This is a very involved or a very simple question.

If you know the expected mass of your purified molecule, you can calculate its m/z for different charges (z). If the LC-MS spectrum contains the respective masses, you may have found the molecule.

If you triggered MS/MS fragmentation analysis for those masses than you may have a fragmentation spectrum of the precursor (your molecule). Of course, only, if the molecule fragments under your conditions.

From a crude extract you can usually program the instrument to look at a particular mass or a select number of masses (SRM, MRM).

If you have problems in the data analysis part, refer to OpenMS.

Dear Dr. Allmer,

As you have mentioned, i can understand your answer for a known compound. But i would like to identify the mass of unknown molecule from a purified and from a crude extract. I am not able to identify which peak is major component peak or which one is fragment peak from an impure compound mixture.

Hi Iniyan,

1. If you want to know the molecular ion (m/z), you should employ a soft ionisation technique such as APCI or ESI MS method for the first MS. This would provide you molecular ion (m/z) most of the time. However, this may not provide a good fragmentation pattern.

2. Using a tandem mass spectrometry, employ, say a second MS with Electron Impact (EI) mode to generate a good fragmentation pattern (which may not provide you the m/z as this is a strong ionisation technique) to figure out what the compound is.

I assume you are talking about unknown molecules in crude extracts?

The only safe way to get the mass for an unknown (bioactive) compound is to purify that compound and measure it again. This will give you in most cases when using soft ionization techniques the required mass [M+H]+. Once you have this mass you can search your crude extract (should appear at the same retention time when same LC-Gradient has been used).

If you see a UV and no mass the detection range might not cover the mass of the molecule.

Sometimes molecules fragment even when soft ionization techniques has been used (loss of carbohyfrates, peptide fragmentation e.g.) try to use negative ionization in addition. UV and a mass +1 and -1 at the same retention time sometimes make things more clear.

Use different techniques like NMR to make sure your compound is really pure, a proton spectra is very useful in that case.

If you dont have NMR available use different HPLC columns (or separation techniques at the purification step) to make sure no compounds co-elute with your "pure" substance and give you the wrong mass.

Never trust mass-spec allone, only a full characterisation of the structure of the compound (NMR, crystalisation) will give you the ultimate proof that you are not running after something wrong and that the measured mass really fits to your molecule.

Yes, I agree with Stephan. If you have an unknown molecule, you need to predict the structure first with NMR. Try both ionisation methods , +ve and -ve, as we do not know for sure the nature of your compounds. For example alkaloids, usually gives better M+H. But better to run both ok.

If you want to just have a guess of what you might have from the crude: preliminary knowledge of what you may have, then run LC MS-MS. From fragmentation patterns one may guess the type of compounds. But of course, do a literature search first on the kind of compounds your plant or sample has. Check from species to genus.

All the best.

I agree with Khalijah and with Stephan. But if don't have access to NMR, then use the LC to optimize a separation where you obtain resolved peaks. Run then ve+ and ve- and use the MS/MS; the second MS with a ramp of 10 up to 40. Locate the peak in the TIC with the first MS (low voltage), you will see there the M-H and M+H.

To be able to figure out the corresponding molecular formula (and perhaps molecular structure) on a unknown compound in a mixture you will need to use HRMS, like TOF or Orbitrap-technology. Using only MS or MS/MS is to crude (you have too low resolution) to reliable find out what a unknown compound is without access to a reference compound.

NMR is a good technique for analyzing unknown compounds but it is limited in that it usually needs a bit larger amounts, in microgram-scale at least, and it should be 95%+ pure unless you want to make your work harder than necessary.

In LC mass case, you will always get a peak fro dimer of your compound. Usually this peak is minor compared to your base peak. But in some cases (like I encountered), the dimer peak is way intense than your molecular ion peak. So I agree with all the above suggestions.

First have pure compound then have atleast two techniques (NMR and HRMS) to know the structure of your compound.

I am agreed with Stephan. Identification a compound is  a very difficult task by relying only on LCMS data. 

In addition to the above answers, you may maximise your chances of identifying whether a peak in your spectrum corresponds to your analyte by increasing the amount of qualitative information you acquire in the LC-MS run. There are a number of ways to do this.

MS analysis - As previously mentioned, using an MS instrument which allows you to toggle between +ve and -ve polarity modes may help define the peak (although not necessarily since the compound will preferentially ionise according to the heteroatoms in it's structure). If you can do this on an HRMS instrument, you may also obtain an empirical formula for your peak, increasing the confidence in the assignment. You should also obtain fragmentation as well as molecular ion data. Each of these conditions can be set up within a single MS acquisition on certain MS instruments.

Secondary analysis/collection - Another option is to pass your LC eluent across a secondary detector (UV, ELSD, CLND) and then split your LC flow between the MS and a secondary fraction collector, meaning that you will collect the peak of interest for further analysis (usually by nanospray if you have that inlet).

Each of the above can be configured within a single LC-MS run, meaning you have a much more information-rich dataset to base your decisions upon (+ve/-ve ion data with accurate mass, molecular ion and fragmentation data, secondary analog data and have the fraction of interest concentrated and 'purified' for subsequent analysis).

These are all system that can easily be 'bolted on' to your single LC-MS analysis and have been used routinely used in qualitative analyses.

Is there anyone who can interpret my LCMS and NMR data ? i need some ideas regarding the compound . Can any expert help in this regard?

The answers given above are all relevant because the crude extract contains a lot of things so the purification should be done and follow the protocol for structural elucidation. For the (M+H) can be obtained from HRESIMS. mostly it is the most intense peak if the molecule is well purified. but branching and homologus series have effect on the it.

With the fragmentation of peaks in LC-MS/MS , one can identify the molecule as per m/z ratios...

Any online software is available to identify the changes/ modification in known compounds with LC/MS mass /peaks.

Ragunathan S: are you looking for this ? http://www.ymdb.ca/spectra/ms/search