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Introduction The in-depth characterization of biopharmaceuticals and the analysis of impurities in drug product formulations are from increasing interest in the pharmaceutical environment.The analysis of biopharmaceuticals like monoclonal antibodies(mAbs)requires an analytical tool with highest separation power due to the complex nature of the sample.Furthermore,the analysis of impurities in pharmaceutical drugs is of high importance to define the safety of the final product.For a reliable impurity profiling,selective and sensitive analytical methods are needed to separate co-eluting compounds and detect low level impurities.Comprehensive and(multiple)heart-cutting 2D-LC is a powerful analytical tool for both tasks.This poster will describe the use of a single 2D-LC platform for the analysis of a complex mAbs sample and of impurities in pharmaceutical substances.Methods For the characterization of mAbs,an antibody was digested analyzed using comprehensive 2D-LC coupled to Q-TOF LC/MS.For the 2D-LC analysis HILIC was used as first dimension and RPLC for the second dimension.For impurity analysis,2D-LC equipped with a DAD was used in Multiple Heart-Cutting(MHC)mode with two different pharmaceutical compounds.For both compounds RPLC was used as first dimension and for the second dimension a chiral column or RP column were used.Results During the comprehensive 2D-LC analysis of mAbs,the combination of HILIC and RPLC provided good orthogonality.The effluent of the first dimension was split.One part was used to acquire a UV-chromatogram and the second part was transferred to the second dimension.Due to reduced volume entering the second dimension,good retention on the RP column was obtained.With this setup a comprehensive picture of the mAb digest was obtained.To enable an impurity analysis with simultaneous determination of the enantiomeric composition,Thalidomide was analyzed using 2D-LC.RPLC in the first dimension resulted in a good separation of Thalidomide from the impurities and with a chiral column in the second dimension a separation of the Thalidomide enantiomers were possible.However,in comparison to the peptide mapping,where the whole effluent from the first dimension was transferred to the second dimension only the Thalidomide peak was transferred to the second dimension(heart-cutting mode).For a second impurity analysis,a pharmaceutical compound with several impurities was analyzed using multiple heartcutting mode.This mode enabled the storage of multiple peaks from the first dimension in loops and sequential analysis in the second dimension.The second dimension revealed three co-eluting impurities with the main compound.Novel Aspect With the described setup a comprehensive analysis and profiling of impurities on a single 2D-LC platform is possible.