Thanks for getting back to me. I am using an untargetted metabolomic approach to begin with. Molecules of interest include amino acids, sugars and phenolic/aromatic compounds. These are what i believe to be in the extraction solvent (from preliminary NMR analysis-but as we know the relative sensitivity and resolution of UPLC-MS compared with NMR is a lot greater-so generally all polar molecules are my primary interest). I am undertaking a 2 stage extraction. One to extract all the polar metabolites and stage two to look at the fatty acid methyl esters (using GC-MS which works great). The primary extraction would ideally be split into two, one for NMR analysis and one to be run on our qTOF. Such that the methodologies can be directly compared for the same samples. I am hoping that this will help increase the predictability of my model when all the data is combined into one.
I have been told about using a HILIC column for this analysis but from reading the literature i went ahead with a HSS T3 (most widely used?). Jules any help with the chromatography side would be great. Is there a specific type of HILIC column you would suggest? The selection available is quite vast.
I am in need of a little advice/help. I am currently undertaking a metabolomic study on brain tissue (analysing the polar metabolome) using UPLC-qTOF-MS and I think that I am struggling with the choice of column or the extraction procedure.
The extraction procedures that I have used thus far include a standard perchloric acid extraction (0.6 M extraction using 5 ml/g tissue (approx 0.3 g tissue), neutarlised to pH 7 using K2CO3-which works perfectly for NMR analysis), 50 % methanol (which I have seen reported for analysing liver-I used 100 mg sample) and an ice cold acetone extraction (100 mg sample). For the three extractions I have seen relatively nothing using the first two methods and a small number of metabolites using the ice cold acetone extraction but nowhere near what I expect. Can anyone suggest a better form of extraction and maybe clean up prior to analysis?
I am currently running the samples on an Acquity UPLC coupled with a Xevo G2 qTOF mass spectrometer using an Acquity HSS T3 1.8 um, 2.1 x 150 mm column. Are the compounds too polar and shooting straight through the column? Can anyone suggest another column for such an experiment if this is the case?
