LTQ Orbitrap进行未知化合物的表征

前言：

To show a workflow for the characterization of an unknown compound (compound X), compound X was provided and analyzed qualitatively and quantitatively. The following steps were taken to provide data:

• Accurately determine the mass of compound X and determine sum formulae proposals without any knowledge about the compound prior to mass analysis

• Exclude false positive hits by comparing the proposed sum formulae to the theoretical isotope patterns without any knowledge about the compound prior to mass analysis

• Confirm the correct hit by the Ring Double Bond equivalent value (RDB equivalent value)

• Investigate and confirm the compound’s identity further by MS/MS using CID and HCD by gaining structural information

• Carry out a quantitative analysis by means of dilution series of compound X.

The study follows an analytical approach in which the compound was unknown in the beginning; subsequently it was identified by accurate MS and MS/MS data and finally quantitatively analyzed down to sub pg loads onto column.

仪器：LTQ Orbitrap

结论：

• An accurate mass measurement of compound X combined with RDBE evaluation and isotope pattern recognition revealed a sum formula C19H22O4N1S2 for compound X.

• An Internet accessible database search using the previously determined sum formula revealed Tiotropium as single hit.

• MS/MS information obtained by Collision Induced Dissociation (CID) and Higher Energy C-trap Dissociation (HCD) combined with the fragmentation rules implemented in Mass Frontier confirmed Tiotropium. CID and HCD fragments are assigned by Mass Frontier.

• A dilution series - applying a targeted MS/MS @ m/z 392 method detecting the fragments at m/z 152 and m/z 170, respectively, in the ion trap or the Orbitrap analyzer - revealed that reliable calibration curves can be acquired down to total column load of 200 fg of Tiotropium. This is equivalent to a 20 μL full loop load of 10 fg/μL solution of Tiotropium.