I have a fungal isolate of which I need to do the secondary metabolite profiling. Which of the two analytical techniques should I go for - HPLC or LC-MS? Should I directly go for the LC-MS of the culture filtrate, or should I get the HPLC analysis done first?
I am not sure what is meant by "culture filtrate". We use EtOAc extracts to examine the secondary metabolites.
We routinely use HPLC-PDA-ESIMS for both our initial fungal and bacterial crude extracts. The samples are often submitted at 20 mg/ml in 90:10 MeOH:H2O with an LC protocol of 10-100% acetonitrile over 15 minutes. For our pipeline, interesting crude extracts from fungi and bacteria are identified from bioactivity assays and subsequently analyzed with LCMS (using the exact extract that gave the positive bioactive result). The LCMS results, coupled with bioactivities, are used for dereplication to ensure that we are not wasting our time on known compounds.
I wish you the best of luck.
It depends on what you're aims are. If you want to know the number of compounds I suggest you start with thin layer chromatography using a silica gel plate and at least a polar and a non polar solvent system. If you have bioactive compounds you can also carry out a bioautogram to identify the active spot(s). The next step is deciding how you will treat your filtrate depending on the polarity of the compounds you are interested in. Extraction with a non polar water-immiscible solvent is best for non polar compounds or ion exchange for a polar charged molecule.
HPLC analysis coupled with a 1. UV-Vis or 2. PDA or 3. MS will tell you 1. The number of compounds that elute from your sample using your particular solvent system; 2. The UV -VIS profile of each of the eluting peaks; and 3. The molecular wt (ion) of each of the eluting peaks. Then you need access to a good database that will allow you to decipher the UV-Vis or Mol wt data.
Good luck,
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