Development of Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Coupled to Quadrupole-Orbitrap High Resolution Mass Spectrometry for Simultaneous Determination of Aflatoxin B 1 , B 2 , G 1 , G 2 , and Ochratoxin A in Snus, a Smokeless Tobacco Product.

The combination of multiple heart-cutting two-dimensional liquid chromatography (MHC-LC/LC) and quadrupole-orbitrap high-resolution mass spectrometry (HRMS) for simultaneous determination of the aflatoxins and ochratoxin A in snus is presented in this work. A C18 capillary column was used as the first dimension (1D) to isolate the aflatoxins and ochratoxin A from the complex matrices; then, a 2-position/10-port high-pressure valve equipped with two 60 μL loops was employed to transfer the heart-cuts of 1D-LC into a pentafluorophenyl (PFP) column for the second dimension (2D) separation. With the better separation of the MHC-LC/LC system, the sensitivity of the method was improved, which is essential for the trace mycotoxins analysis. Furthermore, HRMS performed in parallel reaction monitoring mode was employed to eliminate the interferences, and the sample pretreatment procedure was simplified. A new approach utilizing ethyl acetate with 1% formic acid/water solution was adopted to extract aflatoxins and ochratoxin A in snus, which provided parallel recoveries for aflatoxins and ochratoxin A with higher responses in comparison with the QuEChERS method. A dynamic range between 0.2 and 20 μg/kg was achieved with LOQs of 0.05 μg/kg for aflatoxin B ₁ , 0.1 μg/kg for aflatoxin B ₂ , G ₁ , G ₂ , and 1.0 μg/kg for ochratoxin A in dry mass of product. The results revealed that the established method exhibited good repeatability and recovery and could be used as a rapid and reliable approach for routine analysis of aflatoxins and ochratoxin A in snus.