For non-targeted analysis, you'll want to start with just the LC-TOF analysis, using high resolution and exact mass data to identify compositions for candidate compounds. Of course, data mining can be tricky for complex samples, and there are tricks using mass defects to look for compounds with compositions that differ from the background. MS/MS would then be used to obtain structural information for candidate compounds of interest. If you expect families of compounds with related substructures, you can use precursor scans or neutral loss scans. Selected reaction monitoring is generally reserved for quantitative analysis of targeted compounds.

Sample prep. must be the first step to consider for untargeted analysis. Depending on the sample matrix and compound class of interest, several approaches could be selected. I can suggest a very common method; chloroform-MeOH-water tertiary extraction system you may find as Folch method. This will enable to extraction of different classes of compounds from lipids to proteins, from salts to sugars and etc..

Selecting the chromatographic conditions and accurate consumables for lipidome profile, protein/peptide profile and polar metabolite (water-soluble) profile is also critical. Both RP (separated columns could be needed such as C8 for lipidome, C18 for mid polar compounds) and HILIC conditions (amide bonded for organic acids and carbohydrates, etc) must be set for orthogonal coverage of the whole wastewater.

Mass spec is the biggest part of it for sure. Both negative and positive ESI data should be acquired and mobile phase additives buffers must be chosen regarding the ionization states. A couple of injections will give hints about the MS conditions like desolvation gas flows temperatures, cone/capillary voltages, etc. We can also further discuss the MS/MS options, molecular identification tools, and software options, statistical evaluations to discriminate the marker compounds.

Thank you so much I will review your comment in detail and will reply accordingly very useful!!

Your suspect/NTS approach should be very much dependent on the question and answer you would like to get. We put some thoughts and suggestions here Article The strength in numbers: comprehensive characterization of h...

Thank you very much I have downloaded this paper and going to go through it. Thank you for your support!!!

I would like to know more about sample preparation for wastewater samples analysing with LC-MS - TOF non targeted approach. Some Instruments has in-built SPE automatization but others don't. So, in literature, sometimes SPE is missed when describing sample preparation. I would be glad to hear some suggestions if you can share some knowledge with me? thank you

There is a number of papers that could be of use. Please look into papers from for example Juliane Hollender or Nikos Thomaidis research groups. This will give you some ideas.