionization tech can ionize all the compounds.

If anyone wants to look into this, I suggest you do some first-hand trials here ( I have no any affiliation with this web). You will see what you may not be able to materialize in mind.

http://maltese.dbs.aber.ac.uk:8888/hrmet/search/gr.html

As I mentioned in above post, if the compound is not a novel one, you have a great chance to figure out what it is with isotope pattern, and more, fragmentation pattern. It's also based on the assumption that you can find the right molecular ions (of course there are some tricks helping you find the right one)

At least some preliminary structural information will make subsequent NMR much more easier.