A facile and sensitive mass spectrometric method has been developed for the dereplication of natural products. The method provides information about the molecular formula and substructure of a precursor molecule and its fragments, which are invaluable aids in dereplication of natural products at their early stages of purification and characterization. Collision-induced MS/MS technique is used to fragment a precursor ion into several product ions, and individual product ions are selected and subjected to collision-induced MS/MS/MS analysis. This method enables the identification of the fragmentation pathway of a precursor molecule from its first-generation fragments (MS/MS), through to the nth generation product ions (MSn). It also allows for the identification of the corresponding neutral products released (neutral losses). Elements used in the molecular formula analysis include C, H, N, O, and S, as most natural products are constituted by these five elements. High-resolution mass separation and accurate mass measurements afforded the unique identification of molecular formula of small neutral products. Through sequential add-up of the molecular formulas of the small neutral products, the molecular formula of the precursor ion and its productions were uniquely determined. The molecular formula of the precursor molecule was then reversely used to identify or confirm the molecular formula of the neutral products and that of the productions. The molecular formula of the neutral fragments allowed for the identification of substructures, leading to a rapid and efficient characterization of precursor natural product. The method was applied to paclitaxel (C47H51NO14; 853 amu) to identify its molecular formula and its substructures, and to characterize its potential fragmentation pathways. The method was further validated by correctly identifying the molecular formula of minocycline (C23H27N3O7; 457 amu) and piperacillin (C23H27N5O7S; 517 amu).

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Automated software was developed to analyze the molecular formula of organic molecules and peptides based on high-resolution MS/MS spectroscopic data. The software was validated with 96 compounds including a few small peptides in the mass range of 138-1569 Da containing the elements carbon, hydrogen, nitrogen and oxygen. A Micromass Waters Q-TOF Ultima Global mass spectrometer was used to measure the molecular masses of precursor and fragment ions. Our software assigned correct molecular formulas for 91 compounds, incorrect molecular formulas for 3 compounds, and no molecular formula for 2 compounds. The obtained 95% success rate indicates high reliability of the software. The mass accuracy of the precursor ion and the fragment ions, which is critical for the success of the analysis, was high, i.e. the accuracy and the precision of 850 data were 0.0012 Da and 0.0016 Da, respectively. For the precursor and fragment ions below 500 Da, 60% and 90% of the data showed accuracy within #0.001 Da and #0.002 Da, respectively. The precursor and fragment ions above 500 Da showed slightly lower accuracy, i.e. 40% and 70% of them showed accuracy within #0.001 Da and #0.002 Da, respectively. The molecular formulas of the precursor and the fragments were further used to analyze possible mass spectrometric fragmentation pathways, which would be a powerful tool in structural analysis and identification of small molecules. The method is valuable in the rapid screening and identification of small molecules such as the dereplication of natural products, characterization of drug metabolites, and identification of small peptide fragments in proteomics. The analysis was also extended to compounds that contain a chlorine or bromine atom.