Hello.

My answer is entirely based on the assumption you're using ESI as ionization technique.

Be very careful when using data bases to obtain qualitative info about LC-MS. All the chromatographic conditions should be exactly the same and the same instruments involved. Furthermore, you always have the risk of forming aductors of other species (Na, K, NH4, Cl, OH, etc), and the identification is entirely based on the ion map of your chromatogram. It is perfectly possible that two different peaks have the same m/z (as for position isomers for example) and, depending on the chromatographic conditions, are identified as the same compound in different data bases.

I would advise you to get the pure standards for your target compounds and run standard solutions with the same matrix to base your analysis. You will have the retention times for your system, and with the Orbitrap (HRMS) plenty of qualitative info to conclude about real samples.

Good luck.

Hello.

My answer is entirely based on the assumption you're using ESI as ionization technique.

Be very careful when using data bases to obtain qualitative info about LC-MS. All the chromatographic conditions should be exactly the same and the same instruments involved. Furthermore, you always have the risk of forming aductors of other species (Na, K, NH4, Cl, OH, etc), and the identification is entirely based on the ion map of your chromatogram. It is perfectly possible that two different peaks have the same m/z (as for position isomers for example) and, depending on the chromatographic conditions, are identified as the same compound in different data bases.

I would advise you to get the pure standards for your target compounds and run standard solutions with the same matrix to base your analysis. You will have the retention times for your system, and with the Orbitrap (HRMS) plenty of qualitative info to conclude about real samples.

Good luck.

Dear Ambika,

When you perform LC-MS analysis, you are using different, orthogonal properties of the analyte to increase the chances of accurate identification. The retention time of an analyte is a property of is physico-chemical characteristics and its interaction with the stationary and mobile phases, where as the m/z of the analyte is a property of the mass and its ability to acquire/retain charge.

As Mário Silva mentioned, trying to match retention time of an analyte from your method to that of a database can be misleading if exactly the same LC and chromatographic conditions are not used. Since you are using a HRMS system, it should give you a much higher specificity on the m/z of the analyte and that should help mitigate the redundancy in retention time. Using m/z as the primary identifier, followed by retention time as the secondary/confirmatory identifier may be useful.

Another way to decode the retention time mystery might be to look at the pattern of other analytes in your sample and their elution pattern. If you are using the similar type of column (for eg any C18 column) and same type of mobile phases (for eg Water/ACN) as any of the database methods, the sequence in which the analytes will elute will be the same. The exact retention time may differ, but the sequence should not. Additionally, if you know exactly the analyte you are interested in, you can perform MS/MS or MSe to get a low energy and high energy spectrum and compare that to the database. The probability of 2 compounds having the same precursor m/z and the same product ion pattern is pretty low, unless its an isomer/chiral compound.

Develop your knowledge in area of mass spectrometry and isotopic chemistry and then you can see your compound looking on masspecta.

Mário Silva Thank you for you reply, your assumption was correct we did ionization with positive ESI. Thank you for the answer and suggestion to use standard and to compare the retention time.

Nikunj Tanna Karen A. Darbinyan : Thank you for your comments and suggestion as Karen A. Darbinyan suggested i think i need to learn more basics to understand about the chemical and isotopic chemistry well then i can solve the problem for future.

Thank you everyone for your support

Have a great day

This is not an easy issue. With such a sensitive instrument, it's not surprising to have several compounds in each peak. Also based only on parent accurate mass, you can see the same parent mass with different retention time, they are simply isomers. You need to do data-dependent MS2 scan to help database search. Once you get putative identity of some compounds, using standards to confirm your identification.

Zhiqian Liu : Thank you for your suggestion. As far as comparing with standard is concerned I did that and I was able to interpret the compounds. Just I was concerned with the multiple hit.

Thank you once again