You can always try to improve the chromatographic separation to see if there are any coeluting compounds. Violacein is indole base pigment maybe simple chromatograph separation with 580 nm detection will give you an idea if there is violacein in Your extract.

However, if the molecular ion with the same retention time gives different fragmentation pattern is probably a different compound.

In order to have a precise confirmation you would need an MS/MS to determine the structure of 'daughters' or MRM (multiple reaction mode). Alternatively, maybe an FTIR scan if you have enough sample.

Mr Adam Zmysłowski , actually previously I had some problems separating properly the compounds of the extract so it could likely still be an issue. Regarding the fragmentation pattern, it seems that the molecule couldn't be properly fragmented so I was unsure of the results, expecially because in the previous full scan proviolacein was decteted. Mr Bruce Neagle and Mr Adam Zmysłowski thank you both for kindly giving me those suggestions, hopefully with your help I'll have clearer results.

What does it means that the molecule couldn't be properly fragmented? Is there a molecular ion from violacein present in your sample? If I am correct the previolacein differs from violacein in double bond position so it should be separated in chromatography. Additionally fragmentation pattern should be different, maybe not in case of fragments but in fragment intensity.

chromatographic separation to see if there are any coeluting compounds

Mr. Adam Zmysłowski I meant that my molecule seems to be a" compact block", That's why I was courious to ask : How does coeluting modify the fragmentation pattern? Regarding violacein, my very first analysis has been a full scan and, among others compounds, violacein and proviolacein molecular ions were dected. Proviolacein detection "reassured " me of violacein presence since it's a precursor, that's why I was surprised with the results of the analysis with the standard. I hope my explanation is clear enough, I'm a complete novice in this field so I apologize If it isn't.

MS is 'just' a detector. It is the chromatography (the column and mobile phases) that separates the molecules and prevents co-elution. If co-elution is present you can obtain multiple fragments for that time and thus make the 'wrong' identification of the molecule. MS cannot tell you if an enzyme is active or denatured since both molecules have the same atomic weight.

MSD (mass selective detector) was developed to detect and quantify molecules that have no UV spectrum like ions.

Hi Elisa,

do you have the possibility to record NMR spectra? If yes, then record NMR spectra from your compounds. If you have very little substance, you can use a microprobe head. We do this routinely in our laboratory to validate the structure of purified compounds and it works very well.

Regards

Markus

Using MS scan in order to determinate the presence of a substance, especially in low concentration is very difficult and in most cases inaccurate. Such analysis should be executed in MS/MS mode, where molecular ion is fragmented for fragmentation pattern which can be compared with the pattern of the standard. It seems to me that Your equipment is single quadrupole and You are able to make MS scan or SIM, which in this case the chromatographic separation in order to achieve a single peak of violacein is essential. Changing the mobile phase composition (formic acid, acetic acid, ammonium formate etc.), pH of the mobile phase, the column selectivity or brand is in my opinion way to go. The violacein is mostly aromatic compound, which using chromatographic columns which aromatic stationary phase (e.g. phe-hex) should give good results.

by applying collision energy in MRM scan mode you will get fragments of your compound. you may go for NMR also.

Mr Adam Zmysłowski thank you very much for your help, I'll try to improve my chromatography separation changing my mobile phase as You suggested, If it won't work I'll go for the column. Mr Sharad Medhe and Mr Markus Christ thank you for answering my question, unfortunately I can't use NMR spectroscopy, but maybe I could try Ms/Ms. Mr Bruce Neagle thank you, your explanation was very clear.

I'm really grateful for all your answers, they really helped me a lot to understand this field better and where to focus to improve my work.