HPLC/MS/MS can be done for both known and unknown compounds. For the automated MS/MS analysis of unknown compounds, several of our LC-MS systems have a powerful software control system to enable the instrument to perform data dependent acquisition (DDA), switching from the MS to MS/MS mode and then returning to the MS mode using data dependent criteria.
This method obviates the need to analyze the sample in MS mode to identify the target precursor ions and then re-run the sample in MS/MS mode to acquire the MS/MS data from each of these precursors. This is particularly valuable in the analysis of unknown samples using on-line chromatography where the target precursor ions and their retention times may be different for each sample.